Abstract Book 2010 - CIMT Annual Meeting

067 Hirschhaeuser | New targets & new leads
Efficacy of catumaxomab in tumor spheroid killing is mediated
by its trifunctional mode of action
Franziska Hirschhaeuser 1 , Kirsten Dettmar 2 , Judith Atz 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
112
Catumaxomab is an intact trifunctional bispecific
antibody targeting human EpCAM (epithelial cell
adhesion molecule) and CD3 with further binding to
activating Fcγ receptor type I, IIa and III. We chose
multicellular tumor spheroids (MCTS) of human
EpCAM-positive FaDu tumor cells in co-culture with
human peripheral blood mononuclear cells as an adequate
three-dimensional in vitro model for pharmacological
testing of catumaxomab.
Besides catumaxomab, we used the F(ab`)2 fragments
of catumaxomab and the parental antibodies 26/II/6
(anti-human CD3) and HO-3 (anti-human EpCAM), as
well as BiLu (anti-human EpCAM x anti-mouse CD3)
which is not able to bind to human T cells. We assessed
the spheroid volume growth, cytokine release (IL 2, IL
6, IFN γ, TNF α), and immunohistological stainings for
different immune cell types and for proliferation.
After 6 days, spheroids cultured with the EpCAMtargeting
antibody HO-3 showed almost the same
volume as control spheroids without antibody. Combination
of 2.5 ng/ml HO-3 with 2.5 ng/ml 26/II/6
resulted in spheroid volumes which were reduced by
61%; and approaches with 5.0 ng/ml 26/II/6 showed a
70% volume reduction. Catumaxomab induced a 91%
decrease in spheroid volume. Comparing the effects of
26/II/6 and HO-3 to catumaxomab, demonstrates less
efficacy of the parental antibodies either used alone or
in combination. The importance of the CD3 binding
site was emphasized by further control experiments
with BiLu. Similar to HO-3, BiLu showed no tumor
spheroid killing. The essential function of the intact
Fc region was demonstrated in control experiments
using the F(ab’)2 fragment of catumaxomab: The
bispecific antibody fragment lacking this functional
binding site resulted in a dose-dependent volume reduction
of FaDu spheroids ranging from 54-81% for
concentrations of 1.0, 2.5 and 10.0 ng/ml, while catumaxomab
caused a decrease of 90-100% within the
same concentration range. The highest concentrations
of secreted IL 2 and IFN γ were observed in the setting
with tumor spheroids, PBMCs and catumaxomab.
Cultures of PBMC and 5.0 ng/ml 26/II/6 yielded the
highest levels for IL 6 and TNF α. In contrast, incubation
with BiLu did not induce cytokine secretion,
and the incubation with F(ab’)2 resulted in increased
cytokine levels only for 10.0 ng/ml, but several-fold
less than induced by catumaxomab. The immunological
stainings showed that the infiltrating lymphocytes
are mainly CD8+ T cells in spheroids treated with
catumaxomab and its F(ab’)2 fragment, whereas the
total amount of CD45+ cells in catumaxomab treated
spheroids was higher.
In summary, the results show, that all binding partners
of the postulated tricell complex have to be
present to exert catumaxomab’s full mode of action.
These distinct effects of catumaxomab are based on
the unique composition of the trifunctional bispecific
antibody.
Acknowledgements
The technical assistance of S. Dahms-Prätorius and R. Santos is
greatly acknowledged.
Supported by Fresenius Biotech GmbH and TRION Pharma GmbH
(Munich, Germany).