Consequences of sequence variants for the expression of a dual targeting novel format antibody construct A Thesis Submitted to The University of Manchester for the Degree of Doctor of Philosophy 2014 Claire Gaffney CONTENTS CONTENTS ........................................................................................................................... 2 LIST OF FIGURES .................................................................................................................. 7 LIST OF TABLES ..................................................................................................................10 ABSTRACT ..........................................................................................................................12 DECLARATION ....................................................................................................................13 COPYRIGHT STATEMENT ...................................................................................................13 ACKNOWLEDGEMENTS .....................................................................................................14 DEDICATION ......................................................................................................................14 ABBREVIATIONS ................................................................................................................15 CHAPTER 1: INTRODUCTION ..............................................................................................19 1.1 Introduction to biopharmaceuticals ........................................................................20 1.2 Antibodies as therapeutics .......................................................................................23 1.2.1 Natural antibody structure ...................................................................................23 1.2.2 Chimeric, humanized and fully human antibodies ...............................................27 1.2.3 Novel format antibodies ......................................................................................28 1.3 Technologies in novel format antibody generation .................................................32 1.3.1 Phage display ........................................................................................................33 1.3.2 Construction of a phage library ............................................................................37 1.3.3 Selection and amplification of phage against a desired antigen .........................38 1.4 Expression systems for the production of natural and engineered antibodies .......41 1.4.1 HEK cells as expression hosts ...............................................................................44 1.4.2 CHO cells as expression hosts ..............................................................................45 1.4.2.1 Selection and amplification systems in CHO cells .............................................47 1.4.3 Cell-free protein expression .................................................................................49 1.5 Molecular events that affect recombinant antibody expression in mammalian cells ........................................................................................................................................52 1.5.1 Gene integration and transcriptional silencing ....................................................54 1.5.2 Post-transcriptional processing, localization and stability of mRNA ...................56 1.5.3 Translation and translocation to the ER ...............................................................58 1.5.4 Protein folding and post-translational modifications ..........................................61 1.5.5 The unfolded protein response ............................................................................63 2 1.5.6 Protein degradation pathways .............................................................................65 1.5.7 Secretion ..............................................................................................................67 1.5.8 Amino acid variations in antibody domains affect mAb expression ....................68 1.6 Project summary and aims and objectives ..............................................................71 CHAPTER 2: MATERIALS AND METHODS ..........................................................................75 2.1 Materials and equipment .........................................................................................76 2.1.1 Sources of chemicals, reagents and equipment ..................................................76 2.1.2 Preparation of Solutions .......................................................................................76 2.2 Generation of novel-format antibodies ...................................................................77 2.2.1 Phage libraries used for the selection of anti-hen egg white lysozyme (HEWL) domain antibodies (dAbs) ....................................................................................77 2.2.2 Passive Phage Selection .......................................................................................78 2.2.3 Soluble phage selection ........................................................................................79 2.2.3.1 Biotinylation of Hen Egg White Lysozyme (HEWL) for soluble selection .............79 2.2.3.2 Buffer exchange of biotinylated HEWL .............................................................80 2.2.3.3 Soluble phage selection of anti-HEWL dAbs using biotinylated HEWL .............82 2.2.4 Determination of eluted phage titre ....................................................................83 2.2.5 Amplification of eluted phage in TG1 E.coli .........................................................83 2.2.6 Precipitation of amplified eluted phage ...............................................................84 2.2.7 Overnight deep-well culture of selected phage-infected TG1 E.coli ....................85 2.2.8 Determination of binding specificity of selected phage by ELISA ........................85 2.2.9 Determination of selected phage diversity ..........................................................86 2.2.9.1 Colony PCR of phage-infected TG1 bacteria .....................................................86 2.2.9.2 DNA purification of phage DNA from colony PCR .............................................87 2.2.10 Extraction of phage DNA from selected phage amplified in TG1 E.coli ..............87 2.3 Generation and purification of plasmids..................................................................88 2.3.1 Preparation of competent DH5α E.coli ................................................................88 2.3.2 Bacterial growth medium and agar selection plates ............................................88 2.3.3 Transformation of DH5α competent E.coli cells ..................................................89 2.3.4 DNA preparation from bacterial and phage culture ............................................89 2.3.5 Generation of glycerol stocks ...............................................................................89 2.3.6 Determination of DNA concentration and purity .................................................90 2.3.7 Restriction enzyme digests of plasmid DNA .........................................................90 2.3.8 Agarose gel electrophoresis .................................................................................92 3 2.3.9 Gel purification of DNA bands from 1 [w/v] % agarose .......................................92 2.3.10 DNA ligations for plasmid generation ................................................................93 2.3.11 Generation of pTT5 heavy chain novel-format antibody vectors ......................93 2.3.12 Synthesis and cloning of CDR3 swapped dAb sequences ..................................94 2.3.13 Preparation of plasmid DNA for stable transfection ..........................................94 2.3.14 DNA sequence analysis.......................................................................................95 2.3.14.1 DNA sequencing in 96-well PCR plates............................................................95 2.3.14.2 DNA sequencing in 1.5ml microcentrifuge tubes ............................................96 2.4 Mammalian cell culture ............................................................................................97 2.4.1 General cell culture techniques............................................................................97 2.4.1.1 Determination of cell density and viability .......................................................97 2.4.1.2 Cryopreservation of cells ...................................................................................97 2.4.1.3 Revival of cells from liquid nitrogen ..................................................................98 2.4.1.4 Intracellular protein extraction .........................................................................98 2.4.1.5 Sampling of cell culture medium .......................................................................98 2.4.2 HEK cell lines .........................................................................................................99 2.4.2.1 HEK2936E suspension cell maintenance ...........................................................99 2.4.2.2 HEK2936E suspension cell transient transfection .............................................99 2.4.2.3 Adherent HEK293 transient transfection ........................................................100 2.4.3