Development and characterization of monoclonal antibodies to GDF-15 for potential use in cancer therapy Die Entwicklung und Charakterisierung monoklonaler Antikörper gegen GDF-15 zur potenziellen Anwendung in der Krebstherapie Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section: Infection and Immunity Submitted by Markus Erich Junker from Ludwighshafen/Rhein, Germany Würzburg 2015 Submitted on:……………………………………………………………………………... Office stamp Members of the Promotionskomitee: Chairperson: Professor Dr. Caroline Kisker Primary Supervisor: Professor Dr. Roland Benz (Since 29th July 2015) (From 3rd November 2008 - 20th July 2015 Professor Dr. Jörg Wischhusen) Supervisor (Second): Professor Dr. Roland Benz (Until 29th July 2015) Supervisor (Third): Professor Dr. Thomas Hünig Date of Public Defence:…………………………………………………………………... Date of Receipt of Certificates:…………………………………………………………... Table of contents Table of contents Table of contents ........................................................................................................................ 4 Zusammenfassung ...................................................................................................................... 8 Abstract .................................................................................................................................... 11 1 Introduction ........................................................................................................................... 13 1.1 Cancer ............................................................................................................................. 13 1.1.1 Development of Cancer ........................................................................................... 13 1.1.2 Tumor Progression and Metastasis ......................................................................... 15 1.1.3 Elimination of Cancer Cells by the Immune System .............................................. 16 1.1.3.1 Natural killer Cells ............................................................................................... 16 1.1.3.2 Cytotoxic T Lymphocytes .................................................................................... 18 1.1.3.3 Leukocyte Recruitment ........................................................................................ 20 1.1.4 Immune Evasion ...................................................................................................... 22 1.2 The TGF- Superfamily ................................................................................................. 24 1.2.1 TGF- ...................................................................................................................... 25 1.2.2 TGF- and Cancer ................................................................................................... 26 1.2.3 GDF-15 .................................................................................................................... 27 1.2.3.1 GDF-15 - Structure and Biochemistry ................................................................. 28 1.2.3.2 Expression of GDF-15 ......................................................................................... 30 1.2.3.3 GDF-15 and its Function ...................................................................................... 31 1.2.3.4 GDF-15 in Cancer ................................................................................................ 32 1.3 Treatment of Cancer with Targeted Therapy ................................................................. 34 1.3.1 Antibodies in the Clinic ........................................................................................... 35 1.3.2 Antibody Structure .................................................................................................. 36 1.3.3 Antibody Humanization .......................................................................................... 37 1.3.4 Mode of Actions of Antibodies ............................................................................... 39 1.4 The Aim of the Thesis .................................................................................................... 40 2 Material and Methods ............................................................................................................ 41 2.1 Material .......................................................................................................................... 41 2.1.1 Devices .................................................................................................................... 41 2.1.2 Chemicals and reagents ........................................................................................... 42 2.1.3 Material for Immnunohistochemistry ...................................................................... 43 2.1.4 Material for protein biochemistry ........................................................................... 44 4 Table of contents 2.1.5 Material for molecular biology ............................................................................... 45 2.1.6 Kits and kit contents ................................................................................................ 46 2.1.7 Reagents, buffers for methods to generate monoclonal antibodies ......................... 47 2.1.8 Antibodies ............................................................................................................... 48 2.1.9 Oligonucleotides ...................................................................................................... 49 2.1.10 Cell lines ................................................................................................................ 50 2.1.11 Plasmids ................................................................................................................ 50 2.1.12 Reagents for FACS staining .................................................................................. 51 2.1.13 Cytokines ............................................................................................................... 51 2.1.14 Standard DNA and protein ladder ......................................................................... 52 2.2 Methods .......................................................................................................................... 53 2.2.1 Immunohistochemical staining ............................................................................... 53 2.2.2 Thawing of cells ...................................................................................................... 54 2.2.3 Cryopreservation of cells ........................................................................................ 54 2.2.4 Isolation and preparation of human immune cells .................................................. 54 2.2.4.1. Isolation of human peripheral lymphocytes from whole blood .......................... 55 2.2.5 Flow cytometry ....................................................................................................... 56 2.2.6 Adherence Assay ..................................................................................................... 57 2.2.7 Woundhealing assay ................................................................................................ 57 2.2.8 Determination of cell viability using the WST-1 assay .......................................... 58 2.2.9 Protein biochemical methods .................................................................................. 58 2.2.9.1 Preparation of cell lysates from human PBMCs, tumor cells and tissues ............ 58 2.2.9.2 Determination of the total amount of protein by the Bradford method................ 59 2.2.9.3 Immunoblotting .................................................................................................... 59 2.2.10 Methods for gene expression analysis ................................................................... 61 2.2.10.1 Isolation of RNA from immune cells and HUVEC cells ................................... 61 2.2.10.2 Determination of RNA concentration ................................................................ 61 2.2.10.3 Synthesis of cDNA from isolated RNA ............................................................. 62 2.2.11 Polymerase Chain Reaction (PCR) ....................................................................... 63 2.2.11.1 Isolation of genomic DNA from mouse ear punches ......................................... 63 2.2.11.2 Mouse GDF-15 genotyping ................................................................................ 63 2.2.11.3 DNA gel electrophoresis .................................................................................... 64 2.2.11.4 Quantitative Realtime Polymerase Chain Reaction (qPCR) .............................. 64 2.2.12 DNA-Microarray (Affymetrix) on human PBMCs and HUVECs ........................ 66 2.2.13 Cloning .................................................................................................................. 66 2.2.13.1 Ligation .............................................................................................................. 66 2.2.13.2 Transformation in C2988 bacteria ...................................................................... 67 2.2.13.3 Preparation of plasmids ...................................................................................... 68 5 Table of contents 2.2.13.4 Restriction digest of pJET1.2/blunt (Fermentas)