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

12 th Congress of the European Hematology Association
significantly in the different groups. Iimmunized mice showed high levels
of both, IgM and IgG antibodies with a mean value of luminescence
of 690000 in WT1 injected mice vs 7500 in control mice and high levels
of CTLs against WT1 as compared to mice injected with PBS or GST
alone. Importantly, in control mice injected with TRAMP-C the mean
size of the tumor was 1,6±0,4 cm and 2±0,5 cm in control mice injected
with C1498. By contrast in vaccinated mice the tumour mass was significantly
smaller (0,3±0,1 cm) after 8 weeks of follow-up. Conclusions.
The full length protein vaccination approach is able to elicit both, umoral
and cytotoxic response and it results in a significant antitumor effect in
vivo. Although further studies are required to compare the efficacy of this
approach to the peptide based vaccine, WT1 full length protein could
represent a valid vaccination strategy allowing to overcome the HLA
restriction limit of the peptide approach
0199
DETECTION OF HUMORAL IMMUNE RESPONSES AGAINST WILMS TUMOR GENE (WT1)
PRODUCT IN PATIENTS AFFECTED BY HAEMATOLOGICAL MALIGNANCIES
D. Cilloni, P. Nicoli, I. Defilippi, E. Girola, A. Bondi, E. Messa,
A. Roetto, S. Carturan, F. Messa, F. Arruga, R. Catalano, V. Rosso,
E. Bracco, G. Saglio
University of Turin, TURIN, Italy
Introduction. The Wilms tumor gene (WT1) is expressed at high levels
in many types of haematological malignancies including Acute Myeloid
Leukaemia (AML), Acute Lymphoid Leukaemia (ALL), Chronic Myeloid
Leukaemia (CML), Ph negative Myeloproliferative disorders (CMPD)
and Myelodysplastic Syndromes (MDS). Moreover WT1 gene is overexpressed
in many types of solid tumours. Humoral immune responses
against WT1 product has been described in Acute Leukemias and in
Chronic Myeloid Leukemia (CML). At present, many clinical trials using
WT1 peptides are ongoing. Aims. The aim of the study was to investigate
the presence of a humoral response against WT1 protein in patients
affected by haematological malignancies in order to explore the possibility
to elicit an immune response against WT1 with vaccination using
WT1 peptides or protein. Methods. After informed consent sera and
Peripheral Blood samples were collected from 98 patients: 30 Myelodysplastic
syndromes (MDS), 11 Acute Myeloid Leukaemia (AML), 4 Acute
Lymphoblastic leukaemia (ALL), 23 Multiple Myeloma (MM), 20 Chronic
Myeloid Leukemia (CML), 6 Idiopatic Myelofibrosis (IM), 4 Chronic
Myelomonocytic Leukaemia (CMML). In addition 20 healthy subjects
were evaluated as control. Using dot blot technique we analyzed the
presence of WT1 IgG and IgM antibodies. WT1 transcript amount was
evaluated by quantitative Real Time PCR in PB samples. Results. We
detected a significant levels of IgG in 64% of MDS, 54% of AML, 66%
of ALL, 70% of CML, 70% of MM, 100% of CMML and 66% of IM.
The levels of IgG antibodies were significantly higher in chronic myeloproliferative
disorders as compared as acute leukemias. A significant
level of IgM antibodies were present in 60% of MDS. Among them
they were present in 75% of RA, 30% of RAEB and 25% of s-AML. 10%
of de novo AML, 10% of ALL, 20% of CML, 220% of MM, 100% of
CMML and 50% of IM. By contrast IgG and IgM were undetectable in
healthy subjects. Regression analysis showed that the levels of antibodies
were not correlated with WT1 expression levels (r=0,41). Conclusions.
These data demonstrate that humoral immune responses against
the WT1 protein could be elicited in patients with haematological malignancies.
Moreover, the data suggest that strong and persistent stimulation
by WT1 antigen, which usually occurs in patients with a large
amount of leukemic cells is needed to generate immunoglobulin isotype
class switching from IgM to IgG. Finally, although Multiple Myeloma
patients present low levels of WT1 transcript in BM, they have significant
amount of antibodies in the serum. These data suggest that
patients affected by haematological malignancies, including Multiple
Myeloma patients could be candidate for WT1 based immunotherapy.
72 | haematologica/the hematology journal | 2007; 92(s1)
0200
γ-GLOBIN GENE TRANS-ACTIVATION IN K562 CELLS BY A SYNTHETIC ZINC-FINGER
ACTIVATOR GENE TRANSFER
E. Stavrou, 1 E. Lagadinou, 2 E. Papapetrou, 3 C. Barbas III, 4
N. Zoumbos, 2 A. Athanassiadou3 1 University of Patras, PATRAS, Greece; 2 Dep of Hematology, University of
Patras, PATRAS, Greece; 3 Dep of Biology, University of Patras, PATRAS,
Greece; 4 The Scripps Research Institute, LA JOLLA, USA
The activation of the γ-globin gene pharmacologically or by γ-globin
gene transfer has been considered a valid alternative strategy for treatment
of diseases arising from mutations in this locus, such as of sicklecell
anemia and thalassaemia versus the transfer of the normal β-globin
gene. Lately (Graslund et al 2005) the development of a selective, synthetic
activator of γ-globin gene, Zif-VP64, based on zinc-finger DNA
binding proteins was presented, producing potentially therapeutic levels
of Haemoglobin F. K562 cells express the γ-globin gene and are trisomic
for chromosome 11, the aim, therefore, is to establish an increase
in the γ-globin in the transfected cell lines, as compared to the untransfected
ones, using a non-viral episomal vector. We constructed an episomal
vector containing: the activator Zif-VP64; the reporter gene eGFP,
driven by the CMV promoter; the S/MAR element, which enables the
plasmid to establish itself in the nucleus of the host cell. We transferred
this new, episomal vector of γ-globin activator, Zif-VP64-Ep1, into
human hematopoietic K562 cells, by electroporation. Routinely 1x107
K562 cells were used for every experiment and transfection efficiencies
were around 55%. Our results show that Zif-VP64-Ep1 is maintained as
a stable episome in K562 cells without integrations in the genome as
shown with Southern Blot and plasmid rescue experiments, running
into the tenth month of continuous culture with and without selection
pressure and without loss in cell viability or in expression of eGFP, as
observed under Fluorescent Microscope and documented by Flow
Cytometry. RT-PCR, Western blotting and Intracellular Flow Cytometry
were employed to investigate γ-globin mRNA and Haemoglobin F
protein levels in transfected K562 cells. Our data indicate a significant
increase in Haemoglobin F protein of up to 350% of its level in the
untransfected K562 cells, at list 200 generations post-transfection. This
is the first time that an episomal vector for gene transfer is applied for
the trans-activation of specific gene expression.