Anti-tumor activity, pharmacology and toxicology of EpCAM-, CD3-bispecific single-chain antibodies [Elektronische Ressource] / vorgelegt von Maria Amann

Maria Amann

Sujets

• Biologie

Informations

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

Extrait

ANTI-TUMOR ACTIVITY, PHARMACOLOGY AND
TOXICOLOGY OF EPCAM/CD3-BISPECIFIC SIN-
GLE-CHAIN ANTIBODIES


Dissertation der

Fakultät für Biologie der
Ludwig-Maximilians-Universität
München



Vorgelegt von

Diplom-Biologin
Maria Amann

Ehrenwörtliche Erklärung
Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte
Hilfe angefertigt habe. Ich habe weder anderweitig versucht, eine Dissertation einzureichen
oder eine Doktorprüfung durchzuführen, noch habe ich diese Dissertation oder Teile dersel-
ben einer anderen Prüfungskommission vorgelegt.


München,


Die vorliegende Arbeit wurde zwischen Juli 2005 und Februar 2009 unter der Anleitung von
Prof. Dr. Patrick A. Bäuerle in der Firma Micromet AG, München, durchgeführt.

Wesentliche Teile dieser Arbeit sind veröffentlicht in:
Amann M, Brischwein K, Lutterbuese P, et al. Therapeutic window of MuS110, a single-
chain antibody construct bispecific for mouse EpCAM and mouse CD3. Cancer Res. 2008;
68: 143-51.
Amann M, Friedrich M, Lutterbuese P, et al. Therapeutic window of an EpCAM/CD3-
specific BiTE antibody in mice is determined by a subpopulation of EpCAM-expressing lym-
phocytes that is absent in humans. Cancer Immunol Immunother. 2009; 58: 95-109.
Amann M, dÁrgouges S, Lorenczewski G, et al. Anti-tumor activity of an EpCAM/CD3-
bispecific BiTE antibody during long-term treatment of mice in the absence of T cell anergy
and cytokine release. J Immunother. in press.
Teile dieser Arbeit (Text und Abbildungen) sind daher diesen Veröffentlichungen entlehnt.

Promotionsgesuch eingereicht am: 23.05.2009
Tag der mündlichen Prüfung: 15.12.2009
Erster Gutachter: Prof. Dr. Elisabeth Weiß
Zweiter Gutachter: Prof. Dr. Berit Jungnickel
Sondervotum: Prof. Dr. A. Patrick Bäuerle
- TABLE OF CONTENTS - I

CONTENTS
1 SUMMARY....................................................................................................................1
2 INTRODUCTION.........................................................................................................3
2.1 Epithelial Cell Adhesion Molecule (EpCAM) and its role in cancer.....................3
2.1.1 Structure and tissue distribution............................................................3
2.1.2 Biological functions of EpCAM...........................................................4
2.1.3 EpCAM as a prognostic carcinoma marker and its role in cancer..........7
2.1.4 Therapeutic antibodies targeting EpCAM in cancer..............................8
2.2 The concept of bispecific T cell engagers (BiTEs).............................................10
2.2.1 Structure of BiTEs..............................................................................10
2.2.2 Mode of action of BiTEs....................................................................11
2.2.3 MT110 and muS110...........................................................................13
3 AIM OF THIS STUDY................................................................................................17
4 MATERIAL AND METHODS...................................................................................18
4.1 Material.............................................................................................................18
4.1.1 Cell lines............................................................................................18
4.1.2 Mouse strains.....................................................................................20
4.1.3 Reagents, media and enzymes.............................................................20
4.1.4 Kits.....................................................................................................22
4.1.5 Buffer.................................................................................................23
4.1.6 Antibodies..........................................................................................23
4.1.7 BiTEs.................................................................................................24
4.1.8 Equipment..........................................................................................25
4.1.9 Software.............................................................................................26
4.2 Methods............................................................................................................27
4.2.1 Cell culture.........................................................................................27
4.2.2 Preparation of leukocytes....................................................................27
4.2.2.1 Isolation of PBMC from human and mouse blood 27
4.2.2.2 Isolation of mouse leukocytes from spleen and lymph nodes 28
4.2.2.3 Enrichment of CD3 cells 28
4.2.2.4 Negative isolation of CD3 cells 28
4.2.2.5 Stimulation of T cells 29
4.2.3 Cell staining and FACS analysis.........................................................29
4.2.3.1 FACS analysis of cell surface proteins 30
4.2.3.2 FACS analysis of intracellular proteins 30
4.2.3.3 FACS analysis of whole blood 31
4.2.4 Labeling of target cells.......................................................................31
4.2.4.1 PKH staining 31
4.2.4.2 CFSE labeling 31
4.2.5 Cytotoxicity assay..............................................................................32
4.2.5.1 ToxiLight assay 32
4.2.5.2 Caspase assay 33
4.2.5.3 FACS based assay 34
4.2.6 Determination of cytokine concentration............................................35 - TABLE OF CONTENTS - II

4.2.7 TGF-b enzyme linked immuno sorbent assay (ELISA).......................36
4.2.8 Determination of whole blood cell count............................................36
+ 4.2.9 Depletion of EpCAM cells from mouse splenocytes and PBMC........37
4.2.10 Analysis of BiTE serum concentrations..............................................38
4.2.10.1 Bioassay based BiTE quantification 38
4.2.10.2 BiTE quantification with Meso Scale Discovery (MSD) 39
4.2.11 Saturation binding analysis and calculation of receptor occupancy......39
4.2.12 Histopathology...................................................................................40
4.2.13 Mouse models and in vivo work..........................................................41
4.2.13.1 Housing 41
4.2.13.2 Monitoring of health, body weight and temperature 41
4.2.13.3 Application techniques 42
4.2.13.4 Blood sampling 42
4.2.13.5 Tumor models 43
4.2.13.6 Dissection, isolation of organs and fixation 44
5 RESULTS.....................................................................................................................46
5.1 In vitro characterization of muS110...................................................................46
5.1.1 Binding affinities of muS110 to CD3 and EpCAM.............................46
5.1.2 Redirected lysis of respective target cells mediated by muS110..........47
5.1.3 T cell activation mediated by muS110................................................49
5.1.3.1 Cell proliferation in cytotoxicity assays 49
5.1.3.2 Up-regulation of T cell activation marker induced by muS110 51
5.1.3.3 Cytokine release induced by muS110 55
5.2 Pharmaco-toxicological effects of muS110 in vivo.............................................57
5.2.1 Characterization of single dose administration....................................57
5.2.1.1 Adverse effects induced by a single dose of muS110 57
5.2.1.2 Systemic T cell activation induced by a single dose of muS110 58
5.2.1.3 Transient systemic cytokine release induced by a single dose of muS110 59
5.2.1.4 Peripheral lymphocytopenia induced by a single dose of muS110 61
5.2.1.5 Comparison of pharmaco-toxicological effects of muS110, KT3 and 145-2C11 63
5.2.2 Histopathological analysis of mice repeatedly treated with muS110....66
+5.2.3 EpCAM mouse lymphocytes.............................................................67
+5.2.3.1 Presence of EpCAM cells in mouse but not in human lymphocytes 67
+5.2.3.2 Dependence of muS110-induced T cell activation on EpCAM lymphocytes 69
5.2.4 Characterization of multiple dose administrations...............................72
5.2.4.1 Adverse effects induced by repetetive doses of muS110 72
5.2.4.2 Cytokine release induced by repetitive muS110 treatment 73
5.2.4.3 Cytotoxic T cells induced by repetitive doses of muS110 76
5.2.5 Increased muS110 tolerability and immune modulation......................78
+5.2.5.1 Tolerability increase of muS110 after in vivo depletion of EpCAM
lymphocytes 78
5.2.5.2 Ex vivo T cell activation after repeated administration of muS110 82
5.2.5.3 Cytotoxic and proliferative capacity of T cells after repeated administration of
muS110 84
5.2.6 Impact of glucocorticoids on muS110 tolerability in vivo....................87
5.3 In vivo efficacy..................................................................................................92
5.3.1 MuS110 efficacy and toxicity in mouse pulmonary metastasis models92
5.3.2 MuS110 efficacy and toxicity in an orthotopic 4