SYNTHETIC PROTEIN DESIGN & ENGINEERING OF A MONOCLONAL ANTIBODY MAXIMILIAN ...

222 Pages·2015·5.24 MB·English

SYNTHETIC PROTEIN DESIGN & ENGINEERING
OF A MONOCLONAL ANTIBODY

MAXIMILIAN KLEMENT
(B.S., University of California, Irvine)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY
DEPARTMENT OF CHEMICAL &
BIOMOLECULAR ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE

2014
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Declaration

I hereby declare that this 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 the thesis.

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

Maximilian Klement
30th December, 2014

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Acknowledgments
My PhD journey has been one with many ups and many downs, but
one which has taught me so much. I would like to extend my sincerest thanks
to my supervisors, Prof Miranda Yap, Prof Lee Dong-Yup and Dr Dave Ow, I
wish Prof Yap all the best in her continued recovery. Special thanks to Prof
Lam Kong Peng and Bioprocessing Technology Institute (BTI), A*STAR for
the financial support. I would also like to thank all members of my qualifying
exam and thesis committee.
The work in this would not have been possible without the help and
encouragement of my friends and colleagues at BTI and NUS. I am particular
grateful to Jiyun Zheng and Chengcheng Liu, who contributed so much of
their time and effort to my project. I would like to extend my thanks to Dr
Andre Choo, Jeremy Lee, Pek Han Bin, lab members of Stem Cells 1,
Analytics, Proteomics, Bioinformatics and Microarray for their help along my
PhD journey. And for those times when work was not on my mind, I am
grateful to my lunch, coffee, and sports friends; Daniel Ng, Eddy Tan, Fong
Guo Feng, and Ang Kok Siong.
Above all, my family has always been there, at times waiting to jump
in when I needed their help or at others giving me the freedom to pursue my
dreams. My mom was always willing to listen, no matter how grumpy or bad
tempered I was, and the pasta salads were a lunch I always looked forwards to,
no matter how many times in a row.
Thank you all very much!!

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Table of Contents
Declaration ................................................................................................... ii
Acknowledgments ....................................................................................... iii
Table of Contents ......................................................................................... iv
Summary ................................................................................................... viii
List of Publications & Conference Presentations ........................................ x
List of Tables ............................................................................................... xi
List of Figures .............................................................................................xii
List of Symbols and Abbreviations ........................................................... xiv
Chapter 1: Introduction ............................................................................... 1
1.1 Motivation............................................................................................. 2
1.2 Hypothesis ............................................................................................ 4
1.3 Objectives ............................................................................................. 6
1.4 Thesis Organization............................................................................... 8
Chapter 2: Literature Review .................................................................... 10
2.1 Antibody engineering .......................................................................... 11
2.1.1 Organization of antigen-binding domain ....................................... 15
2.1.2 Linker engineering ........................................................................ 18
2.1.3 Engineering multivalent antibody fragments ................................. 30
2.1.4 Complement-independent cytotoxic antibodies ............................. 34
2.2 Microbial host production systems for producing engineered antibody
fragments ............................................................................................ 38
2.2.1 Escherichia coli ............................................................................ 40
2.2.2 Pichia pastoris .............................................................................. 46
2.2.3 Codon optimization....................................................................... 50
2.3 Applications of antibodies for stem-cell based regenerative medicine .. 54
2.3.1 Cell replacement therapy............................................................... 54
2.3.2 Organ replacement ........................................................................ 56
2.3.3 Limitations and solutions of hESC-based therapy ......................... 57
Chapter 3: Bioprocess Optimization of Engineered Antibody
Fragments……………………………………………………………………60
3.1 Abstract ............................................................................................... 61
3.2 Introduction ......................................................................................... 62
3.3 Materials and Methods ........................................................................ 65
3.3.1 Vector construction and cloning .................................................... 65
3.3.2 Expression, isolation, and purification of antibody fusion proteins 66

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3.3.3 Protein quantification .................................................................... 67
3.3.4 Gel electrophoresis and Western blot analysis ............................... 68
3.3.5 Analytical Size-Exclusion Chromatography (SEC) ....................... 69
3.3.6 Fluorescence Activated Cell Sorting (FACS) ................................ 70
3.4 Results and Discussion ........................................................................ 71
3.4.1 Cloning and expression of the engineered antibody fragments ...... 71
3.4.2 Optimization of the expression of engineered antibody fragments . 77
3.4.3 Co-expression of chaperones......................................................... 82
3.4.4 Purification and analysis of the engineered antibody fragments ..... 83
3.5 Conclusion .......................................................................................... 88
Chapter 4: Codon Optimization for Designing a Synthetic Gene ............. 89
4.1 Abstract ............................................................................................... 90
4.2 Introduction ......................................................................................... 92
4.3 Materials and Methods ........................................................................ 95
4.3.1 Implementation of codon optimization frameworks ....................... 95
4.3.2 Gene synthesis and cloning ........................................................... 97
4.3.3 Expression and isolation of invertase in E. coli ............................. 99
4.3.4 Expression of trimeric engineered antibody fragments in P. pastoris
................................................................................................... 100
4.3.5 Bradford assay ............................................................................ 100
4.3.6 Gel electrophoresis and Western blot analysis ............................. 101
4.3.7 Enzyme activity assay for invertase............................................. 102
4.3.8 Fluorescence activated cell sorting .............................................. 103
4.4 Results and Discussion ...................................................................... 104
4.4.1 Codon context fitness to improve protein expression in heterologous
hosts ........................................................................................... 104
4.4.2 Sequence alignment and protein expression ................................ 106
4.4.3 Comparison of codon-optimized invertase using an enzyme activity
assay ........................................................................................... 108
4.4.4 Production of engineered antibody fragments in P. pastoris ........ 111
4.4.5 Comparison of the expression levels of engineered antibody
fragment in P. pastoris................................................................ 114
4.4.6 Binding of trimeric engineered antibody fragments ..................... 116
4.5 Conclusion ........................................................................................ 118
Chapter 5: Effect of Linker Flexibility and Length on the Functionality of
a Cytotoxic Engineered Antibody Fragment ........................................... 119
5.1 Abstract ............................................................................................. 120
5.2 Introduction ....................................................................................... 121

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5.3 Materials and Methods ...................................................................... 124
5.3.1 Plasmid construction ................................................................... 124
5.3.2 Expression, isolation, and purification......................................... 124
5.3.3 Analytical size-exclusion chromatography .................................. 126
5.3.4 Fluorescence activated cell sorting .............................................. 126
5.3.5 Homology modeling ................................................................... 127
5.3.6. Molecular dynamics simulation.................................................. 128
5.3.7 Differential scanning calorimetry ................................................ 129
5.4 Results and Discussion ...................................................................... 130
5.4.1 Engineering and expression of antibody fragments with various
linkers......................................................................................... 130
5.4.2 Effect of linkers in engineered antibody fragments on cytotoxicity
................................................................................................... 136
5.4.3 Molecular dynamic simulations to quantify the physical differences
of the linkers upon the engineered antibody fragments ................ 141
5.4.4 Protein stability of engineered antibody fragments ...................... 144
5.5 Conclusion ........................................................................................ 147
Chapter 6: Antibody Engineering of a Cytotoxic Monoclonal Antibody
84: Investigating the Effects of Valency on Cytotoxicity ......................... 149
6.1 Abstract ............................................................................................. 150
6.2 Introduction ....................................................................................... 152
6.3 Materials and Methods ...................................................................... 156
6.3.1. Plasmid construction .................................................................. 156
6.3.2 Expression, isolation, and purification......................................... 156
6.3.3 Analytical size-exclusion chromatography .................................. 158
6.3.4 Flow cytometry analysis ............................................................. 158
6.3.5 Embryoid body generation and penetration studies...................... 159
6.4 Results and Discussion ...................................................................... 161
6.4.1 Engineering and expression of multivalent engineered antibody
fragments .................................................................................... 161
6.4.2 The effect of valency of engineered antibody fragments on binding
and cytotoxicity .......................................................................... 166
6.4.3 Penetration of multivalent engineered antibody fragments into cell
clusters ....................................................................................... 172
6.5 Conclusion ........................................................................................ 175
Chapter 7: Conclusion and Future Work ................................................ 176
7.1 Conclusion ........................................................................................ 177
7.2 Future work ....................................................................................... 180

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7.2.1 Improvements in production and properties of the engineered
antibody fragments ..................................................................... 180
7.2.2 Tools to aid in the design and engineering of antibodies .............. 181
7.2.3 The development of next generation antibody therapeutics ......... 182
Bibliography ............................................................................................. 186

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Summary
Antibody engineering has enabled the directed modification of
immunoglobulins to improve their biological action, for example in terms of
protein affinity, stability, or pharmacokinetics. This field of engineering has
also enabled the construction of smaller, customized antibodies, termed
antibody fragments. More sophisticated modifications may be achieved by
genetic fusion of two protein domains. Engineered antibody fragments, are
formed by fusing an antibody fragment and a functional domain, i.e. a
multimerization domain, using a peptide linker region. These synthetic
proteins exploit the distinct functions derived from each of their component
moieties. Besides their wide applications in biological research, they have also
become an important category of biopharmaceuticals.
Specifically, we are interested in the application of engineered
antibody fragments in the growing field of regenerative medicine. This field
has gained attention due to the potential of stem cells for cell therapy;
however, one of the most pertinent concerns using differentiated cells from
embryonic stem cells is the presence of residual undifferentiated embryonic
stem cells because they carry a risk of teratoma formation in vivo, which can
be cancerous to patients. Earlier work discovered a monoclonal antibody
(mAb84) which specifically targets and kills undifferentiated human
embryonic stem cells; however, mAb84 is limited by its large size which
limits penetration into cell clusters. We are interested in developing smaller
engineered antibody fragments to overcome the limitations of mAb84 while
maintaining its functionality. We hypothesized that steric hindrance of the

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binding sites and the number of binding sites (valency) plays an important role
in undifferentiated human embryonic stem cells death.
This thesis reports on the construction of a number of engineered
antibody fragments, their expression in microbial hosts Escherichia coli and
Pichia pastoris and optimization to improve protein titer. Secondly, we
investigated the effects of the linker on the structural properties and
functionality of the engineered antibody fragment. Interestingly, we observed
the amino acid composition affecting linker rigidity/flexibility is of greater
importance than linker length on the functional activity. We further
substantiated this observation by using molecular dynamic simulations and
calorimetry to study the different constructs, and deduced that longer and
flexible linkers are more likely to decrease the antibody fragment stability
within the protein. Thirdly, using engineered antibody fragments with a
valency ranging from one to four, we observed an inverse exponential
relationship of valency versus cytotoxicity, indicating that valency is the
deciding factor in increasing cytotoxicity. Finally, upon consideration of
penetration into cell clusters, it was deduced that the trimeric engineered
antibody fragment was the best protein for targeting undifferentiated hESCs.
In conclusion, these results have direct applications in regenerative medicine
to be able to generate in vitro tissues from human embryonic stem cells, and
have potential implications in the fields of imaging and cell-cell separations.

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List of Publications & Conference Presentations
Klement, M., Zheng, J., Liu, C., Choo, A. B. H., Lee, D. Y., and Ow, D. S.
W. (2015). Antibody engineering of a cytotoxic monoclonal antibody 84:
investigating the effects of valency on cytotoxicity (submitted).
Klement, M., Liu, C., Loo, B. L. W., Choo, A. B. H., Ow, D. S. W., and Lee,
D. Y. (2015). Effect of linker flexibility and length on the functionality of a
cytotoxic engineered antibody fragment. Journal of Biotechnology, 199:90-97.
Pek, H. B.*, Klement, M.*, Ang, K. S.*, Chung, B. K. S., Ow, D. S. W., and
Lee, D. Y. (2015). Exploring codon context bias for designing a synthetic
thermostable invertase gene in Escherichia coli. Enzyme Microbial
Technology, 75-76:57-63.
Chung, B. K. S., Lakshmanan, M., Klement, M., Mohanty, B., Lee, D.-Y.
(2013). Genome-scale in silico modeling and analysis for designing synthetic
terpenoid-producing microbial cell factories. Chemical Engineering Science,
103:100-108.
Chung, B. K. S., Lakshmanan, M., Klement, M., Bun, C. C. and Lee, D.-Y.
(2013). Metabolic reconstruction and flux analysis of industrial Pichia yeasts.
Applied Microbiology and Biotechnology, 97(5):1865-1873.
Klement, M., Chung, B. K. S., Ow, D. S. W., and Lee, D. Y. (2014). Proof of
concept study of a novel codon optimization algorithm on antibody fragments:
Insights into the importance of codon context. Pichia 2014 Protein Expression
Conference (poster).

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