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

month is not relevant to chronic GvHD or GvL. The presence of mH epitope
specific T-cells is also not linked to the GvHD but very interestingly
is associated to the GvL effect (GvL p″0.01). Conclusions. Our results
suggest that monitoring of mH responses on a restricted panel of antigens
could have interest very early following hemapoietic stem cell transplantation.
This a could give arguments for early therapeutic intervention
for patients with a high risk of relapse and when no detectable
alloreactivity could be observed during the first 3 months post transplant.
0196
HIGH ALLOREACTIVE POTENTIAL OF SUICIDE GENE EXPRESSING CENTRAL MEMORY T
LYMPHOCYTES CULTURED WITH HOMEOSTATIC Γ-CHAIN CYTOKINES FOR THE CURE OF
HEMATOLOGICAL MALIGNANCIES
S. Kaneko, 1 S. Mastaglio, 1 A. Bondanza, 1 M. Ponzoni, 1 L. Aldrighetti, 1
S. Toma, 2 M. Radrizzani, 2 S. La Seta-Catamancio, 2 F. Ciceri, 1
T. Nagasawa, 3 C. Bordignon, 1 C. Bonini1 1 Scientific Institute San Raffaele, MILAN, Italy; 2 MolMed, MILAN, Italy; 3 University
of Tsukuba, TSUKUBA, Japan
Background. Alloantigen targeting adoptive immunotherapy is a powerful
therapeutic approach to increase the graft-versus-leukemia (GvL)
effect mediated by allogeneic hematopoietic stem cell transplantation,
for the cure of hematological malignancies. Although alloreactivity mediated
by donor lymphocytes plays a crucial role in treating and preventing
disease relapse, its extensive exploitation is limited by the risk of a
life-threatening complication: Graft-versus-host disease (GvHD). To
solve this double bind, we’ve investigated the therapeutic potential of
donor lymphocytes retrovirally transduced to express the suicide gene
thymidine kinase of Herpes Simplex virus (TK cells) in patients affected
by leukemia, and showed that the infusion of TK cells induces GvL and
allows to control severe GvHD by ganciclovir (GCV). Initial clinical experience
showed that infused TK cells have a reduced alloreactivity and are
mainly CD45RA – /CD62L – effector memory (EM) T cells, known to have
limited survival potential. This functional phenotype is possibly due to
the ex vivo manipulation based on the enforced expansion of lymphocytes
by polyclonal stimulation (soluble anti-CD3 antibodies) and high
concentration of IL-2. Aims. The aim of the study is to maximize the
alloreactive potential of TK cells for the cure of leukemia, while controlling
severe GvHD. Methods. We hypothesized that culture with anti-
CD3/CD28Ab coated magnetic beads (3/28b) and homeostatic γ-chain
cytokines may allow to fully maintain alloreactivity on TK cells, while
permitting retroviral transduction. We tested IL-7, a central regulator of
the survival and maintenance of naïve and memory T lymphocytes, and
IL-15, a regulator of the initiation, clonal expansion, contraction, and
maintenance of memory cells. We used the SFCMM3 vector for T cells
transduction. Phenotypes, antigen reactivity, and survival of TK cells
were analysed in vitro and in vivo; using a murine model based on TK cells
infusions in NOD/Scid mice, previously transplanted with allogeneic
human skin. Results. The combination of 3/28b, IL-7 and IL-15 generated
high numbers of CD45RA – /CD62L + central memory (CM) TK cells
with preserved CD4/CD8 ratio, a g-IFN/IL-2 secretion profile, and persistent
expression of high levels of IL-7R-α, a molecule associated to
memory lymphocytes survival. In mixed lymphocytes cultures, CM TK
cells showed higher alloreactivity than EM TK cells, and maintained a
CM phenotype after multiple allogeneic stimulations. Moreover, CM
TK cells were eliminated by GCV as efficiently as EM TK cells. in vivo,
infused CM TK cells were engrafted and expanded more than EM TK
cells, showing preserved CD4/CD8 ratio and persistent expression of
high levels of IL-7R-α. Most importantly, CM TK cells were more potent
than EM TK cells in inducing both xenogeneic and allogeneic GvHD, as
documented by higher engraftment and a more extensive infiltration of
TK lymphocytes in the allogeneic human skin (grade 3 vs grade 1 allo-
GvHD). Summary. This study shows that CM TK cells, generated by
CD3/CD28 activation and culture in the presence of homeostatic
cytokines combine a high alloreactive potential with the selective sensitivity
to GCV-mediated cell death, providing a tool for maximal antileukemia
activity and controlled GvHD.
12 th Congress of the European Hematology Association
0197
SELECTIVE DEPLETION OF ALLOREACTIVITY AND PRESERVING OF ANTI-TUMOR ACTIVITY
OF SPECIFIC T CELL CLONES IN PATIENS WITH LEUKEMIA AND RENAL CARCINOMA
E. Matejkova, Z. Hrotekova, A. Stejskalova, D. Kyjovska, P. Vidlakova,
J. Michalek
Masaryk University, BRNO, Czech Republic
Background. A major challenge in the field of hematopoietic stem cell
transplantation (HSCT) is to prevent the alloreactivity of donor T-cells
which leads to acute graft-versus-host disease (GVHD) while preserving
graft-versus-tumor (GVT) effect. GVHD is leading cause of morbidity
and mortality after HSCT. Aims. Selective depletion using anti-CD25
immunotoxin (IT) can eliminate harmful alloreactive T-cells while preserving
other donor T-cells with antileukemic/antitumor reactivity. Methods.
We have used irradiated peripheral blood mononuclear cells (PBMC)
from cancer patients and healthy donor PBMC as responder cells in primary
mixed leukocyte reaction (MLR).To prepare GVL/GVT-specific Tcells,
alloreactive T-cells in primary MLR were depleted with anti-CD25
IT. The remaining T-cells had insignificant alloreactivity in secondary
MLR. Allodepleted donor cells were then repeatedly stimulated using
purified leukemia/tumor cells from the same cancer patient.
Leukemia/tumor-reactive donor T-cells were purified immunomagnetically
on the basis of INF-γ production. Results. 17 MLRs (10 with
leukemic and 7 with renal carcinoma cells) were performed. Selective
depletion of alloreactive donor T-cells with anti-CD25 IT led to more
than 2log depletion. Graft-versus-leukemia (GVL) effect of donor T-cells
was well preserved while the graft-versus-host (GVH) reactivation of
donor cells was negligible ever after repeated stimulation with patient’s
PBMC. In the case of renal carcinoma GVT-effect was less dominant
and GVH-reactivation of donor cells led to significant amount. Summary.
In conclusion, it is possible to selectively deplete donor alloreactive
T-cells with anti-CD25 IT. In the case of patients with leukemia, the
GVL-effect can be separated from GVHD, but in case of renal carcinoma
severe GVHD-effect re-appeared.
Supported by The Czech Ministry of Education, Youth and Sport, NPVII-
2B06058.
0198
WT1 FULL LENGTH PROTEIN VACCINATION SHOWS HIGH IMMUNOGENICITY AND
SIGNIFICANT ANTI-TUMOUR ACTIVITY IN THE MOUSE MODEL
D. Cilloni, I. Defilippi, P. Nicoli, A. Roetto, A. Bondi, E. Girola,
F. Arruga, F. Messa, S. Carturan, V. Rosso, R. Catalano, R. Taulli,
E. Bracco, G. Saglio
University of Turin, TURIN, Italy
Background. The Wilms’ tumor gene (WT1) is overexpressed in many
types of haematological malignancies including Acute Myeloid
Leukaemia (AML), Acute Lymphoblastic Leukaemia (ALL), Chronic
Myeloid Leukaemia (CML) and Ph negative myeloproliferative disorders.
WT1 holds great promise for immunotherapy of leukaemia since
it is expressed at high levels in blast cells but not in normal tissues, it is
involved in the maintenance of malignant phenotype and it is spontaneously
immunogenic. Many clinical trials using MHC class I-restricted
WT1 peptide, have been recently performed in patients affected by
AML, MDS, lung and breast cancer with satisfactory clinical results and
without relevant toxicity. Aims. the aim of the study was to set up a vaccination
approach in the mouse model using the WT1 full length protein
and to test the safety and efficacy of the vaccine. Methods. We purified
the WT1 full length protein. Complete coding sequence was cloned in
a pGEX bacterial expression vector for the production of the fusion protein
GST-WT1 that was subsequently purified by glutathione conjugated
beads. The murine leukemic cell line C1498 was transduced with
lentiviral vector PKG WT1 (kindly provided by Dr. L. Naldini) in order
to obtain a syngeneic cell line expressing WT1. 40 C57BL7/6 mice were
immunized with 50 µg of purified WT1 protein and 50 µL of Freund
adjuvant every 15 days, for a total of 3 immunizations; 40 mice were
injected with GST and adjuvant and 40 with PBS alone as control. 2
weeks after the last administration, 10 vaccinated mice and 10 controls
have been injected with 200.000 TRAMP-C2 cells and 10 of each group
with C1498. 10 mice from for each group were sacrificed and lymphocytes,
cultured in presence of interleukin-2 to perform a cytotoxicity
assay. Antibodies against WT1 have been evaluated. Long term toxicity
has been evaluated. Blood values have been constantly tested. Results.
Toxicity on normal tissues has been ruled out using the histochemical
analysis of different tissues. After mice vaccination, no organ toxicity has
been observed. White blood cell, Hb and platelets counts did not change
haematologica/the hematology journal | 2007; 92(s1) | 71