6 Sequences
6.12 scDb-EHD2-scTRAIL (pSecTagFLAG)
ScDb-EHD2-scTRAIL constructs were generated by insertion of the respective scDb sequences (see 6.5) as KpnI-NotI fragments into the sequence represented in 6.10 (after KpnI-NotI digestion).
List of Figures
Figure 1.1: Crystal structure of TRAIL in complex with TRAIL-R2. ... 16
Figure 1.2: Apoptosis, non-canonical TRAIL signaling, and possible levels of interference of bortezomib. ... 18
Figure 1.3: Requirements for efficient apoptosis induction. ... 22
Figure 2.1: Schematic representation of relevant vector parts for cloning of scFv-EHD2 and scFv-Fc molecules. ... 48
Figure 2.2: Schematic representation of the relevant vector part for cloning of Db- and dsDb-scTRAIL molecules. ... 49
Figure 2.3: Schematic representation of relevant vector parts for cloning of EHD2-containing scTRAIL fusion proteins. ... 49
Figure 2.4: Schematic representation of relevant vector parts for cloning of Fc-containing scTRAIL molecules. 51 Figure 3.1: Overview of different recombinant antibody formats. ... 64
Figure 3.2: Biochemical characterization of EGFR-targeting recombinant antibodies. ... 65
Figure 3.3: Binding analysis of EGFR-targeting recombinant antibodies and effects on Colo205 cells. ... 66
Figure 3.4: Biochemical characterization of recombinant antibodies directed against HER2. ... 67
Figure 3.5: Binding analysis of HER2-targeting recombinant antibodies and effects on Colo205 cells. ... 68
Figure 3.6: Biochemical characterization of 3M6-based recombinant antibodies. ... 69
Figure 3.7: Binding analysis of 3M6-based recombinant antibodies and effects on Colo205 cells. ... 70
Figure 3.8: Characterization of scFv3-43-Fc. ... 72
Figure 3.9: Characterization of scFv323/A3hu3-Fc. ... 73
Figure 3.10: Overview of scTRAIL molecules of different formats. ... 75
Figure 3.11: Biochemical characterization of EGFR- and non-targeted scTRAIL molecules. ... 76
Figure 3.12: Binding of EGFR- and non-targeted scTRAIL molecules to target proteins in ELISA. ... 78
Figure 3.13: Binding of EGFR- and non-targeted scTRAIL molecules to cell surface-expressed receptors. ... 79
Figure 3.14: Cell death induction of EGFR- and non-targeted scTRAIL molecules on Colo205 cells. ... 81
Figure 3.15: Cell death induction of EGFR- and non-targeted scTRAIL molecules on Colo205 cells in the presence of cetuximab. ... 82
Figure 3.16: Cell death induction of EGFR- and non-targeted scTRAIL molecules on HCT116 cells. ... 83
Figure 3.17: Effects of treatment length on cell death induction of Dbhu225-scTRAIL on Colo205 cells... 84
Figure 3.18: Induction of caspase-8 activity in Colo205 cells. ... 85
Figure 3.19: Induction of caspase-8 and -3/7 activity in Colo205 cells. ... 86
Figure 3.20: Plasma stability and in vivo pharmacokinetics of EGFR- and non-targeted scTRAIL molecules. ... 87
Figure 3.21: Effects of bortezomib in vivo. ... 89
Figure 3.22: In vivo activity and PK of different EGFR-targeting scTRAIL formats and Fc-scTRAIL in combination with bortezomib. ... 90
Figure 3.23: Anti-tumor effects and PK of scFvhu225-Fc-scTRAIL and Fc-scTRAIL in combination with bortezomib. ... 92
Figure 3.24: In vivo effects and PK of different formats of EGFR-targeting scTRAIL fusion proteins and Fc-scTRAIL in the absence of bortezomib. ... 93
Figure 3.25: Biochemical characterization of HER2-targeting scTRAIL fusion proteins. ... 94
Figure 3.26: Binding studies of HER2-targeting scTRAIL molecules. ... 95
Figure 3.27: Cell death induction of HER2-targeting scTRAIL molecules on Colo205 cells. ... 96
Figure 3.28: Cell death induction of HER2-targeting scTRAIL molecules on HCT116 cells. ... 97
Figure 3.29: Biochemical characterization of HER3-targeting scTRAIL fusion proteins. ... 99
Figure 3.30: Binding studies of HER3-targeting scTRAIL molecules. ... 100
Figure 3.31: Cell death induction of HER3-targeting scTRAIL molecules on Colo205 cells. ... 101
Figure 3.32: Cell death induction of HER3-targeting scTRAIL molecules on HCT116 cells. ... 102
Figure 3.33: Biochemical characterization of EpCAM-targeting scTRAIL fusion proteins. ... 103
Figure 3.34: Binding studies of EpCAM-targeting scTRAIL molecules. ... 104
Figure 3.35: Cell death induction of EpCAM-targeting scTRAIL molecules on Colo205 cells. ... 106
Figure 3.36: Cell death induction of EpCAM-targeting scTRAIL molecules on HCT116 cells. ... 106
Figure 3.37: Binding of scFv-Fc-scTRAIL molecules and Fc-scTRAIL to target proteins in ELISA. ... 109
Figure 3.38: Flow cytometry studies of scFv-Fc-scTRAIL molecules and Fc-scTRAIL on Colo205 and HCT116 cells. ... 110
Figure 3.39: Blocking studies of scFv323/A3hu3-Fc-scTRAIL-induced cell death. ... 113
Figure 3.40: Effects of varying bortezomib concentrations on cell death induction on Colo205 cells. ... 114
Figure 3.41: Analysis of potential correlation of cell death induction and antigen binding. ... 115
Figure 3.42: In vivo activity and PK of scFv-Fc-scTRAIL molecules and Fc-scTRAIL. ... 116
Figure 3.43: Schematic representation and generation of scDb-EHD2-scTRAIL molecules. ... 118
Figure 3.44: Biochemical characterization of scDb-EHD2-scTRAIL molecules. ... 119
Figure 3.45: Binding studies of scDb-EHD2-scTRAIL molecules. ... 120
Figure 3.46: Cell death induction of scDb-EHD2-scTRAIL molecules. ... 122
Figure 4.1: Visualization of positively charged patches at the surface of TRAIL. ... 134
List of Tables
Table 1.1: Variety of TRAIL fusion proteins exploiting active targeting and/or increased valency. ... 26
Table 2.1: Antibodies and sera used in ELISA, flow cytometry, blocking studies, and PK. ... 35
Table 2.2: Primers used for cloning, PCR screening, and sequencing. ... 41
Table 2.3: Composition of SDS polyacrylamide gels. ... 56
Table 3.1: Biochemical and binding properties of EGFR-targeting recombinant antibodies. ... 66
Table 3.2: Biochemical and binding properties of HER2-targeting recombinant antibodies. ... 68
Table 3.3: Biochemical and binding properties of recombinant antibodies directed against HER3. ... 71
Table 3.4: Biochemical and binding properties of scFv323/A3hu3-Fc. ... 73
Table 3.5: Biochemical properties of EGFR- and non-targeted scTRAIL molecules. ... 77
Table 3.6: Binding properties of EGFR- and non-targeted scTRAIL molecules. ... 80
Table 3.7: EC50 values of cell death induction of EGFR- and non-targeted scTRAIL molecules. ... 84
Table 3.8: Pharmacokinetic properties. ... 88
Table 3.9: Biochemical and binding properties of HER2-targeting scTRAIL fusion proteins. ... 95
Table 3.10: EC50 values of cell death induction of HER2-targeting scTRAIL molecules. ... 97
Table 3.11: Biochemical and binding properties of HER3-targeting scTRAIL molecules. ... 100
Table 3.12: EC50 values of cell death induction of HER3-targeting scTRAIL molecules. ... 102
Table 3.13: Biochemical and binding properties of EpCAM-targeting scTRAIL molecules. ... 105
Table 3.14: EC50 values of cell death induction of EpCAM-targeting scTRAIL molecules. ... 107
Table 3.15: Binding properties of scFv-Fc-scTRAIL molecules and Fc-scTRAIL. ... 111
Table 3.16: EC50 values of cell death induction of scFv-Fc-scTRAIL molecules and Fc-scTRAIL. ... 112
Table 3.17: Biochemical and binding properties of scDb-EHD2-scTRAIL molecules. ... 121
Table 3.18: EC50 values of cell death induction of scDb-EHD2-scTRAIL molecules. ... 123
Danksagung
Allen voran gilt mein Dank Prof. Dr. Roland Kontermann für die hervorragende Betreuung, die vielen Anregungen und hilfreichen Diskussionen, die Unterstützung insbesondere bei widersprüchlich erscheinenden Daten sowie das Vertrauen in meine Arbeit. Danke, dass ich in den letzten Jahren Teil deiner Gruppe sein durfte und es noch sein darf.
Herzlichen Dank an Prof. Dr. Ralf Takors für die Übernahme des Zweitgutachtens dieser Arbeit.
Darüber hinaus danke ich Prof. Dr. Klaus Pfizenmaier, der wertvolle Anregungen zu dieser Arbeit geliefert hat, sowie Dr. Dafne Müller für hilfreiche Unterstützung in sowohl organisatorischen als auch wissenschaftlichen Fragen.
Weiterer Dank gilt unseren Kooperationspartnern für die inspirierende Zusammenarbeit im Rahmen des vom Bundesministerium für Bildung und Forschung finanzierten Projekts PREDICT, insbesondere Dr. Kristin Dickschen, Dr. Michael Block, Dr. Jens Schmid sowie Dr.
Thomas Mürdter.
Ganz besonders danke ich meinen Kollegen und Freunden Sina Fellermeier, Dr. Felix Unverdorben, Dr. Oliver Seifert und Dr. Fabian Richter sowie allen „Kontermännern“, die wesentlich dazu beigetragen haben, die letzten Jahre zu einer ganz besonderen Zeit werden zu lassen. Liebe Sina, lieber Felix, danke für die vielen schönen Abende, die wir zu sechst außerhalb des Labors verbracht haben. Ich freue mich auf die vielen, die noch folgen werden.
Liebe Sina, ich mache es mir an dieser Stelle einfach und schließe mich dir an: „von der besten Kollegin zu einer meiner wichtigsten Freundinnen.“ Mehr Worte braucht es wirklich nicht.
Liebe Silvi, du hast mich am ersten Tag an dieser Uni angesprochen und seitdem nicht mehr aufgehört zu reden. Danke dafür, für deine Freundschaft und die vielen motivierenden Worte.
Zudem danke ich unseren TAs für ihre Unterstützung, insbesondere Nadine Heidel und Doris Göttsch. Vielen Dank an Alexandra Kraske und Beatrice Reiser sowie an alle Tierpflegerinnen, die im Laufe dieser Doktorarbeit für das Wohl meiner Mäuse gesorgt haben. Liebe Alex, danke für die Unterstützung im und außerhalb des Tierstalls.
An dieser Stelle möchte ich mich bei den wichtigsten Menschen bedanken, bei Harun und unseren Familien. Danke an meine Eltern. Danke für eure permanente bedingungslose Unterstützung, euer Verständnis und euren Glauben an mich. Ohne euch würde es diese Arbeit nicht geben. Danke an meine Schwester Tina, dafür ein Vorbild für mich zu sein und dafür mir die wundervollsten Patenkinder Yvonne und Ben geschenkt zu haben. Yvonne und Ben, ihr bereichert mein Leben und seid eine unglaubliche Motivation. Danke, Harun. Es gibt keine passenden Worte, die all deiner Unterstützung, deinem Verständnis, deiner Geduld, deinem Vertrauen in uns und deinem Glauben an mich gerecht werden. Çok teşekkür ederim.
Erklärung
Hiermit erkläre ich, dass die vorgelegte Dissertation und die darin gezeigten Daten von mir persönlich und ohne unrechtmäßige Hilfe angefertigt wurden. Alle genutzten Quellen sind entsprechend zitiert.
Declaration
I hereby declare that this thesis and the work presented in it are my own and have been prepared without any illegitimate help. All sources used have been quoted adequately.
Meike Hutt
Stuttgart, March 19, 2017