DEVELOPMENT OF A NAÏVE HUMAN FAB ANTIBODY

LIBRARY TO GENERATE MONOCLONAL ANTIBODIES

AGAINST BmSXP RECOMBINANT ANTIGEN

NOORSHARMIMI BINTI OMAR

UNIVERSITI SAINS MALAYSIA

2015

DEVELOPMENT OF A NAÏVE HUMAN FAB ANTIBODY

LIBRARY TO GENERATE MONOCLONAL ANTIBODIES

AGAINST BmSXP RECOMBINANT ANTIGEN

by

NOORSHARMIMI BINTI OMAR

Thesis submitted in Fulfillment of the Requirements

For the Master of Science

SEPTEMBER 2015

ii

ACKNOWLEDGEMENTS

In the name of Allah S.W.T, the most Gracious and the most Merciful.

Thank you Allah! For the strength given throughout my master research life. I would

like to address my appreciation to Dr Lim Theam Soon as my supervisor for his

helping hand, guidance, support and patience throughout my research work, also

many thanks to Professor Rahmah Noordin and Dr Choong Yee Siew from Institute

for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia. My

acknowledgment also goes to all the administration and laboratory staffs in

INFORMM for their cooperation.

For my laboratory mates (Hidayah, Bee Nar, Siang Tean , Qiuting , Chai Fung,

Anizah, Faezee, Soo Khim and Hamizah) sincere thanks for the knowledge shared,

assistance and best moments together. Plenty of thanks and love to my parents,

husband, siblings and in-laws for giving spiritual support, encouragements and

prayers during my study. Lastly, my lovely son you are my courage to continue the

success in life.

I would like to acknowledge the contribution of Pusat Sejahtera Universiti Sains

Malaysia, Penang during the blood collection process and My Brain Scholarship

awarded to me from Ministry of Higher Education (MOHE) in order to complete this

research.

Finally, this project was funded by the Malaysian Ministry of Higher Education,

Centre of Excellence Program (311/CIPPM/44001005).

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS

ii

TABLE OF CONTENTS iii

LIST OF TABLES

xii

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xvi

ABSTRAK xix

ABSTRACT xxi

CHAPTER 1 INTRODUCTION

1.1 The immune system 1

1.2 Antibody development and its diversity 1

1.3 Antibody structure 4

1.4 Phage display technology 5

1.4.1 Structure of bacteriophage 7

1.4.2 Infection mechanism 7

1.4.3 Display formats 9

1.4.4 Antibody libraries by phage display 11

1.4.4.1 Recombinant antibody libraries 11

1.5 Selection process (Bio-panning) 12

1.6 Applications of recombinant antibody 15

1.6.1 Application of Fab fragments 19

1.7 Model target – BmSXP recombinant antigen 19

iv

1.8 Statement of problem and rationale of study 20

1.9 Research objectives 21

CHAPTER 2 MATERIALS AND METHODOLOGIES

2.1 Materials for blood collection and isolation of B –

lymphocytes

22

2.1.1 Blood samples 22

2.1.2 Related materials 22

2.2 Materials for molecular biology work 23

2.2.1 Enzymes and corresponding buffers 23

2.2.2 Bacterial strains and phages 24

2.2.3 Primers 25

2.2.3.1 Primers design for vector construction 25

2.2.3.2 Primers design for DNA sequencing 25

2.2.3.3 Primers design for amplification of VH and VL

gene repertoire

26

2.2.3.4 Restriction endonucleases (RE) site primers –

secondary PCR

27

2.2.4 Plasmids and antigen 28

2.2.5 Culture media and additives 29

2.2.5.1 Culture media 29

2.2.5.2 Media preparations 29

2.2.5.3 Additives 29

2.2.6 Chemicals, buffers and solutions 30

2.2.6.1 Chemicals 30

2.2.6.2 Buffers for agarose gels 31

v

2.2.6.3 Buffers and solutions for phage library and

phage display

31

2.2.6.4 Chemicals, buffers and solutions for protein

expression and purification

32

2.2.6.5 Buffers and solutions for SDS-PAGE, native-

PAGE and Western blot

32

2.2.7 Prefabricated materials 33

2.2.7.1 Kits 33

2.2.7.2 DNA ladders 33

2.2.7.3 Protein ladders 34

2.2.7.4 Recombinant proteins, affinity columns and

magnetic particles

34

2.2.8 Laboratory consumable 34

2.2.9 Laboratory equipment 35

2.2.10 Software 36

2.3 Methodologies 36

2.3.1 Blood Experimental Section 36

2.3.1.1 Whole blood collection 36

2.3.1.2 In Vitro isolation of human total lymphocytes

from whole blood

37

2.3.2 Preparation of RNA and its integrity analysis 37

2.3.2.1 Total RNA extraction from lymphocytes cells 37

2.3.2.2 RNA fragments quality control and quantitation 38

2.3.3 Production of double-stranded DNA (dsDNA) 40

2.3.3.1 First strand cDNA synthesis 40

2.3.4 Production of V-gene DNA library 41

2.3.4.1 Primer design of the V-gene amplification 41

vi

2.3.4.2 Reconstitution of the primer for PCR 41

2.3.5 V-gene amplification by PCR 42

2.3.5.1 Restriction site amplification of the V-gene 42

2.3.6 Electrophoresis of the amplified V-gene/DNA 43

2.3.6.1 Preparation of agarose gel 43

2.3.6.2 DNA separation by electrophoresis and

visualization

43

2.3.6.3 DNA extraction and PCR purification 44

2.3.6.4 DNA purification 44

2.3.7 Modification of phagemid vector 45

2.3.7.1 Vector modification 45

2.3.7.1.1 Amplification of modified components

for pFb-Fab

45

2.3.7.1.2 Digestion of amplified DNA by

restriction enzyme

47

2.3.7.1.3 pLABEL-Fab cloning process 47

2.3.8 Preparation of V-gene DNA template for cloning 48

2.3.8.1 First step of cloning 48

2.3.8.2 Second step of cloning 48

2.3.8.3 Digestion of phagemid and V-gene DNA by

restriction enzyme

49

2.3.8.4 Dephosphorylation of cleaved phagemid 49

2.3.8.5 Ligation of cleaved DNA fragments 50

2.3.9 Ligation of cleaved DNA fragments 50

2.3.9.1 Plasmid – DNA from E.coli culture 50

2.3.9.2 Mini and Maxi preparation of plasmid – DNA 50

vii

2.3.9.3 Ethanol precipitation of plasmid – DNA 51

2.3.9.4 Colony PCR for insert determination 51

2.3.10 DNA sequencing 51

2.3.11 Molecular biology work using E.coli cells 52

2.3.11.1 Preparation of electrocompetent E.coli cells 52

2.3.11.2 Culturing of E.coli cells liquid media 52

2.3.11.3 Culturing of E.coli cells on solid media 53

2.3.11.4 Transformation of E.coli cells by

electroporation

53

2.3.12 Harvesting E.coli culture from assay plates 53

2.3.13 Library size estimation 54

2.3.14 Storage of E.coli cells 54

2.3.15 Molecular work on phage display 54

2.3.15.1 Helper phage preparation 54

2.3.15.2 Titration of helper phage 55

2.3.15.3 Preparation of phage library 56

2.3.15.4 Titration of phage library 56

2.3.16 Pre-selection, selection and post-selection process 57

2.3.16.1 Coating of antigens on streptavidin beads 57

2.3.16.2 Coating of antigens on microtitre well for

panning

57

2.3.16.3 Coating of antigens on microtitre well for

ELISA

57

2.3.16.4 Pre-selection of phage library with PTM 58

2.3.16.5 A selection process (Panning) 58

viii

2.3.16.5.1 Automated panning by King Fisher

Flex

58

2.3.16.5.2 Conventional panning by microtitre

well

60

2.3.17 Panning phage titration 61

2.3.18 Polyclonal phage ELISA 61

2.3.19 Picking of monoclonal phages 62

2.3.20 Monoclonal phage ELISA 63

2.3.21 Evaluation of monoclonal Fab antibody 63

2.3.21.1 Soluble expression of monoclonal Fab

antibody

63

2.3.21.2 Preparation of periplasmic recombinant

protein

63

2.3.21.2.1 Periplasmic extraction of

recombinant bacteria pellet

63

2.3.21.2.2 Protein purification by 1 mL Ni-

NTA ‗fast-flow

64

2.3.21.3 Analysis of recombinant antibody by gel

electrophoresis Sodium dodecyl sulphate

polyacrylamide (SDS-PAGE) and

native-PAGE

64

2.3.21.3.1 SDS-PAGE 64

2.3.21.3.2 Native-PAGE 65

2.3.21.4 Antigen ELISA for antibody detection test 66

2.3.21.5 Western blotting 66

2.3.21.5.1 Preparation of nitrocellulose

membrane

66

2.3.21.5.2 Protein transfer from gel to

nitrocellulose membrane

67

2.3.21.5.3 Recombinant antibody detection

using BmSXP antigen

67

ix

2.3.21.5.4 DAB preparation for HRP

detection

67

CHAPTER 3 RESULTS

3.1 Antibody library cloning 69

3.1.1 Modification of Fab library vector 69

3.1.2 Amplification of model 1 in pFb-Fab and stuffer gene

1&2 in Anti-eGFP

73

3.1.3 Ligation of stuffer gene 1 into model 1 75

3.1.4 Ligation of model 2 to stuffer gene 2 75

3.1.5 Cloning of model 3 into pFb-Fab and analysis 79

3.1.5.1 Sequencing result 79

3.2 RNA fragments quality control and quantitation 82

3.3 Production of heavy chain and light chain as insert for Fab

library

83

3.3.1 Restriction site amplification of HC and LC 85

3.4 Preparation of Fab library 86

3.4.1 Preparation of HC library 86

3.4.2 Preparation of full Fab library 89

3.4.3 Sequencing result 91

3.5 Phage library panning 91

3.5.1 Analysis of recombinant antigen BmSXP by SDS-

PAGE

93

3.6 Panning 100

3.6.1 Phage enrichment and polyclonal phage ELISA 100

3.6.2 Monoclonal phage ELISA 101

3.6.3 Quality control of monoclonal Fab antibody 104

x

3.6.4 Amplicon sequencing result of monoclonal Fab

antibody

108

3.6.5 Sodium dodecyl sulphate-polyacrylamide (SDS-PAGE)

and Native-PAGE of recombinant Fab antibody

108

3.6.6 Analysis of soluble Fab by biotinylated antigen ELISA 112

3.6.7 Western blotting 112

3.6.7.1 Recombinant antibody detection using

recombinant antigen BmSXP

112

3.6.7.1.1 Detection of Fab antibody in

denaturing form (SDS-PAGE)

112

3.6.7.1.2 Detection of Fab antibody in non-

denaturing form (native-PAGE)

117

CHAPTER 4 DISCUSSION

4.1 Generation of vector pLABEL-Fab 121

4.2 RNA quality control 122

4.3 Fab insert generation (V-gene repertoire generation) 123

4.4 Fab library preparation 125

4.5 Phage display 126

4.6 Soluble monoclonal Fab evaluation 130

4.6.1 Protein expression and purification 130

4.6.2 Specificity study of soluble Fab by biotinylated antigen

ELISA

132

4.6.3 Specificity study of soluble Fab by Western blot 132

CHAPTER 5 CONCLUSION

5.1 Conclusion 134

5.2 Limitations to study 135

5.3 Future studies 136

xi

REFERENCES 138

LIST OF

PUBLICATIONS

143

APPENDICES

146

xii

LIST OF TABLES

Page

Table 3.1 Analysis of 90 donors RIN values using Bioanalyzer

instrument

84

Table 3.2 V-D-J analysis of 10 random Fab fragment clones by

VBASE 2

94

Table 3.3 CDR analysis of 10 random Fab fragment clones by

VBASE2

95

Table 3.4 V-D-J analysis of P2E1 and P1E4 monoclonal antibodies

against BmSXP recombinant antigen of HC and LC analysis

by VBASE2

110

Table 3.5 CDRs analysis of P2E1 and P1E4 monoclonal antibodies

against BmSXP recombinant antigen of HC and LC analysis

by VBASE2 and IMGT/V-Quest

111

xiii

LIST OF FIGURES

Page

Figure 1.1

Organization of immunoglobulin heavy chain gene

segments rearrangement

3

Figure 1.2 Typical structure of an antibody molecule 6

Figure 1.3 A representation of filamentous phage particle 8

Figure 1.4 The mechanism of the bacterial infection by filamentous

phage

10

Figure 1.5 Antibody libraries type 14

Figure 1.6 A scheme of bio-panning strategy by phage display 16

Figure 1.7 Schematic representation of different antibody formats 18

Figure 2.1 Schematic diagram of pLABEL-Fab vector 46

Figure 3.1 Overview of the pFb-Fab and pLABEL-Fab vectors 71

Figure 3.2

Flow chart of vector modification strategy of the pFb-Fab

to pLABEL-Fab for phage display Fab fragment library

72

Figure 3.3 Overview of the model derived from pFb-Fab and stuffer

genes derived from anti-egfp in pIT2

74

Figure 3.4 Agarose gel electrophoresis images of amplified model

1and stuffer genes

76

Figure 3.5 A schematic representations of amplification of model 2

and model 3

77

Figure 3.6 Agarose gel electrophoresis images of amplified model 2

and model 3

78

Figure 3.7 Agarose gel electrophoresis images of colony PCR of the

model 3 into pFb-Fab.

80

xiv

Figure 3.9 Agarose gel electrophoresis images of donor 0035 HCs and

LCs amplifications

87

Figure 3.10 Agarose gel electrophoresis images of HCs and LCs

restriction site amplification of pooled donor 66-70

88

Figure 3.11 Agarose gel electrophoresis images of HC-mini library

colony PCR

90

Figure 3.12 Agarose gel electrophoresis images of full Fab library

colony PCR

92

Figure 3.13 Overview of the donors pooling strategy of HC/ LC

repertoires and cloning pooling strategy

98

Figure 3.14 SDS-PAGE profile of purified biotinylated BmSXP

recombinant antigen and conjugated biotinylated BmSXP

on streptavidin magnetic beads

99

Figure 3.15 Graphs of recovery phages and polyclonal phage ELISA

of each semi-automated panning round

102

Figure 3.16 Graphs of recovery phages and polyclonal phage ELISA of

each microtitre plate panning round.

103

Figure 3.17 Monoclonal ELISA result of monoclones readout at OD405 105

Figure 3.18 Agarose gel electrophoresis images of the seven

monoclonal binders by PCR

109

Figure 3.19 Native-PAGE and SDS-PAGE profiles of periplasmic

extractions

106

Figure 3.20 SDS-PAGE profile of purified recombinant monoclonal

antibody

114

xv

Figure 3.21 Native-PAGE profile of purified recombinant monoclonal

antibodies

115

Figure 3.22 Graph of monoclonal antibody-antigen ELISA 116

Figure 3.23 Western blot analysis of SDS-PAGE antibody separation 118

Figure 3.24 Western blot analysis of native-PAGE antibody separation 119

xvi

LIST OF ABBREVIATIONS

A Absorbance

ABTS 2,2‘-azino-bis(3-ethylbenzenethiazoline-6-sulphonic acid)

bp Base pair

C Carboxyl

cDNA Complementary deoxyribonucleic acid

CDR Complement determining region

CH1 Constant heavy chain 1

CH2 Constant heavy chain 2

CH3 Constant heavy chain 3

CH4 Constant heavy chain 4

CL Constant light chain

D Diversity/Diameter

DNA Deoxyribonucleic acid

dH20 Distilled water

dNTP Deoxynucleotide triphosphate

dsDNA Double stranded DNA

E.coli Escherichia coli

EDTA Ethylene diamine tetraacetic acid

eGFP Enhanced green fluorescence protein

ELISA enzyme-linked immuno assay.

EtBr Ethidium bromide

EtOH Ethanol

Fab Fragment antigen binding

Fr Framework

xvii

Fv Fragment variable

HC Heavy chain

His Histidine

HRP Horseradish peroxidase

IgA Immunoglobulin A

IgD Immunoglobulin D

IgE Immunoglobulin E

IgM Immunoglobulin M

IgG Immunoglobulin G

IgG4 Immunoglobulin G4

IPTG Isopropyl-beta-D-thiogalactopyranoside

J Junction

KF King Fisher

kDa kiloDalton

lacZ β-galactosidase gene

LC Light chain

N Amino

N1 Amino 1

N2 Amino 2

OD Optical density

pI-pXI Phage coat protein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11

PBS Phosphate buffer saline

PBS-T Phosphate buffer saline containing Tween 20

PTM Milk powder in PBST

RBS Ribosome binding site

xviii

RE Restriction endonuclese

RIN RNA integrity number

RNA Ribonucleic acid

U Unit

V-gene Variable gene

V Variable/Voltan

VH Variable heavy chain

VL Variable light chain

xix

PEMBANGUNAN PERPUSTAKAAN FAB ANTIBODI NAIF MANUSIA

UNTUK MENGHASILKAN ANTIBODI MONOKLONAL TERHADAP

ANTIGEN REKOMBINAN BmSXP

ABSTRAK

Antibodi adalah glykoprotein yang dihasilkan oleh sel B terhadap jasad molekul

asing. Penyelidikan ini bertujuan menghasilkan antibodi Fab (fragmen ikatan

antigen) manusia yang specifik terhadap antigen. Fragmen Fab terbahagi kepada satu

domain malar dan domain manipulasi yang terletak pada rantai berat dan rantai

ringan. Protokol optimum telah dicapai bagi menghasilkan perpustakaan Fab

antibodi rekombinan daripada penderma tidak berimunisasi yang terdiri daripada

tiga kumpulan etnik majoriti (Melayu, Cina dan India) di Malaysia. Kami

menggunakan seluruh RNA dari limfosit periferal darah daripada 90 penderma bagi

mendapatkan gen-gen manipulasi cDNA yang spesifik. Fragmen antibodi Fab yang

mengekod bagi kelas IgM diamplifikasi secara individu melalui proses PCR

menggunakan kepelbagaian set-set primer yang merujuk kepada bahagian manipulasi

rantai berat (µ) dan rantai ringan (κ,λ). Kepelbagaian perpustakaan antibodi dicapai

melalui gabungan-gabungan campuran semua rantai berat dan rantai ringan secara

rawak. Kepelbagian perpustakaan antibodi naif yang tinggi adalah sangat berguna

untuk saringan antibodi terhadap beberapa panel antigen. Saringan faj dilakukan

terhadap antigen BmSXP meggunakan dua strategi ‗panning‘ yang berbeza untuk

memahami keberkesanan strategi ‗panning‘. BmSXP adalah antigen rekombinan bagi

Wuchereria Bancrofti yang spesifik untuk penyakit limfatik Bancroftian filaria dan ia

digunakan untuk kegunaan diagnostik bagi jangkitan limfatik filaria. Persembahan

kedua-dua strategi ‗panning‘ menunjukkan kesukaran ‗panning‘ perpustakaan Fab

secara proses automatik manakala cara ‗panning‘ konvensional berjaya

xx

mengasingkan gabungan-gabungan positif. Dua Fab antibodi monoklonal terhadap

antigen BmSXP telah dikesan dan disahkan. Walaupun penghasilan perpustakaan

Fab naif adalah sukar, namun ia berguna untuk penghasilan Fab antibodi

monoklonal untuk aplikasi-aplikasi hiliran seperti diagnostik. ‗Panning‘

perpustakaan Fab naif secara paparan faj boleh menghasilkan antibodi-antibodi

monoklonal terhdadap pelbagai target antigen dengan berkesan. Oleh demikian, ini

menyediakan penyelesaian yang menarik untuk penghasilan antibodi-antibodi

manusia rekombinan.

xxi

DEVELOPMENT OF A NAÏVE HUMAN FAB ANTIBODY LIBRARY TO

GENERATE MONOCLONAL ANTIBODIES AGAINST BmSXP

RECOMBINANT ANTIGEN

ABSTRACT

Antibodies are glycoproteins produced by B-cells against molecules foreign to the

body. This study focused on the production of antigen specific human The Fab

region consists of one constant and one variable domain which are both located at the

heavy chain and the light chain. An optimized protocol for the production of a

recombinant Fab library of unimmunized donors from 3 major ethnic groups

(Chinese, Malay and Indian) in Malaysia was carried out. We used peripheral blood

lymphocytes total ribonucleic acids (RNA) from 90 donors to obtain variable genes

(V-gene) specific cDNA. The Fab-encoding regions of IgM repertoire were

individually amplified by polymerase chain reaction (PCR) using a diverse set of

primers corresponding to the different variable region of the heavy (µ) and the light

chains (к,λ). The diversity of the library is created via random combinatorial mixing

of all heavy and light V-genes. A highly diverse naïve library is useful for the

selection of antibodies against a panel of antigens. Phage selection was carried out

against BmSXP using two different panning strategies to understand the efficiency of

Fab library panning with different strategies. BmSXP is a recombinant antigen of

Wuchereria Bancrofti which is specific for Bancroftian lymphatic filarial disease and

is used for diagnostic of lymphatic filarial infection. The performance of both

panning strategies suggests the difficulty for Fab library panning with an automated

process as only the conventional panning method was able to isolate positive binders.

Two monoclonal Fab antibodies against BmSXP were identified and confirmed.

Although difficult, the production of a Fab naïve library is useful for the generation

xxii

of monoclonal Fab antibodies for downstream applications such as diagnostics.

Phage display panning of a naïve Fab library is able to select monoclonal antibodies

against the target antigens efficiently. This provides an attractive solution for the

generation of recombinant human antibodies.

1

CHAPTER 1

INTRODUCTION

1.1 The Immune system

The human immune system is a complex protection mechanism that involves several

different cellular mechanisms. There are two natural mechanisms involved in

fighting off infection and diseases in the human body, which are the innate and

adaptive immune response (Kindt et al,. 2007). A major feature of the innate

immunity is the ability to block the infiltration of infections by providing a physical

barrier to wade off pathogens and ensure they never gain access into the host. This

mechanism does not lead to any memory recognition of the antigens. On the other

hand, the adaptive immune response is capable of specifically killing foreign

microorganisms and differentiate self from non-self antigens. Unfortunately,

pathogens use several strategies to escape elimination from the adaptive system for

example by reducing its antigenicity, mimicking the host cell surfaces, selectively

suppress the immune responses, and continual variation of antigen surfaces. The

adaptive immunity provides a line of defense that eliminates a specific pathogen and

generates a memory towards it whenever the body is invaded by the similar pathogen

again. The natural cooperation between the innate and adaptive immune response in

human provides a critical defense against organisms causing diseases, but such

responses are not sufficient to completely protect a person from widespread

infectious diseases (Janeway et al., 1999).

1.2 Antibody development and its diversity.

The human adaptive immune system has the capability to produce a highly diverse

collection of antibody specificities due to the immunoglobulin genes encoded in the

2

germline. The maturation process is carried out by a gene assembly process that

produces a collection of new genes through site-specific recombination. Variable

region gene of the antibody consists of multiple germline segments that are classified

as variable (V), diversity (D) and junction (J) segments.

Human antibody variable heavy gene contains 48 VH, 23 D and 6 JH. The

immunoglobulin (Ig) κ gene contains 41Vκ and 5 Jκ while Igλ contains 34 Vλ and 5

Jλ (Kindt et al., 2007). These different segments are joined by recombination to

produce a full antibody VDJ segment whereas light chain genes are able to undergo

similar recombination excluding the D segment gene as outlined in figure 1.1. The

random gene rearrangement of V-J and V-D-J combination contributes to the

generation of a vast diversity of antibody repertoire (Lewis, 1994).

A part of the antibody diversity also involves the pairing association between heavy

and light chains. The combination of the associated heavy and light chain will lead to

a higher complexity resulting in higher diversification of antibodies. Apart from that,

antibody diversity is also associated by junctional flexibility caused by changes of

amino acid at CDR3 of heavy and light chain. It is a process where nucleotides can

either be lost or added (P / N –addition) during V-D-J and V-J rearrangements

(Parslow, 2001). Antibody diversity could be generated in rearranged variable region

gene units called somatic hypermutation after stimulation of B-lymphocyte by

antigen encounter. As a result, nucleotides in VJ or VDJ units are substituted with

alternatives and thus altering the specificity of the encoded antibodies further

(Raaphorst et al., 1997).

3

Figure 1.1 Organization of immunoglobulin heavy chain gene segments

rearrangement.

4

1.3 Antibody structure

Antibodies are produced through secretion by plasma cells to act as the front line

defense mechanism of our immune system. The common structure of an

immunoglobulin molecule consists of four subunits that is made up of two heavy

chain polypeptide chains held together by disulphide links and non-covalent

interactions (Davies et al., 1990). Antibodies are heterodimers composed of 2 heavy

chains and 2 light chains. There are two different light chains known as kappa (κ)

and lambda (λ), either one of these will be associated with the heavy chain as one

antibody, never both. The light chain consists of only a variable domain (VL) and a

constant domain (CL).

The heavy chain is made up of a variable domain, either three or four constant

domains juxtaposed to its variable domain (CH1, CH2, CH3 or CH4) and a hinge

region depending on the antibody class (Kindt et al., 2007). The classes of the

antibody are IgM, IgG, IgA, IgD, IgE or IgG where the heavy chain of each class

will have either a κ or λ light chains. For IgG, IgA and IgD isotypes the constant

domains involved are CH1, hinge, CH2 and CH3 while for IgM and IgE the domains

are CH1, CH2, CH3 and CH4. Therefore, each class of antibody is determined by the

proportion of heavy chain constant domain (Parslow, 2001).

There are two significant parts of an antibody that is the antigen binding and effector

functions located at the amino (N) - terminal and carboxyl (C) - terminal respectively

Janeway et al. (1999). The general Y-structure is the same for all antibodies,

however only the heavy and the light chain are responsible for recognizing antigens.

The variability calculations (number of different amino acids at particular position /

frequency of the most common amino acid at particular position), is able to show the

5

variation at the formation of the loop at the β-strand called the hypervariable region

mostly known as complementary determining regions (CDRs) (Kindt et al., 2007).

The remaining region with the least amount of variation is the framework (Fr)

region. The variations among antibodies are located at the three CDRs that make up

the binding site of the antigen epitope. It is this particular diversification that a lot of

the antibodies are produced that can distinguish any protein target. These CDRs are

separated by four relatively constant gene sequences as the framework (Janeway et

al., 1999). Figure 1.2 shows the antibody Y-shaped structure and its ribbon drawing

structure presentation.

1.4 Phage display technology

Antibodies are in high demand as they are suitable for diagnostic applications.

Conventional methods for monoclonal antibody generation are lengthy and tedious

(Li et al., 2006, Cambrosio and Keating, 1988). The introduction of phage display

allowed for rapid generation of monoclonal antibodies (Willats, 2002). The method

is based on a bacteriophage that is harmless to humans but only infects bacteria as a

reproduction host. The viral genomic material is able to enter the bacteria host cell

and directs the production of progeny phage. The progeny phage would infect other

bacteria cells and secrete a new phage in the cytoplasm of the cell. However, a

filamentous phage assembles its progeny on the cell surface by a special secretory

mechanism and leaves the cell viable for the next round of infection (Marvin, 1998).

It has been reported that the insertion of a foreign DNA into the phage genome is

capable of yielding a phage particle with the foreign peptide sequence displayed on

the phage coat protein (Smith, 1985). By exploiting the morphology and secretory

mechanism of the filamentous phage, it is possible to physically link genotype and

phenotype together (Barbas et al., 2004). Therefore, DNA sequence encoding

6

Figure 1.2 Typical structure of an antibody molecule. A) Antibody consists of two

heavy chains and two light chains which are linked together by disulphide bonds.

One bond linked together VH and VL, two bonds linked two VHs and several bonds

linking antibody subdomains. B) Finger-like loops of a single light chain whereby

binding of antigen occur. Figure adapted from (Alberts et al., 2013).

7

the foreign molecules can be retrieved through DNA sequencing as first reported by

George Smith in 1985 leading to the innovation of phage display technology.

1.4.1 Structure of bacteriophage

Filamentous bacteriophage belongs to a type of related viruses that contains single-

stranded viral DNA that infects only gram-negative bacteria. It is 2000 nm in length

and 6-10 nm in diameter enclosing all viral DNA genome consisting of 11 genes

(Zimmermann et al., 1986). The functions of the 11 genes are: p (III, VI – IX) for

coat proteins, p (I , IV and XI) for phage assembly, p (II and X) for DNA replication

and lastly pV for binding ssDNA. The viral mass consists mainly of about 2700

copies of the pVIII major coat protein, about 3-5 copies of pVII and pIX at one side

which are assembled first and 3-5 copies of pIII and pVI on the other side are for

phage particle stability and infection (Russel et al., 1997). Figure 1.3 shows the basic

filamentous phage particle consisting of its major coat proteins.

1.4.2 Infection mechanism

Filamentous phage is dependent on the F-pilus for infection and it does not kill its

host (Marvin, 1998). The pIII minor coat protein consists of three domains which are

carboxyl terminal (C), amino terminal 2 (N2) and amino terminal 1(N1) which are

separated by a glycine-rich linker region (Deng et al., 1999). Initially, the N2 domain

of the phage particle binds strongly with the F-pilus of E.coli and mediate the

binding of the N1 domain to the bacteria protein TolA (Click and Webster, 1997). By

natural forces, it pulls the phage particle closer to the bacteria. Consequently, the N1

domain is released from the N1N2 complex of the pIII after the F-pilus binding.

Next, free N1 domain will interact with TolA receptor (Karlsson et al., 2003). TolA

is a membrane bound protein which was believed to incorporate other membrane

8

Figure 1.3 A representation of filamentous phage particle. The major coat proteins

of the filamentous phage such as M13KO7 ; pIII, pVI, pVII, pVIII and pIX.

9

proteins, TolQ and TolR to form a complex (Click and Webster, 1997). This protein

complex facilitates the pIII and pVIII into the cytoplasmic region of the bacteria.

Finally, anchored TolA at the cytoplasm will enter the periplasm where phage

infection occurrs. At this point, the viral ssDNA is secreted into the cytoplasm and

gets replicated into dsDNA format; making it ready for the expression of the coat

proteins. The new phage particles are assembled and will be secreted into the

medium (Rakonjac et al., 1999). Figure 1.4 shows the infection mechanism of the

filamentous phage.

1.4.3 Display formats

There are two commonly used coat protein for phage display which is pVIII and pIII

(Smith and Petrenko, 1997). The major coat protein, pVIII exist in thousands of

copies that make up the long cylindrical phage particle while pIII consists of three

functional domains (Webster, 1996). The presentation of any foreign sequence is

usually inserted at the C - terminus of the pVIII and pIII coding sequences while the

linker sequences are inserted at the N - terminus on a phagemid vector. Protein pVIII

only allows short peptides (6 - 8 residues) to be displayed on it as it could interrupt

the interactions of the pVIII molecules during assembly (Qi et al., 2012). However,

there are reports on the fusion of antibody fragments on pVIII (Kang et al,

McCafferty et al). On the other hand, pIII coat proteins are able to tolerate large

foreign molecules on it and its infectivity still remained active. This makes pIII the

ideal coat protein for the presentation of foreign proteins for display.

10

Figure 1.4 The mechanism of bacterial infection by filamentous phage. First, the pIII

coat protein N2 domain interacts with the bacterial F-pilus followed by recognition

of N1 domain with TolA. Next, retraction of the F-pilus brings the phage closer and

opening of the phage head to release phage DNA. Figure adapted from (Karlsson et

al., 2003)

11

1.4.4 Antibody libraries by phage display

Phage display has become a powerful technology that allows for in vitro mimicking

of the natural human production of antibodies in vivo. Fully human monoclonal

antibodies (mAbs) production is efficient for downstream application for humans due

to the highe risk associated with the use of murine derived mAbs (Vaughan et al.,

1998). Phage display is a robust scientific research and development for antibody

selection.

1.4.4.1 Recombinant antibody libraries

There are four types of antibody libraries that can be generated for scientific

applications depending on the source of the antibody genes obtained; naïve (non-

immunized), immunized, semi-synthetic and fully synthetic library (Ponsel et al.,

2011, Marks and Marks, 1996). Libraries derived from immunized and non-

immunized individuals are directly influenced by the natural human antibody gene

diversity while semi-synthetic and synthetically design libraries are based on the

designed gene to exhibit similar diversities found in natural human antibody

repertoires.

Naïve libraries can be built using peripheral blood or bone marrow of healthy

individuals. These libraries are used to harvest the natural binding sites of the

antibodies (Løset et al., 2005). The antibody repertoire is constructed from natural

rearranged V-genes of the B-cells representing in vivo IgM antibodies that are

commonly produced by developing B-cells that do not show any preference to any

particular antigen that provides higher percentage of success when isolating new

binders.

12

Immunized antibody libraries are made using genetic materials from individuals that

have contracted or have been exposed to a particular disease (de Carvalho Nicacio et

al., 2002). This will skew the antibody producing cells to generate antibodies against

immunogenic antigens related to the disease. This can prove to be useful to generate

high affinity and specific antibodies against antigens of that particular disease.

Therefore the preferred antibody gene isotype for immunized libraries is usually IgG.

The synthetic antibody libraries are made up of antibody gene variation that can be

designed and generated through degenerate oligonucleotides to produce fully

synthetic heavy and light chains with different properties. The entire gene fragment

of a synthetic antibody is chemically synthesized in vitro (Knappik et al., 2000).

Such libraries allow massive customization in terms of framework preferences and

even CDR designs. As the randomization in such libraries generates highly diverse

repertoires, the library will have the tendency to exhibit unskewed repertoire much

like those of the naïve libraries. Semi-synthetic libraries are a fusion of the naïve and

synthetic library designs (De Kruif et al., 1995). This is because in semi-synthetic

libraries, the genetic information can be obtained from donors and the randomized

regions or framework regions can be synthesized. This allows semi-synthetic

antibody libraries to present a naïve repertoire too. Therefore semi- and fully

synthetic libraries can function to generate antibodies against any antigen target.

Figure 1.5 shows the four different types of antibody libraries can be generated for

phage display.

1.5 Selection process (Bio-panning)

A heterogeneous mixture of the phage clones or phage library is produced with

standard molecular biology techniques involving the exploitation of DNA vectors to

accommodate V-gene inserts and accommodate packaging to mature phage particles

13

by appropriate E.coli hosts (Galanis et al., 1997) . In order for the antibody to be

presented on the phage particle, the V-genes need to be tethered to the phage coat

protein so that the presentation will be a fusion of both proteins. The integrity of the

antibody presented is maintained as it is physically placed on the surface of the

phage particle. This physical interaction between the antibody and phage particle

allows for easy in-vitro selection of specific antibodies against immobilized target

antigens (Romanov, 2003). This selection process is commonly known as bio-

panning. The selection process is a collection of repetitive rounds that allows

antibody displaying phages to bind immobilized antigens on solid phases. The

antigens can be immobilized onto (1) plastic tubes (Marks et al., 1991), (2) columns

with target-activated matrix (McCafferty et al., 1990), (3) biotinylated antigens

selection (Hawkins et al., 1992), (4) an automated selection whereby the antigens are

coated on the magnetic particles (Konthur and Walter, 2002), (5) immunopins (Lou

et al., 2001) and (6) microtitre plate (Krebs et al., 2001). Each solid phase provides a

different set of advantages and disadvantages. The choice of the best suitable solid

phase is mainly dependent on the target antigen and selection conditions. During the

panning process, a repetitive cycle of physical binding and removal of unwanted

phage particles is carried out. The initial binding step allows for specific binders to

capture the target antigens and interact with it in a suitable environment. The wash

steps are introduced to remove unbound phages and contaminants (Goodchild et al.,

2005). The panning process can be customized to allow for different stringency. This

will affect the end product of the panning whereby antibody selectivity can be tuned

with different competing conditions. The antibody bound to the target can be eluted

using several different techniques such as acid/basic elution (Hoogenboom et al.,

1998, Marks et al., 1991), enzymatic cleavage (Ward et al., 1996) or reducing agents

14

Figure 1.5 Antibody libraries type A) Generation of naïve libraries are from the B-

cells isolation of non-immunized donor. B) Generation of immunized libraries are

from the B-cells isolation of non-immunized donor. C) Generation of synthetic

libraries are fully based on the computational design of the gene sequence D)

Generation of semi- synthetic libraries are combination of pre-designed segments

and natural source of gene sequence. Figure adapted from (Ponsel et al., 2011).

15

(Kretzschmar and von Rüden, 2002). The rescued phage clones are then infected

with E.coli allowing the selected phage to be amplified as input phage for the next

round of panning (Goodchild et al., 2005). To identify monoclonal binders, single

clones have to be picked from a pool of enriched binders and analyzed individually.

Figure 1.6 shows the bio-panning process by phage display technology.

1.6 Applications of recombinant antibody

The main characteristic for the antibodies to be useful for various applications is

dependent on the high affinity and specificity towards an antigen. In 1975, Köhler

and Milstein (Köhler and Milstein, 1975) reported the revolutionary method to

generate antibodies by fusing murine antibody-producing cells with myelomas from

bone marrow to produce hybridomas. Hybridoma technology has been applied

successfully to produce monoclonal antibodies against many antigens. The method

involves the isolation of hybridomas producing antibodies against the selected

antigen by screening each individual hyridoma (Cambrosio and Keating, 1992). One

major complication associated with hyridoma-based antibodies is the potential

allergic reaction accustomed to the application of animal derived antibodies in

humans. The allergic reaction is a result of the human immune system reacting

against the foreign animal derived antibody as a potential threat (Khazaeli et al.,

1994). Therefore, antibodies derived using hybridoma technology was not able to

achieve its intended benefits. Therefore, to circumvent this issue various

modifications have been introduced. The modifications lead to the introduction of

chimeric, humanized and ultimately fully human antibodies. Another major

consideration for recombinant antibodies is the ability to increase production with

microbial expression systems. For example, Escherichia coli (E.coli) as an

expression host has become a widely used technique that is able to produce

16

Figure 1.6 A scheme of bio-panning strategy by phage display. In phage display

panning where antibodies are presented on the phage particles and add into

immobilized target antigens for binders. This is followed by several wash steps to

remove unbound phages and elution and propagation of the bounded phages. Figure

adapted from (Goodchild et al., 2005).

17

monoclonal antibodies due to its fast growth rate, the ability to accept a vector

carrying specific genetic material and a high transformation rate (Plückthun, 1992).

The production of full-length antibody is relatively difficult by expression in the

E.coli system if compared to the expression of smaller antibody fragments (Simmons

et al., 2002). For instance, the first antibody produced is the fragment variable (Fv)

domain antibodies which comprises of either the VH or VL domains expressed in the

periplasmic environment of the bacterial cells (Skerra and Pluckthun, 1988). As the

binding characteristics of an antibody is associated to the ability of both the heavy

and light chain domains to work in tandem, a modified fragment namely the single

chain Fragment variable (scFv) was introduced. scFv fragments consist of both

variable domains to be expressed as one fragment with the help of a peptide linker

(Bird et al., 1988). Even so, variations in the length of the linker sequence have been

reported to promote aggregation during expression (Whitlow et al., 1993).

On the other hand, the stabilization of Fab molecules is generally associated to the

disulphide bond between the variable heavy and the light chain. The production of

Fab molecules requires the transportation of the Fab to the periplasmic space for

disulphide bond formation. However, expression levels of Fab are difficult due to the

need for both fragments to be independently expressed and form a disulfide bond in

the periplasmic space (Skerra and Plückthun, 1991). However, Fab molecules are

important fragments for antibody engineering. This is because Fab fragments are

suitable for direct transfiguration to the larger full immunoglobulin molecule without

the loss of binding efficiency (Rader, 2009). This would make full antibody molecule

production easier and more efficient. Figure 1.7 shows the different recombinant

antibody formats for phage display.

18

Figure 1.7 Schematic representation of different antibody formats. Fv (fragment

variable) comprises of VH and VL. scFV (single chain fragment variable) is included

of VH and VL connected by a linker. Fab (fragment antigen binding) comprises of

VH and VL with a constant region (CH1 and CL respectively) connected by a

disulphide linkage. scFab (single chain fragment antigen binding) consists of Fab but

with a linker connection.

19

1.6.1 Application of Fab fragments

The developments in antibody generation technology have enabled recombinant

antibodies to be made fully of human fragment (Yan and Xu, 2006, Barbas et al.,

2004). The most widely used recombinant antibodies with respect to the antigen

binding fragments are the scFV and Fab. Both recombinants are biologically and

physically different in properties. Fab in general has a better stability comparison.

The interaction of the HC and LC by an interchain disulfide bond is the major

contribution towards the stability of a Fab fragment (Röthlisberger et al., 2005) .

Apart from that, a synergy between the VH, VL, CH1 and CL domains had shown

significant stabilization effects within the interfaces by the intrachain disulfide

bonding. These were reported by Shimba et al. which discussed Fab stability in

comparison to Fv based on thermodynamics study (Shimba et al., 1995). Another

study by Daniela Röthlisberger et al. discussed the stabilizing effects of Fab domain

interactions and compared that with the scFv. The Fab domain showed several key

advantages such as better mutual stabilization of both HC and LC, better stability due

to the presence of the interchain disulfide bond producing and kinetic stabilization

(Röthlisberger et al., 2005). As such the application of Fab monoclonal antibodies

are still used due to the wide benefits it is able to provide in terms of format

conversion even with the challenges posed in handling.

1.7 Model Target – BmSXP Recombinant Antigen

In this work, the model antigen selected was BmSXP antigen used for detection of

bancroftian filariasis. It is a recombinant protein of Wuchereria Bancrofti - a filarial

nematode that lives in the human lymphatic system. BmSXP is a highly specific

recombinant antigen used for the detection of specific IgG4 antibodies for

bancroftian worms (Khoo et al., 2010). BmSXP recombinant protein was an ORF of

20

SXP1 gene from Brugia Malayi cDNA library which its sensitivity is 95% in

detection of bancroftian filariasis infection. The used of this recombinant antigen

with BmR1 recombinant antigen (specific for brugian filariasis detection) was found

to enhance the specificity and sensitivity of lymphatic filariasis for diagnostic

purposes. (Ab Rahman, 2008) Therefore, there is a necessity to produce antibodies

against it for future downstream applications, mainly for immunopurification. A

common challenge in the production of the diagnostic kit is the purification of the

antigen with high specificity. As the current diagnostic kit is based on antibody

detection, the generation of monoclonal antibodies could also pave the way for the

development of an antigen detection system.

1.8 Statement of problem and rationale of study

The antibody molecule is an integral part of many proteomics, diagnostic and

therapeutic research due to its ability to specifically bind a target antigen. Production

of human antibodies has been explored widely in a race to find unique binders. In

vitro antibody production through phage display technology is an interesting

alternative to conventional methods for antibody development. Phage display allows

for a faster and easier approach to generate antibodies with the generation of an

antibody library mimicking the natural antibody diversity. Antibody variable regions

(variable heavy chain/variable light chain) are easily manipulated and displayed on

the phage particle. In order to develop this technology in-house to overcome the

complications of antibody production, a naïve Fab library was developed and used to

identify monoclonal antibodies against BmSXP. Fab antibody still possess a lot of

challenges as a tool for detection of diagnostic and therapeutic studies by

development of phage display technology. Bypassing the use of animals, phage

display is a well-established technology for antibody isolations of fully human small

21

antibody format such as scFV and Fab. Generation of Fab antibody by this

technology was a challenge due to two significant reasons. First, Fab fragment is

larger compare to scFv fragment to be expressed in E.coli system. Second, the

presentations of the Fab on the phage particle was a synergy between two

independent chains endeavoring for each other in E.coli periplasm which always

leading to low yield of phage presenting full Fab fragment. Therefore, the generation

of a highly diverse naive Fab library for phage display was indeed required to

produce quality Fab antibodies.

1.9 Research Objectives

1. To generate a naïve fragment antigen- binding library (Fab fragment library)

2. To produce human monoclonal Fab antibodies by phage display against BmSXP

recombinant antigen

3. To evaluate the specificity of the monoclonal antibodies generated

22

CHAPTER 2

MATERIALS AND METHODOLOGIES

2.1 Materials for blood collection and isolation of B – lymphocytes

2.1.1 Blood samples

90 donors (Malay, Chinese and Indian)

2.1.2 Related materials

K2 EDTA Blood collection tubes 3.0 mL BD Vacutainer , USA

Ficoll – Paque TM

PLUS GE Healthcare Life

Sciences , UK

β – Mercaptoethanol (β-ME) Sigma Aldrich, USA

23

2.2 Materials for molecular biology work

2.2.1 Enzymes and corresponding buffers

NcoI NEB3/BSA New England Biolabs,

USA

MluI NEB3/BSA New England Biolabs,

USA

SalI HF NEB4/BSA New England Biolabs,

USA

NheI HF NEB4/BSA New England Biolabs,

USA

Pfu DNA

polymerase

10X Pfu Buffer (MgSO4) Thermo Scientific, USA

Vent DNA

Polymerase

10X ThermoPol buffer New England Biolabs,

USA

T4 DNA Ligase T4 DNA ligase buffer New England Biolabs,

USA

24

2.2.2 Bacterial strains and phages

DH10B T1

Phage

Resistant

Cells

F-mcrA Δ(mrr-hsdRMS-mcrBC)

Φ80lacZΔM15

ΔlacX74 recA1 endA1araD139Δ

(ara, leu)7697 galU galK λ-

rpsL nupG tonA

Invitrogen,

Carlsbad,

USA

TG1

supE thi-1 ∆(lac-proAB) ∆(mcrB-

hsdSM)5 (rK–mK–) [F´ traD36 proAB

lacIqZ∆M15]

Agilent

Technologies,

USA

XL1 Blue

recA1 endA1 gyrA96 thi-1 hsdR17

supE44 relA1 lac [F´ proAB lacIqZ∆M15

Tn10 (Tetr)]

Agilent

Technologies,

USA

M13KO7

helper

phage

knr

New England

Biolabs, USA