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Engineering Von Bindungsproteinen Gegen Hsp70 Als Werkzeuge Zur Visualisierung Und Therapie Von Tumoren

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Engineering Von Bindungsproteinen Gegen Hsp70 Als Werkzeuge Zur Visualisierung Und Therapie Von Tumoren Lehrstuhl für Biologische Chemie Forschungsdepartment für Biowissenschaftliche Grundlagen Engineering von Bindungsproteinen gegen Hsp70 als Werkzeuge zur Visualisierung und Therapie von Tumoren Dipl.-Biol. Lars Friedrich Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Prof. Dr. Michael Schemann Prüfer der Dissertation: 1. Prof. Dr. Arne Skerra 2. Prof. Dr. Gabriele Multhoff 3. Prof. Dr. Iris Antes Die Dissertation wurde am 16.03.2017 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 09.01.2018 angenommen. Inhalt 1 Einleitung ........................................................................................................................... 1 1.1 Das 70 kDa Heat Shock Protein HSPA1A/B als Zielstruktur auf Tumorzellen ................ 1 1.2 Antikörperfragmente und alternative Protein-Scaffolds für Tumor-Imaging und -Therapie ..................................................................................................................... 5 1.3 Mikrobielles Surface Display für die Selektion von Bindungsproteinen ........................ 12 1.4 Zielsetzung der Arbeit...................................................................................................... 18 2 Material und Methoden.................................................................................................... 19 2.1 Material ............................................................................................................................ 19 Bakterienstämme und Phagen .................................................................................. 19 Zelllinien und Versuchstiere .................................................................................... 19 Plasmide ................................................................................................................... 20 Oligodesoxynukleotide ............................................................................................ 20 Antikörper und sonstige Proteinreagenzien ............................................................. 21 Chemikalien ............................................................................................................. 22 Standards und Kits ................................................................................................... 24 Geräte ....................................................................................................................... 25 Sonstiges Material .................................................................................................... 27 Medien, Antibiotika und allgemeine Lösungen ....................................................... 28 Software ................................................................................................................... 32 2.2 Molekularbiologische Methoden ..................................................................................... 33 Kultivierung und Konservierung von E. coli-Stämmen ........................................... 33 Transformation nach der CaCl2-Methode ................................................................ 33 Transformation mittels Elektroporation ................................................................... 34 DNA-Isolierung aus E. coli ...................................................................................... 35 Isolierung von RNA aus Säugerzellen ..................................................................... 35 Gelelektrophorese und Reinigung von DNA ........................................................... 36 In vitro-Modifizierung von DNA ............................................................................. 36 2.3 Anticalin-Präsentation und -Selektion ............................................................................. 39 Herstellung von Anticalin-Bibliotheken zur Präsentation auf E. coli ...................... 39 Affinitätsanreicherung durch Adsorption an paramagnetische Partikel .................. 40 Affinitätsanreicherung mittels Fluorescence Activated Cell Sorting (FACS) ......... 41 Cytofluorimetrische Einzelklonanalyse ................................................................... 42 II 2.4 Produktion rekombinanter Proteine in E. coli ................................................................. 42 Anzucht, Induktion und Ernte von Kulturen im Schüttelkolben ............................. 42 Anzucht, Induktion und Ernte von Kulturen im Laborfermenter ............................ 43 2.5 Proteinchemische Methoden ............................................................................................ 45 Chromatographische Verfahren ............................................................................... 45 SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE) ............................................ 47 N-terminale Proteinsequenzierung (Edman-Abbau) ................................................ 47 Bestimmung von Proteinkonzentrationen ................................................................ 48 Amino-selektive chemische Modifizierung von Proteinen ...................................... 48 Chemische Modifizierung von Cystein-Seitenketten .............................................. 51 Herstellung von Proteinkonjugaten mit Zirkonium und Lutetium .......................... 52 2.6 Immunologische Methoden und Zytotoxizitätstests ........................................................ 53 Enzyme-Linked Immunosorbent Assay (ELISA) .................................................... 53 Epitop-Kartierung mit dem SPOT-Verfahren .......................................................... 55 Immunofluoreszenz-Mikroskopie ............................................................................ 56 Durchfluss-Cytofluorimetrie von Zelllinien ............................................................ 57 Zytotoxizitätstest ...................................................................................................... 58 2.7 Biophysikalische Methoden ............................................................................................. 58 Circulardichroismus-Spektroskopie (CD) ............................................................... 58 Oberflächenplasmon-Resonanzspektroskopie (SPR) .............................................. 59 Massenspektrometrie ............................................................................................... 60 2.8 Tierexperimentelle Methoden .......................................................................................... 61 Tumor-Xenotransplantation ..................................................................................... 61 Positronenemissions-Tomographie (PET) und Computer-Tomographie (CT) ....... 61 Szintillationsradiometrische Biodistributionsuntersuchung .................................... 61 Autoradiographie von Tumor-Dünnschnitten .......................................................... 62 3 Ergebnisse ........................................................................................................................ 63 3.1 Herstellung eines Fab-Fragments gegen Hsp70 in E. coli und dessen funktionelle Analyse ............................................................................................................................ 63 Subklonierung der V-Regionen eines Hsp70-spezifischen Antikörpers und Versuche zur Produktion des entsprechenden Fab-Fragments in E. coli ................. 63 De novo-Sequenzierung der VH- und VL-kodierenden Bereiche des cmHsp70.1-Transkripts ............................................................................................ 66 III Versuche zur Verbesserung der funktionellen Proteinausbeute in E. coli ............... 68 Untersuchung der Antigenbindung mittels ELISA und Oberflächenplasmon- Resonanzspektroskopie ............................................................................................ 73 Epitop-Bestimmung des monoklonalen Antikörpers cmHsp70.1 im Vergleich zum rekombinanten Fab-Fragment Hsp70ch/m durch SPOT-Analyse ................. 75 Nachweis der Bindung von Antigen auf der Oberfläche von Tumorzellen ............. 78 3.2 Herstellung und Reinigung von humanem HSP70.1, dessen Substrat-Bindedomäne (SBD) sowie der SBD von humanem BiP in E. coli ........................................................ 79 Konstruktion eines Expressionsvektors, Produktion und Reinigung von humanem Hsp70.1 ................................................................................................... 79 Konstruktion von Expressionsvektoren, Produktion und Reinigung der Substrat-Bindedomänen von humanem Hsp70.1 und BiP ....................................... 80 Konstruktion eines Expressionsvektors, Produktion und Reinigung der Hsp70.1-Substrat-Bindedomäne in Fusion mit eGFP .............................................. 84 3.3 Bakterielles Surface Display zur Selektion von Anticalinen gegen Hsp70 ..................... 86 Herstellung einer Anticalin-Bibliothek und Affinitätsanreicherung durch Adsorption von E. coli-Zellen an paramagnetische „Partikel“ ................................ 86 Selektion von hHsp70.1-bindenden Anticalinen mittels FACS ............................... 88 3.4 Affinitätsmaturierung des Hsp70-Anticalins BBG10 ..................................................
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