Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation Entwicklung CD40 gerichteter bifunktioneller scFv-TRAIL Fusionsproteine die TRAILR1- und TRAILR2-spezifischen Zelltod und dendritischen Zellaktivierung induzieren Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg Section Biomedicine submitted by Mohamed El-Sayed Ahmed Mohamed El-Mesery PhD German-Egyptian Research Long Term Scholarship (GERLS) holder from Biochemistry Department, Faculty of Pharmacy Mansoura University, Mansoura, Egypt Würzburg 2014 I Submitted on: …………………………………………………………… Members of the promotion committee: Chairperson: Prof. Dr. Jörg Schultz Primary Supervisor: Prof. Dr. Harald Wajant Supervisor (Second): Prof. Dr. Ralf Bargou Supervisor (Third): Prof. Dr. Thomas Müller Date of Public Defence: …………………………………………….………… Date of Receipt of Certificates: ………………………………………………. II Affidavit I hereby confirm that my thesis entitled “Development of CD40-targeted bifunctional scFv- TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation” is the result of my own work. I did not receive any help or support from commercial consultants. All sources and/or materials applied are listed and specified in the thesis. Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form. Würzburg,…………………………………………………………………… Place, Date Signature Eidesstattliche Erklärung Hiermit erkläre ich an Eides statt, die Dissertation “Entwicklung CD40 gerichteter bifunktioneller scFv-TRAIL Fusionsproteine die TRAILR1- und TRAILR2-spezifischen Zelltod und dendritischen Zellaktivierung induzieren” eigenständig, d.h. insbesondere selbständig und ohne Hilfe eines kommerziellen Promotionsberaters, angefertigt und keine anderen als die von mir angegebenen Quellen und Hilfsmittel verwendet zu haben. Ich erkläre außerdem, dass die Dissertation weder in gleicher noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat. Würzburg,…………………………………………………………………… Datum Unterschrift III The current work was achieved in the period from 1. April 2011 till 30. April 2014 in the Division of Molecular Internal Medicine, University Hospital of Würzburg under the supervision of Prof. Dr. Harald Wajant. IV Acknowledgments I am greatly thankful to my main supervisor Prof. Dr. Harald Wajant who accepted me in his research group in the Division of Molecular Internal Medicine, University Hospital of Würzburg and I would like to express my deep gratitude for his kind supervision, valuable guidance, constructive criticism and continuous follow up during the course of my PhD study. I was extremely fortunate to work under his supervision. I would like to thank the German Academic Exchange Service (DAAD) and the Egyptian Ministry of Higher Education who awarded me with the PhD German-Egyptian Research Long Term Scholarship (GERLS) and financed my stay in Germany during my PhD study. I am indebted to Prof. Dr. Ralf Bargou and Prof. Dr. Thomas Müller for their support and follow up during my PhD study as second and third supervisors. I would like to express my deep thanks to all my colleagues and members of the Division of Molecular Internal Medicine, University Hospital of Würzburg for their continuous help and kind support. I am also thankful to the members of the Graduate School of Life Sciences (GSLS), Würzburg University for their continuous help, support and interesting programs and workshops during my PhD study. I am also indebted to Mrs. Renate Jung and Mr. Werner Tiltz, who rented me an apartment in their house in Würzburg and took care with my wife and my child during my PhD work. They were like our second family and helped us to integrate in the German society. I am deeply thankful to my parents for their hearty patience, strong motivation and support all over my life and especially during my PhD study in Germany away from Egypt. Finally, I would like to express my sincere thanks to my wife Nada and my son Adham who stayed with me in Germany during my PhD study for more than 3 years. I really appreciate their patience for my extra work at weekends and in vacations and their continuous motivation to finish this current work in a reasonable time. Mohamed El-Mesery V For my parents, my wife and my children VI Table of contents Table of contents 1. Introduction.......................................................................................................................11 1.1. Receptors and ligands of the TNF family ................................................................................. 11 1.1.1. TNF receptor superfamily (TNFRSF) ................................................................................. 11 1.1.2. TNF ligand family ............................................................................................................. 11 1.2. TRAIL/TRAILR-system and activation of apoptosis................................................................... 14 1.2.1. Classification of TRAILRs .................................................................................................. 14 1.2.2. Mechanisms of TRAIL DR-induced apoptosis .................................................................... 15 1.2.3. Clinical trials for TRAIL DR targeting therapies.................................................................. 17 1.2.4. Mechanisms of resistance against TRAIL-induced apoptosis............................................. 18 1.3. Exogenous activation of TNFRs ............................................................................................... 19 1.4. Response of TRAIL DRs to soluble recombinant TRAIL and strategies to improve its activity ... 19 1.5. CD40 and its role in cancer immunotherapy ........................................................................... 21 1.6. The role of DCs in immune system and the effect of tumors on their function ........................ 22 1.6.1 The role of DCs in immune system .................................................................................... 22 1.6.2. Effect of tumors on DCs function ..................................................................................... 23 1.7. Aim of the work...................................................................................................................... 24 2. Materials ...........................................................................................................................25 2.1. Chemicals, reagents and cell culture mediums for the cell culture .......................................... 25 2.2. Enzymes ................................................................................................................................. 26 2.3. Antibodies .............................................................................................................................. 26 2.4. Kits ......................................................................................................................................... 27 2.5. Instruments and disposable materials/equipments ................................................................ 28 2.6. Preparations and buffers ........................................................................................................ 29 2.7. Cells ....................................................................................................................................... 30 2.7.1. Eukaryotic cells ................................................................................................................ 30 2.7.2. Dendritic cells (DCs) ......................................................................................................... 31 7 Table of contents 2.7.3. Prokaryotic cells .............................................................................................................. 31 2.8. Plasmids ................................................................................................................................. 31 3. Methods ...........................................................................................................................32 3.1. Cell culture ............................................................................................................................. 32 3.2. Cloning and production of the expression plasmids ................................................................ 32 3.3. Protein production ................................................................................................................. 32 3.4. Protein purification ................................................................................................................ 33 3.5. FACS analysis .......................................................................................................................... 33 3.5.1. Detection of cell surface markers ..................................................................................... 33 3.5.2. scFv:G28-TRAIL binding to CD40-transfectant cells .......................................................... 34 3.6. Equilibrium binding studies .................................................................................................... 34 3.6.1. Binding studies in HEK293 cells transiently transfected with the various TRAILRs ............. 34 3.6.2. Binding studies