Haematologica 2003 - Supplements

decreased after 96h (pre:0.12%,post:0.07%), when CD25+ γδT
cells levels were higher. We did not find significative differences
in this study between the different groups, although this could be
due to the small size of the subgroups. Conclusions: MM and PD
have the same percentage of γδ T cells, but the activation was
higher in MM. After pamidronate, the induction was rapid in
MM: after 72h, MM did not show any increase in CD69+, but
higher levels of CD25+. Stimulation was delayed in PD, with
higher levels of CD69+ but no increase in CD25+. These results
show that pamidronate stimulate γδT cells in MM and PD, but
further studies are needed to assess this antitumor role.
068
T-cells rendered to express chimeric immunoreceptors
targeting B-cell antigens lyse multiple myeloma cells
Max S. Topp, Horst-Dieter Hummel, Christina Neff,
Gabriele Kuntz, Laurence J.N. Cooper, Michael C. Jensen,
Hermann Einsele
Department of Hematology and Oncology, Universität
Tübingen,Tübingen,GermanyPediatric Hematology-Oncology, City
of Hope National Medical Center, USA
There is increasing evidence that multiple myeloma patients may also
harbour circulating multiple myeloma progenitors, which express
early/intermediated B-cell antigens such as CD19 and CD20 but lack
expression of the plasma cell antigen CD138. Transplantation of
purified CD19+ PBMC form multiple myeloma patients into
irradiated NOD/SCID mice results in multiple myeloma engraftment
(Pilarski et. al; Exp Hematol. 2002.; Matsui et al, ASH 2002).
Furthermore multiple myeloma progenitors cells are also
characterized by over-expression of the multi drug resistance gene
and may therefore be resistance to standard high dose chemotherapy
followed autologous stem cell transplantation.
Thus novel treatment strategies targeting such multiple myeloma
cells effectively are highly warranted.
An attractive approach of combing the dynamic properties of
cytolytic potential of T-cells with the development of high affinity
specific for B-cell antigens is the construction and expression of
chimeric immunoreceptors in T-cells. This was accomplished by the
genetic modification of CTL to express a chimeric immunoreceptor
composed of either the CD19 or the CD20-specific single-chain
immunoglobulin extra cellular targeting domain fused to a CD3-ζ
intracellular signalling domain. CD19- and CD20-specific CD8+ T-
cells clones were established through limiting dilution and the
surfaces expression of chimeric immunoreceptors determined by
flow-cytometry with an anti-Fc polyclonal antibody. T-cell clones
with the highest expression were then used as effectors in standard
Cr-release assays with the CD19+ multiple myeloma cell lines
CD19+ ARH-77 and HS-Sultan and as a control the CML cell line
CD19- K562. Both the CD19 and CD20-specific CD8+ T-cell clones
could specifically lyse the MM cell lines ARH-77 and HS-Sultan (up
to 65% at E/T of 1:1) whereas the CD19- targets where not
recognized. Lysis was abrogated by preincubation of targets with
anti-MHC-I antibody, demonstrating the MHC-I independent
recognition of the targets. Similarly T-cells stimulated with the either
MM cell line ARH-77 or HS-Sultan readily produced IFN-γ (ELISA)
whereas incubation with the CD19- cell lines K562 no IFN-γ
production was detected in collected supernatants.
In summary, construction and expression of either CD19 or
CD20-specific chimeric immunoreceptors in CD8+ T-cells results
in specific recognition of CD19+ multiple myeloma cells. These
pre-clinical observation helps justify further investigation in to
the potential of this novel concept in preventing the engraftment
of multiple myeloma in NOD/SCID mice.
069
Myeloma Infiltrating Lymphocytes (MILS) as a Strategy
for Enhancing Adoptive Immunotherapy
Kimberly A. Noonan, William Matsui, Hyam I. Levitsky, Ivan
Borrello
Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins,
Baltimore, MD 21231
T cell mediated immune responses can mediate a tumor-specific
response in the appropriate setting. Unfortunately, the severe
defects underlying immune responses in cancer-bearing hosts
limit the efficacy of adoptive T cell transfer from such hosts. Exvivo
activation of T cells with beads conjugated to CD3 and
CD28 antibodies has been capable of activating and augmenting
peripheral T cells in a non-specific polyclonal manner from
cancer-bearing hosts. However, a major concern is the absence
of tumor-specificity of this approach.
T cells can be obtained from the tumor micro-environment with a
heightened tumor specificity as compared to peripheral blood. In
comparing T cells obtained from these two different
compartments, we have demonstrated oligoclonal restriction of
myeloma infiltrating lymphocytes (MILS) obtained from marrow
aspirates. Utilizing the anti-CD3/CD28 conjugated beads, the
MILS showed a greater expansion and an enhanced response to
or plasma cells compared to peripheral blood lymphocytes, in
several assays, suggestive of the activation of a memory/effector
response with heightened tumor specificity.
This strategy provides the ability to enhance the efficacy of
adoptive immunotherapy through the selective isolation and
expansion of tumor-specific cell populations with a greater
antigenic specificity compared to tumor antigen stimulated cells.
The ability to infuse these polyclonal myeloma specific T cells
and ultimately integrate the adoptive transfer of these cells with
myeloma-specific vaccinations are the goals of the clinical
implementation of such an approach.
070
Ex Vivo Activation and Expansion of T Cells from the
Peripheral Blood of Multiple Myeloma Patients Using
the XcellerateTM Process
Vescio, Robert(1); Berenson, James(1); Vij, Ravi(2);
DiPersio, John(2); Borrello, Ivan(3); Rowley, Scott(4);
Siegel, David(4); Martin, Thomas(5); Rasmussen,
Angela(6); Kalamasz, Dale(6); Roehrs, Heather(6);
Markham, Eric(6); Green, Cherie(6); Berenson, Ronald(6);
Bonyhadi, Mark(6); Frohlich, Mark(6)
1)Cedars Sinai Medical Center, Los Angeles, CA; 2)Washington
University, St. Louis, MO; 3)Johns Hopkins University, Baltimore,
MD; 4)Hackensack University, Hackensack, NJ; 5)University of
California, San Francisco, CA; 6)Xcyte Therapies, Inc., Seattle, WA
Previous studies in multiple myeloma have demonstrated a
significant association between survival and baseline levels of T
cells prior to chemotherapy (Kay et al., Blood 2001) as well as
lymphocyte recovery to >500/mm3 fifteen days following
autologous transplantation (Porrata et al., Blood 2001).
Therefore, infusion of large numbers of T cells post transplant
may improve clinical results.
We have developed the XcellerateTM Process for the ex vivo
activation and expansion of T cells using immobilized anti-CD3
and anti-CD28 antibodies covalently linked to magnetic beads
(Dynabeads® M-450 CD3/CD28 T). Adding the beads to
peripheral blood mononuclear cell cultures results in the rapid coligation
of the T cell receptor/CD3 complexes and CD28
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