Parameters affecting phage display library design for improved generation of human antibodies

192 Pages·2009·9.8 MB·English

Parameters affecting phage display library design for
improved generation of human antibodies
Inaugural-Dissertation
to obtain the academic degree
Doctor rerum naturalium (Dr. rer. nat.)
submitted to the Department of Biology, Chemistry and Pharmacy
of Freie Universität Berlin

by
THEAM SOON LIM
from
Penang, Malaysia
June, 2009

I hereby declare all works and writting contained within this thesis was conducted by myself,
all references used are cited accordingly and any personal assitance acknowledge by name.
Theam Soon Lim
Berlin, 2009
All the work for this PhD thesis was conducted from October 2006 to April 2009 in the working
group of Dr. Zoltán Konthur at the Department of Vertebrate Genomics under the tutelage of
Prof. Dr. Hans Lehrach in the Max Planck Institute for Molecular Genetics.
1st Reviewer: Prof. Dr. Hans Lehrach
2nd Reviewer: Prof. Dr. Markus C. Wahl
Date of defence: 3rd September 2009

To my parents, wife and beloved daughter.
Table of Contents
1.0 ntroduction 1

1.1 Antibodies 1
1.2 Recombinant antibody technology 8
1.3 Antibody format engineering 13
1.4 Antibody library generation 16
1.5 Phage display panning procedures 21
2.0 Objective 24
3.0 Materials 26

3.1 Consumables 26
3.2 Laboratory equipment 27
3.3 Softwares 28
3.4 Chemicals, buffers and solutions 31
3.5 Media 31
3.6 Additives 32
3.7 Microorganisms and Eukaryotic cell lines 32
3.8 Plasmids 35
3.9 Recombinant Proteins, Affinity Columns and Magnetic Beads 35
3.10 Antibodies 35
3.11 DNA and Protein Markers 35
3.12 Kit Systems 36
3.13 Enzymes and Molecular Biology Materials 37
3.14 Oligonucleotides 38
3.15 Antibody library repertoire 39
3.16 Magnetic Particle Processor 40
4.0 Methods 41
4.1 Molecular Biology based methods 41
4.1.1 Medium for E. coli cultivation 41

4.1.2 Storage conditions and regrowth of bacterial cultures 41
4.1.3 OD measurements of cultures 41
4.1.4 Preparation of electrocompetent cells 41
4.1.5 Purification of Plasmid 42
4.1.6 Gel electrophoresis separation of DNA 42
4.1.7 DNA extraction from gel electrophoresis 42
4.1.8 DNA concentration with Ethanol precipitation 42
4.1.9 DNA concentration determination 43
4.1.10 Restriction enyzme digestion 43
4.1.11 Dephosphorylation of digested plasmids 43
4.1.12 Ligation of DNA fragments 43
4.1.13 Transformation of E. coli by electroporation 44
4.1.14 DNA sequencing 44
4.1.15 PCR protocols 44
4.1.16 Subcloning of Antibody Library Repertoire 48
4.1.17 First strand cDNA synthesis 48
4.1.18 PBMC preparation and RNA isolation 48
4.1.19 Evaluation of RNA samples 49
4.1.20 Estimation of library size 50
4.2 Protein based methods 52
4.2.1 Expression of in-vivo biotinylated antigens 52
4.2.2 Preparation of proteins under native conditions 52
4.2.3 Preparation of proteins under denatured conditions 52
4.2.4 Preparation of periplasmic proteins 53
4.2.5 His Tag purification using Ni-NTA columns
under native conditions 53
4.2.6 His Tag purification using Ni-TED columns
under native conditions 53
4.2.7 Purification of biotinylated proteins using
Monoavidin columns 53
4.2.8 Protein L purification of antibody fragments 54
4.2.9 Protein concentration determination 54
4.2.10 SDS PAGE analysis 54

4.2.11 SDS PAGE Staining and Destaining 55
4.2.12 Silver Staining of SDS PAGE gels 55
4.2.13 Western Blotting 56
4.2.14 mmunostaining 56
4.2.14 Mass spectrometry identification of proteins 56
4.3 Phage display based methods 57
4.3.1 Helper phage preparation 57
4.3.2 Phage library preparation 58
4.3.3 Phage precipitation 58
4.3.4 Titration of phage particles 58
4.3.5 Panning of phage antibody libraries 59
4.3.5.1 Loading of Magnetic Beads 59
4.3.5.2 Semi-automated Panning on Magnetic
Particle Processor 59
4.3.5.3 Packaging of Phagemids 62
4.3.5.4 Magnetic Particle ELSA of Polyclonal
Antibody Phage 63
4.3.6 Evaluation of phage selections 65
4.3.6.1 Picking monoclonal antibody fragment
presenting cells 65
4.3.6.2 Monoclonal scFv expression and periplasmic
preparation in microtitre plate format 66
4.3.6.3 Monoclonal antibody fragment expression
in culture 66
4.3.6.4 Preparation of monoclonal antibody fragment
presenting phages 66
4.3.6.5 Monoclonal ELSA with soluble antibody
fragments 67
4.3.6.6 Monoclonal ELSA with antibody fragment
presenting phages 68
4.3.7 Antibody fragment presentation assay with
Protein L beads 68

4.4 The Diversity (DiVE) Assay 69
5.0 Results 71
5.1 Construction of pTSL phagemid vector series 71
5.1.1 Amplificaiton of constant regions of IgD 71
5.1.2 Amplificaiton of ccdB cassette 71
5.1.3 Subcloning of inserts to phagemid vector 72
5.1.4 pTSL Vector series 73
5.1.5 Evaluation of CcdB cassette activity 74
5.2 Generation of semi-synthetic antibody repertoire for
library generation 76
5.2.1 Generation of Variable Region Repertoire 76
5.2.2 PCR assembled 1-step Cloning Strategy 76
5.2.3 Semi synthetic Library Generation 80
5.3 Antigen preparation 86
5.3.1 Evaluation of antigen purification strategies 86
5.3.2 Evaluation of recombinantly expressed antigens 90

5.4 Antibody fragment presentation assay 94
5.5 Diversity Visualization by Endonuclease – A rapid assay to
monitor diverse nucleotide libraries 96
5.8.1 Optimisation of S1 nuclease incubation temperature 96
5.8.2 Titration of S1 nuclease enzyme units for digestion 98
5.8.3 Optimisation of S1 nuclease incubation time 98
5.8.4 Application of DiVE Assay to monitor phage display
panning rounds 98
5.8.5 Batch sequencing of amplicons from all rounds 100
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5.6 Antibody selection process 103

5.5.1. Coupling of biotinylated antigens to streptavidin beads 103
5.5.2. Semi-automated selection of antibodies 103
5.5.3. Polyclonal ELSA evaluation of panning rounds 103
5.5.4. Monoclonal ELSA evaluation of selected clones 108
5.7 Format switching 112
5.8 Amplification of V-gene repertoire from B-cells 114

5.8.1 Bioinformatic analysis of V-gene specific primers 114
5.8.2 Evaluation of two different reverse transcriptases
for cDNA synthesis 119
5.8.3 Evaluation of different polymerases for PCR
amplification 120
5.8.4 Effects of addition of Extreme Thermostable
Single-Strand DNA binding protein (ET SSB) on
PCR efficiency 122
5.8.5 Antibody isotype and idiotype amplification 122
5.8.6 V-gene repertoire optimization for library generation 125
6.0 Discussion 127
6.1 Phagemid vector construction 127
6.2 Generation of semi-synthetic antibody library 128
6.3 Antigen preparation 130
6.4 Antibody format presentation efficiency assay 131
6.5 Diversity Visualization by Endonuclease (DiVE) assay 132
6.6 Phage display selection with semi-synthetic libraries 134
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