USE OF ANTIBODY ENGINEERING TO IMPROVE THE PROPERTIES OF ANTIBODIES AS ...

128 Pages·2015·15.48 MB·English

USE OF ANTIBODY ENGINEERING TO IMPROVE
THE PROPERTIES OF ANTIBODIES AS
REAGENTS

ENG HUIYAN
(B.Sc.), National University of Singapore

A thesis submitted for the degree of Doctor of Philosophy
Department of Medicine
National University of Singapore

Acknowledgements
It is my great honor to be able to work alongside some of the most brilliant minds in
the field during the course of my PhD study.
First and foremost, I would like to thank Professor Sir David Lane for taking me
under his wing, allowing me to explore the mysteries of antibody engineering in the
p53 Laboratory, and giving me much appreciated advice and encouragement
throughout the course.
A/Prof Paul MacAry and Dr Andre Choo for their relentless optimism with regards to
my projects, useful advice and encouragement at our frequent TAC meetings. Special
thanks to A/Prof Paul MacAry for helping to smooth out many doubts we had with
regards to the administrative matters during my course.
Dr Farid Ghadessy for his invaluable insights on my projects, especially on the
development of a novel platform based on in-vitro compartmentalization. He has also
been a source of much encouragement throughout my course and his optimism with
respect to experimental results has been contagious. His help with multiple critical
proof-readings of this thesis is also very much appreciated.
Dr Wang Cheng-I, Dr Patricia Ng Miang Lon, and Ms Lee Chia Yin for sharing their
invaluable knowledge on antibody recombination techniques and bio-layer
interferometry with me. Without their patient teaching, the engineering recombinant
anti-mouse p53 antibody project may not have been possible.
Mr Hendrick Loei for patiently and generously sharing with me the principles
underlying the bio-layer interferometry technology, and providing very helpful advice
on experimental optimizations and data analysis.
Dr Xue Yue Zhen for her patient sharing of her immunohistochemical (IHC) staining
techniques with me. Also, Ms Chiam Poh Cheang for her bright smiles everyday and
providing me with tons of expertly sectioned mouse intestinal and spleen tissue
sections for my IHC staining work.
Dr Hwang Le-ann, Ms Koh Xin Yu and Ms Koh Xiao Hui for their patience and
generosity in sharing their knowledge of immunology with a beginner like me.
Special thanks to Ms Koh Xiao Hui for sharing aliquots of her purified bacterial
expressed mouse and human recombinant full-length p53 proteins with me.
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Dr Li Ling for her patience in teaching me data analysis, especially in the calculation
of real-time PCR-generated datasets.
Dr Julin Wong for patiently teaching me the basics of antibody cloning.
Dr Tan Ban Xiong for generously sharing the tips and tricks in formatting a thesis fit
for submission for a Doctorate of Philosophy degree.
Fellow p53 laboratory colleagues and friends for their generous help, understanding
(of me being missing-in-actions for weeks for rotating laboratory duties), advice and
encouragement. Special thanks to Dr Cynthia Coffill for helping to proof-read my
thesis within days and at a very short notice. Special thanks to Ms Siau Jia Wei for
generously sharing her primer sequences, reagents and data on another in-vitro
display platform she was working on with me; Ms Goh Hui Chin for her daily
uplifting cheekiness; Ms Sharon Chee Min Qi for sharing aliquots of her purified
bacterial expressed human p53 (N-terminus only) proteins with me, and dragging me
off for lunch and home for the night; and all three of them for taking over the extra
burden of my laboratory duty role while I was away.
I would also like to thank Ms Joey Xu, my fellow course-mate cum friend, for her
company, support, encouragement, empathy when experiments did not work out well,
and dragging me off for dinners. Special thanks to Mr Tang Chin Huat, my fiancé, for
his comforting warm embraces, lending me his shoulders when I needed them, and
patiently waiting five long years till course completion for me; my life-time buddies
for their understanding that I needed to disappear from the face of the Earth for days,
weeks or months to focus on my course; and my family for putting up with my
unstable temperaments during stressful periods, my noisy typing sounds till the wee
hours each day, and my sisters for taking care of my parents on my behalf when I
needed to focus 100% in my course.
Without these special people in my life, this course would have been impossible to
complete. Thank you for taking part in this difficult journey with me.

Last but not least, thank you examiners for taking precious time off to read this thesis.

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Contents
1. INTRODUCTION
1.1 Antibodies and Adaptive Immunity 1
1.1.1 Antibody Production 2
1.1.2 Antibody Engineering 4
1.1.2.1 To combat ‘HAMA’ effects 4
1.1.2.2 To overcome limitations with immunisation technology 5
1.1.2.3 To increase affinity or alter specificity 6
1.1.2.4 For other purposes 6
1.2 Display Platforms 7
1.2.1 Genotype-Phenotype Linkages with the help of Cells 8
1.2.1.1 Phage Display 8
1.2.2 Totally In-Vitro Genotype-Phenotype Linkages 9
1.2.2.1 Ribosome display 10
1.2.2.2 in-vitro Compartmentalization 12
1.2.2.3 DNA display – STABLE 13
1.2.2.4 Dendrimer-like DNA (“DL-DNA”) display: SNAP 15
1.2.3 High-throughput selection methods 16
1.3 Future Prospects of Antibody as Reagents 18
1.4 Antigen of interest: p53 protein 19
1.5 Scope of thesis 21

2. MATERIALS AND METHODS
2.1 General Buffers and Solutions 22
2.2 General Molecular Techniques 23
2.2.1 Polymerase chain reactions, PCR 23
2.2.1.1 High-fidelity PCR 23
2.2.1.2 Non-high-fidelity; high-fidelity with low template 23
2.2.1.3 Real-time quantitative PCR (qPCR) 24
2.2.2 In-Fusion HD cloning (Clonetech Laboratories) 25
2.2.3 Cloning for sequencing (TOPO® TA cloning, Invitrogen) 26
2.2.4 (Preparations for) DNA sequencing 26
2.3 Antibody Maturation/ Epitope Mapping 27
2.3.1 Library construction 27
2.3.1.1 For antibody maturation of DO-1 single chain variable fragment 27
(DO1scFv)
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2.3.1.2 For epitope mapping 28
2.3.2 Selections 29
2.3.2.1 Antibody maturation 30
2.3.2.2 Epitope mapping 31
2.3.3 Functional assays 34
2.3.3.1 Enzyme-linked immunosorbent assay, ELISA (DO1scFv 34
maturation only)
2.3.3.2 Immunoprecipitation, IP 35
2.3.3.3 Western blot (only for Epitope Mapping) 35
2.4 Engineering Recombinant Anti-mouse p53 Antibody 36
2.4.1 Characterizing parental PAb242 antibody 36
2.4.1.1 ELISA 36
2.4.1.2 Western blot 38
2.4.1.3 Immunohistochemical (IHC) staining of paraffin-embedded 39
sections
2.4.1.4 Determining critical residues in epitope sequence 41
2.4.1.4.1 Phage-displayed peptides epitope mapping 41
2.4.1.4.2 Alanine-scan ELISA 42
2.4.2 Cloning 242scFv from PAb242 hybridoma cells 43
2.4.2.1 Phage-displayed 242scFv and DO1scFv antibody construction 43
and expression
2.4.2.2 MH242 antibody cloning, expression and purification 46
2.4.2.3 Characterizing chimeric MH242 antibody 48
2.4.2.3.1 Affinity constants (bio-layer interferometry) 48

3. DEVELOPMENT OF NOVEL PLATFORMS BASED ON IN-VITRO
COMPARTMENTALISATION (IVC)
3.1 Introduction 50
3.2 Results 52
3.2.1 DO1scFv Maturation 52
3.2.1.1 Optimisations and Proof of principle selections 52
3.2.1.2 Library Construction 77
3.2.1.3 Selections and secondary assays 81
3.2.2 Epitope Mapping 86
3.2.2.1 Protein G beads 86
3.2.2.2 Epoxy beads 99
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3.2.2.2.1 Library Construction 99
3.2.2.2.2 Experimental selection and secondary assay(s) 102
3.3 Discussion 106
3.4 Conclusions and Suggestions for Future Work 109

4. ENGINEERING RECOMBINANT ANTI-MOUSE P53 ANTIBODY
4.1 Introduction 112
4.2 Results and Discussions 112
4.2.1 Characterizing parental PAb242 antibody 112
4.2.1.1 Functionality test/ applications 113
4.2.1.1.1 ELISA assays 113
4.2.1.1.2 Immunohistochemical (IHC) staining 119
4.2.1.2 Determining critical residues in epitope sequence 125
4.2.1.2.1 Epitope mapping (phage-displayed peptides) 125
4.2.1.2.2 Alanine-scan ELISA 129
4.2.2 Cloning from hybridoma cells 132
4.2.2.1 Hybridoma antibody-expression test 132
4.2.2.2 Cloning of PAb242 variable regions from the hybridoma cell 134
line
4.2.2.3 Functionality test (Phage ELISA) 144
4.2.3 Mouse-Human Chimeric Antibody, MH242 146
4.2.3.1 MH242 construction 145
4.2.3.2 Characterizing the chimeric MH242 antibody 146
4.2.3.2.1 ELISA 146
4.2.3.2.2 Western Blots 150
4.2.3.2.2.1 Purified recombinant p53 proteins 150
4.2.3.2.2.2 Endogenous p53 proteins 153
4.2.3.2.3 Immunohistochemical (IHC) staining 155
4.2.3.2.3.1 Mouse intestines (p53 R172H, with γ-irradiation) 155
4.2.3.2.3.2 Mouse intestines (p53 R172H, without γ-irradiation) 162
4.2.3.2.3.3 Mouse spleen (p53 R175H, with γ-irradiation) 165
4.2.3.3 Affinity constant determination by bio-layer interferometry 171
4.3 Further Discussion and Conclusions 180

5. OVERALL CONCLUSION 186

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REFERENCES
APPENDICES
i. List of Oligonucleotides
ii. List of Peptides

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Summary
The aim of this project is to generate a best-in-class antibody for use against mouse
p53 with particular focus on the immunohistochemical (“IHC”) staining of paraffin-
embedded mouse tissue sections. The first approach undertaken was to engineer a
highly efficient antibody targeting human p53 (DO-1) to bind the homologous
epitope in mouse p53 which differs by a single amino acid residue only. To this end,
a well-characterised anti-human p53 antibody (DO-1) was randomly mutated and
subjected to selection against the homologous mouse p53 epitope sequence using a
novel selection platform, “SBP-display”. The SBP-display platform is a totally in-
vitro selection platform where the phenotype-genotype linkage is achieved via a
streptavidin-streptavidin binding peptide (“SBP”) linkage established inside ‘water-
in-oil’ emulsion compartments. Model selections indicated that both the phenotype-
genotype linkage and the developed platform worked. However, experimental
selections for DO1scFv variants that bind the homologous mouse p53 epitope
sequence failed, primarily due to high unspecific background binding. Despite
numerous attempts, this issue could not be readily resolved.
Hence, another strategy was instigated to meet our purpose. It has been previously
reported that using ubiquitous primary antibodies of mouse origin to detect mouse
proteins results in high off-target background binding due to anti-mouse secondary
antibodies binding to endogenous immunoglobulins (“Ig”) that can be present in
mouse tissue sections. These can have profound effects on signal-to-noise ratios in
IHC staining, thus confounding experimental observations and conclusions. An anti-
mouse p53 antibody was engineered with the scFv region of a mouse anti-p53
antibody (PAb242) grafted onto a human IgG1 scaffold. The resultant hybrid anti-
mouse p53 antibody (MH242) retained both the affinity and specificity of the parental
antibody in ELISA and Western Blot applications. IHC staining of mouse intestinal
tissue sections showed that the MH242 antibody was able to detect mouse p53 protein
at varying protein expression levels with similar affinity to the parental PAb242
antibody but with reduced background binding. IHC staining of mouse spleen tissue
sections (containing a much higher level of contaminating endogenous Ig) showed
that the MH242 antibody was able to detect mouse p53 protein with almost-zero
background as compared with the PAb242 antibody. The off-target background
binding observed in IHC staining on mouse tissue sections using mouse origin
primary antibodies was confirmed to be due to anti-mouse secondary antibody
staining endogenous Ig. It was observed that while affinity is one critical property in
determining the detection sensitivity of the MH242 antibody when used for Western
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blotting. However, affinity was not as important in determining detection sensitivity
when MH242 was applied in the IHC staining assay. More experiments need to be
done to confirm if this observation is applicable to all antibodies and also to delineate
critical antibody properties for each type of assay.
In summary, this project explored two different antibody engineering strategies to
obtain a specific and functional antibody applicable in various assays to detect mouse
p53 protein. The successfully engineered MH242 antibody may be used as a reagent
in future ELISA and IHC experiments. The successful antibody recombination
protocol may be applied directly in future antibody engineering projects with a
similar purpose of improving an antibody specifically for use as an IHC reagent. Also,
experience gained from both the in-vitro selection and antibody recombination
studies, together with future experiments that delineate antibody properties critical for
each assay type, would allow improved strategic formulations in future antibody
engineering projects aimed at developing application-specific antibodies.

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