Generation and Characterization of heavy chain antibodies derived from Camelids Katrin Schmidthals München 2013 Generation and Characterization of heavy chain antibodies derived from Camelids Katrin Schmidthals Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades an der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Katrin Schmidthals aus Penzberg München, den 18. April 2013 Erstgutachter: Prof. Dr. Heinrich Leonhardt Zweitgutachterin: Prof. Dr. Angelika Böttger Eingereicht am: 18. April 2013 Tag der mündlichen Prüfung: 13. Juni 2013 Table of Contents Table of Contents Summary............................................................................................................................... 6 Zusammenfassung ................................................................................................................ 7 1 Introduction ................................................................................................................. 9 1.1 Antibodies .......................................................................................................... 10 1.1.1 Structure and Function of Antibodies ............................................................ 11 1.1.2 Areas of Application ....................................................................................... 15 1.2 Display Technologies and Recombinant Antibody Formats .............................. 17 1.2.1 Recombinant Antibody Formats ..................................................................... 18 1.2.2 Display Technologies ...................................................................................... 20 1.3 Heavy chain antibodies ...................................................................................... 24 1.3.1 Structure of Camelidae heavy chain antibodies ............................................. 24 1.3.2 Genetic background of functional heavy chain antibodies ............................ 25 1.3.3 Unique features of V Hs ................................................................................. 29 H 1.3.4 Applications of V Hs ....................................................................................... 31 H 1.4 Aims and Objectives ........................................................................................... 34 2 Materials and Methods ............................................................................................. 35 2.1 Material .............................................................................................................. 35 1 Table of Contents 2.1.1 Consumables ................................................................................................... 35 2.1.2 Solutions and Chemicals ................................................................................. 37 2.1.3 Instruments..................................................................................................... 40 2.1.4 Cell lines .......................................................................................................... 41 2.1.5 Antibodies ....................................................................................................... 42 2.1.6 Primer ............................................................................................................. 42 2.1.7 Bacterial strains .............................................................................................. 43 2.2 Methods ............................................................................................................. 44 2.2.1 Molecular biological Methods ........................................................................ 44 2.2.2 Cell Culture Methods ...................................................................................... 48 2.2.3 Biochemical Methods ..................................................................................... 50 2.2.4 Immunological Methods ................................................................................. 54 2.2.5 Phage Display Technology .............................................................................. 59 3 Results ........................................................................................................................ 67 3.1 Generation and selection of antigen specific V Hs ............................................ 69 H 3.1.1 Antigen selection ............................................................................................ 69 3.1.2 Amplification of the V H repertoire ............................................................... 72 H 3.1.3 Cloning of V H library in pHEN4 ..................................................................... 75 H 2 Table of Contents 3.1.4 Phage Display .................................................................................................. 81 3.1.5 Selection of antigen specific V HS by Phage ELISA ...................................... 100 H 3.1.6 Selection of antigen specific V Hs by the F2H-Assay ................................... 111 H 3.2 Characterization of antigen specific V Hs ........................................................ 117 H 3.2.1 Biochemical characterization ....................................................................... 118 3.2.2 Intracellular characterization ....................................................................... 134 4 Discussion ................................................................................................................ 139 4.1 V H libraries ..................................................................................................... 140 H 4.1.1 Immunogenicity of Antigens......................................................................... 140 4.1.2 V H Library Size and Diversity ...................................................................... 142 H 4.2 V H Selection .................................................................................................... 144 H 4.2.1 A new method to select V Hs for in vivo applications ................................. 144 H 4.3 Compensation of V -V combination ............................................................... 147 H L 4.4 CDR3, disulfide bonds and functionality .......................................................... 148 4.5 Outlook ............................................................................................................. 151 5 Annex ....................................................................................................................... 153 5.1 References ........................................................................................................ 153 5.2 Abbreviations ................................................................................................... 166 3 Table of Contents 5.3 Eidesstattliche Erklärung .................................................................................. 169 5.4 Acknowledgement............................................................................................ 170 5.5 Publications/Patent applications ..................................................................... 172 5.5.1 Publications .................................................................................................. 172 5.5.2 Patent applications ....................................................................................... 172 4 5 Summary Summary Antibodies and antibody fragments are essential tools in basic research, diagnostics and therapy. Conventional antibodies consist of two heavy and two light chains with both chains contributing to the antigen-binding site. In addition to these conventional antibodies, camelids (llamas, alpacas, dromedaries and camels) possess so-called heavy chain antibodies (hcAbs) that lack the light chains. The antigen binding site of these unusual antibodies is formed by one single domain only, the so called V H domain. The V H domain represents H H the smallest intact antigen binding fragment (~ 15 kDa) and is characterized by very high stability, solubility and specificity. These unique features render V Hs a promising alternative H to conventional antibodies and antibody fragments with a multitude of possible applications. In the course of this thesis, we aimed to generate and characterize V Hs suitable for H different applications ranging from biochemical studies to immunofluorescence assays and live cell imaging. In order to meet the diverse requirements of the intended downstream applications, a new selection method differing from traditional phage display, called native panning, was developed and established. In combination with the protein-protein interaction fluorescent two-hybrid (F2H) assay, a new process to identify antigen specific V Hs functional inside living cells was developed. H We used the immune system of alpacas to generate V H libraries against various H antigens, ranging from small peptides to large proteins. The libraries were screened by phage display and antigen specific V Hs were identified by phage ELISA. We were able to H identify antigen specific V Hs against different antigens which were characterized and their H functionality was tested in various applications. Furthermore, we could demonstrate that the selection method influences which V Hs are identified and therefore needs to be chosen H very carefully with regard to the intended biochemical and cell biological application. In summary, we developed and established an efficient and versatile process to screen and identify antigen specific V Hs suitable for different downstream applications. H 6