Abstract Book 2010 - CIMT Annual Meeting

089 Sha | Tumor biology & interaction with the immune system
Influence of activation of human immune effector cells
on the penetration into tumor spheroids
Weixiao Sha 1 , Franziska Hirschhaeuser 1 , Stefan Walenta 1 , Kirsten Dettmar 2 and Wolfgang
Mueller-Klieser 1
1 Institute of Physiology and Pathophysiology, University Medical Center of the Johannes Gutenberg-University
Mainz, Germany
2 Fresenius Biotech GmbH, Munich, Germany
138
The trifunctional bispecific antibody catumaxo-
mab (anti-EpCAM x anti-CD3) was designed for
anti-tumor immunotherapy by targeting the tumor-associated
antigen human EpCAM (epithelial
cell adhesion molecule) on tumor cells and human
CD3, expressed on T lymphocytes. In addition, catumaxomab
recruits simultaneously Fcγ receptorpositive
accessory cells, to promote the formation
of a so-called “tri-cell-complex” which finally leads
to the destruction of tumor cells by immune cells.
MCTS (multicellular tumor spheroids) of the FaDu
cell line (over-expressing EpCAM) were co-cultured
with PBMCs (peripheral blood mononuclear cells)
and incubated with the test agents in microtiter
plates. With the objective to compare the influence
of the different binding sites of catumaxomab we
additionally used the F(ab`)2 fragments of catumaxomab
lacking a Fc-region and BiLu (anti-human
EpCAM x anti-mouse CD3) which is unable to bind
to human T cells. Furthermore, LPS (Lipopolysaccharid)
and con A (Concanavalin A) were applied
to the test system. To asses the immune cell infiltration,
rapidly frozen spheroids were cryosectioned
and stained for immunohistology. Antibodies
against CD45, CD14, CD2, CD4 and CD8 were used
to discriminate different subtypes of infiltrating
leukocytes in MCTS. Cytokine release (IFN γ, TNF
α, IL 2, IL 6) gave further information on the type of
immune response. Besides immunological parameters,
the metabolic effects of the treatment with the
different agents were analyzed by measurement of
the extracellular lactate production in co-culture
supernatants.
Catumaxomab-mediated stimulation of PBMCs led
to a massive penetration of spheroids by CD2+ T
cells after 3 days and the volumes of the MCTS
were significantly decreased. CD8+ T lymphocytes
were identified as the dominating subtype of tumor
infiltrating T cells. Higher levels of IL-2 through catumaxomab
application were detected after 1 day,
while no IL-2 was detectable after 3 days. Incubation
with the F(ab`)2 fragment of catumaxomab resulted
in similar results. Catumaxomab stimulation
of co-cultures also resulted in increased concentrations
of extracellular lactate. On the other side, IFN
γ and TNF α secretion as well as lactate concentration
was elevated in PBMC cultures stimulated
with catumaxomab in the absence of tumor cells,
whereas incubation with F(ab`)2 showed no detectable
effect. These catumaxomab-mediated effects
were enhanced in the presence of PBMC and tumor
cells. Furthermore, BiLu did not reveal any effects
in co-cultures of tumor cells with human PBMC.
In summary, it was demonstrated that the catumaxomab-mediated
stimulation of PBMCs in MCTS
co-culture plays a pivotal role for tumor infiltration
by cytotoxic T lymphocytes and secretion of antitumoral
cytokines, thus underscoring the importance
of the trifunctional mode of action for the
initiation of an effective anti-tumoral response.
Supported by Fresenius Biotech GmbH and TRION Pharma GmbH
(Munich, Germany).