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Immuntherapeutische Ansätze Beim Rhabdomyosarkom

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Immuntherapeutische Ansätze Beim Rhabdomyosarkom Immuntherapeutische Ansätze beim Rhabdomyosarkom: Therapeutische Antikörper und Phagozytose Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Delia Herrmann aus Ulm Tübingen 2011 Tag der mündlichen Qualifikation: 29.11.2011 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatter: Prof. Dr. Sorin Armeanu-Ebinger 2. Berichterstatter: Prof. Dr. Olaf Rieß Inhaltsverzeichnis I Inhaltsverzeichnis Inhaltsverzeichnis .................................................................................................... I Abkürzungsverzeichnis ......................................................................................... V 1. Einleitung ........................................................................................................... 1 1.1 Das Rhabdomyosarkom (RMS) .......................................................................... 1 1.1.1 “Klassisches” ERMS ................................................................................ 2 1.1.2 ARMS ...................................................................................................... 2 1.2 Tumorimmunologie ............................................................................................. 4 1.2.1 Spezifische Tumorerkennung .................................................................. 4 1.2.2 Unspezifische Mechanismen ................................................................... 8 1.2.3 Immunevasion von Tumoren ................................................................. 10 1.3 Krebsimmuntherapie ......................................................................................... 11 1.3.1 Tumorvakzinierung ................................................................................ 11 1.3.2 Adoptive zelluläre Immuntherapie ......................................................... 12 1.3.3 Antikörpertherapie ................................................................................. 12 1.4 Zielsetzung der Arbeit ....................................................................................... 14 2. Material und Methoden ................................................................................... 15 2.1 Materialien ........................................................................................................ 15 2.1.1 Chemikalien ........................................................................................... 15 2.1.2 Reagenziensysteme (Kits) und Fertiglösungen ..................................... 16 2.1.3 Lösungen und Puffer ............................................................................. 17 2.1.4 Zellkulturlösungen und verwendete Zellen ............................................ 21 2.1.5 Vektoren, Proteinstandard und Antikörper ............................................. 23 2.1.6 Patientenmaterial und Versuchstiere ..................................................... 25 2.1.7 Geräte und Hilfsmittel ............................................................................ 26 2.1.8 Verbrauchsmaterial ............................................................................... 28 2.1.9 Computerprogramme ............................................................................ 29 2.2 Zellbiologische Methoden ................................................................................. 31 2.2.1 Zellkultur ................................................................................................ 31 2.2.2 Transfektion und Transduktion .............................................................. 34 2.2.3 Durchflusszytometrie ............................................................................. 36 2.2.4 GLuc-Messung ...................................................................................... 36 2.2.5 Bestimmung der Vitalität (MTT-Test) ..................................................... 37 2.2.6 Immunzytochemie ................................................................................. 38 2.2.7 Antikörpervermittelter Zytotoxizitätstest (ADCC) ................................... 38 2.2.8 Phagozytosetest .................................................................................... 39 II Inhaltsverzeichnis 2.3 Histologische Methoden ................................................................................... 41 2.3.1 Fixieren und Einbetten .......................................................................... 41 2.3.2 Herstellen von Gewebeschnitten .......................................................... 41 2.3.3 Immunhistochemie ................................................................................ 42 2.3.4 Hämatoxylin-Eosin-Färbung ................................................................. 43 2.4 Molekularbiologische Methoden ....................................................................... 45 2.4.1 Isolierung von RNA aus Geweben und Zelllinien .................................. 45 2.4.2 RNA Quantitäts- und Qualitätsbestimmung .......................................... 45 2.4.3 Mikroarray Expressionsanalyse ............................................................ 46 2.4.4 Funktionelle Analyse von Expressionsprofilen ...................................... 47 2.5 Proteinbiochemische Methoden ....................................................................... 48 2.5.1 Proteinextraktion aus Zelllinien ............................................................. 48 2.5.2 Bestimmung der Proteinkonzentration .................................................. 48 2.5.3 SDS-Polyacrylamid-Gelelektrophorese ................................................. 49 2.5.4 Western Blot und Immundetektion ........................................................ 50 2.5.5 Silberfärbung ........................................................................................ 51 2.5.6 Immunpräzipitation ............................................................................... 51 2.5.7 Proteinidentifikation mittels LC/MS-Analyse ......................................... 52 2.6 Tierexperimentelle Methoden ........................................................................... 54 2.6.1 Xenotransplantation .............................................................................. 54 2.6.2 Blutentnahme und Plasmagewinnung .................................................. 54 2.6.3 GLuc-Bestimmung im Plasma .............................................................. 55 2.6.4 In vivo Bildgebung: Lumineszenz und Fluoreszenz .............................. 55 2.6.5 In vivo Bildgebung: PET/MRT ............................................................... 56 2.6.6 Tötung und Tumorexplantation ............................................................. 57 3. Ergebnisse ...................................................................................................... 59 3.1 Evaluierung von Cetuximab bei RMS ............................................................... 59 3.1.1 Zielstrukturen bei RMS für eine Antikörpertherapie .............................. 59 3.1.2 Aktivierung von EGFR durch EGF und Cetuximab ............................... 61 3.1.3 Cetuximab-vermittelte Zytotoxizität gegen RMS-Zellen ........................ 63 3.2 Etablierung eines monoklonalen Antikörpers ................................................... 65 3.2.1 Charakterisierung monoklonaler Antikörper .......................................... 65 3.2.2 Expression des 58B1A2-Antigens in RMS ............................................ 66 3.2.3 58B1A2-vermittelte Zytotoxizität gegen RMS-Zellen ............................ 68 3.2.4 Charakterisierung des unbekannten Antigens ...................................... 69 3.3 Phagozytose bei RMS ...................................................................................... 71 3.3.1 Expression von CD47 und Calreticulin in RMS ..................................... 71 3.3.2 CD47-vermittelte Zytotoxizität gegen RMS-Zellen ................................ 74 3.3.3 CD47-modulierte Phagozytose ............................................................. 75 3.3.4 Alternative Mechanismen zur Modulation der Phagozytose ................. 77 Inhaltsverzeichnis III 3.4 In vivo Modell zur Evaluierung immuntherapeutischer Ansätze ........................ 82 3.4.1 Beobachtung des Tumorwachstums durch Plasma-GLuc ..................... 82 3.4.2 Detektion der Tumoren durch optische Bildgebung ............................... 83 3.4.3 Lokalisation der Tumoren mittels PET/MRT .......................................... 86 3.4.4 Vergleich verschiedener PET-Tracer ..................................................... 88 3.4.5 Optische Bildgebung der exponierten Organe ....................................... 89 3.4.6 Histologie der Tumoren ......................................................................... 92 4. Diskussion ....................................................................................................... 95 4.1 Evaluierung von Cetuximab bei RMS................................................................ 95 4.2 Etablierung eines monoklonalen Antikörpers .................................................... 99 4.3 Phagozytose bei RMS ..................................................................................... 101 4.4 In vivo Modell zur Evaluierung immuntherapeutischer Ansätze ...................... 104 5. Zusammenfassung ......................................................................................
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