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Dual Antigen-Restricted Complementation of a Two-Part Trispecific Antibody for Targeted Immunotherapy of Blood Cancer

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Dual Antigen-Restricted Complementation of a Two-Part Trispecific Antibody for Targeted Immunotherapy of Blood Cancer Dual Antigen-Restricted Complementation of a Two-Part Trispecific Antibody for Targeted Immunotherapy of Blood Cancer Von zwei Antigenen abhängige Komplementierung eines zweiteiligen trispezifischen Antikörpers zur gezielten Immuntherapie von Blutkrebs Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Agnes Banaszek aus Gdynia (dt. Gdingen), Polen Würzburg 2013 Declaration Declaration I hereby assure that I have carried out the presented study independently and without using any other sources or help, except where stated. Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form. I further declare that I have not acquired or tried to acquire any other academic degrees, except my diploma degree. Würzburg, 20 September 2013 ......................................................................... Agnes Banaszek Eingereicht am: …………………………………………………………… Mitglieder der Promotionskommission: Vorsitzender: …………………………………………………………… Gutachter: Prof. Dr. rer. nat. Harald Wajant (Med. Fakultät) Gutachter: Prof. Dr. med. Thomas Dandekar (Biol. Fakultät) Tag des Promotionskolloquiums: ………………………………………….. Doktorurkunde ausgehändigt am: ………………………………………….. Agnes Banaszek ii Acknowledgements Acknowledgements First of all, I would like to express my gratitude to Prof. Dr. Harald Wajant, who gave me the opportunity to carry out the major part of this study within his group and laboratory. Thank you for your willingness to be my supervisor, for your valuable advice and encouraging support, for always having an open door for me, for the helpful discussions and suggestions, and, finally, for proofreading. Of course, this study wouldn’t have been possible without Dr. med. Gernot Stuhler, who had the brilliant idea for this project. Thank you for guiding this project through all its ups and downs, for granting me the opportunity to work on such an innovative and challenging research issue, for giving me the freedom I needed to make my own decisions, and, finally, for providing the necessary funding. I would also like to thank Prof. Dr. Thomas Dandekar, who agreed to supervise me during this study as a second assessor. Prof. Dr. med. Stefan Gattenlöhner deserves thanks for providing the scFv against fAChR and for the good collaboration on the rhabdomyosarcoma concerning part of this study. Furthermore, I wish to thank Dr. Kurt Bommert and his group for providing me with a workplace at his laboratory for the first one and a half years of my doctoral studies, for their support and the nice working atmosphere. I am also grateful to Dr. med. Dirk Hönemann, who supervised me at the very beginning of this project, before leaving for the other side of the world. Special thanks go to Daniela Weisenberger for cloning a few fusion proteins (CD3- Gaussia Luciferase) and for the preparation and monitoring of some of the bacterial cultures for antibody expression. In addition, I am indebted to Jacqueline Maar and Hannes Schloßnagel for helping me a few times with the bacterial production and purification of the 18 antibody constructs. Furthermore, I am grateful to Nadine Vornberger for performing the cytotoxicity assays presented in this study. They all worked very diligently. Moreover, I would like to thank the members of MIM ( Molekulare Innere Medizin ) for their support, advice, the pleasant working atmosphere in the laboratory, and their friendly welcome. Special thanks go to my parents for their support throughout my entire studies and my sister Paulina, an English expert, for proofreading. I would particularly like to thank Martin for all his love, his patient and tireless support throughout my studies and work on this thesis, and for thorough proofreading. Finally, thank you to everyone else who supported me during the last one and a half years as best they could. Agnes Banaszek iii Table of Contents Table of Contents Declaration.................................................................................................................. ii Acknowledgements ....................................................................................................iii Table of Contents .......................................................................................................iv 1 Abstract ............................................................................................................... 1 Kurzbeschreibung ..................................................................................................................... 3 2 Introduction ......................................................................................................... 5 2.1 Cancer Prevalence and Motivation for the Project ...................................................... 5 2.2 Targets in Tumour Therapy ......................................................................................... 5 2.2.1 Fetal Acetylcholine Receptor and Rhabdomyosarcoma ........................................ 6 2.2.2 HLA-A2 and the Function of MHC Molecules ........................................................ 8 2.2.3 CD45 – Leucocyte-Common Antigen .................................................................. 11 2.3 Therapeutic Antibodies in Cancer Therapy ............................................................... 13 2.3.1 The Concept of Bispecific Antibodies .................................................................. 14 2.3.2 Recombinant Bispecific Antibody Formats .......................................................... 14 2.4 Bispecific Antibodies for Targeting Immune Effector Cells ........................................ 17 2.4.1 Activation of Naive T Cells ................................................................................... 17 2.4.2 Primary Signal for T-Cell Activation ..................................................................... 19 2.4.3 Bispecific Antibodies for the Retargeting of T Cells ............................................. 20 2.5 Dual Targeting with Bispecific Antibodies .................................................................. 23 2.6 Thesis Aims and the Concept of a Dual Antigen-Restricted Trispecific Antibody ..... 24 3 Materials and Methods .......................................................................................27 3.1 Materials .................................................................................................................... 27 3.1.1 Instruments........................................................................................................... 27 3.1.2 Special Implements .............................................................................................. 28 3.1.3 Chemicals, Solutions, and Media ......................................................................... 28 3.1.4 Antibodies and their Working Dilution .................................................................. 30 3.1.5 Antigens, Enzymes, Kits, and Markers ................................................................ 31 3.1.6 Oligonucleotides ................................................................................................... 32 3.1.7 Vectors ................................................................................................................. 34 3.1.8 E. coli Strains ....................................................................................................... 36 3.1.9 Mammalian Cell Lines .......................................................................................... 37 3.2 Strategies for Cloning of Recombinant Antibody Constructs..................................... 38 3.2.1 Construction of Recombinant Bispecific AChR γ × CD3 taFvs ............................. 38 3.2.2 Construction of a Two-Part Trispecific HLA-A2 × CD3 × CD45 Antibody ........... 39 3.2.3 Construction of Bispecific HLA-A2 × CD3 taFvs .................................................. 41 3.3 Cloning of Recombinant Antibody Constructs ........................................................... 41 3.3.1 Polymerase Chain Reaction (PCR) ..................................................................... 41 3.3.2 Restriction Digestion ............................................................................................ 42 3.3.3 Agarose Gel Electrophoresis and DNA Gel Extraction ........................................ 42 3.3.4 Ligation ................................................................................................................. 43 3.3.5 Preparation of Chemically Competent Cells ........................................................ 43 3.3.6 Transformation of Chemically Competent Cells................................................... 43 3.3.7 Screening of Positive Transformants by PCR ...................................................... 44 3.3.8 Plasmid DNA Isolation (Mini and Midi Preparation) ............................................. 44 3.3.9 Sequence Analysis ............................................................................................... 45 Agnes Banaszek iv Table of Contents 3.4 Expression and Purification of Recombinant Antibodies ........................................... 45 3.4.1 Periplasmic Protein Expression ........................................................................... 45 3.4.2 Immobilised-Metal Affinity Chromatography (IMAC) ............................................ 45 3.4.3 Determination of Protein Concentration ..............................................................
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