Aspects of the mode of action of bispecific T cell engager (BiTE) antibodies [Elektronische Ressource] / vorgelegt von Cornelia Hauff

Thilo.Hauff2

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Aspects of the mode of action of bispecific
T cell engager (BiTE) antibodies






Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg



vorgelegt von

Cornelia Hauff
aus Augsburg


Würzburg 2009
























Eingereicht am: ........................................................................................................................

Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr. M. J. Müller
Gutachter: Prof. Dr. P. A. Baeuerle
Gutachter: Prof. Dr. G. Krohne

Tag des Promotionskolloquiums: ............................................................................................

Doktorurkunde ausgehändigt am: ............................................................................................



























"Der erste Trunk aus dem Becher der Naturwissenschaft macht atheistisch,
aber auf dem Grund des Bechers wartet Gott."

Werner Heisenberg
Table of contents I
TABLE OF CONTENTS

1 Introduction .......................................................................................................................... 1
1.1 Monoclonal antibodies in cancer therapy ..................................................................... 1
1.2 Bispecific T cell engager in cancer therapy.................................................................. 2
1.3 Lysis of pathogenic cells by T cells.............................................................................. 3
1.4 Role of granzyme B in T cell-mediated cell death........................................................ 5
1.5 Role of caspases in T cell-mediated cell death ............................................................. 6
1.6 Tumor escape mechanisms ........................................................................................... 8
1.7 The target antigen EpCAM......................................................................................... 10
1.7.1 Structure ............................................................................................................... 10
1.7.2 Tissue distribution ................................................................................................ 11
1.7.3 Binding partners and function .............................................................................. 12
1.8 The target antigen CD19............................................................................................. 13
1.9 Finding the initial dose for a first-in-man clinical trial............................................... 13
1.10 Objective of this thesis................................................................................................ 15
2 Materials............................................................................................................................. 16
2.1 BiTE molecules........................................................................................................... 16
2.2 Chemicals.................................................................................................................... 16
2.3 Antibodies ................................................................................................................... 17
2.4 Analytical kits ............................................................................................................. 18
2.5 Eucaryotic cell lines.................................................................................................... 18
2.6 Laboratory consumables ............................................................................................. 18
2.7 Equipment ................................................................................................................... 19
2.8 Software for data analysis, and data base ................................................................... 20
3 Methods.............................................................................................................................. 21
3.1 Cell culture.................................................................................................................. 21
3.2 Passaging of cells........................................................................................................ 21
3.3 Cryoconservation of cells ........................................................................................... 22
3.4 Effector cell preparation.............................................................................................. 22
3.5 Stimulation of effector cells........................................................................................ 22
3.6 CD3 T cell enrichment................................................................................................ 23
3.7 Fluorescent labeling of cells ....................................................................................... 23
3.8 Membrane-labeling of target cells .............................................................................. 24 Table of contents II
3.9 Surface labeling of cells with fluorescence-conjugated antibodies ............................ 24
3.10 Intracellular staining of cells with fluorescence-conjugated antibodies ..................... 24
3.11 Cytotoxicity assay....................................................................................................... 26
3.12 Analysis of caspase 3/7 activation .............................................................................. 27
3.13 Release of adenylate kinase ........................................................................................ 28
3.14 Analysis of PARP cleavage ........................................................................................ 28
3.15 ELISA for cleaved PARP ........................................................................................... 29
3.16 Analysis of DNA fragmentation ................................................................................. 29
3.17 Analysis of cytokine production ................................................................................. 30
3.18 Estimation of the minimal anticipated biological effect level .................................... 31
4 Results ................................................................................................................................ 32
4.1 Effects of the BiTE molecule MT103......................................................................... 32
4.1.1 Lysis of tumor cells by PBMC............................................................................. 32
4.1.2 Granzyme B expression in T cells in response to stimulation ............................. 33
4.1.3 Activation of caspases.......................................................................................... 34
4.1.4 Perforin expression in T cells in response to stimulation..................................... 34
4.1.5 Expression of activation markers CD69 and CD25 ............................................. 35
4.1.6 Expression of adhesion molecules CD2 and LFA-1 ............................................ 38
4.1.7 Dexamethasone as a co-medication in cancer therapy......................................... 39
4.1.7.1 Influence of dexamethasone on expression of activation markers............... 40
4.1.7.2 Influence of dexamethasone on expression of adhesion molecules ............. 41
4.1.7.3 Influence of dexamethasone on expression of cytokines ............................. 42
4.2 Mode of action of the BiTE molecule MT110............................................................ 44
4.2.1 Lysis of tumor cells by PBMC............................................................................. 44
4.2.2 Lytic potential of pre-stimulated T cells .............................................................. 46
4.2.3 Granzyme B expression in T cells in response to stimulation ............................. 48
4.2.4 Perforin expression in T cells in response to stimulation..................................... 49
4.2.5 Release of adenylate kinase during lysis of tumor cells....................................... 50
4.2.6 Expression of CD25, CD2 and LFA-1 ................................................................. 50
2+4.2.7 Importance of Ca ions for lysis of tumor cells .................................................. 52
4.2.7.1 Influence of EGTA on lysis of tumor cells .................................................. 53
4.2.7.2 Influence of EGTA on activation of caspases .............................................. 54
4.2.8 Importance of caspases for lysis of tumor cells ................................................... 55
4.2.9 Importance of granzyme B for lysis of tumor cells.............................................. 57 Table of contents III
4.2.10 Importance of soluble factors for lysis of tumor cells.......................................... 59
4.3 Correlation between TGF-β and the efficiency of T cells