The Regulation of Peptide Mimotope/Epitope Recognition by Monoclonal Antibodies by Richard Gary Smith B.Sc. (Hons) Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy, March 2002. TABLE OF CONTENTS Abstract………………………………………………………………...……….…….8 Acknowledgements…………………………………………………………………...9 Glossary of terms……………………………………………………………………10 Chapter 1 Antibody Purification…………………………………………………..11 1.1 Introduction ......................................................................................................11 1.1.1 Antibodies ................................................................................................11 1.1.2 Monoclonal and engineered Antibodies...................................................12 1.2 Examples of a therapeutic and a commercially applied monoclonal antibody17 1.2.1 C595 monoclonal antibody (NCRC48)...................................................17 1.2.2 mAb 4155.................................................................................................18 1.3 Approaches to antibody purification................................................................20 1.3.1 Column chromatography..........................................................................20 1.3.2 Fluidized bed and expanded bed adsorption ............................................20 1.3.3 High performance Tangential Flow Filtration (HPTFF)..........................21 1.3.4 Magnetic beads.........................................................................................21 1.4 Physical separation techniques.........................................................................22 1.4.1 Ion exchange ............................................................................................22 1.4.2 Gel filtration .............................................................................................23 1.4.3 Hydrophobic Interaction Chromatography (HIC)....................................23 1.5 Affinity Purification Techniques......................................................................23 1.5.1 Fc Binding Proteins..................................................................................24 1.5.2 Recombinant Protein Tags .......................................................................24 1.5.3 Paratope specific binding ligands.............................................................25 1.5.4 Non-proteinaceous, paratope specific affinity ligands.............................30 1.6 Summary ..........................................................................................................32 Chapter 2 Regulation of peptide antibody interactions……………………….…33 2.1 Introduction ......................................................................................................33 2.1.1 Library Technologies ...............................................................................35 2.1.2 The distinction between screening and selection in library technologies 36 2.2 Synthetic Peptide Libraries ..............................................................................40 2.2.1 Pepscan – The application of synthetic peptides in Epitope mapping. ....40 2.2.2 Replacement Net (RNET) analysis ..........................................................40 2 2.2.3 Combinatorial peptide libraries................................................................41 2.3 Biological libraries ...........................................................................................42 2.3.1 In vivo libraries.........................................................................................42 2.3.2 In vitro libraries........................................................................................47 2.4 Other non-peptide ligands ................................................................................51 2.4.1 Synthetic Libraries ...................................................................................51 2.4.2 Biological Libraries..................................................................................51 2.5 Mutagenesis......................................................................................................52 2.6 Aims and Objectives of the research................................................................55 Chapter 3 Materials and Methods…………………………………………………57 3.1 Reagents ...........................................................................................................57 3.2 Enzymes ...........................................................................................................59 3.3 Buffers and Solutions.......................................................................................60 3.4 Methods............................................................................................................65 3.4.1 Monoclonal Antibody Production............................................................65 3.4.2 Biopanning of phage library.....................................................................65 3.4.3 PCR analysis of individual phage clones .................................................66 3.4.4 Direct sequencing of PCR products .........................................................67 3.4.5 Phage Capture ELISA ..............................................................................68 3.4.6 Sepharose Bead Assay .............................................................................69 3.4.7 Peptide Affinity Chromatography............................................................69 3.4.8 Sodium dodecyl sulphate polyacrylamide gel electrophoresis ................70 3.4.9 Circular Dichroism...................................................................................71 3.4.10 Fluorescence Quenching Immunoassay...................................................72 3.4.11 Conjugation of peptides to BSA using glutaraldehyde ............................73 3.4.12 Production of a BSA-Estrone Conjugate .................................................73 3.4.13 Enzyme linked immunosorbent assay (ELISA).......................................73 3.4.14 Surface Plasmon Resonance (SPR) analysis of the C595 – mucin core peptide APDTRPAPG interaction............................................................76 3.4.15 SPR analysis of the interaction between peptide conjugate BSA- (D)DYFLG, and the anti E3G monoclonal antibody 4155. .....................79 3.4.16 Production of pCANTAB-5E [C595scFvGFP]. A Phagemid expression vector containing a C595 ScFv – Green Fluorescent Protein Gene fusion. ..................................................................................................................81 3 3.4.17 Protein expression ....................................................................................88 3.4.18 Western blot analysis ...............................................................................90 3.4.19 ELISA of Supernatant, Periplasmic Extract, Whole Cell Extract and fractions from affinity purification of supernatant...............................................90 3.4.20 Random mutagenesis of the C595-ScFv gene – analysis of a selection of mutants. ................................................................................................................91 3.4.21 Evaluation of pSKGFP expression system...............................................92 Chapter 4 - Results. Phage Display of Peptides…………………………………..93 4.1 Introduction ......................................................................................................93 4.2 The T7Select415 phage display library............................................................94 4.3 Determination of initial phage library titer and stability of the library............97 4.4 Analysis of passive adsorption of proteins on the surface of NUNC immunotubes by ELISA method..............................................................................98 4.5 Removal of non-specifically binding phage.....................................................99 4.6 Biopanning - Screening of library against 4155 mAb....................................100 4.6.1 Determination of the extent of enrichment that can be observed...........100 4.7 Screening of library against C595 mAb.........................................................101 4.7.1 Determination of the extent of enrichment that can be observed...........101 4.7.2 Capture ELISA of phage populations from biopanning rounds.............102 4.7.3 Capture ELISA of phage clones from rounds one and four of screening. …………………………………………………………………………104 4.8 SPR analysis of selected phage clones...........................................................106 4.9 PCR analysis of phage clones from round four of initial screen....................109 4.10 Sequencing of phage clones .......................................................................110 4.10.1 Sequencing of clones from the fourth round of biopanning...................110 4.10.2 Sequence analysis of all four biopanning rounds...................................114 4.11 Summary ....................................................................................................121 Chapter 5 - Results. Analysis of peptides derived from phage display………..123 5.1 Introduction ....................................................................................................123 5.2 ELISA of BSA – peptide conjugates..............................................................124 5.3 Sepharose bead assay .....................................................................................125