12th Congress of the European Hematology ... - Haematologica

that the incidence of JAK2 mutation in Asian ET patients is lower than
in Caucasian ET patients. Their disease profile may be similar to that of
their wild-type counterparts and different from the Caucasian patients.
0947
THE PREPARATION OF MONOCLONAL IMMUNOGLOBULIN LOADED DENDRITIC CELLS
VACCINE FOR MYELOMA PATIENTS UNDER GMP CONDITIONS: PRECLINICAL AND FIRST
CLINICAL RESULTS OF A PHASE I/II CLINICAL TRIAL
D. Ocadlikova, 1 L. Zahradova, 2 L. Kovarova, 1 J. Smejkalova, 1 L. Pour, 2
P. Vidlakova, 1 D. Kyjovska, 1 A. Stejskalova, 1 H. Novotna, 3 M. Penka, 4
J. Michalek, 1 R. Hajek2 1 2 Cell Immunotherapy Center, BRNO; Department of Hematooncology, BRNO;
3 4 Department of Clinical Biochemistry, BRNO; Department of Clinical Hematology,
BRNO, Czech Republic
Background: Adjuvant immunotherapy with antigen-loaded dendritic
cells (DCs) represents a novel and relatively non-toxic treatment modality
for multiple myeloma (MM). Malignant cells in MM produce a monoclonal
immunoglobulin (idiotypic protein) which is considered a tumorspecific
antigen and can be used for the induction of T lymphocytes. To
enhance the anti-myeloma immune response, the idiotypic protein (Idprotein;
Idiotype) can be loaded into autologous DCs and used for vaccination.
Aims. The aim of this study was preclinical evaluation of to
evaluate DC-based vaccine and clinical demonstration of the safety and
immune response of the vaccine in patients with MM. Patients and Methods.
Pre-clinical testing was performed in 8 patients with MM. DC loaded
with autologous myeloma cells were used for autologous T cell stimulation
in vitro. After successful preclinical testing, we have vaccinated 4
patients with stable disease or asymptomatic slow progressive disease
after prior stem cell transplant (SCT) according to EBMT criteria. DC precursors
were isolated as an adherent fraction from peripheral blood of
the myeloma patients. DCs were prepared in vitro and loaded with Idprotein
under GMP conditions as previously described (Ocadlikova et al.
Med Oncol 2006, 23: 377-384). Patients were vaccinated every 4 weeks
subcutaneously with 6 doses, each containing 1,46-18,1×10 6 (mean
9,52×10 6 ) DCs. The immune response was evaluated by flow cytometry,
Elispot and the skin test of hypersensitivity. Results. IFN-gamma production
of T cells stimulated with autologous myeloma cell loaded DC
was observed. After successful pre-clinical testing a clinical phase I/II
trial was initiated. A total of 24 vaccines were applied to 4 patients so
far (January 2007). The viability, number and functional characteristics
of in vitro matured DCs loaded with Id-protein were satisfactory with
50,10-99,3% (mean 87,08%) of HLADR/CD86+ cells. Each vaccination
was well tolerated with only mild fever in 1 patient. No grade II-IV toxicity
appeared. The clinical trial is ongoing and a total of 12 patients is
planned to be enrolled. Conclusions. Feasibility and safety of vaccination
with Id-protein loaded autologous dendritic cells was proved in patients
with multiple myeloma.
Supported by IGA MZCR NR 8945-4.
0948
ISOLATION AND EXPANSION OF ALLOGENEIC MYELOMA-SPECIFIC T CELLS PRODUCING
INTERFERON-GAMMA
D. Ocadlikova, 1 L. Zahradova, 2 L. Kovarova, 1 A. Stejskalova, 1
M. Penka, 3 R. Hajek, 2 J. Michalek1 1 Cell Immunotherapy Center, BRNO; 2 Department of Hematooncology, BRNO;
3 Department of Clinical Hematology, BRNO, Czech Republic
Background. Multiple myeloma (MM) is a malignant disease characterized
by clonal proliferation of plasma cells in the bone marrow. Highdose
chemotherapy with autologous stem cell transplantation is recently
considered a standard therapy for myeloma. Despite of the high number
of complete remissions achievement is the median relapse-free survival
3 years and median overall survival of 4 to 5 years. Adoptive
immunotherapy is a promising approach in the treatment of multiple
myeloma in the last 20 years. Aims. The aim of this study was to identify
and characterize autologous myeloma-reactive T cells in vitro and to
evaluate their cytotoxic effect. Methods. Irradiated myeloma cell line
ARH 77 has been used as tumor antigen to stimulate allogeneic CD4 + and
CD8+ T lymphocytes. Peripheral blood mononuclear cells of 8 healthy
volunteers and 10 MM patients were used for repeated stimulation of T
lymphocytes. Activated myeloma-specific T cells that produced interferon-gamma
(IFN-γ) were isolated using immunomagnetic separation (Miltenyi
Biotech) and further expanded in vitro by phytohemaglutinin and
high concentrations of interleukin 2. Cytotoxicity against the original
myeloma cells has been tested after the T cell expansion with propidi-
12 th Congress of the European Hematology Association
um iodide or 7-amino actinomycin D. Activated T cells were labeled by
a 5-(6-) carboxyfluoresceine diacetate succinimidyl ester. Allogeneic T
cells and IFN-γ negative fraction of T cells served as controls. Results.
Myeloma-reactive IFN-γ positive T cells in healthy donors were enriched
from 2.83% (1.97%; 4.58%) to 48.57% (15.14%; 82.98%) and from
1.91% (1.14%; 3.4%) to 73.14% (3.9%; 88.75%) (median, min., max.)
for CD3 + CD4 + and CD3 + CD8 + T cells after immunomagnetic separation.
IFN-γ positive T cells were further expanded in vitro from 0.5×10 6
(0.5×10 6 ; 0.6×10 6 ) to 160×10 6 (150×10 6 , 420×10 6 ) (median, min., max.)
cells within 4 weeks. The percentage of killed ARH 77 myeloma cells
was 69.17% (38.04%; 78.23%) (median, min., max.). Cytotoxicity
against the third-party PBMC was negligible. IFN-γ negative fraction of
T cells demonstrated also negligible cytotoxicity against ARH 77 myeloma
cells. The percentage of myeloma-reactive IFN-γ positive cells in MM
patients was enriched from 1.12% (0.27%; 6.2%) to 7.85% (0.42%;
12.6%) and from 1.9% (0.37%; 14.4%) to 14.7% (1.28%; 71.4%) (median,
min., max.) for CD3 + CD4 + and CD3 + CD8 + T cells after immunomagnetic
separation. IFN-γ positive T cells were expanded in vitro from 0.12
×10 6 (0.05 ×10 6 ; 0.4 ×10 6 ) to 88.5 ×10 6 (35 ×10 6 ; 226 ×10 6 ) (median, min.,
max.) within 8-12 weeks. The percentage of killed autologous multiple
myeloma cells was 41,5% (15,6-60,5%) (median, min., max.). For the 2:1,
10:1 and 50:1 E:T ratios, the median killing of ARH 77 myeloma cells was
21.78%, 34.31% and 62.33% after 4 hours; 60.84%, 69.82%, and
73.49% after 24, and 57.75%, 71.85% and 59.69% after 48 hours, respectively.
Third-party reactivity of myeloma-reactive T cells from patients
with MM was negligible. Conclusion: This study demonstrates the feasibility
of identification and isolation of myeloma-specific T cells in
healthy donors as well as in patients with MM. Myeloma-specific T
cells can be further expanded and used as adoptive immunotherapy.
Supported by IGA MZCR NR 8945-4.
0949
ASSOCIATION OF ZAP-70 EXPRESSION WITH PLASMA LEVELS OF ANGIOGENIC
ACTIVATORS IN CHRONIC LYMPHOCYTIC LEUKEMIA
L. Smolej, C. Andrys, V. Vroblova, J. Novosad, D. Belada, P. Zak,
J. Krejsek, M. Hrudkova, O. Siroky, J. Maly
University Hospital and Medical School, HRADEC KRALOVE, Czech Republic
Background. Chronic lymphocytic leukaemia (CLL) is a disease with an
extremely variable clinical course. Several studies have shown that angiogenesis
is increased in CLL and may potentially serve as a new prognostic
factor. Zeta-associated protein of 70 kilodaltons (ZAP-70) is an intracellular
tyrosin kinase belonging to modern powerful prognostic markers
in CLL with significant impact on clinical course. Aims. to assess
potential relationship of ZAP-70 and angiogenic signaling in CLL. Methods.
We analyzed ZAP-70 expression using flow cytometry in CLL cells
from peripheral blood of 32 patients. Furthemore, we quantified plasma
concentrations of angiogenic activators (vascular endothelial growth factor
- VEGF, basic fibroblast growth factor - bFGF) in peripheral blood
plasma of the same CLL patient group and 80 healthy donors. Commercially
available sandwich ELISA kits (RD Systems) were used for bFGF
and VEGF measurement. ZAP-70 expression was quantified using PEconjugated
ZAP-70 monoclonal antibody (Caltag); cut-off level of 20%
positivity was used as recommended by literature. Results. Both angiogenic
cytokines were significantly increased in CLL patients when compared
to controls (bFGF, p