Gene-immunotherapy with TRAIL and bispecific antibody EpCAMxCD3 for the selective induction of apoptosis in advanced pancreatic and prostate cancer [Elektronische Ressource] / presented by Ariane Groth

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Gene-immunotherapy with TRAIL and bispecific antibody EpCAMxCD3 for the selective induction of apoptosis in advanced pancreatic and prostate cancer Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Ariane Groth Master of Science born in: Wolfen, Germany Oral-examination:................................................ Referees: First referee: PD Dr. Philipp Beckhove Second referee: Prof Dr. Ingrid Herr Declaration by candidate I hereby confirm that this work submitted for assessment is my own and is expressed in my own words. Any uses made within it of the works of other authors in any form (i.e. ideas, equations, figures, text, tables, programs) are properly acknowledged at the point of their use. A full list of the references employed has been included. Where other sources of information have been used, they have been indicated or acknowledged.

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Published 01 January 2010
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Gene-immunotherapy
with TRAIL and bispecific antibody EpCAMxCD3
for the selective induction of apoptosis
in advanced pancreatic and prostate cancer

























































Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences

























































presented by Ariane Groth
Master of Science

born in: Wolfen, Germany
Oral-examination:................................................




















Referees: First referee: PD Dr. Philipp Beckhove
Second referee: Prof Dr. Ingrid Herr


























Declaration by candidate

I hereby confirm that this work submitted for assessment is my own and is expressed in my
own words. Any uses made within it of the works of other authors in any form (i.e. ideas,
equations, figures, text, tables, programs) are properly acknowledged at the point of their use.
A full list of the references employed has been included. Where other sources of information
have been used, they have been indicated or acknowledged.




Signature:

1
Acknowledgement


This dissertation was carried out under the supervision of Prof Dr. rer. nat. Ingrid Herr and
PD Dr. med. Philipp Beckhove, from September 2006 till March 2010 as a cooperative
project of the German Cancer Research Center and the Department of Molecular
OncoSurgery at the University Hospital of Heidelberg.
My first acknowledgement goes to my doctoral supervisor Ingrid Herr for her guidance,
constant support and encouragement during my doctoral research. I am very grateful for the
opportunity to work in her group, for her constant support, advice and guidance in my
dissertation and the freedom she granted me in my research.
Further, I am very grateful to PD Dr. Philipp Beckhove for taking on the task of the first
referee of my dissertation and for the reviews of this work.
I owe a huge debt of gratitude to Dr. Alexei Salnikov and PD Dr. Gerhard Moldenhauer for
their constant guidance, assistance and advice. They greatly contributed to my dissertation by
sharing ideas and suggestions at every stage of my work. A special thank you goes to Alexei
Salnikov for his support, the time and the patience in reading, evaluating and commenting my
writings. I learned a great deal from him and thank him for many fruitful discussions and the
time in the lab with him.
I extend my thanks to all current and former members of my lab group, especially Vanessa
Rausch, Benjamin Beckermann, Georgious Kallifatidis, Sabine Schlesinger and Jury
Gladkich. They all contributed in various forms of support to my work and the time in this
lab. I especially want to acknowledge the work of Jury Gladkirch and Sabine Schlesinger for
their help in the animal experiments and with immunohistochemistry.
Next, I wish to thank Isabel Vogler and Prof. Dr. Manuel Grez, for their time in Frankfurt and
their cooperation. I am also very grateful for the scientific advice of Prof Dr. Wolfgang
Uckert, his contribution was essential for the vector construction.
I also want to express my gratitude to all the people who have willingly donated blood for the
numerous experiments with lymphocytes.
Particular thanks go to Martin Mollenhauer for advice in vector cloning and in reading and
commenting the dissertation.
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Most importantly, I want to thank my parents to for their love, encouragement and constant
support. They have always believed in me and helped me reach my goals.
My final acknowledgement goes to Sandro Giannattasio, who has been my emotional anchor
during the preparation and writing of this dissertation. His unselfish support kept me focused
and well in balance during the major phase of my doctoral thesis. Thank you.

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Abstract

Patients with pancreatic and prostate cancer have a poor survival rate and new therapeutic
strategies are needed. Epithelial cell adhesion molecule (EpCAM), suggested as a marker for
cancer stem cells, is over-expressed on most pancreatic and prostate tumor cells but not on
normal cells and may be an ideal therapeutic target. The anti-tumor efficiency of bispecific
EpCAMxCD3 antibody linking tumor cells and T lymphocytes was evaluated. Furthermore, it
was tested whether the combination of bsAb EpCAMxCD3 with lymphocytes over-
expressing the death ligand TRAIL, inducing apoptosis in cancer cell but not on normal cells,
would be effective. In
NOD/SCID
mice,
EpCAMxCD3
had
a
long
serum
half ‐life
(t 1/2
∼
7

days).
 Co-transplanted pre-activated lymphocytes and EpCAMxCD3 effectively retarded the
tumor growth of BxPc-3 pancreatic and PC-3 prostate xenografts. TRAIL-over-expressing
lymphocytes strongly enhanced the efficacy of very low doses of EpCAMxCD3 and retarded
the tumor growth. Furthermore, TRAIL-over-expressing lymphocytes enhanced
EpCAMxCD3 treatment in a BxPc-3 xenograft model with developed tumors. Tumor growth
retardation was associated with a reduced tumor cell proliferation and with a reduced blood
vessel density. In 3D in vitro tumor reconstructs, TRAIL-over-expressing lymphocytes
dramatically increased the expression of apoptosis-related proteins, compared to pre-activate
lymphocytes and EpCAMxCD3. For mimicking a pancreatic cancer microenvironment in
vitro, we used a three-dimensional tumor reconstruct system, in which lymphocytes were co-
cultured with tumor cells and fibroblasts in a collagen matrix. In this in vivo–like system,
EpCAMxCD3 potently stimulated production of the effector cytokines IFN-γ and TNF-α by
extracorporally pre-activated lymphocytes. Moreover, compared with a bivalent anti-CD3
antibody, EpCAMxCD3 more efficiently activated the production of TNF-α and IFN-γ by
non-stimulated peripheral blood mononuclear cells. Most excitingly, we demonstrate for the
first time that EpCAMxCD3 induces prolonged contacts between lymphocytes and tumor
cells, which may be the main reason for the observed anti-tumor effects. In addition,
EpCAMxCD3 stimulated the expression of multiple chemokines in 3D tumor reconstructs.
Those chemokines could potentially contribute to the increased infiltration of the tumor
+ +tissues with CD68 and F4/80 macrophages. Conclusively the combination of TRAIL-
transduced lymphocytes and the bispecific antibody EpCAMxCD3 was very efficient in
pancreatic and prostate xenograft models in vivo and in vitro. This combination of gene
therapy with immunotherapy may open a way to improve the immune response and treatment
outcome in patients with pancreatic, prostate cancer and most likely other tumor entities.
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