engineering anti-allergen antibodies as candidate therapeutics for allergic disease 2016

196 Pages·2016·6.19 MB·English

ENGINEERING ANTI-ALLERGEN
ANTIBODIES AS CANDIDATE THERAPEUTICS
FOR ALLERGIC DISEASE

CHAN JIN HUI SHERLYNN
B.Sc. (Hons), NUS

A THESIS SUBMITTED FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY

NUS GRADUATE SCHOOL FOR INTEGRATIVE
SCIENCES AND ENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2016
Declaration

I hereby declare that the thesis is my original work and it has been written by me in
its entirety. I have duly acknowledged all the sources of information which have
been used in thesis.

This thesis has also not been submitted for any degree in any university previously.

______________________________

Chan Jin Hui Sherlynn

2016

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Acknowledgements

I would like to extend my heartfelt gratitude to my supervisor, A/P Paul MacAry,
for his time and guidance throughout the span of my PhD. I thank him also for his
contagious optimism which renews my confidence in my work and the motivation
to push on when the going gets hard.
I thank my co-supervisor, Prof. Kemeny for his constructive feedback on the
various aspects of my project. To the lab members of DMK lab: Benson, thank you
for your mouse model and your help. Ka Hang, for never hesitating to lend me a
helping hand with my experiments and a listening ear. Kenneth, for your Blo t 5
purification protocol, your blood and advice. Finally, Kazuki, who has ever-so-
patiently taught me the animal sacrifice techniques necessary for the completion of
my in vivo experiments, I cannot thank you enough.
I would also like to thank Brendon and his team for giving me the opportunity to
learn the antibody engineering and phage display techniques. To Conrad &
Angeline, thank you for the kind assistance.
Thanks to A/P Chew Fook Tim, for the intellectual discussions regarding my
project and also access to the reagents and patient samples from his lab.
To my fellow past & present PAM lab members: Voja, thank you for your
mentorship and for scaling this steep learning curve with me. Chien tei, thank you
for unselfishly imparting your knowledge on all aspects of the hybridoma
technology. This project would be nothing without your guidance. Emma, thank
you for teaching me flow cytometry, helping me overcome the fear of using that
daunting Fortessa machine and for being such a pleasant desk neighbour. Fatimah,
thank you for processing my POs promptly, for the random chit-chat sessions
during my incubation times, for listening to my complaints and also for the
emergency help when I first started using the FPLC machine. Jacklyn, thank you
for proof-reading parts of my thesis, for your constructive input of ideas & for
being a friend. The constant lunch kakis - Jacklyn, Wei Jian Low & Saket, thank
you all for the laughter during our re-fuelling sessions. The rest of PAM lab - Yan
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Ting, Wei Jian Tan, Junyun, Youjia, Gu Yue, She Yah, Adrian, Olivia, Min Zin
Oo, Gosia & Maria for making this PhD stint an enjoyable one.
Special thanks to Enwei & Anna, thank you both so very much for your friendship
and for the wonderful times we've spent together, during and outside of office
hours.
To members of A/P Sylvie Alonso's lab: Li Ching, thank you for your reassurance,
advice, and for volunteering your help with my animal experiments. Issac, Pei
Xuan & Jieling, thank you all for the fond memories.
I would also like to extend my appreciation to friends in and outside of office who
have lent their support in one way or another throughout this journey - Amaury,
Debbie, Luling, Xun, Hong Yong, Whit, Lau, Nana, Deming & clique.
I'm hugely indebted to my parents: papa & mummy, I wouldn't be here today
without the both of you. Thank you for your unconditional love and relentless
support all these years, for all the home-cooked meals that fill my tummy and
warm my heart at the end of every working day. I also thank the rest of my family
for their understanding and generous encouragement.
Last but not least, I thank you, Andrew, for being my bestest friend, my strength
and my number 1 fan. Thank you for your constant love, patience and faith in me. I
could never have come this far without you.

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Table of Contents
Declaration .............................................................................................................. ii
Acknowledgements ............................................................................................... iii
List of Figures .......................................................................................................... x
List of Tables ....................................................................................................... xiii
List of Abbreviations ........................................................................................... xiv
List of Publications .............................................................................................. xvi
Summary .............................................................................................................. xvii

Chapter 1: Introduction
1.1 Asthma ............................................................................................... 1
1.1.1 Epidemiology ......................................................................... 2
1.1.2 Pathophysiology of allergic asthma ....................................... 4
1.1.2.1 Airway hyper-responsiveness .................................... 4
1.1.2.2 Airway obstruction..................................................... 4
1.1.2.3 Airway remodelling ................................................... 5
1.1.3 Immunological mechanisms .................................................. 8
1.1.3.1 Sensitization ............................................................... 8
1.1.3.2 Early phase ............................................................... 11
1.1.3.3 Late phase ................................................................ 14
1.1.3.4 Chronic inflammation .............................................. 18
1.2 House dust mite (HDM) .................................................................. 20
1.2.1 Blomia tropicalis .................................................................. 22
1.2.1.1 Blo t 5 ....................................................................... 23
1.3 Biologics in allergic asthma treatment ............................................. 27
1.3.1 Targeting IL-4 & IL-13 ........................................................ 27
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1.3.2 Targeting IL-5 ...................................................................... 28
1.3.3 Targeting IgE ....................................................................... 30
1.4 Allergen-specific Immunotherapy ................................................... 33
1.4.1 Common methods in allergen-SIT ....................................... 35
1.4.1.1 Recombinant allergens ............................................. 35
1.4.1.2 Route of administration............................................ 36
1.4.1.3 Peptide immunotherapy (PIT).................................. 37
1.4.2 Antibody responses in allergen-SIT ..................................... 39
1.5 Key Project Objectives .................................................................... 44

Chapter 2: Materials & Methods
2.1 Buffers & solutions .......................................................................... 47
2.1.1 General buffers .................................................................... 47
2.1.2 Buffers for bacteria cultures & protein expression .............. 47
2.1.3 Buffers for hybridoma & cell cultures ................................. 48
2.1.4 Buffers for western blot ....................................................... 48
2.1.5 Buffers for DNA gel electrophoresis ................................... 49
2.1.6 Buffers for in vivo study ...................................................... 49
2.2 Primers ............................................................................................ 50
2.3 Blo t 5 Purification ........................................................................... 51
2.3.1 Bacterial Cultures ................................................................ 51
2.3.2 Induction of Blo t 5 protein expression ............................... 51
2.3.3 Inclusion bodies isolation ................................................... 52
2.3.4 Ammonium sulfate precipitation ........................................ 52
2.3.5 Purification of recombinant Blo t 5 by FPLC ..................... 53
2.3.6 Measurement of protein concentration ............................... 53
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2.4 Generation of monoclonal antibodies .............................................. 54
2.4.1 Immunization of mice .......................................................... 54
2.4.2 Preparation of macrophage feeder layers ............................. 54
2.4.3 Harvesting of NS-1 cells ...................................................... 55
2.4.4 Fusion ................................................................................... 55
2.4.5 Sub-cloning by limiting dilution .......................................... 56
2.4.6 Screening for clones producing anti-Blo t 5 antibodies by
ELISA ................................................................................... 56
2.4.7 Validation of monoclonality ................................................ 57
2.5 Characterization of Anti-Blo t 5 mAb ............................................. 59
2.5.1 SDS-PAGE gel .................................................................... 59
2.5.2 Native gel ............................................................................ 60
2.5.3 Western blot ........................................................................ 60
2.5.4 Antibody isotyping .............................................................. 62
2.5.5 Immuno-dot blot ................................................................. 62
2.5.6 Affinity determination by ELISA ....................................... 63
2.5.7 Generation of Blo t 5 double mutants ................................. 63
2.6 in vivo study of anti-Blo t 5 mAb..................................................... 65
2.6.1 Animals ............................................................................... 65
2.6.2 Sera collection by cardiac puncture .................................... 65
2.6.3 Bronchoalveolar lavage (BAL) fluid analysis .................... 65
2.6.4 Measurement of total IgE and Blo t 5-specific IgE ............ 66
2.6.5 Measurement of cytokines in BAL fluid by ELISA ........... 66
2.6.6 Lung histology and scoring ................................................. 67
2.7 in vitro study of anti-Blo t 5 mAb .................................................... 68
2.7.1 Generating chimeric variants of anti-Blo t 5 mAb .............. 68
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2.7.2 Expression and purification of chimeric anti-Blo t 5 IgGs . 68
2.7.3 Basophil activation test (BAT) ........................................... 69
2.8 Statistical analysis ............................................................................ 70

Chapter 3: Generation & Characterization of Anti-Blo t 5 Monoclonal
Antibody
3.1 Introduction ..................................................................................... 72
3.2 Results ............................................................................................. 73
3.2.1 Generation of anti-Blo t 5 monoclonal antibody ................ 73
3.2.2 Purification of anti-Blo t 5 monoclonal antibody ............... 77
3.2.3 Characterization of anti-Blo t 5 mAb .................................. 79
3.2.3.1 Isotype & sequence analysis .................................... 79
3.2.3.2 Affinity determination by ELISA ............................ 83
3.2.3.3 Epitope mapping ...................................................... 84
3.2.3.4 Blo t 5 mutants at positions K54 and R57 ............... 86
3.2.4 Antagonistic potential of anti-Blo t 5 mAb ......................... 88
3.3 Discussion ....................................................................................... 91

Chapter 4: Application of anti-Blo t 5 mAb in a murine model of severe
allergic asthma
4.1 Introduction ..................................................................................... 94
4.2 Results ............................................................................................. 95
4.2.1 Anti-Blo t 5 mAb lowers inflammatory cell infiltration in
BAL fluid of asthmatic mice ............................................... 95
4.2.2 Anti-Blo t 5 mAb reduces total IgE & Blo t 5-specific IgE in
the sera of treated mice ........................................................ 99
4.2.3 Anti-Blo t 5 mAb treatment does not alter the inflammatory
cytokine profile of BAL except Eotaxin ........................... 102
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4.2.4 Anti-Blo t 5 mAb ameliorates airway inflammation, mucus
hyper-secretion and airway remodelling ........................... 104
4.3 Discussion ..................................................................................... 110

Chapter 5: in vitro application of anti-Blo t 5 mAb - Human Basophil
Activation
5.1 Introduction ................................................................................... 115
5.2 Results ........................................................................................... 116
5.2.1 Cloning and expression of chimeric anti-Blo t 5 antibodies
............................................................................................ 116
5.2.2 Affinity determination of purified chimeric antibodies ...... 118
5.2.3 Basophil activation test (BAT) on human basophils .......... 120
5.2.4 Application of chimeric anti-Blo t 5 antibodies in BAT .... 124
5.3 Discussion ..................................................................................... 129

Chapter 6: Generation of fully human anti-allergen antibodies by phage
display
6.1 Introduction ................................................................................... 134
6.2 Results ........................................................................................... 135
6.2.1 Construction of a human immune library ........................... 135
6.2.2 Panning of the human immune library ............................... 140
6.3 Discussion & Future Work ........................................................... 142

Chapter 7: Final discussion & future directions ............................................... 145

Chapter 8: References ......................................................................................... 156

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List of Figures

Figure 1.1: Global prevalence of asthma ................................................................. 3
Figure 1.2: Mortality rates of asthma ....................................................................... 3
Figure 1.3: Sensitization to allergens in the airway ............................................... 10
Figure 1.4: Immediate hypersensitivity ................................................................. 13
Figure 1.5: Late phase of an allergic airway inflammation ................................... 17
Figure 1.6: Chronic inflammation of allergic airways ........................................... 19
Figure 1.7: Phylogenetic tree depicting the clinically important mites ................. 21
Figure 1.8: Different views of the NMR structures of Blo t 5 ............................... 24
Figure 1.9: The amino acid residues constituting a major linear IgE binding
epitope .................................................................................................. 25
Figure 1.10: Mechanisms of action of Omalizumab .............................................. 32
Figure 1.11: IgG subclasses and the Fab arm exchange phenomenon .................. 41
Figure 1.12: Mechanisms of action of IgG4 as blocking antibodies ..................... 42
Figure 3.1: Expression and purification of recombinant Blo t 5 protein ............... 75
Figure 3.2: Overall percentage of hybridoma clones positive for anti-Blo t 5
responses .............................................................................................. 76
Figure 3.3: Representative binding profiles of polyclonal candidates deemed
positive for anti-Blo t 5 responses ........................................................ 76
Figure 3.4: Purification of anti-Blo t 5 antibodies by FPLC .................................. 78
Figure 3.5: Isotype test for anti-Blo t 5 specific monoclonal antibody .................. 81
Figure 3.6: IgG template & sequence analysis of anti-Blo t 5 mAb ...................... 82
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