Generation and Characterization of heavy chain antibodies derived from Camelids

176 Pages·2013·14.23 MB·English

by  Katrin Schmidthals

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
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1.3.4 Applications of V Hs ....................................................................................... 31
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1.4 Aims and Objectives ........................................................................................... 34
2 Materials and Methods ............................................................................................. 35
2.1 Material .............................................................................................................. 35
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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
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3.1.1 Antigen selection ............................................................................................ 69
3.1.2 Amplification of the V H repertoire ............................................................... 72
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3.1.3 Cloning of V H library in pHEN4 ..................................................................... 75
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Table of Contents

3.1.4 Phage Display .................................................................................................. 81
3.1.5 Selection of antigen specific V HS by Phage ELISA ...................................... 100
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3.1.6 Selection of antigen specific V Hs by the F2H-Assay ................................... 111
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3.2 Characterization of antigen specific V Hs ........................................................ 117
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3.2.1 Biochemical characterization ....................................................................... 118
3.2.2 Intracellular characterization ....................................................................... 134
4 Discussion ................................................................................................................ 139
4.1 V H libraries ..................................................................................................... 140
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4.1.1 Immunogenicity of Antigens......................................................................... 140
4.1.2 V H Library Size and Diversity ...................................................................... 142
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4.2 V H Selection .................................................................................................... 144
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4.2.1 A new method to select V Hs for in vivo applications ................................. 144
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4.3 Compensation of V -V combination ............................................................... 147
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4.4 CDR3, disulfide bonds and functionality .......................................................... 148
4.5 Outlook ............................................................................................................. 151
5 Annex ....................................................................................................................... 153
5.1 References ........................................................................................................ 153
5.2 Abbreviations ................................................................................................... 166
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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
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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
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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
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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
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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.
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We used the immune system of alpacas to generate V H libraries against various
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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
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identify antigen specific V Hs against different antigens which were characterized and their
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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
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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.
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