Struktur und molekulare Interaktionsanalyse von monoklonalen Antikörpern in Komplex mit Rezeptor-Tyrosinkinasen [Elektronische Ressource] / von Judith Schmiedel

X131852

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Struktur und molekulare Interaktionsanalyse von monoklonalen
Antikörpern in Komplex mit Rezeptor-Tyrosinkinasen





Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften





vorgelegt beim Fachbereich 14
Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main






von
Judith Schmiedel
aus Berlin





Frankfurt 2009
D30






vom Fachbereich 14 „Biochemie, Chemie und Pharmazie“ der Johann
Wolfgang Goethe-Universität Frankfurt am Main als Dissertation
angenommen.





Dekan: Prof. Dr. Dieter Steinhilber


1. Gutachter: Prof. Dr. Volker Dötsch
2. Gutachter: Prof. Dr. Martin Pos







Datum der Disputation:


































Table of contents
Table of contents

1. ZUSAMMENFASSUNG................................................................................................... 5
2. AIM OF THE THESIS..................................................................................................... 11
3. RECEPTOR TYROSINE KINASES............................................................................... 13
3.1. Introduction.......................................................................................................... 13
3.2. Structures of RTKs............................................................................................... 14
3.3. RTK activation 16
3.4. Signaling mechanisms downstream of activated RTKs....................................... 17
3.5. RTKs and cancer .................................................................................................. 19
4. MATERIALS & METHODS........................................................................................... 21
4.1. Molecular Biology................................................................................................ 21
4.1.1. EGFR................................................................................................................ 21
4.1.2. sEGFRvIII........................................................................................................ 22
4.1.3. sIGF-1R............................................................................................................ 23
4.1.4. Generation of recombinant baculovirus ........................................................... 23
4.2. Protein expression 23
4.2.1. sEGFR and sEGFRvIII..................................................................................... 23
4.2.2. sIGF-1R 24
4.3. Protein purification............................................................................................... 25
4.3.1. sEGFR.............................................................................................................. 25
4.3.2. sEGFRvIII, sIGF-1R domain I-III and domain II............................................ 25
4.3.3. Fab fragments................................................................................................... 25
4.3.4. Receptor:Fab complexes.................................................................................. 26
4.4. Molecular interactions and biophysics................................................................. 27
4.4.1. Dynamic light scattering 27
4.4.2. Static light scattering........................................................................................ 27
4.4.3. Surface plasmon resonance .............................................................................. 27
4.4.4. Analytical ultracentrifugation.......................................................................... 29
4.4.5. Isothermal titration calorimetry........................................................................ 29
4.4.6. Small angle X-ray scattering............................................................................ 30
4.5. Protein Crystallography........................................................................................ 32
4.5.1. sEGFR:Fab72000............................................................................................. 32
4.5.2. Fab72000.......................................................................................................... 32
4.5.3. sEGFRd3:Fab72000......................................................................................... 33
4.5.4. sEGFRvIII........................................................................................................ 35
4.5.5. Fab1159476...................................................................................................... 36
5. Matuzumab binding to EGFR prevents the conformational rearrangement required for
dimerization..................................................................................................................... 37
5.1. Introduction 37
5.1.1. Ligand-induced EGFR activation..................................................................... 39
5.1.2. Structures of ErbB receptor family extracellular domains............................... 40
5.1.3. ErbB receptor dimerization at the cell surface ................................................. 41
5.1.4. EGFR and cancer ............................................................................................. 42
5.1.5. Anti-EGFR antibodies...................................................................................... 43
5.2. Results.................................................................................................................. 46
5.2.1. Matuzumab binding to sEGFR......................................................................... 46
5.2.2. Ligand competition analysis of matuzumab..................................................... 47
5.2.3. Matuzumab binding prevents receptor dimerization........................................ 48
5.2.4. The matuzumab epitope ................................................................................... 49
1 Table of contents
5.2.5. The matuzumab epitope is distinct from the ligand binding site on domain III
of sEGFR.......................................................................................................... 52
5.3. Discussion............................................................................................................ 54
5.3.1. Matuzumab binding characteristics to soluble and cell surface EGFR............ 54
5.3.2. The matuzumab epitope on sEGFR domain III ............................................... 55
5.3.3. Matuzumab and ligand epitopes do not overlap on sEGFR domain III........... 56
5.3.4. The matuzumab inhibition mechanism ............................................................ 57
5.3.5. Matuzumab binding properties interpreted with structural information .......... 59
5.3.6. Implications for the therapeutic application of matuzumab............................. 60
5.4. Conclusion 63
6. Antibody binding and dimerization properties of the mutant EGFR variant III
ectodomain ...................................................................................................................... 65
6.1. Introduction.......................................................................................................... 65
6.1.1. EGFRvIII in-frame deletion............................................................................. 65
6.1.2. EGFRvIII signaling activity 67
6.1.3. EGFRvIII down-regulation.............................................................................. 67
6.1.4. Therapeutic strategies against EGFRvIII ......................................................... 68
6.2. Results.................................................................................................................. 70
6.2.1. Expression and purification sEGFRvIII........................................................... 70
6.2.2. sEGFRvIII dimerization properties.................................................................. 71
6.2.3. Antibody and ligand binding properties of sEGFRvIII.................................... 73
6.2.4. The sEGFRvIII structure.................................................................................. 74
6.2.5. The sEGFRvIII solution structure.................................................................... 76
6.3. Discussion............................................................................................................ 78
6.3.1. Antibody and ligand binding characterisitics to soluble EGFRvIII................. 78
6.3.2. The structure of EGFRvIII domain III and IV is unaffected by the mutation . 79
6.3.3. sEGFRvIII in solution ...................................................................................... 80
6.3.4. sEGFRvIII dimerization and activation ........................................................... 80
6.3.5. Implications for a therapeutic approach against EGFRvIII driven cancers ..... 81
6.4. Conclusion 83
7. Characterization of the antibody EMD1159476 bindi