Developing novel strategies for oncolytic adenovirus therapy by host cell gene expression profiling and arming with therapeutic antibodies [Elektronische Ressource] / presented by Dominik Dorer

-

English
162 Pages
Read an excerpt
Gain access to the library to view online
Learn more

Description

INAUGURAL DISSERTATION submitted to the Combined Faculties for the Natural Sciences and thfoer mMatiacs of the Ruperto-Carola University of Heidelberg, Ger many for the degree of Doctor of Natural Sciences presented by Dominik Dorer Diplom Molekular Mediziner born in: Stuttgart, Germany Date of oral examination: ………………………. DEVELOPING NOVEL STRATEGIES FOR ONCOLYTIC ADENOVIR USTHERAPY BY HOST CELL GENE EXPRESSION PROFILING AND ARG MINWITH THERAPEUTIC ANTIBODIE.S This work was carried out at the German Cancer Research Center (DKFZ), Applied Tumor Virology First referee: PD. Dr. Suat Özbek Faculty of Biosciences, Heidelberg University Second referee: PD Dr. Dirk M. Nettelbeck Helmholtz University Group “Oncolytic Adenoviruses”, DKFZ ad n Department of Dermatology, Heidelberg University Hospital THESIS DECLARATION Hereby, I declare that the submitted work has been complemted , byt he undersigned, and that I have not used any other than permitted reference ss oorur cematerials nor engaged in any plagiarism. All references and other sources used by vme e behaen appropriately acknowledged in the work. I further declare that the work han s submnotitte d before the purpose of academic examination, either in its original or asirm iflorm, anywhere else. Heidelberg, 10.02.

Subjects

Informations

Published by
Published 01 January 2011
Reads 18
Language English
Document size 9 MB
Report a problem



INAUGURAL DISSERTATION

submitted to the

Combined Faculties for the Natural Sciences and thfoer mMatiacs
of the Ruperto-Carola University of Heidelberg, Ger many

for the degree of

Doctor of Natural Sciences



presented by


Dominik Dorer
Diplom Molekular Mediziner
born in: Stuttgart, Germany


Date of oral examination: ……………………….




DEVELOPING NOVEL STRATEGIES FOR ONCOLYTIC ADENOVIR US
THERAPY BY HOST CELL GENE EXPRESSION PROFILING AND ARG MIN
WITH THERAPEUTIC ANTIBODIE.S







This work was carried out at the
German Cancer Research Center (DKFZ), Applied Tumor Virology







First referee: PD. Dr. Suat Özbek
Faculty of Biosciences, Heidelberg University

Second referee: PD Dr. Dirk M. Nettelbeck
Helmholtz University Group “Oncolytic Adenoviruses”, DKFZ ad n
Department of Dermatology, Heidelberg University Hospital



THESIS DECLARATION


Hereby, I declare that the submitted work has been complemted , byt he undersigned, and
that I have not used any other than permitted reference ss oorur cematerials nor engaged in
any plagiarism. All references and other sources used by vme e behaen appropriately
acknowledged in the work. I further declare that the work han s submnotitte d before the
purpose of academic examination, either in its original or asirm iflorm, anywhere else.



Heidelberg, 10.02.2011
Dom ini k D or er

























“Time is a companion that goes with us on a jo urInte yr.eminds us to cherish each moment,
because it will never come again. What we leave db eish inot as important as how we have
lived.”
Jean-Luc Picard


Abstract



ABSTRACT
Oncolytic adenoviruses are promising candidates for treatmoefn tv arious cancer entities with a
favorable safety profile and therapeutic efficacy in inddiuavil cases. However, it has been
neglected so far to investigate whether the observed vary ingefficacy in clinical trials could be
due to differences in the cellular environment, between tumor lcse land the virus’s natural host
cells, which may influences successful virus replication. aMimy was to analyze wild type
adenovirus 5 infections in primary human bronchial epithelial lsc ealnd various tumor cells by
microarray analysis in order to reveal pathways that differteianlly affect oncolytic adenovirus
replication. Kinetics of early viral gene expression and DNA replication together with cytotoxicity
assays showed that primary bronchial epithelial cells optim aslluypported adenovirus infection.
In contrast, infection of the two melanoma cell lines SK-MEL- 2a8nd Mel624 revealed delayed
early viral gene expression and slower DNA replication and decreas ed cytotoxicity. Microarray
analysis of these melanoma cells versus primary bronchial helpiialt cells led to the
identification of differentially expressed genes and activate d cellular pathways. The most
prominent one, called the G1/S-phase transition pathway, was ongstrly activated in the primary
bronchial epithelial cells but not in melanoma. The data helope ded vtelop a strategy which
enhanced viral replication in SK-MEL-28 cells by combining cheotmherapy using temozolomide
and viral oncolysis.
Another aim of my work was to enhance therapeutic efficacy of yonctic oladenoviruses
by combining viro- and antibody therapy. Therefore, I armed t-umseloerctive adenoviruses with
a gene encoding a recombinant single-chain antibody directed againsthet well-established
carcinoembryonic antigen. As effector domain the constant domaoin f immunoglobulin type G
2a was selected which is able to mediate anti-tumor immunell caectivation by antibody-
mediated cytotoxicity. First, different tools to facilita tefficient antibody production via the
adenoviral major late promoter were compared, identifying natne rnai l ribosomal entry site and
a splice acceptor from the human adenovirus 40 as the most po otenents. Then, antibodies
expressed after infection with recombinant adenoviruses wearena lyzed by Western Blot, ELISA,
and flow cytometry. These assays demonstrated that the recombinantly produced antibodies
were fully functional regarding their ability to bind the carcinoembryonic antigen in-vitro and on
living tumor cells.
i

Zusammenfassung


ZUSAMMENFASSUNG
Die virale Onkolyse oder Virotherapie ist ein vielversprencdeher Ansatz zur Behandlung von
Krebserkrankungen und wurde unter anderem klinisch getestet füronkolytische Adenoviren.
Jedoch schwankt die therapeutische Wirksamkeit von Fall zu Fall ,was daran liegen könnte, dass
Krebszellen nicht die natürlichen Wirtszellen von Adenoviren sind. Ziel meiner Doktorarbeit war
es Adenovirus 5 Infektionen in ihrem natürlichen Umfeld preimr äBrronchialepithelzellen und
verschiedener Krebszellen zu untersuchen, um Gene und zelluläre gSnai lwege zu finden, die
einen Einfluss auf die adenovirale Replikation haben. Unter deanrem konnte ich zeigen, dass in
Bronchialepithelzellen die frühe virale Genexpression und DNA Replikation sehr schnell einsetzt,
verbunden mit einer hohen Zytotoxizität. Dagegen war die früheir avle Genexpression als auch
die DNA Replikation in zwei Melanomzelllinien stark verlangsamt, verbunden mit einer geringen
Zytotoxizität. Genexpressionsanalysen mittels Microarray in nfiizierten Bronchialepithelzellen
und jener Melanomzelllinien konnten zeigen, dass in den jewegielin Zelltypen unterschiedliche
Expressionsmuster einzelner Gene und zellulärer Signalwege vlaorgen. Der am stärksten
beeinflusste Signalweg reguliert den Eintritt in die S-eP hadess Zellzyklus und war in den
primären Bronchialepithelzellen aktiviert, aber nicht in den Melanomzelllinien. Anhand der
Daten konnte eine Strategie zur verbesserten Onkolyse in einzelnen Melanomzelllinien
entwickelt werden. Dafür wurden Adenovirus infizierte Melanomzellen uszätzlich mit dem
Chemotherapeutikum Temozolomid behandelt.
Ein weiteres Ziel meiner Arbeit war es, die Wirksamkeitol yonktischer Adenoviren durch
die Expression von therapeutischen Antikörpern zu verbessern. Daf ür habe ich tumor-
spezifische Adenoviren mit einem rekombinanten „single-chain“ nAtikörper-Gen ausgerüstet.
Die Antikörper erkennen das Tumor-assoziierte karzinoembrylonae Antigen und verfügen
außerdem über die Effektordomäne des Typ G Immunglobulins 2a, um eineAntikörper
vermittelte Immunantwort gegen Krebszellen zu richten. Zuerst wurden verschiedene
Expressionskonstrukte für eine optimale Antikörperproduktion midet m adenoviralen „major
late promoter“ getestet. Dabei wurden zwei effiziente Exsprioensskonstrukte identifziert, die
sogenannte „internal ribosomal entry site“ und ein Spleikßz-eAptor des humanen Adenovirus
40. Anschließend wurden die rekombinant hergestellten Antikörpeirm Western Blot, ELISA und
mittels Durchflusszytometrie auf ihre Funktionalität geprt.üf Eine erfolgreiche Antikörper-
Antigen Reaktion konnte in-vitro als auch anhand lebender Tumorzellen gezeigt werden.
ii

Table of contents



TABLE OF CONTENT S
Abstract ............................................................................................................................................. i
Zusammenfassung ............................................................................................................................ i i
Table of contents .............................................................................................................................. ii i
Abbreviations ..................................................................................................................................vii
I Introduction .............................................................................................................................. 1
1 History of cancer ..................................................................................................................... 1
2 Gene therapy and viral oncolysis .......................................................................................... 2
3 Human adenoviruses and their application for cancer thepray ................................................. 5
3.1 Structure and genome organization ..................................................... .7. ....
3.2 Adenoviral life cycle ........................................................................ 8
3.3 Targeting human adenoviruses for cancer therapy. ............................. .1.3. ..................
3.4 Limitations and implications for adenoviral oncosl y.si......................................... .1.7. ...............
4 Gene expression profiling of adenovirus infections ............................................................. 21
5 Objectives ............................................................................................................................ 24
II Results .................................................................................................................................... 25
1 Comparison of HAdV-5 infection in various cell typ..e..s. ....................................................... 25
1.1 Analysis of lung cells as model system ............................................................................. .2.5 .....
1.2 Cytotoxicity assays of primary cells and establishuemdo tr cell lines ................ 2.7. .................
1.3 Transduction capacity of HAdV-5 .................................................. .2.9 .....
1.4 Insights into the HAdV-5 life cycle ........................................................ .31
1.5 E1A promoter assay ................................................................................................................. 34
1.6 Production of virus progeny .................................................................................................... 37
2 Analysis of host cell gene expression after HAdV-5 itniofenc ................................................. 39
2.1 Microarray hybridization and analysis ........................................... .39. .........
2.2 Data interpretation by diverse computational too.ls. .............................. .4.4 .................
2.3 S-phase reporter gene assay ................................................................................................ .5.0.
2.4 Candidate gene expression in a wider melanoma cell eplan ...................... 5.3. ........................
3 Identification of strategies to enhance HAdV-5 replicoant in cancer cells .............................5..7
3.1 Overexpression of regulatory genes ................................................ .5.7 ......
3.2 Knockdown of cell cycle inhibitors ...................................................... .6.0.
3.3 Impact of chemotherapeutics on adenovirus replicoatn i.................................. 6.3. ...................
4 Arming of oncolytic adenoviruses with therapeutic antiboieds .............................................. .69
4.1 Virus design ............................................................................ 69
4.2 Antibody expression and cytotoxicity .............................................. .7.1. ......
iii

Table of content s


4.3 Kinetics of antibody expression ............................................................................................... 74
4.4 Binding of secreted antibodies to CEA on the cel lsurface ................................. 7.6. .......
4.5 Influence of scFv -Fc on phagocytosis of CEA positive tumor cells by macorphages ........... 78 CEA
III Discussion ............................................................................................................................... 81
IV Materials ................................................................................................................................. 91
1 Chemicals ............................................................................................................................. 91
2 Prokaryotic cells, eukaryotic cells, and viruses ............................................................... 92
2.1 Bacterial strains ............................................................................ 92
2.2 Human primary cells and established cell lines .......................................... .9.3. .......
2.3 Human adenoviruses ............................................................................................................... 94
3 Media .................................................................................................................................. 95
3.1 For bacteria ................................................................................. 95
3.2 For eukaryotic cells .......................................................................... 95
4 Buffers and solutions........................................................................................................... 96
4.1 For common use ...................................................................................................................... 96
4.2 For gel electrophoresis of nucleic acids ... . . . ............................................. .9.6
4.3 For gel electrophoresis for proteins ........................................................ 97
4.4 For Western Blot analysis and enzyme linked immunosrbent assays .................................. 97
4.5 For protein purification .................................................................... 98
4.6 For fluorescent cell activated sorting ..................................................................................... .98
4.7 For HAdV purification and analysis...................................................... .9.8. .
5 Nucleic acids, oligonucleotides and plasmids ................................................................... 99
5.1 For cloning and PCR ........................................................................ 99
5.2 For quantitative PCR .............................................................................................................. 100
5.3 Small interfering RNAs ................................................................... 100
5.4 Plasmids .................................................................................. 101
6 Antibodies and recombinant proteins .............................................................................. 103
V Methods ................................................................................................................................ 104
1 Recombinant DNA and RNA techniques ............................................................................... 104
1.1 Gel electrophoresis of DNA fragments .................................................. .1.0.4. ..
1.2 Cloning of DNA fragments into plasmid vectors ....................................... .1.0.4. .........
1.3 Production of transformation-competent bacteria. .................................. .1.0.5. ............
1.3.1 For heat shock ............................................................................................................... 105
1.3.2 For electroporation ............................................................... 105
1.4 Heat shock transformation ............................................................. 106
1.5 Transformation by electroporation .................................................... .1.0.6. ..
1.6 Generation of recombinant adenoviral genomes by homologous recombination............... 106
iv