Université Libre de Bruxelles Faculté de Médecine
Institut de Recherche Interdisciplinaire en Biologie Humaine et Moleculaire Laboratoire de Cytologie et de Cancérologie Expérimentale
Development of cancer immunotherapy
based on parvoviral vectors and hybrid cell vaccination
Thèse présentée en vue de l’obtention du grade de Docteur en Sciences Biomédicales
Siew Chiat Cheong
Promoteurs : Thierry Velu
Annick Brandenburger
Année 2004-2005
Members of the Jury:
Prof. Philippe LEBRUN (President of the jury) Prof. Thierry VELU (Promoter)
Dr Annick BRANDENBURGER (Co-promoter) Prof. Yvan DE LAUNOIT
Prof. Michel GOLDMAN Prof. Philippe MARTIAT
Prof. Kris THIELEMANS Vrije Universiteit Brussel (External member) Prof. Pierre COULIE Université Catholique de Louvain (External member)
Index
Acknowledgements 7
List of abbreviations 10
Résumé 11
Summary 12
Introduction 13
I Principles of tumour immunity 15
I.1 Tumour associated antigens 15
I.2 Innate and adaptive immunity 16
I.3 Antigen presentation 17
I.4 T-cell immunity 18
I.5 Tolerance 18
I.6 Mechanisms to escape from tumour surveillance 19 I.7 Cancer immunotherapy strategies 19
II DC-based vaccines 21
II.1 Co-culture of DCs with tumour cells 21 II.2 Loading DCs with tumour peptides or lysates 22 II.3 Transduction of DCs with DNA/RNA encoding tumour antigens 22 II.4 Generation of dendritic cell / tumour cell hybrids 22
III Fusion via viral fusogenic peptides for cancer therapy 26
IV Cancer therapy through the use of viral vectors 28
Results 31 I A novel method for the titration of recombinant virus stocks 32
I.1 Titration of MVM vectors by in situ hybridization 32
I.2 ELISPOT titration method I 33
I.3 ELISPOT titration method II 37
I.4 IL2 production kinetics 38
I.5 Sequencing cDNA of infected cells 39
I.6 P38 transactivation 39
II Generation of dendritic cell - tumour cell hybrids via the expression of a viral
fusogenic membrane glycoprotein 41
II.1 Fusion via the expression of GaLV-FMG 41 II.2 Hybrid formation after transient transfection of human tumour cells with GaLV-FMG 42 II.3 Dendritic cell/tumour cell hybrid formation after transduction with recombinant AAV 44 II.3.1 Transduction efficiency of different AAV serotypes in melanoma cells 44 II.3.2 Kinetics of transgene expression after AAV2 infection 45 II.3.3 Construction and production of AAV2-FMG vector 46 II.3.4 DC/TC hybrid formation after AAV2-FMG infection of tumour cells 47 II.3.5 Hybrid formation between tumour cells after AAV2-FMG infection 47
II.4 Hybrid formation with an inducible FMG expressing cell line 49 II.4.1 Establishment of inducible 518Tet-on cell lines 50 II.4.1.1 Kinetics of Tet-on induction 51 II.4.1.2 Establishment of a stable 518Tet-FMG cell line 52 II.4.2 Establishment of a silenced inducible 518Tet-on cell line 53 II.4.2.1 Kinetics of Tet-on induction in the presence of a silencer 54 II.4.2.2 Establishment of a stable 518Tet-FMGsil cell line 55
II.5 Towards unambiguous hybrid detection 56 II.5.1 Evaluation of the background of double-labelled cells in the absence of fusion 56 II.5.2 Optimization of cell staining 57 II.5.3 Hybrid formation using transiently transfected CHO cells 58 II.5.4 Establishment of a stable CHO-FMG cell line 59
II.6 Optimization of the fusion conditions 66
II.6.1 Adherence 66
II.6.2 Ratio of fusion partners and cell density 66
II.6.3 Incubation time 68
II.6.4 Percentage of FMG expressing cells 68
II.6.5 Addition of polybrène 70
II.7 Hybrid formation between CHO-FMG and human dendritic cells 71 II.7.1 Hybrid formation at different DC ratios 73 II.7.2 Hybrid formation using different DC types 73
II.8 CHO-FMG cells as fusogenic cell line for the formation of human DC/TC hybrids 76 II.8.1 Formation of tri-parental hybrids via CHO-FMG cells 76 II.8.2 Optimization of tri-parental hybrid generation 78 II.8.3 Human DC/TC fusion via CHO-FMG membranes 79
II.9 Generation and characterization of GaLV-FMG specific antibodies 81 II.9.1 Presence of αFMG specific antibodies 81 II.9.2 Fusion blocking ability of αFMG antibodies 84
Discussion 87
Material and Methods 95
References 109
Curriculum Vitae 121
Annexes 123 I Cheong SC et al (2003)
A novel method for the titration of recombinant virus stocks by ELISPOT assay.
J Virol Meth 109:119-124
II Cheong SC et al (submitted)
Generation of multiparent cell hybrids via a fusogenic cell line
III Phan V et al (2003)
A new genetic method to generate and isolate small, short-lived but highly potent dendritic cell-tumour cell hybrid vaccines.
Nat Med 9:1215-1219
IV El Bakkouri K et al (2005)
In vivo anti-tumour activity of recombinant MVM parvoviral vectors carrying the human interleukin-2 cDNA.
J Gene Med (2005) 7:189-197
V Schagen FHE et al (1999)
Photodynamic treatment of adenoviral vectors with visible light: an easy and convenient method for viral inactivation.
Gene Ther 6:873-881
VI Griscelli et al (2000)
Combined effects of radiotherapy and angiostatin gene therapy in glioma tumor model PNAS 97:6698-6703
