TOWARDS THE DEVELOPMENT OF NOVEL BISPECIFIC ANTIBODIES TO INHIBIT KEY CELL SURFACE RECEPTORS INTEGRAL FOR THE GROWTH AND MIGRATION OF TUMOUR CELLS

Andrew Lai Bachelor of Science, UNSW 2008 Master of Biotechnology, QUT 2010 Bachelor of Applied Science (Hons), QUT 2012

Submitted for the degree of

Doctor of Philosophy

Institute of Health and Biomedical Innovation Faculty of Health Queensland University of Technology 2016

Keywords

Breast cancer, extracellular matrix, insulin-like growth factor, metastasis, migration, therapeutics, phage display, single chain variable fragments, vitronectin

Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells i

Abstract

Metastatic breast cancer, or breast cancer which has spread from the primary tumour to distal secondary sites, remains a major killer of women today. Researchers have observed that the relationship between tumour cells and its surrounding environment plays an important role in cancer progression. One such interaction is between the Insulin-like growth factor (IGF) system and the system, which has been demonstrated to be involved in cancer cell metabolic activity and migration. Therefore, the aim of this project was to translate this knowledge into the generation of bispecific antibody fragments (BsAb) targeting both systems, in order to disrupt their roles in cancer growth and metastasis. To screen for IGF-1R and αv integrin binding ScFv, a phage display enrichment procedure using the Tomlinson ScFv libraries was conducted. After the panning cycles, 192 clones were screened for binding using ELISA, of which 16 were selected for sequencing. Analysis of the results revealed 1 IGF-R and 3 αv integrin unique binding ScFv, which were all subsequently expressed in a bacterial expression system. The ScFv were purified to a high degree using Ni-NTA and protein-L agarose affinity chromatography and were further assessed using size exclusion chromatography to detect the presence of any ScFv aggregates. Functional characterisation studies using MCF-7 breast cancer cells indicated that the IGF-1R targeting ScFv was able to inhibit both IGF-I and VN dependent metabolic activity and migration. Cell-based assays were also used to characterise the anti-αv integrin ScFv, however, no inhibition of cell attachment or migration was observed. Investigation into possible reasons for the inability to inhibit cell processes revealed the original αv integrin sample used for panning may not have been fit for purpose. Thus, while the anti-IGF-1R ScFv shows potential, the lack of a functional anti-αv integrin ScFv made the generation of a bi-specific antibody fragment unachievable within this project. In light of these results, further characterisation should be conducted to assess the potential therapeutic treatment of breast cancer using the generated ScFv.

ii Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells

Table of Contents

Keywords ...... i Abstract ...... ii Table of Contents ...... iii List of Figures ...... vii List of Tables ...... ix List of Abbreviations ...... x Statement of Original Authorship ...... xv Acknowledgements ...... xvi Literature Review ...... 1 1.1 Breast cancer ...... 2 1.2 Targeted cancer therapies ...... 3 1.3 Insulin-like growth factor system ...... 6 1.4 ...... 8 1.5 IGF-1R and αv integrin signalling pathway interaction ...... 9 1.6 IGF-1R and VN binding integrin targeting strategies ...... 10 1.7 Antibody structure and fragmentation ...... 14 1.8 Bispecific antibodies ...... 15 1.9 Antibody generation ...... 18 1.10 Phage display ...... 19 1.10.1 KM13 phage physiology and structure ...... 21 1.10.2 Phage display selection ...... 22 1.11 Conclusion and knowledge gaps ...... 23 1.12 Project outline ...... 24 1.12.1 Hypothesis ...... 24 1.12.2 Aims ...... 24 Materials and Methods ...... 26 2.1 Introduction ...... 27 2.2 Materials ...... 27 2.3 Cloning ...... 27 2.3.1 General PCR ...... 27 2.3.2 Mini-prep plasmid isolation ...... 28 2.3.3 Sanger sequencing of plasmids ...... 29 2.4 Mammalian cell culture ...... 30 2.4.1 Propagation of MCF-7 cell lines ...... 30 2.4.2 Cell counting ...... 30 2.4.3 Cryopreservation of cell lines ...... 30 2.4.4 Mycoplasma Testing ...... 31 2.5 Protein isolation from adherent cell cultures ...... 32

Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells iii

2.5.1 Denaturing conditions ...... 32 2.5.2 Non-denaturing conditions ...... 32 2.6 Protein Quantification ...... 33 2.7 Protein detection ...... 33 2.7.1 SDS-PAGE and Chemiluminescence Western ...... 33 2.7.2 Quantitative Westerns ...... 34 2.7.3 Silver staining ...... 34 Phage Display selection and screening...... 36 3.1 Introduction ...... 37 3.2 Experimental procedures ...... 40 3.2.1 Materials ...... 40 3.2.2 Phage display libraries ...... 40 3.2.3 E. coli strain, media, and culture conditions ...... 40 3.2.4 KM13 helper phage amplification ...... 42 3.2.5 Amplification of phage library repertoire ...... 44 3.2.6 Phage titre determination ...... 44 3.2.7 Library ‘I’ selection of IGF-1R binders ...... 45 3.2.8 Phage selection against αv integrin with library ‘I’ and ‘J’ ...... 46 3.2.9 Polyclonal phage ELISA ...... 46 3.2.10 Monoclonal soluble ELISA ...... 47 3.2.11 Sequencing of positive binders ...... 47 3.3 Results ...... 48 3.3.1 rIGF-1R can be passively coated onto MaxiSorp® immunotubes ...... 48 3.3.2 Panning and polyclonal phage ELISA of IGF-1R binders ...... 51 3.3.3 Soluble ScFv IGF-1R expression and Monoclonal ELISA ...... 52 3.3.4 Panning and monoclonal ELISA of αv integrin ScFv binders ...... 54 3.3.5 Sequencing of IGF-1R binding clones ...... 56 3.3.6 Sequencing of αv integrin binding ScFv clones ...... 59 3.4 Discussion ...... 61 ScFv expression and purification ...... 67 4.1 Introduction ...... 68 4.2 experimental procedures ...... 70 4.2.1 Materials ...... 70 4.2.2 Site directed mutagenesis ...... 70 4.2.3 Preparation of competent HB2151 E. coli ...... 73 4.2.4 Transformation...... 74 4.2.5 Induction time pilot study ...... 74 4.2.6 Detection of the expressed ScFv by Western blotting ...... 74 4.2.7 Large scale ScFv expression ...... 75 4.2.8 Periplasmic extraction ...... 75 4.2.9 Precipitation of ScFv from culture medium using PEG ...... 75 4.2.10 Purification using Ni-NTA affinity chromatography ...... 76 4.2.11 Small scale purification using protein-L agarose ...... 76 4.2.12 Large scale protein-L agarose expression and purification from culture medium ...... 77 4.2.13 Expression of αIGF-1R ScFv using EnPresso B ...... 77 4.2.14 Protein Quantification ...... 77 4.3 Results ...... 78 4.3.1 Amber stop codon modification using Site-directed mutagenesis ...... 78

iv Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells

4.3.2 Post-induction ScFv expression time course ...... 80 4.3.3 Precipitation of proteins from culture medium using PEG formed insoluble products ...... 81 4.3.4 Optimisation of Ni-NTA purification with the addition of urea...... 84 4.3.5 Large scale Ni-NTA purification of ScFv from periplasmic extracts ...... 86 4.3.6 Verification of the final Ni-NTA affinity chromatography purified ScFv ...... 88 4.3.7 Improved purification using protein-L agarose ...... 89 4.3.8 Large scale purification of ScFv from medium using protein-L agarose ...... 91 4.3.9 Expression and purification of αIGF-1R ScFv using EnPresso® medium ...... 93 4.4 Discussion ...... 95 Biophysical characterisation ...... 99 5.1 Introduction ...... 100 5.2 experimental procedures ...... 101 5.2.1 Materials ...... 101 5.2.2 Analytical size exclusion chromatography ...... 101 5.2.3 BIAcore ...... 102 5.3 Results ...... 103 5.3.1 Generation of a SEC molecular weight calibration curve ...... 103 5.3.2 Optimisation of SEC resolution of α-IGF-1R ScFv ...... 106 5.3.3 Fractionation of α-IGF-1R ScFv ...... 108 5.3.4 SEC characterisation of α-αv ScFv ...... 110 5.3.5 Affinity of α-IGF-1R ScFv to IGF-1R ...... 112 5.3.6 Affinity of α-αv integrin ScFv to αvβ3 integrin ...... 114 5.4 Discussion ...... 116 Functional characterisation ...... 121 6.1 Introduction ...... 122 6.2 Experimental procedures ...... 124 6.2.1 Materials ...... 124 6.2.2 Cell culture ...... 124 6.2.3 Metabolic assay ...... 124 6.2.4 Migration assay ...... 125 6.2.5 Attachment assay ...... 126 6.2.6 Isolation of functional Insulin-like growth factor receptor...... 126 6.2.7 SDS-PAGE and Western immunoblotting ...... 128 6.2.8 Receptor in vitro phosphorylation assay ...... 129 6.3 Results ...... 130 6.3.1 The effect of IGF-1R targeting ScFv on MCF-7 cell metabolic activity ...... 130 6.3.2 The effect of IGF-1R targeting ScFv on MCF-7 cell migration ...... 132 6.3.3 Effect of αv-integrin targeting ScFv on MCF-7 cell attachment ...... 133 6.3.4 The effect of αv-integrin targeting ScFv on MCF-7 cell migration ...... 134 6.3.5 Isolation of IGF-1R was successful using WGA agarose ...... 136 6.3.6 Development of a functional screen for in vitro IGF-1R activation ...... 140 6.3.7 High throughput of IGF-1R stimulation assay ...... 142 6.4 Discussion ...... 144 General discussion ...... 147 Rational design of IGF-1R/αv integrin bi-specific antibodies .. 167 8.1 Introduction ...... 168

Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells v

8.2 Experimental procedures ...... 176 8.2.1 Selection of ScFv encoding sequences ...... 176 8.2.2 Construction and synthesis of the BsAb constructs ...... 177 8.2.3 Cloning of BsAb constructs into the expression vector pET-scFv-T ...... 179 8.2.4 Transformation of BsAb constructs ...... 180 8.2.5 Expression of BsAb ...... 181 8.2.6 Affinity purification of BsAb using protein-L agarose ...... 181 8.2.7 Expression and purification of αv integrin ...... 182 8.2.8 Fractionation of BsAb monomers ...... 182 8.2.9 Surface Plasmon Resonance (SPR)-based affinity determination ...... 183 8.2.10 N-Terminal sequencing ...... Error! Bookmark not defined. 8.2.11 Metabolic assessment of the BsAb on MCF-7 cells ...... 183 8.2.12 Migration assay ...... 184 8.2.13 Attachment assay ...... 184 8.2.14 Cell signalling ...... 185 8.2.15 3D tumour spheroid assays ...... 185 8.2.16 Xenograft model and stability testing ...... 186 8.2.17 Statistical analyses ...... 187 8.3 Discussion ...... 188 References ...... 195 Appendices ...... 245

vi Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells

List of Figures

Figure 1-1: Genomic and transcriptomic alteration of IGF system genes in breast cancer samples ...... 7 Figure 1-2: Genomic and transcriptomic alteration of αv integrin genes in breast cancer samples ...... 9 Figure 1-3: Various IGF-1R and αv targeting strategies...... 11 Figure 1-4: Examples of intact IgG and its fragments...... 15 Figure 1-5: Selected classes of bispecific antibodies and examples of their unique configurations...... 18 Figure 1-6: Schematic of a KM13 phage particle ...... 22 Figure 3-1: Tomlinson libraries (I+J) phagemid...... 39 Figure 3-2: The helper phage KM13 encodes for a trypsin sensitive phage protein pIII...... 43 Figure 3-3: Efficiency of IGF-1R passive adsorption onto polystyrene...... 50 Figure 3-4: Library ‘J’ polyclonal phage ELISA...... 51 Figure 3-5 Soluble IGF-1R ScFv ELISA...... 53 Figure 3-6 Selection of αv integrin binding ScFvs...... 55 Figure 3-7: Sequence map for IGF-1R binding clone...... 57 Figure 3-8: DNA and translated amino acid sequence for αv integrin clones A2, B1 and F3 ScFv...... 60 Figure 4-1: Site-Directed Mutagenesis workflow...... 72 Figure 4-2 Modification of amber stop codon sequence from ScFvs...... 79 Figure 4-3: Time course trial of ScFv expression...... 81 Figure 4-4 Precipitation of ScFv by PEG 6000...... 83 Figure 4-5: Improved Ni-NTA affinity purification of α-IGF-1R ScFv with the addition of 1 M urea...... 85 Figure 4-6: Purification of ScFv from E. coli periplasmic extracts using Ni- NTA...... 87 Figure 4-7: Verification of the final Ni-NTA purified ScFv by silver- stained SDS-PAGE and Western blotting...... 88 Figure 4-8: Two step purification of α-BSA ScFv using Ni-NTA and protein-L conjugated agarose...... 90 Figure 4-9: Large scale purification of ScFv using protein-L agarose...... 92 Figure 4-10: Expression of α-IGF-1R ScFv using EnPresso® medium and purification using protein-L agarose ...... 94 Figure 5-1: Size exclusion chromatography of reference proteins...... 105

Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells vii

Figure 5-2: Size exclusion chromatography of α-IGF-1R ScFv...... 107 Figure 5-3: Fractionation of α-IGF-1R ScFv monomers and dimers using HPLC...... 109 Figure 5-4: Characterisation of α-αv ScFv by SEC...... 111 Figure 5-5: BIAcore affinity determination of ScFv and IgG targeting IGF-1R...... 113 Figure 5-6: BIAcore analysis of αv targeting ScFv and IgG to αvβ3 integrin...... 115 Figure 6-1: MCF-7 proliferation with IGF-1R inhibitors...... 131 Figure 6-2: MCF-7 migration with IGF-1R inhibitors...... 133 Figure 6-3: MCF-7 attachment with αv integrin inhibitors...... 134 Figure 6-4: MCF-7 migration with αv integrin inhibitors...... 135 Figure 6-5: Wheat Germ Agglutinin (WGA) purification of IGF-1R from MCF-7 cell lysate...... 137 Figure 6-6: Concentration determination of WGA purified fractions using quantitative Western immunoblotting...... 139 Figure 6-7: IGF-I stimulation on WGA purified IGF-1R...... 141 Figure 6-8: IGF-I stimulation on WGA purified IGF-1R...... 143 Figure 7-1: Antibody discovery platform and their clinical trial status...... 151 Figure 7-2 Alternative workflows for the generation of BsAb against αv integrin and IGF-1R...... 162 Figure 8-1:Proposed tandem bispecific antibody constructs against IGF- 1R and αv integrin...... 178 Figure 8-2 Cloning workflow of BsAb encoding fragment into the pET- ScFv-T vector...... 180

viii Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells

List of Tables

Table 1-1: Monoclonal Cancer therapeutic antibodies approved in the United States ...... 5 Table 1-2: Expression of αvβ3 and αvβ5 in various cancers and cancer ...... 8 Table 1-3: Select antibody encoding libraries for phage display ...... 21 Table 2-1: PCR reaction setup for One Taq master mix ...... 28 Table 2-2: PCR reaction setup for Q5 master mix...... 28 Table 2-3: Sanger sequencing primers for the pIT2 vector ...... 29 Table 2-4: Nested PCR primers for Mycoplasma detection...... 31 Table 2-5: Mycoplasma PCR cycling conditions ...... 31 Table 2-6: Silver staining of SDS-PAGE gels using the Pierce™ Silver Stain Kit ...... 35 Table 3-1 E. coli strains used in this project ...... 42 Table 3-2 Sequencing primers for the phagemid vector pIT2 ...... 48 Table 3-3: IGF-1R binding clones above threshold ...... 54 Table 3-4: αv integrin binding clones above threshold ...... 56 Table 4-1: Mutagenic primers for Site-Directed Mutagenesis ...... 73 Table 4-2: Site-Directed Mutagenesis PCR cycling conditions ...... 73 Table 5-1: Concentration series of analyte used in BIAcore affinity experiments ...... 103 Table 8-1: The amino acid sequence of the IGF-1R, BSA and αv integrin targeting ScFv...... 177

Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells ix

List of Abbreviations

% Percent

°C Degrees Celsius

µM Micromolar

2D Two-dimensional

2xYT Two times yeast extract tryptone

2xYT A Two times yeast extract tryptone ampicillin

2xYT AG Two times yeast extract tryptone ampicillin glucose

AIHW Australian Institute of Health and Welfare

ANOVA Analysis of variance

Asn Asparagine

Asp Aspartic acid

BiTE® Bi-specific T-Cell Engager

BSA Bovine Serum Albumin

BsAb Bispecific Antibodies

CD19 Cluster of differentiation 19

CD3 Cluster of differentiation 3

CDS Coding sequence

CO2 Carbon dioxide

CSPS Comparative shotgun protein sequencing

DMEM/F12 Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

E. coli Escherichia coli

x Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells

ECM Extracellular matrix

EGFR Epidermal growth factor receptor

ELISA Enzyme-linked immunosorbent assay

EMA European medicine agency

EpCAM Epithelial cell adhesion molecule

ERK Extracellular signal-regulated kinase

Fab Antigen binding fragment

FAK Focal Adhesion Kinase

FBS Foetal bovine serum

Fc Crystallisable fragment

FDA Food and Drug Administration

g Gravity

GlcNAc N-acetyl-D-glucosamine

Gly Glycine

GM Growth media

h Hour

His Histidine

HRP Horseradish peroxidase

IGF-1R Insulin-like growth factor 1 receptor

IGFBP Insulin-like growth factor-binding protein

IGF-I Insulin-like growth factor 1

IGF-II Insulin-like growth factor 2

IgG Immunoglobulin G

IPTG Isopropyl β-D-1-thiogalactopyranoside

IR Insulin receptor

kDa Kilo Dalton

Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells xi

L Litre

M Molar

MAPK Mitogen-activated protein kinase

MBC Metastatic breast cancer

MCF-7 Michigan Cancer Foundation-7

min Minute

mL Millilitre

MPBS Milk phosphate buffered saline

MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt

MW Molecular weight

NaCl Sodium chloride

ng/mL Nanograms per millilitre

NHMRC National Health and Medical Research Council

Ni-NTA Nickel-nitrilotriacetic acid

PBS Phosphate buffered saline

PBST Phosphate buffered saline Tween 20

PCR Polymerase chain reaction

PEG Polyethylene glycol

PFS Progression-free survival

PI3K Phosphatidylinositol 3-kinase

PSMA Prostate-specific membrane antigen

QUT Queensland University of Technology

RIPA Radio-Immunoprecipitation Assay

RPM Revolutions per minute

xii Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells

ScFv Single-chain variable fragment

SDS-PAGE Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis

SEM Standard error of the mean

Ser Serine

SFM Serum free media

SMP Skim milk powder

TBS Tris buffered saline

TBST Tris buffered saline tween 20

Thr Threonine

TMB 3,3′,5,5′-Tetramethylbenzidine

Tris 2-Amino-2-hydroxymethyl-propane-1,3-diol

Triton X-100 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol

TRR Tissue Repair and Regeneration

Tween 20 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy]ethyl dodecanoate

TYE Tryptone yeast extract

TYE AG Tryptone yeast extract agarose glucose

UNSW University of New South Wales

v/v Volume to volume

VH Variable heavy

VL Variable light

VN Vitronectin

w/v Weight to volume

WGA Wheat germ agglutinin

α Alpha

Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells xiii

β Beta

μg/mL Micrograms per millilitre

μL Microlitre

ρ p-value

xiv Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.

QUT Verified Signature

Signature:

Date: ______05/10/2016______

Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells xv

Acknowledgements

Foremost, I would like to thank my principal supervisor Dr Derek Van Lonkhuyzen for your continuous encouragement and support throughout this long doctorate journey. Your research excellence and mentorship has been critical not only to the success of this project, but also for my scientific development. To my associate supervisor Dr Gary Shooter, your wealth of knowledge in proteins was vital, particular during the purification process. Finally, thank you A/Pro. Terry Walsh as my associate supervisor, for the feedback given during the thesis writing process.

In addition, I would like to acknowledge the IHBI support staff. Especially to Dod for bringing my reagents in a timely manner and also miscellaneous bits and pieces that I might need. Furthermore, I would like to thank the TRR group for the positive experience. I was glad to be part of such a diverse group of talented students and researchers.

Finally, to my loving wife Jay, thank you for your patience and understanding for the many hours I spent away in the lab. Going home to your smiling face makes it all worthwhile

xvi Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells

Literature Review

Chapter 1: Literature Review 1

1.1 BREAST CANCER

Breast cancer is a major burden globally, with the probability of 1 in 8 females developing the disease. Furthermore, breast cancer is the second most common cause of cancer related death in Australian women (Australian Institute of Health and Welfare, 2014). In 2012, close to a third of all new cancer cases in women were attributed to breast cancer (Siegel, Ma, Zou, & Jemal, 2014). Being a heterogeneous disease, breast cancer presents with variable clinical diagnosis and progression, and the selection of treatments proves very difficult (Bertucci, 2008). The Tumour, Node and Metastases (TNM) classification system is a 5 point scale which defines the progression of breast cancer, from the pre-invasive stage 0 to metastasised stage 4 (National Cancer Institute, 2011). As the name suggests, the algorithm used to calculate the TNM score is based on the three parameters of tumour size, lymph node status and metastatic state (National Breast and Ovarian Cancer Centre, 2010). The tumour component, scored from 1-4, factors in the size and depth of the tumour. The node component incorporates the presence of cancerous cells found in sentinel and surrounding lymph nodes and is scored from 1-3 in severity. The metastases score is based on the detection of secondary tumours which have spread from the primary site. Upon dissemination from the primary tumour to secondary organs, predominantly bone and lung, the term metastatic breast cancer (MBC) is used (Minn et al., 2005; Solomayer, Diel, Meyberg, Gollan, & Bastert, 2000). Despite advances in treatments, translating into an average five-year survival rate of 99% and 84% for localised and regional breast cancer respectively, MBC remains incurable, with an average five year survival rate of 24% (Siegel, et al., 2014). Given the poor prognosis of MBC compared to localised breast cancer, therapies that disrupt the metastatic process would likely improve patient prognosis and survival.

Traditional breast cancer treatments can be divided into four main categories: surgery, chemotherapy, radiation, and hormone therapies, combinations of which may be used depending on disease progression (National Cancer Institute, 2011). The limitations of current treatments include their invasive nature, undesirable side effects, prohibitive costs, and potential drug resistance. Surgery is a common treatment option in Australia with close to 32,000 breast cancer related surgical procedures performed in hospitals during 2006-2007 (Cancer Australia and Cancer Council Australia, 2010).

2 Chapter 1: Literature Review

Two main surgical procedures are performed, depending on the stage of breast cancer. Breast-conserving surgery (lumpectomy) removes the tumour and surrounding margin, whereas the more invasive mastectomy aims to remove the whole breast and associated lymph nodes. For early stage breast cancer (where the tumour can be removed surgically in its entirety) no survival difference was found between breast conserving surgery followed by radiotherapy compared to full mastectomy (Early Breast Cancer Triallists’ Collaborative Group, 1995). The inherent invasiveness of surgery can cause physical and psychological stress, with 82% of patients reporting swelling, weakness and pain in their arms (Maunsell, Brisson, & Deschenes, 1993). In addition to surgical interventions, chemotherapy is often prescribed as an adjuvant to the primary surgical treatments. The two main classes of chemotherapy drugs most commonly administered are the taxanes and anthracyclines. The taxanes target the microtubule network, leading to the disruption of mitosis. Another class of chemotherapy drugs, anthracyclines disrupts DNA repair and replication (Bilardi, Kimura, Phillips, & Cutts, 2012). The use of cytotoxic drugs can have severe side effects, including hair loss, nausea, fatigue, and emotional stress (Love, Leventhal, Easterling, & Nerenz, 1989). In particular, the use of anthracyclines has been linked with an increased frequency of heart failures (Volkova & Russell, 2011). Due to the current limitations associated with breast cancer treatments, there is a driven effort by researchers to develop novel targeted approaches to breast cancer therapy.

1.2 TARGETED CANCER THERAPIES

Recently, targeted cancer therapies have been developed that preferentially target tumour cells based on specific molecules on the surface of, or within the cell. A wide range of molecules expressed in tumour cells can be targeted, including growth factor receptors, adhesion molecules, and signalling pathways associated with cell proliferation and differentiation (Munagala, Aqil, & Gupta, 2011). One of the best- known drugs to combat breast cancer is (Herceptin), a targeting Human Epidermal Growth Factor Receptor 2 (HER2). It was approved in 1998 and has been shown to be effective in treating HER2+ breast cancers (Vogel et al., 2002). The use of monoclonal antibodies dominates the biopharmaceutical industry, indeed, six of the top ten selling products in 2013 were antibody based, with Humira (adalimumab) generating global sales of $USD 11 billion

Chapter 1: Literature Review 3

(Walsh, 2014). The monoclonal antibody targeting HER2 in the treatment of breast cancer, Herceptin (trastuzumab), was ranked 7th with total sales of $USD 6.91 billion. The current monoclonal antibodies approved by the Food and Drug Administration (FDA) for the treatment of cancer are listed in Table 1-1. Given that one of the indicators of cancer is growth dysregulation, it is not surprising that a large proportion of the approved antibodies bind to growth factor receptors and their cognate ligands such as epidermal growth factor receptor (EGFR), Vascular endothelial growth factor receptor 2 (VEGFR2) and human epidermal growth factor receptor (HER2) in order to inhibit the growth of cancer cells. Another growth factor system which is an omission from the approved therapeutic table is the Insulin-like growth factor (IGF), which has been linked to various malignancies, including breast cancer (Christopoulos, Msaouel, & Koutsilieris, 2015).

4 Chapter 1: Literature Review

Table 1-1: Monoclonal Cancer therapeutic antibodies approved in the United States

Trade name International Target; Format Indication first Approval nonproprietary approved or reviewed year name Tecentriq PD-L1; Human Bladder cancer 2016 IgG1 Portrazza EGFR; Human Non-small cell lung 2015 IgG1 cancer Empliciti SLAMF7; Multiple myeloma 2015 Humanized IgG1 Darzalex CD38; Human Multiple myeloma 2015 IgG1 Unituxin GD2; Chimeric Neuroblastoma 2015 IgG1 Opdivo PD1; Human IgG4 , non-small cell 2014 lung cancer Blincyto CD19, CD3; Acute lymphoblastic 2014 Murine bispecific leukemia tandem scFv Keytruda PD1; Humanized Melanoma 2014 IgG4 Cyramza VEGFR2; Human Gastric cancer 2014 IgG1 Gazyva CD20; Humanized Chronic lymphocytic 2013 IgG1; leukemia Kadcyla Adotrastuzumab HER2; humanized Breast cancer 2013 emtansine IgG1; Perjeta HER2; humanized Breast Cancer 2012 IgG1 Adcetris Brentuximab CD30; Chimeric Hodgkin lymphoma, 2011 vedotin IgG1; systemic anaplastic large cell lymphoma Yervoy CTLA-4; Human Metastatic melanoma 2011 IgG1 Arzerra CD20; Human Chronic lymphocytic 2009 IgG1 leukemia Vectibix EGFR; Human Colorectal cancer 2006 IgG2 Avastin VEGF; Humanized Colorectal cancer 2004 IgG1 Erbitux EGFR; Chimeric Colorectal cancer 2004 IgG1 Bexxar - CD20; Murine Non-Hodgkin lymphoma 2003 I131 IgG2a Zevalin Ibritumomab CD20; Murine Non-Hodgkin lymphoma 2002 tiuxetan IgG1 MabCampath, CD52; Humanized Chronic myeloid 2001 Campath-1H; IgG1 leukemia; multiple Lemtrada sclerosis Mylotarg Gemtuzumab CD33; Humanized Acute myeloid leukemia 2000 ozogamicin IgG4

Chapter 1: Literature Review 5

Herceptin Trastuzumab HER2; Humanized Breast cancer 1998 IgG1 MabThera, CD20; Chimeric Non-Hodgkin lymphoma 1997 Rituxan IgG1 Source: Modified from Janice M. Reichert, Reichert Biotechnology Consulting LLC (May 26, 2015) with information from FDA, New Molecular Entity and New Therapeutic Biological Product Approvals for 2015, 2016. Last accessed 3/10/16

1.3 INSULIN-LIKE GROWTH FACTOR SYSTEM

The insulin-like growth factor (IGF) system consists of ligands (IGF-I and IGF- II), receptors (IGF-1R, IGF-2R) and IGF binding proteins (IGFBP 1-6) (Denley, Cosgrove, Booker, Wallace, & Forbes, 2005). Upon the binding of IGF-I, the tyrosine kinase receptor (IGF-1R) autophosphorylates at tyrosine residues 1131, 1135 and 1136, resulting in the activation of two main signalling pathways (Riedemann & Macaulay, 2006). The first pathway is mediated by the recruitment and phosphorylation of insulin receptor substrate 1 (IRS-1), followed by activation of phosphoinositide 3-kinase (PI3K) and eventual AKT stimulation (Denduluri et al., 2015). The activation of PI3K/AKT pathway confers a potent anti-apoptotic effect (Kennedy et al., 1997). The second signalling pathway resulting from IGF-1R stimulation is the Ras/Mitogen-activated protein kinases (MAPK) pathway, which through the actions of mediators such as Raf induces an increase in mitogenicity, motogenicity and anti-apoptotic effects in cells (Dhillon, Hagan, Rath, & Kolch, 2007).

Research has shown that the IGF system plays an intimate role in somatic and cancer cell survival, proliferation and migration/metastasis (Samani, Yakar, LeRoith, & Brodt, 2007). Studies have demonstrated that IGF-1R is over expressed in many different cancer tissues, including breast, gastrointestinal, stromal, multiple myeloma and cervical cancers (Chng, Gualberto, & Fonseca, 2005; Papa et al., 1993; Resnik, Reichart, Huey, Webster, & Seely, 1998; Steller, Delgado, Bartels, Woodworth, & Zou, 1996; Tarn et al., 2008). The alteration in the expression of IGF-1R can be explained by investigating genomic and molecular changes of IGF related genes in breast cancer samples. Analysis of The Cancer Genome Atlas (TCGA) dataset

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(Ciriello et al., 2015) using cBioPortal (http://www.cbioportal.org/) was conducted on the IGF system’s related genes (IGF1R, IGF2R, IGF-I, IGF-II, IGFBP 1-6). The resulting genomic and expression data shown in Figure 1-1 revealed that 38% of TCGA breast cancer samples (n= 367/971) contained molecular alteration in at least one member of the IGF system. Of particular interest is IGF-1R, which was amplified, deleted, overexpressed and/or mutated in 9 % of tumours. The genomic alteration of IGF-1R related genes in breast cancer commonly result in IGF-1R overexpression. A study conducted on 930 primary breast cancer samples using tumour microarrays demonstrated that IGF-1R was overexpressed in 87% of all cases tested (Nielsen et al., 2004). In a follow up study, it was demonstrated that phosphorylated IGF-1R/insulin receptor was detected in all breast cancer subtypes, including the difficult to treat triple negative breast cancers (Law et al., 2008).

Figure 1-1: Genomic and transcriptomic alteration of IGF system genes in breast cancer samples Data from the 2015 TCGA dataset analysed using cBioPortal (http://www.cbioportal.org/) for disruption to IGF system related genes. Included for analysis are the genes encoding for Insulin-like growth factor ligands I and II (IGF-I, IGF-II), their cognate receptors (IGF1R, IGF2R) and Insulin-like growth factor binding proteins (IGFBP 1-6). Each row represents a different gene and the percentage of alteration among the tested samples. Each grey box represents an individual tumour sample. Genetic alteration includes amplification and deletion of the indicated gene (filled in red and blue boxes respectively), mRNA upregulation and downregulation (red and blue outline respectively) and somatic base pair mutation (green dot). Tumours without any alteration in the IGF related genes were removed for visualisation.

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1.4 INTEGRINS

Integrins are cell surface receptors which interact with the ECM and influence many cellular functions, including growth, migration and adhesion (Jin & Varner, 2004). In humans, the integrin family consists of eighteen α subunits (α1-11, αIIb, αD, αE, αL, αM, αx, and αv) and eight β subunits (β1-β8) forming heterodimers consisting of one α and one β subunit. The αv subfamily, consisting of 5β binding partners (β1, β3, β5, β6, β8), is termed αv because of its ability to bind to the ECM protein vitronectin (VN) (Hynes, 2002). VN binding integrins, in particular αvβ3 and αvβ5, have been the major focus of investigations to determine the roles of αv-integrins in cancer biology. Table 1-2 outlines the expression of αvβ3 and αvβ5 in various types of cancer. In a similar manner to the IGF analysis, αv integrin related genes, encoding for the alpha chain (ITGAV) and beta chains (ITGB1, ITGB3, ITGB5, ITGB6, ITGB8) were submitted for analysis on the TCGA dataset using the cBioPortal. The resulting genomic and expression data shown in Figure 1-2 demonstrate that 28% of TCGA breast cancer (n= 271/971) contained molecular alteration in at least one member of the αv integrin family.

Table 1-2: Expression of αvβ3 and αvβ5 in various cancers and cancer

Adapted from (Desgrosellier & Cheresh, 2010; Meyer, Marshall, & Hart, 1998)

Tumour Type Integrins expressed

Melanoma αvβ3

Breast αvβ3, αvβ5

Prostate αvβ3

Pancreatic αvβ3

Ovarian αvβ3

Cervical αvβ3,

Glioblastoma αvβ3, αvβ5

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Figure 1-2: Genomic and transcriptomic alteration of αv integrin genes in breast cancer samples

Data are from the 2015 TCGA dataset and analysed using cBioPortal (http://www.cbioportal.org/) analysed for disruption to αv integrin related genes. Included for analysis are the genes encoding for the alpha chain (ITGAV) and five beta chains (ITGB1, ITGB3, ITGB5, ITGB6, ITGB8). Each row represents a different gene and the percentage of alteration among the tested samples. Each grey box represents an individual tumour sample. Genetic alteration includes amplification and deletion of the indicated gene (filled in red and blue boxes), mRNA upregulation (red outline), somatic base pair missense and truncating mutation (green and grey dot). Tumours without any alteration in the αv integrin genes were removed for visualisation.

The expression of αvβ3 has been associated with an increased metastatic potential in breast, osteosarcoma and prostate cancers (Cooper, Chay, & Pienta, 2002; Duan et al., 2005; Zhao et al., 2007). In addition, both αvβ3 and αvβ5 interaction with the ECM has been found to be critical in tumour induced angiogenesis (Brooks, Clark, & Cheresh, 1994; Hodivala-Dilke, Reynolds, & Reynolds, 2003). Furthermore, αvβ3 integrin has been detected on the leading edge of migrating tumour cells, supporting its role in cancer metastasis (Gladson & Cheresh, 1991). Moreover, it has been demonstrated in that study on primary breast tumours that αvβ5 expression was detected in nearly all the samples studied (97.6%) (Vogetseder et al., 2013). Given this association, the integrin system may provide a potential target for cancer therapies intended to disrupt not only tumour growth, but more specifically the detachment, migration and re-attachment metastasis process.

1.5 IGF-1R AND αV INTEGRIN SIGNALLING PATHWAY INTERACTION

While the IGF and αv integrin systems both show independent influences on cancer cell biology, studies have also demonstrated there are collaborative mechanisms between the two systems. IGF-I was demonstrated to bind directly to

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αvβ3 integrin and have a direct influence on IGF-I signalling (Saegusa et al., 2009). The mechanism involved is still under investigation, however the current “ligand occupancy” modelling suggests that the binding of integrins such as αvβ3 to ECM proteins (vitronectin) serves to enhance IGF-1R signalling (Clemmons, 2007). In addition, research from our group has shown that there is cross talk between VN- binding integrins and IGF-I activated signalling pathways (Hollier, Kricker, Van Lonkhuyzen, Leavesley, & Upton, 2008; Kashyap et al., 2011; Van Lonkhuyzen, Hollier, Shooter, Leavesley, & Upton, 2007). We have shown that the increase in cancer cell responses, including cell migration and survival, stimulated by VN+IGF-I complexes is associated with synergistic increases in intracellular signalling mediated via the co-activation of the IGF-1R and VN-binding αv integrins (Hollier, et al., 2008; Kashyap, et al., 2011; Van Lonkhuyzen, et al., 2007). In addition, monoclonal antibody treatments against αv integrins, IGF-1R and a combination of both have demonstrated that the stimulatory effect of the VN+IGF-I complex can be abrogated by inhibition of these receptors (Hollier, et al., 2008; Van Lonkhuyzen, et al., 2007). Furthermore, in cell migration studies, dual antibody inhibition of both αv integrins and IGF-1R produced a significantly greater decrease in migration of cancer cells as compared to individual antibody treatment (Hollier, et al., 2008). This is consistent with other studies which concluded that activation of both the IGF-1R and αv integrins is required for maximal cellular response (Doerr & Jones, 1996). The breast cancer cell line MCF- 7 used in previous studies within our group expresses both IGF-1R and αv integrin, making it an ideal cell model for the investigation of IGF and αv integrin interactions.

1.6 IGF-1R AND VN BINDING INTEGRIN TARGETING STRATEGIES

As discussed in previous sections, both the IGF and VN-binding integrin systems play critical roles in breast cancer cell survival and metastasis. The cumulative data identify these systems as potential targets for cancer therapies. A review of recent literature has revealed several strategies that may be used to target the IGF or αv integrin systems ligand binding along with downstream signal transduction pathways (Figure 1-3).

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Figure 1-3: Various IGF-1R and αv targeting strategies.

Disruption of the IGF and integrin systems can be achieved by different strategies. The neutralisation of the ligands using antibodies or modified receptors can reduce circulating ligands. The receptor itself can be targeted with antibodies, preventing the ligand from binding. Finally, the signalling pathway could be inhibited using small molecule inhibitors. Because of the association of IGF-I and VN to form a trimeric complex, inhibiting peptides could also target complex formation. Adapted from: (Askari, Buckley, Mould, & Humphries, 2009; Belfiore & Malaguarnera, 2011)

There has been sustained research on the role of IGF-1R in cancer growth and metastasis (Surmacz, 2000). Indeed, over 30 novel IGF-1R targeting therapies are currently under investigation, the majority of which are IGF-1R targeting antibodies (n=29, Appendix 1) with a small subset consisting of IGF-1R tyrosine kinase inhibitors (TKIs) (Crudden, Girnita, & Girnita, 2015). A search of current clinical trials revealed 30 trials involving IGF-1R alone, or in combination therapies in the United States (U.S National Institude of Health, 2016). Despite the intense research focus on generating therapeutics against IGF-1R, clinical trial results have been less promising. An example of this is (IMC-A12), a monoclonal antibody against IGF- 1R. Preclinical data with MCF-7 breast cancer cells showed that it was able to inhibit IGF-I dependent signalling and demonstrated significant inhibition of tumour growth in breast, renal and pancreatic tumours using xenograft models (Burtrum et al., 2003). Phase I clinical trial data from cixutumumab as a treatment showed the antibody was well tolerated, with no major adverse events (Ma et al., 2013; Wilky et al., 2015). When phase II trials were conducted, it was concluded that cixutumumab exhibited limited clinical efficacy in treating breast (Gradishar, Yardley, et al., 2015) and

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prostate cancers (Hussain et al., 2015). Several potential factors may explain the limited success of IGF-1R inhibitors in a clinical setting. In the case of the stage 2 clinical trial for targeting IGF-1R, patient’s tumour IGF-1R expression status was not assessed during enrolment (Robertson et al., 2013). This could limit Ganitumab efficacy in patients having tumours with low IGF-1R expression. An alternative explanation is that by inhibiting IGF-1R signalling, other pathways such as Epidermal growth factor receptor (EGFR) are activated to compensate, with studies demonstrating their linked roles in drug resistance (Jin & Esteva, 2008; Lee, Wang, James, Jeong, & You, 2015; Wang et al., 2013). Given the involvement of IGF-1R in different pathways, combination therapies targeting multiple systems are being investigated.

Another approach to inhibiting the IGF system is by the use of tyrosine kinase inhibitors (TKI) which block the signal transduction cascade upon ligand binding. Examples of candidates in development are Linsitinib (OSI-906) (Mulvihill et al., 2009), BMS-754807 (Carboni et al., 2009), BVP 51004 (Girnita et al., 2004) and XL228 (Smith et al., 2009). Linsitinib, the leading candidate, is an oral TKI with IGF- 1R and IR targeting functionality. In a similar result to antibody based candidates against IGF-1R, Linsitinib showed promise in preclinical studies, however, results from a phase III study revealed that Linsitinib did not improve overall survival (Fassnacht et al., 2015). The authors suggested that there needs to be improved genetic classification of patients for which IGF-1R or IR targeting therapeutics can derive benefit. In addition, potential as a combination treatment should be explored.

Furthermore, an alternative approach to target the IGF signalling pathway is for the IGF ligands (IGF-I, IGF-II) to be sequestered using antibodies or modified receptors in order to reduce their bioavailability (D'Ambrosio, Ferber, Resnicoff, & Baserga, 1996; Gao et al., 2011a; Wang et al., 2015). The ‘ligand trap’ approach has managed to reduce circulating TNF-α using a TNF-α receptor-FC, and is currently available for use as Enbrel® (etanercept) for the treatment of rheumatoid arthritis. A promising candidate of an IGF ligand trap is MEDI-573, an antibody which can bind to both IGF-I and IGF-II. It has demonstrated anti-tumour activities in preclinical

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trials, and was well tolerated in a phase I trial in patients with advanced solid tumours (Haluska et al., 2014).

Therapies targeting VN-binding integrins have also been investigated, due to its importance in metastasis as outlined above. These include: αv integrin targeting peptides (Witt et al., 2009), antibodies (Trikha et al., 2004), and RGD inhibitors (Russo et al., 2013). Currently, 14 antibody based therapeutic candidates targeting αv integrin are in various stages of development (Appendix 2). A promising candidate is (formerly CNTO 95), a fully humanised monoclonal antibody which binds to αv integrins (Trikha, et al., 2004). It has been evaluated in combination with docetaxel in phase I clinical trials in patients with metastatic prostate cancer (Chu et al., 2011). The results demonstrated Intetumumab’s safety profile and that at 10 mg/Kg has the potential to be therapeutic. The phase II clinical trial involved comparing the progression-free survival (PFS) of a combination of Intetumumab, docetaxel and prednisone in metastatic prostate cancer (Heidenreich et al., 2013). Unexpectedly, the results of the clinical trial showed that Intetumumab was unable to improve PFS, possibly due to the limited sample size.

As discussed previously, synergistic cross-talk can be observed between the IGF-1R and αv integrin systems. Although therapeutic candidates targeting these pathways individually have not been successful, targeting both IGF and integrin systems simultaneously may increase potency by limiting the potential of compensation. This dual pathway targeting approach has been investigated by the addition of α-IGF-1R or αv integrin inhibitory antibodies either alone or as a dual combination treatment on the breast cancer cell line MCF-7 (Hollier, et al., 2008). When stimulated with IGF-I and VN in migration studies, reductions in migration of 22.2% (αv) and 61.6% (IGF-1R) were observed. Importantly, the dual treatment showed increased potency by inhibiting migration by 85.7% (αv±IGF-1R) compared to the uninhibited control. Another approach of targeting both IGF and integrin pathways is by using an agonistic peptide against the formation of the VN-IGFBP- IGF-I complex, therefore inhibiting the co-activation of the pathways (Kashyap et al., 2016). Clearly, a therapy with the ability to inhibit activation of both the IGF-1R and αv integrin may have the potential to arrest tumour growth and metastasis. A possible

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approach for targeting two separate molecules is the generation of bispecific antibodies (BsAb), which will be the platform investigated within this study.

1.7 ANTIBODY STRUCTURE AND FRAGMENTATION

Antibodies are glycoproteins which elicit various functions as part of the adaptive and innate immune system. There are five classes of antibody: IgA, IgD, IgE, IgG and IgM. Each class differs in structure, molecular weight, avidity, and localisation. The most abundant antibody subclass in serum, IgG, makes up ~85% of total serum antibodies (Manz, Hauser, Hiepe, & Radbruch, 2005). In addition to being abundant, IgG is also the most stable, with a serum half-life of 20 days, compared to 5-8 days for IgM and IgA (Brekke & Sandlie, 2003), 2.8 days for IgD (Rogentine Jr, Rowe, Bradley, Waldmann, & Fahey, 1966) and 2 days for IgE (Stone, Prussin, & Metcalfe, 2010). Given its stability and relative abundance, IgG is the principal antibody class used for therapeutic and diagnostic purposes.

IgG is made up of two heavy and two light polypeptide chains joined by disulphide bonds in the classical ‘Y’ arrangement. An IgG molecule can be further divided into the crystallisable fragment (Fc) and the antigen biding fragment (Fab) (Figure 1-4). The Fab contains the variable light (VL) and variable heavy (VH) domains, which determines antigen binding specificity. Because of the modular nature of IgG, it can be fragmented using reducing agents, enzymatic cleavage (using papain/pepsin) or recombinant production, with the resulting products retaining the VH and VL domains giving equal specificity as their full length parental antibody (Vlasak & Ionescu, 2011).

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Figure 1-4: Examples of intact IgG and its fragments.

Full length IgG molecules can be fragmented into Fab by enzymatic means (papain) or can be recombinantly generated by linking the VH and VL domains with a polypeptide linker to form a ScFv. The crystal structure representation of IgG (1IGT), Fab (4M43) and ScFv (1P4I) were obtained from the protein data bank (http://www.rcsb.org) and visualised using VMD 1.9.2.

1.8 BISPECIFIC ANTIBODIES

A novel means to increase the functionality of conventional monospecific antibodies is to develop bispecific antibodies, also known as dual targeting antibodies. Bispecific antibodies are designed to have dual modes of action which can include: 1) Neutralising two ligands, 2) Inhibiting two receptors, 3) Cross-linking of two receptors, or 4) the recruitment of cytotoxic immune cells (Kontermann, 2011). They were initially generated by chemically cross-linking two different targeting antibody fragments. In this approach, Fab’ fragments were generated from the enzymatic cleavage of the two parental IgG, and subsequently reduced to produce reactive SH groups. The SH groups of one Fab were alkylated yielding free maleimide groups. The two halves were combined which allowed the maleimide and SH groups to react, therefore facilitating the formation of a thioester-linked bispecific Fab (Glennie, McBride, Worth, & Stevenson, 1987). An alternative method of producing BsAb was to fuse two different antibody producing hybridoma to form a hybrid

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hybridoma/quadroma, which would secrete various combinations of mono and bispecific species (Milstein & Cuello, 1983). Both methods have disadvantages including being labour intensive, low yield, high cost and producing heterogeneous BsAb species (Staerz & Bevan, 1986). Given the limitations, both techniques were mainly used as a research tool. With the refinement of molecular cloning and recombinant techniques, the interest in BsAb was reignited. Sequences encoding for Ab’s could be isolated and specifically manipulated to form novel BsAb structures relatively easily, at high yield, low cost and with inherent high specificity.

There has been a growing interest in bispecific antibodies (BsAb) since this technology was first reported as a pharmacological strategy in the 1980s with the market totalling >$3.5 billion for the period 2009-2011 (Baeuerle, 2011; Frincke et al., 1986). As of 2015, the number of bispecific antibodies in clinical trials stands at 14, with the majority of them in phase 1 and 2 (Sheridan, 2015). The first bispecific antibody to obtain market approval by the European Medicines Agency (EMA) was Removab () in 2009. It binds to epithelial cell adhesion molecule (EpCAM)-expressing carcinoma cells, CD3 on T cells, and also to the accessory cells (macrophages, dendritic cells and natural killer cells) via the Fc receptor (Walsh, 2010). As a result, the T-cells activate apoptotic and lytic pathways while the accessory cells act via the antibody-dependent cell-mediated cytotoxicity (ADCC) phagocytosis pathway to clear cancer cells (Seimetz, 2011).

Recombinant BsAbs can be engineered into multiple formats, with more than 45 different frameworks in development that can be classified into groups based on their key features (Kontermann, 2012). ScFv based BsAbs consist only of the variable fragment (Fv) of an antibody, the smallest unit of antigen recognition, for each target. As such, they are compact and capable of being expressed in both bacterial and eukaryotic expression systems (Pleass & Holder, 2005). The simplest form of ScFv based BsAb is the tandem single chain format, which is arranged as VH1-VL1-VH2-VL2 and is linked by a polypeptide linker such as (Gly4Ser)n (Figure 1-5). This format was chosen for this project. However, while small antibodies such as the ScFv based format are compact (~60 kDa) and allow for deep tissue penetration, they have a much shorter serum half-life than the larger IgG based antibodies (150 kDa) (Kipriyanov et al.,

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1999). Due to its simplicity, the ScFv format has been used widely, including the creation of the promising Bi-specific T-Cell Engager (BiTE) format® as a potential cancer therapeutic (Nagorsen et al., 2009). A BiTE® antibody consists of two different targeting ScFv linked by a polypeptide linker. One ScFv would target the tumour specific antigen, and the other binds to a T cell specific receptor (T cell:ScFv- ScFv:tumour cell). The mechanism of BiTE® acts firstly to recruit cytotoxic T cells to tumour cells, and upon close physical contact, activates the T cells for the lysis of the targeted cell. The first BiTE® antibody Blincyto® (Blinatumomab) was generated by isolating the VH and VL domains of α-CD3 and α-CD19 IgG and engineered as a single chain BsAb linked by a Gly4Ser linker sequence. Given the success of the phase II clinical trial, in which 43% (n= 81/189) of the treated patients with acute lymphoblastic leukaemia achieved complete remission (CR) or partial haematological recovery of peripheral blood counts (CRh), it was placed in the accelerated approval program and was the first FDA approved BsAb on the market in 2014 (Table 1-1) (Topp et al., 2015). Other tumour antigen markers are also being investigated for use in the BiTE® format, including Epithelial cell adhesion molecule (EpCAM) for use in pancreatic cancer and prostate-specific membrane antigen (PSMA) as a potential prostate cancer therapeutic (Cioffi, Dorado, Baeuerle, & Heeschen, 2012; Friedrich et al., 2012). These studies demonstrate the function of the BsAb arrangement in both in vitro and in vivo, thus such a format will be utilised in the current study.

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Figure 1-5: Selected classes of bispecific antibodies and examples of their unique configurations.

Orange and yellow VH and VL domains indicate different antigen specificity. Wildtype IgG molecule is made up of 2 identical heavy chains and 2 identical light chains. The heavy chain consists of one variable (VH) and three constant (CH1-CH3) domains. The light chain contains the VL and CL domains. The VH and VL domains determine antigen specificity and can be engineered to form fusion molecules via linkers. In the case of ScFv based bispecific antibodies, this linker is often based on a GlySer motif. Adapted from (Baeuerle, 2011)

1.9 ANTIBODY GENERATION

Antibodies against a particular target can be generated in a number of ways. Historically, it was by challenging an animal’s immune system with a purified antigen or hapten. Upon detection by the immune system, it would prompt the generation of a humoral response in which the secretion of antibodies is a component. Because different epitopes can be present in an antigen, the resulting antibody is polyclonal. The antibodies created can be isolated and purified from the host’s blood. Therefore, the choice of animal host is dependent on the total amount of antibody required. For small animals such as mouse and rat, their total blood volume is low; 2.5 mL and 30

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mL respectively. If higher yield is required, a larger animal such as a goat (3000 mL) or sheep (4000 mL) can be used (Hanly, Artwohl, & Bennett, 1995).

The development of the mouse hybridoma technology by Kohler and Milstein in 1975 allowed for the continuous production of monoclonal antibodies, and to a large extent making the need to constantly harvest blood from an immunised animal redundant (Kohler & Milstein, 1975). This was achieved by isolating antibody producing B cells from a host such as a mouse, and fusing it with a cancerous myeloma cell. The resulting immortalised hybridoma can be implanted back into the mouse to form an antibody producing tumour or can be cultured for in vitro production. However, this approached for the generation of a human therapeutic was unsuccessful because the mouse antibody generated an adverse immune response in humans, termed human anti-mouse antibody (HAMA) (Tjandra, Ramadi, & McKenzie, 1990). Subsequently, techniques have been developed to ‘humanise’ murine antibodies in order to reduce immunogenicity (Kettleborough, Saldanha, Heath, Morrison, & Bendig, 1991; Roguska et al., 1994).

The use of animals for scientific purposes requires careful ethical considerations for the welfare of the animals involved. The National Health and Medical Research Council (NHMRC) has set guidelines for the use of animals in monoclonal antibody production (National Health and Medical Research Council, 2008). It recommends that the use of mice will continue to be required, however, improved techniques which can minimise animal harm should be employed. Thus, there is an ever increasing stringency with respect to methods involving animals and it is foreseeable that such techniques will be phased out in time. Consequently, antibody production systems not requiring animals are of significant interest. One such method which by-passes the use of animals and also allows for the screening of fully human antibodies is phage display.

1.10 PHAGE DISPLAY

Phage display was first described by Smith in 1985 as a method to display exogenous protein fused to coat proteins of bacteriophage (Smith, 1985). It is a powerful

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technique that has been used in many protein interaction studies, including: antibody epitope mapping (He et al., 2012), monoclonal antibody generation (Chu, et al., 2011), protein-DNA interactions (Choo & Klug, 1994) and molecule specific diagnostic applications (Deutscher, 2010). Phage display is the oldest and the most applied technique in the field of molecular display, with competing platforms such as bacterial and yeast, ribosome and mRNA display (Levin & Weiss, 2005). The key feature of phage display is the direct linkage between phenotype (function) of the displayed protein, and the genotype. This relationship allows for the screening of libraries encoding for millions of different protein variants and selection based on affinity interactions. One important application of this technology is the ability to screen human antibody encoding libraries against therapeutic targets. A selection of the antibody encoding libraries for phage display is shown in (Table 1-3). Phage display libraries can be broadly divided into 3 categories, depending on the source of the antibody encoding sequences and method of generating diversity: 1) Naïve libraries are produced by harvesting B-cells from human donors, isolating the mRNA and subsequent reverse transcription into cDNA. The variable domain genes are amplified using PCR and cloned into a suitable phage display vector such as pIT2 or pHEN. The resulting library’s diversity is a result of naturally reorganised immunoglobin encoding genes from the human donor/s (Pansri, Jaruseranee, Rangnoi, Kristensen, & Yamabhai, 2009a; Sheets et al., 1998). 2) To further increase a library diversity, the amplified variable domain genes from donors be mutated to form a semi-synthetic library (de Wildt, Mundy, Gorick, & Tomlinson, 2000) and 3) Fully synthetic libraries are diversified by rational design using artificially generated sequences (Knappik et al., 2000; Prassler et al., 2011; Rothe et al., 2008). An example of a phage display library which has been well characterised in the literature are the Tomlinson libraries (I+J), which has been used in many diverse applications, such as for generation of ScFv for diagnostics and imaging (de la Cruz et al., 2013; Kim, Wang, Wahlberg, & Edwards, 2014; Xu, Liu, Brown, Christian, & Hornby, 2012), and a screen for potential therapeutic candidates (Patil et al., 2015; ten Haaf et al., 2015). As a consequence of its popular usage, the libraries were readily obtainable to researchers. Taken together, the Tomlinson libraries was chosen for this project. The antibody libraries are encoded by phagemid vectors which are propagated within E. coli hosts. To produce functional phage molecules displaying the antibody fragment from antibody libraries, additional genetic information is required. Infecting the phage library containing E. coli with a

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helper phage such as KM13 provides the necessary components for packaging the phagemid DNA into phage particles (Kristensen & Winter, 1998).

Table 1-3: Select antibody encoding libraries for phage display All libraries encode for the Fab or ScFv format. The size of a library indicates the total number of unique antibody sequences contained within the library.

Name of library Format Size of library Reference Dyax library Fab 3.5 × 1010 (Hoet et al., 2005) HuCAL® ScFv 2.0 × 109 (Knappik, et al., 2000) HuCAL Gold® Fab 1.0 × 1010 (Rothe, et al., 2008) HuCAL® PLATINUM® Fab 4.5 × 1010 (Prassler, et al., 2011) MedImmune ScFv 1.4 × 1010 (Vaughan et al., 1996) Tomlinson I + J ScFv 2.8 × 108 (de Wildt, et al., 2000)

1.10.1 KM13 phage physiology and structure KM13 is an E. coli specific filamentous bacteriophage that is ~1 μm in length and ~6 nm in diameter (Marvin, Hale, Nave, & Citterich, 1994). Unlike other bacteriophage such as lamda or T4, M13 is non-lytic in its life cycle. This is important in a phage display context as it simplifies phage recovery and purification by reducing contaminating bacterial proteins. Phage particles are composed of supercoiled single stranded DNA (ssDNA) surrounded by 2700 copies of the major coat protein (pVIII), and 5 copies each of the minor coat proteins (pIII, pVI, pVII, pIX) (Figure 1-6). All 5 coat proteins have been used in phage display systems, with fusion to pIII being the most common (Sidhu, 2001).

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pVIII pVII

pIII N1 N2 CT pVI ssDNA pIX

Figure 1-6: Schematic of a KM13 phage particle M13 phage particles contain a ssDNA core surround by the major coat protein pVIII and minor coat proteins pIII, pVI, pVII, pIX. The pIII protein is essential for infection and contains three domains, N1, N2, and CT.

1.10.2 Phage display selection Antibody fragments displayed on the surface of phage can be used to screen against immobilised targets. The antibody phage display enrichment procedure can be summarised as follows (Rouet et al., 2012):

1. Phage generation. The first stage involves the generation of phage particles displaying the ScFv encoded by the phagemid vector housed and propagated in E. coli. The phagemid contained both the ScFv fused to pIII in addition to phage origin of replication. In order to produce phage displaying ScFv, additional genetic materials for phage packaging is provided upon infection with a helper phage. There are many different helper phage strains, in this case, the Tomlinson library system utilises the protease sensitive KM13 helper phage (Chasteen, Ayriss, Pavlik, & Bradbury, 2006; Kristensen & Winter, 1998).

2. Immobilisation of target. In order to select displayed phage, the target of interest is immobilised onto a solid substrate. This step can be facilitated by passive adsorption of the target molecule onto polystyrene materials with different chemistries. For example, Thermo Fisher offers a variety of surfaces to provide optimal binding of the target (Esser, 2010). PolySorp® surface treatment has a high affinity to molecules of hydrophobic nature. Whereas MultiSorp™ surface

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treatment binds more efficiently to hydrophilic molecules. MaxiSorp™ surfaces enable immobilisation with high affinity for molecules with mixed hydrophilic/hydrophobic domains. In addition to passive absorption, the target can also be immobilised onto solid support by affinity systems. One example is by firstly biotinylating the target, followed by immobilisation onto streptavidin conjugated magnetics beads and using this as the matrix through which the phage preparation will be selected (Chames & Baty, 2010).

3. Enrichment of binders. The selection and enrichment of binders requires the completion of step 1 and 2, that is, the generation of phage displaying the antibody fragment library, and the immobilisation of the target onto a solid substrate. To enrich for positive binders, the solid substrate containing the target is firstly incubated commonly with skim milk or BSA blocking agent to reduce non-specific binding. The phage is allowed to mix with the target and a wash step is performed to remove any non-binders. The population of binders will be low at the end of the first panning round, but will increase once the positive binders are allowed to re- amplify by firstly infecting E. coli and additional incubation and wash steps.

1.11 CONCLUSION AND KNOWLEDGE GAPS

Breast cancer currently contributes close to a third of all new cancer cases in women (Siegel, et al., 2014). The treatment of metastatic breast cancer, or cancers which have spread to secondary sites, are limited in success, translating into a low five- year survival rate (Siegel, et al., 2014). In addition, traditional cancer treatment such as surgery and chemotherapy can have severe side effects. This prompted researchers to investigate targeted approaches in treating tumour cells. Two systems have been identified as potential therapeutic targets. The IGF system plays an important role in normal cellular growth processes and has links to various cancers, where the growth checkpoints are dysregulated (Samani, et al., 2007). It has been demonstrated that IGF- 1R is overexpressed in breast cancer tissues and inhibiting the IGF system by blocking ligand binding decreased cancer cell growth (Farabaugh, Boone, & Lee, 2015; Gao et al., 2011b). The integrin system mediates various functions, including angiogenesis, migration, attachment and survival of cells (Avraamides, Garmy-Susini, & Varner,

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2008; Kim, Turnbull, & Guimond, 2011). Like the IGF system, integrins have been linked to cancer progression, in particular metastatic dissemination (Desgrosellier & Cheresh, 2010). Our own research has shown synergism between the systems, and their additive effect on cell migration and proliferation upon co-simulation (Hollier, et al., 2008; Van Lonkhuyzen, et al., 2007). Despite demonstrating functional success in preclinical studies using a singular antibody targeting IGF-1R or αv integrin, results from clinical trials were less promising, potentially due to signalling pathway compensation. Given the roles of both the IGF and integrin systems in cancer progression, an agent which can inhibit both pathways has the potential to be a potent cancer therapeutic. To achieve this goal, the current project will attempt to generate independent function blocking ScFv toward IGF-1R and αv integrin, with the ultimate aim of combining them into a BsAb based on the tandem ScFv format with the potential to inhibit key cancer progression mechanisms.

1.12 PROJECT OUTLINE

1.12.1 Hypothesis The hypotheses explored in this PhD studies were:

1) By using phage display technologies, ScFv which can bind to IGF-1R and αv integrin will be selected and recombinantly expressed.

2) The generated ScFv will inhibit the migration and survival of breast cancer cells upon stimulation by IGF-I and VN; and

3) The BsAb constructed from IGF-1R and αv integrin targeting ScFv would retain the functional activities of the parental ScFv.

1.12.2 Aims To address the above mentioned hypothesese, the following aims were pursued:

1) Selection of IGF-1R and αv integrin function blocking single chain variable fragments (ScFv).

24 Chapter 1: Literature Review

2) Functional characterisation of the generated anti-IGF-1R and anti-αv integrin ScFv in cell based assays (Hollier, et al., 2008; Van Lonkhuyzen, et al., 2007).

3) Selection of functional ScFv and recombinant bispecific antigen binding antibody based protein/s towards IGF-1R and αv integrin.

4) Functional characterisation of the generated BsAb in cell based assays as per aim 2.

Chapter 1: Literature Review 25

Materials and Methods

26 Chapter 2: Materials and Methods

2.1 INTRODUCTION

The materials and methods in the following results chapters are outlined within. Any deviations are stated in the respective chapters as required.

2.2 MATERIALS

All mammalian cell culture materials, including Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12), penicillin-streptomycin, gentamicin, TrypLE™ Express, 0.4% trypan Blue and Fetal bovine serum (FBS) were all purchased from Thermo Fisher Scientific/Life Technologies (Scoresby, Australia). The NuPage SDS-PAGE system, consisting of pre-casted 4-12% Bis-Tris Protein Gels, MES SDS running buffer, LDS sample buffer, and reducing agent were sourced from Thermo Fisher Scientific/Life Technologies (Scoresby, Australia). All oligonucleotides used in both PCRs and sequencing reactions were synthesised by Sigma-Aldrich (St Louis, US). OneTaq® Hot Start Quick-Load® 2X Master Mix and Q5® Hot Start High-Fidelity 2X Master Mix were sourced from New England Biolabs (Massachusetts, US). Monoclonal antibody for phospho-IGF-1R β (Tyr1135/1136), IGF-1R, GAPDH (14C10) and Integrin αv were all sourced from Cell Signalling (Danvers, US). All primers were synthesised from Sigma-Aldrich. The naïve human ScFv libraries ‘I’ and ‘J’ (Tomlinson I+J), KM13 helper phage, Escherichia coli (E. coli) TG1 and HB2151 were purchased from Source Bioscience (Cambridge, UK). Immuno MaxiSorp® 5 mL tubes (cat: NUN444202) and MaxiSorp® 96 well plates (NUN442404) for phage panning were sourced from Thermo Fisher. All general reagents are of the highest quality and were purchased from Sigma-Aldrich unless otherwise stated.

2.3 CLONING

2.3.1 General PCR Reaction setup One Taq reaction setup for the routine non proof read applications, such as mycoplasma testing is given in Table 2-1.

Chapter 2: Materials and Methods 27

Table 2-1: PCR reaction setup for One Taq master mix

Reagent 20 μL Reaction (μL) Final concentration One Taq Master mix (2x) 10 1x DNA sample 1 F primer (10 μM) 1 0.2 μM R primer (10 μM) 1 0.2 μM Water 7 Total 20

Setup for Q5 reactions used in high fidelity applications is given in Table 2-2.

Table 2-2: PCR reaction setup for Q5 master mix

25 μl Final Reaction concentration Q5 Hot Start High-Fidelity 2X Master Mix 12.5 μL 1X 10 μM Forward Primer 1.25 μL 0.5 μM 10 μM Reverse Primer 1.25 μL 0.5 μM Template DNA (1-25 ng/ μL) 1 μL 1-25 ng Nuclease-free water 9.0 μL

Annealing temperature calculations Primer annealing temperatures were calculated per primer set and for each polymerase using the NEB Tm calculator at http://tmcalculator.neb.com/

2.3.2 Mini-prep plasmid isolation Plasmids were isolated from E. coli clones which housed the pIT2 based vector using the QIAprep miniprep spin column kit (Qiagen) according to manufacturer’s high yield protocol. All the required buffers are supplied with the kit and all isolation procedures were conducted at room temperature. Briefly, 5 mL of 2YT medium (16 g bacto-tryptone, 10 g of yeast extract and 5 g of NaCl per litre) was inoculated overnight from either a single colony on an agarose plate or from a frozen glycerol stock. The culture was maintained with shaking at 200 RPM at 37 °C overnight. The cells were then pelleted by centrifugation at 3,200 g for 5 mins and the supernatant discarded.

28 Chapter 2: Materials and Methods

The pelleted cells were resuspended in 250 μL of P1 buffer and transferred to a 1.5 mL microcentrifuge tube. Cells were lysed by the addition of 250 μL of P2 into the microcentrifuge tube, after which the reaction was neutralised by the addition of 250 μL of N3. The reaction was centrifuged at 18,000 g to remove chromosomal DNA contaminants. The supernatant was transferred into a spin column and centrifuged for 1 min, at which point the flowthrough was discarded. The spin column was further washed with 500 μL of buffer PB, and 750 μL of buffer PE. The plasmid DNA were eluted from the spin column using ultrapure water and stored at 4 °C. The yield and quality of the isolated plasmid was assessed using spectrophotometry (Nanodrop ND- 1000).

2.3.3 Sanger sequencing of plasmids Plasmids were submitted to the Australian Genome Research Facility (AGRF) for Sanger sequencing using the purified DNA (PD) service. Samples were submitted based on recommendations from AGRF. The sequencing primers used to sequence clones based on the pIT2 vector are listed in Table 2-3.

Table 2-3: Sanger sequencing primers for the pIT2 vector

Primer name Sequence (5’ to 3’) Binding site Forward LMB3 CAG GAA ACA GCT ATG AC Upstream of RBS Reverse pHEN CTA TGC GGC CCC ATT CA Downstream of myc-tag

In short, each sequencing reaction submitted consists of 600-1500 ng of plasmid, 6.9 pmol of sequencing primer, and ultra-pure water to a final volume of 12 μL. The resulting sequencing chromatograms were organised, analysed and annotated using CLC Main Workbench version 7.6. From the DNA sequence, the protein sequence was translated using the standard translation tables as provided in the software.

Chapter 2: Materials and Methods 29

2.4 MAMMALIAN CELL CULTURE

2.4.1 Propagation of MCF-7 cell lines The human breast adenocarcinoma cell line MCF-7 (HTB-22) was cultured in growth medium (GM) consisting of Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 0.1 mg/mL streptomycin, 50 units/mL penicillin and 1 μg/mL gentamicin. All cell lines were maintained in a humidified incubator at 37 °C with 95% air and 5% CO2. Media was changed every 3-4 days or upon 80% confluence, at which point cells were passaged at a ratio of 1:3. Cells were detached from the tissue culture flasks using TrypLE™ Express, a trypsin replacement dissociation agent. All functional assays were performed using serum free media (SFM), with the same supplements as the GM minus the FBS. The identities of the breast cancer cell line have been validated using short tandem repeat profiling (CellBank Australia, Sydney, NSW, Australia).

2.4.2 Cell counting Cells were manually counted using a hemocytometer and the trypan blue exclusion method. Aliquots of the harvested cell suspension were added to 0.4% trypan blue solution 5 fold. The stained cells were loaded into the hemocytometer chamber and counted using a phase contrast microscope at a total magnification of 400x. The cell concentration was determined from the average of one square using the formula:

= × 10

2.4.3 Cryopreservation of cell lines Cells to be frozen were detached from cell culture flasks using TrypLE™ Express. The excess TrypLE™ was inactivated by the addition of GM and a cell count performed as per 2.4.2. The cell suspension was pelleted by centrifugation at 1000 RPM on a Hettich Universal 320 and resuspended to 2x106 viable cells/mL/Cryovial using ice-cold freezing medium consisting of Growth Media with 5% (v/v) DMSO. The cryovials were cooled at a rate of -1 °C per minute to -80 °C in a freezer container

30 Chapter 2: Materials and Methods

filled with isopropanol and subsequently transferred to liquid nitrogen for long term storage.

2.4.4 Mycoplasma Testing Cell lines were tested for mycoplasma infection using nested polymerase chain reaction (Nested PCR) before freezing down of cells for stock. The PCR reaction was carried out using two sets of primers as listed in Table 2-4:

Table 2-4: Nested PCR primers for Mycoplasma detection. Primers are listed from 5’ to 3’ direction.

Forward 5’ to 3’ Reverse 5’ to 3’ Mycoplasma PCR 1 ACACCATGGGAGCTGG CTTCATCGACTTTCAGA TAAT CCCA Mycoplasma PCR 2 GTTCTTTGAAAACTGA GCATCCACGAAAAACT AT CT

Two independent reactions with the amplicon from the first reaction used as the template for the second. The cycling conditions are listed below in Table 2-5.

Table 2-5: Mycoplasma PCR cycling conditions

STEP TEMP TIME Initial Denaturation 94 °C 300 seconds 35 Cycles 94 °C 30 sec 55 °C 60 sec

72 °C 60 sec Hold 4-10 °C ∞

The resulting products were separated on a 1.5 % agarose gel run at 120 V. The banding patterns of the samples were compared to the Mycoplasma positive samples to assess infection status.

Chapter 2: Materials and Methods 31

2.5 PROTEIN ISOLATION FROM ADHERENT CELL CULTURES

2.5.1 Denaturing conditions Adherent MCF-7 cells were grown until 80% confluent in growth medium (GM) consisting of Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 0.1 mg/mL streptomycin, 50 units/mL penicillin and 1 μg/mL gentamicin. The cultures were maintained in a humidified incubator with

5% CO2 and 95% atmospheric air at 37 °C. Prior to assays, the cells were serum starved for 4 hours by firstly removing the old media, washing once with PBS, then adding serum free DMEM/F12 (SFM) with 0.1 mg/mL streptomycin, 50 units/mL penicillin and 1 μg/mL gentamicin. The media was aspirated and washed twice with ice-cold PBS. Cell lysis was achieved by adding 0.4 mL of complete lysis buffer containing Radioimmunoprecipitation assay (RIPA) buffer, 20 mM Tris-HCl (pH 7.5), 150 mM

NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, (Cell signalling) with the addition of Complete protease inhibitor (Roche) and phosphatase inhibitor (Thermo-Fisher Scientific). After 5 mins incubation with the lysis buffer, the cell lysates were harvested using a cell scraper and transferred into a pre-chilled centrifuge tube. The samples were then placed in a sonication water bath (Soniclean 120 TD) for 4 mins and were subsequently centrifuged for 15 mins at 14,000 g to remove insoluble proteins. The supernatant was reserved and frozen at -80 °C. The protein concentration was estimated using the BCA assay using the Pierce™ BCA protein assay kit according to the manufacturer’s instructions.

2.5.2 Non-denaturing conditions To maintain the native conformation of cellular proteins, a non-denaturing lysis buffer was also used. After the 4 hour serum starve incubation, SFM was aspirated and 2 mL of non-denaturing lysis buffer, consisting of 50 mM Tris (pH 7.4), 100 mM NaCl, 1% Triton X-100 and complete protease inhibitor (Roche), was added to each of the T-175 flasks. The cell lysates were harvested from the cell culture flasks using cell scrapers and transferred into pre-chilled low protein binding microfuge tubes. To assist with cell lysis, the samples were immersed in a sonication water bath at 4 °C for

32 Chapter 2: Materials and Methods

5 mins. The samples were then centrifuged at 14,000 g for 20 mins and the supernatant was retained and stored at -80 °C for protein quantification and purification.

2.6 PROTEIN QUANTIFICATION

Protein concentration was estimated using the Bicinchoninic Acid (BCA) protein assay kit (Pierce) as per the manufacturer's instruction. BSA standards were diluted in either PBS or lysis buffer depending on the protein sample to form a range of concentrations from 2000 µg/mL to 125 μg/mL used as the protein concentration reference. The BCA reaction plate was read at 562 nm using the benchmark plus microplate spectrophotometer system. The resulting standards and unknown absorbance data were corrected for dilution factors and curve fitted using a quadratic formula using the Microplate Manager Software version 6.2 (Biorad)

2.7 PROTEIN DETECTION

2.7.1 SDS-PAGE and Chemiluminescence Western Protein samples were mixed with NuPage® LDS sample buffer and reducing agent to a final concentration of 141 mM Tris base, 2% LDS, 10% Glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G, 0.175 mM Phenol Red, 50 mM DTT, pH 8.5 and heated at 70 °C for 10 mins. The protein samples were separated based on molecular weight on a precast NuPage® 4-12% SDS-PAGE with NuPage® MES-SDS running buffer. The electrophoresis proceeded at a constant 200 V for 35 mins. SDS-PAGE separated proteins were transferred onto a nitrocellulose membrane using the wet tank blotting system (Biorad) filled with Towbin buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol) at a constant 200 mA for 90 mins. The blot was blocked for 1 h at room temperature with 5% (w/v) BSA or 5% (w/v) skim milk powder (SMP) in Tris buffered saline (TBS - 10 mM Tris, 0.5 M NaCl, pH 7.6) with 0.1% Tween-20 (TBST). The blocked membrane was incubated with primary antibody diluted in blocking buffer and incubated overnight at 4 °C. The blot was then washed for 5 mins in TBST, repeating 2 times. After the washes, diluted anti primary species horse radish conjugated antibody (1:5000) in blocking buffer was added and incubated for 1 h at room temperature. An additional 3 washes of 5 mins each in TBST were performed to

Chapter 2: Materials and Methods 33

remove unbound secondary antibodies. The immune complex bound proteins were detected by the addition of ECL prime (GE) and visualised on an X-ray film developed using the AGFA CP 1000 film developer.

2.7.2 Quantitative Westerns Quantitative Westerns were performed using the Li-cor Odyssey® system with a similar workflow to the chemiluminescence Westerns. After transfer, the blot was blocked with Odyssey® Blocking Buffer (OBB) at room temperature for one hour. The blocking buffer was removed and the primary antibody was diluted in OBB and incubated overnight at 4 °C. The blot was then washed for 5 mins in TBST, repeating 2 times. Anti-rabbit or anti-mouse conjugated with DyLight 680 or DyLight 800 secondary antibodies diluted at 1:10,000 in OBB was added. Washes of 5 mins each of TBST and an addition wash of TBS for 5 mins to remove any residual Tween-20. The blot was scanned using a LI-COR® Biosciences Odyssey® Infrared Imaging System and data quantified using Image Studio software version 4.0. The blot was stripped by incubation at room temperature for 15 mins with Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific) if assessing both phosphorylated and non- phosphorylated proteins.

2.7.3 Silver staining Resolved SDS-PAGE gels were stained using the highly sensitive silver stain and the following modified staining protocol listed in Table 2-6.

34 Chapter 2: Materials and Methods

Table 2-6: Silver staining of SDS-PAGE gels using the Pierce™ Silver Stain Kit

Step Time Reagents 1-Wash 2x5 mins MilliQ water 2- Fixing 2x15 mins 30% (v/v) ethanol, 10% (v/v) acetic acid 3- Wash 2x5 mins 10% (v/v) ethanol 4- Sensitise 1 min 10 μL Sensitiser in 5 mL MilliQ water 5- Wash 2x1 mins MilliQ water 6- Stain 30 mins 100 μL Enhancer in 5 mL stain 7- Wash 2x20 sec MilliQ water 8- Develop As required 100 μL Enhancer in 5 mL developer 9- Stop 10 mins 5% (v/v) acetic acid

Chapter 2: Materials and Methods 35

Phage Display selection and screening

36 Chapter 3: Phage Display selection and screening

3.1 INTRODUCTION

Antibodies are glycoproteins produced by the immune system in response to foreign antigens. Due to their high specificity, antibodies have been exploited by biomedical research as molecular probes (Freise & Wu, 2015), and as targeted therapeutics such as Herceptin® (trastuzumab) for the treatment of metastatic breast cancer (Hudis, 2007). Historically, laboratory-based antibody production techniques were developed in the 1970s and 1980s and extensively described by the work of Harlaw and Lane (1988). In brief, animals were challenged with an antigen of interest, eliciting an immune response and the production of antigen-specific antibodies. The generated antibodies can be recovered from the serum of the animal, or antibody producing B-cells harvested and fused with a myeloma cell forming a hybridoma. By immortalising antibody producing B-cells, the hybridoma technique allows for the unlimited production of an antibody without requiring the use of animal serum and the ability to freeze and create an antibody-producing cell bank. However, the underlying principle still requires the immunisation of a host animal. Therefore ethical implications must be considered. Thus, alternative approaches which require no animals were in need of development to overcome these concerns.

Phage display was first described by Smith in 1985 as a method to display protein fused to coat proteins of bacteriophage (Smith, 1985). An application of this technique, called antibody phage display (APD), allowed for the in vitro selection of monoclonal antibodies against virtually any target, including those that were not possible by previous methods, such as animal immunisation, due to their toxic nature. In addition, because of the link between the DNA sequence and the translated protein being displayed, it offers additional flexibility such as recombination expression using various hosts (Miller, Weaver-Feldhaus, Gray, Siegel, & Feldhaus, 2005), interchange among immunoglobulin formats, and the creation of novel bi-specific antibodies and conjugates (Kontermann, 2011).

Human immunoglobulins such as IgG can be truncated into smaller fragments and yet retain antigen specificity. One such fragment, called single chain variable fragment (ScFv) consists of the variable heavy (VH) and viable light (VL) chains joined

Chapter 3: Phage Display selection and screening 37

by a linker sequence. Because of its smaller molecular weight of 28 kDa compared to a full-length IgG molecule of 150 kDa, the ScFv has several favourable properties, including that the ScFv format can be used in phage display, unlike the full length IgG. Prominent examples of a ScFv based phage display library, developed by the laboratory of Dr Winter (Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom), are the Tomlinson I + J libraries. Both libraries were engineered on the pIT2 phagemid vector (Figure 3-1) and encode for human variable heavy chain (VH) based on a single framework (V3-23/DP-47 and JH4b) and variable light chain (VL) (O12/O2/DPK9 and Jκ 1) (de Wildt, et al., 2000). Within the pIT2 phagemid vector, the VH and VL are linked by a (Gly4Ser)3 polypeptide sequence to allow correct conformational folding, and fused in frame with the pIII bacteriophage coat protein. The expression cassette is controlled by the lac promoter and is induced with isopropyl L-D-thiogalactosidase (IPTG) for periplasmic expression in E. coli via the pelB signal sequence. An amber stop codon DNA sequence (TAG) between the

ScFv (VH-VL) and the pIII allows for the ScFv to be fused to pIII during phage display as the stop codon is suppressed in TG-1 E. coli strains, while soluble ScFv without the pIII fusion can be expressed within a non-suppression strain such as HB2151, which recognises the amber stop codon. Polyhistidine and myc tags are incorporated at the end of the ScFv sequence to allow for purification by affinity chromatography.

38 Chapter 3: Phage Display selection and screening

Figure 3-1: Tomlinson libraries (I+J) phagemid. Both libraries were built on the phagemid vector pIT2 and encodes for human antibody fragments VH and VL fused to the phage pIII coat protein and joined by a (Gly4Ser)3 linker. The amber stop codon allows for the expression of VH-VL-pIII in a suppressor E. coli strain. The pelB is an export signal, directing the ScFv to the periplasm. The m13 origin of replication allows for the production of phage while colE1 origin of replication allows phagemid propagation in E. coli strains. The polyhistidine tag (His) is for the affinity purification of the ScFv by Immobilised metal ion affinity chromatography (IMAC). An alternative affinity tag, myc, can also be used for affinity chromatography. The PCR primers LMB3 and pHEN bind upstream of RBS and downstream of the myc tag respectively.

The Tomlinson ScFv libraries have been described and utilised extensively in the literature, including the development of potential cancer therapeutics (Patil, et al., 2015; ten Haaf, et al., 2015) and in the generation of targeted probes for imaging and detection (de la Cruz, et al., 2013; Kim, Wang, et al., 2014; Wang, Zhang, Zhang, Liu, & Liu, 2012). Of particular relevance to this project are studies which target cell receptors with the aim of generating competitive receptor antagonists, examples include: lutropin and thyrotropin receptor (Majumdar, Railkar, & Dighe, 2012), fibroblast growth factor receptor 3 (Martínez-Torrecuadrada et al., 2005) and the leptin receptor (Molek, Vodnik, Štrukelj, & Bratkovič, 2014).

As part of the overall objective of this project, to generate bi-specific function blocking antibody against IGF-1R and αv integrin, the aims of this chapter are:

 Amplification of the Tomlinson ScFv antibody libraries (I, J) and the generation of phage displaying antibody fragment repertoire.

 To independently screen both phage libraries against passively immobilised IGF-1R and αv integrin in order to generate ScFv against both respective targets.

Chapter 3: Phage Display selection and screening 39

3.2 EXPERIMENTAL PROCEDURES

3.2.1 Materials All general reagents were purchased from various companies and are of highest quality. The naïve human ScFv libraries ‘I’ and ‘J’ (Tomlinson I+J), KM13 helper phage, Escherichia coli (E. coli) TG1 and HB2151 were purchased from Source bioscience (Cambridge, UK). Recombinant human IGF-1R containing the extracellular domains was purchased from R&D systems (Minneapolis, US) with Human recombinant αv integrin acquired through Abnova (Taipei, Taiwan). Odyssey® Blocking Buffer (TBS) was purchased from LI-COR Biosciences (Lincoln, US). Anti-IGF-I Receptor β, Phospho-IGF-I Receptor β (Tyr1131) antibody, Anti- rabbit IgG (H+L) (DyLight™ 680 Conjugate) and Anti-mouse IgG (H+L) (DyLight™ 800 Conjugate) were all obtained from cell signalling (Danvers, US). Immuno MaxiSorp® 5 mL tubes (cat: NUN444202) and MaxiSorp® 96 well plates (NUN442404) for phage panning was sourced from Thermo Fisher. All other reagents were sourced from Sigma Aldrich (St Louis, US) unless otherwise stated.

3.2.2 Phage display libraries The Tomlinson ScFv library repertoire was engineered based on the phagemid vector pIT2 and has a complexity of 1.47x108 and 1.37x108 for the ‘I’ and ‘J’ libraries respectively. Both libraries were used sequentially in screening to allow for the greatest number of positive IGF-1R and αv integrin binders to be selected. Included with the libraries are pre-selected E. coli clones encoding for ScFv against bovine serum albumin (BSA) and ubiquitin, which were used as positive controls in early testing to confirm correct expression of the ScFv and the ability to bind to BSA or ubiquitin.

3.2.3 E. coli strain, media, and culture conditions E. coli strains used in this project are listed below in Table 3-1. All strains were routinely propagated in 2xYT broth (16 g tryptone, 10 g yeast extract, 5 g NaCl per litre), supplemented with 4% (v/v) glucose and 200 µg/mL of ampicillin (2xYT AG)

40 Chapter 3: Phage Display selection and screening

as needed. The cultures were grown in disposable Erlenmeyer flasks (Corning, Mount Martha, Vic, Australia) with shaking at 250 RPM at 37 °C. In addition, strains were also grown on TYE agar plates (10 g tryptone, 5 g yeast extract, 8 g NaCl, 15 g agar per litre) supplemented with 4% (v/v) glucose and 200 µg/mL of ampicillin (TYE AG) as required. For the selection of single phage colonies, H top agar (10 g tryptone, 8 g NaCl, 7 g agar per litre) was used. To maintain the F’-episome in TG1 and JM109, which is essential for KM13 phage infection, strains were plated on M9 minimal medium glucose plates (Atlas, 2004) before any procedure involving phage infection. The F’-episome in the strains used for this project have been engineered to contain the pro operon, which was deleted from the chromosome. The loss of F’ will lead to proline biosynthesis deficiency and cell death.

Chapter 3: Phage Display selection and screening 41

Table 3-1 E. coli strains used in this project Strain Genotype Purpose Reference

TG1 K12 (lac-proAB) supE thi hsdD5/F' Phage display Gibson, T.J, 1984 traD36 proA+B lacIq lacZ M15), (Gibson, 1984)

JM109 endA1, recA1, gyrA96, thi, hsdR17 (rk– Phage display Yanisch-Perron, C, , mk+), relA1, supE44, Δ( lac-proAB), 1985 [F´ traD36, proAB, aqIqZΔM15]

HB2151 K12 ara (lac-proAB) thi/F' proA+B Production of Carter et al, 1985 lacIq lacZ M15 soluble ScFv (Carter, Bedouelle, & Winter, 1985)

3.2.4 KM13 helper phage amplification Stock KM13 helper phage from the manufacturer was serially diluted from 10-2 to 10-8 in PBS. Log phase JM109 or TG1 were infected with each dilution and incubated at 37 °C without shaking for 20 mins. Helper phage used in the library ‘I’ selection process was produced in JM109 while TG1 produced helper phage was used for library ‘J’. Each sample was added to 3 mL of melted H-top agar at 42 °C and poured onto pre-warmed TYE agar plates. After overnight incubation at 37 °C, a single ‘plaque’ was picked and log phase E. coli were reinfected and incubated at 37 °C overnight with shaking. The overnight culture was clarified by centrifugation at 4,000 g for 30 mins. To the supernatant, 1/5 volume of PEG/NaCl (20 % (w/v) Polyethylene glycol 6000, 2.5 M NaCl) was added and allowed to incubate on ice for 1 h. The resulting precipitated phage was pelleted by centrifugation at 3,300 g at 4 °C for 30 mins. An additional round of purification was performed by resuspending the pellet in 5 mL of PBS with 1 mL of PEG/NaCl solution and placed on ice for 10 mins. The solution was again pelleted at 3,300 g at 4 °C for 10 mins and resuspended in PBS. A final centrifugation was performed at 14,000 g for 10 mins to remove bacterial debris.

KM13 is a protease sensitive helper phage that has been modified by the insertion of a trypsin cleavage site between the D2 and D3 domains of the pIII coat protein (Kristensen & Winter, 1998). This allows for elution using trypsin to release the substrate bound phage at the end of the panning round in addition to inactivating background phage.

42 Chapter 3: Phage Display selection and screening

Figure 3-2: The helper phage KM13 encodes for a trypsin sensitive phage protein pIII.

KM13 helper phage used in this project has been modified with a trypsin cleavable sequence between the D2 and D3 domain of the pIII coat protein to eliminate background phage lacking displayed proteins. A) Wild type pIII -The bound phage at the end of a panning round can be recovered by the addition of trypsin which cleaves the trypsin cleavage site (EQK-LISEEDLN) within the engineered myc tag. B) Modified pIII – Phage which does not display ScFv are cleaved between the D2 and D3 domain, rendering it non-infective in subsequent panning rounds. C) Modified pIII – When ScFv is correctly displayed, the trypsin cleavage site within the D2/D3 domain is shielded. Adopted from Goletz et al. (2002)

Upon elution with trypsin at the end of a bio-panning cycle, non-antibody displayed phage is rendered non-infective with the removal of domains D1 and D2

Chapter 3: Phage Display selection and screening 43

while antibody-pIII with wild type pIII remains unaffected. The consequence of this is the removal of background phage since 99% of phage does not display antibody. To test for trypsin sensitivity, 45 µL of purified helper phage was treated with 5 µL of trypsin solution (1 mg/mL trypsin in PBS) and allowed to incubate at room temperature for 30 mins. The trypsin treated and non-treated phage was serially diluted and allowed to infect log phase growth E. coli. The mixture was added to melted H- top agar and poured onto a pre-warmed TYE plate and incubated at 37 °C overnight. The titre between the treated and non-treated phage was compared to determine sensitivity.

3.2.5 Amplification of phage library repertoire A frozen ampule of library ‘I’ or ‘J’ was rapidly defrosted and grown in 200 mL of pre-warmed 2xYT AG with shaking at 250 RPM at 37 °C. After 2.5 h, a 50 mL aliquot was taken and 2x1011 KM13 helper phage added. Upon infection by helper phage, kanamycin resistance was conferred to the E. coli host. This was used to deplete non helper phage infected E. coli. The E. coli / phage solution was incubated at 37 °C for 30 mins without shaking in a water bath. The culture was centrifuged at 3,300 g for 10 mins after which the pellet was resuspended in 100 mL of 2xYT supplemented with 100 µg/mL ampicillin, 50 µg/mL kanamycin and 0.1% glucose and shaken overnight with the incubator set at 30 °C. The resulting phage library repertoire was purified using PEG6000/NaCl in a similar manner to the production of the helper phage (Section 3.2.4). In addition to library ‘I’ and ‘J’, preselected phage in E. coli encoding ScFv that are reactive to BSA and ubiquitin was also generated and purified to act as controls.

3.2.6 Phage titre determination The purified helper phage titre was determined by counting clearings on a plate, plates with 30-300 clearings were used for counting, and the titre was calculated based on the dilution of the counted plate:

1000 = / (μ)

44 Chapter 3: Phage Display selection and screening

Library ‘I’ and ‘J’ titre was determined by infecting log phase E. coli with a dilution series of phage and plated on 2xYT AG. The resulting plates were incubated overnight at 37 °C. The number of ampicillin resistant clones was counted on plates with 30-300 colonies and titre calculated using the same equation outlined above.

3.2.7 Library ‘I’ selection of IGF-1R binders Selection of target specific binders involves three rounds of immobilisation of the target antigen, incubation of the phage library repertoire, removing unbound phage, elution of reactive phage, and reinfection. For the first round of selection, a Nunc MaxiSorp immunotube (Scoresby, Vic, Australia)) was coated with 1 mL of 20 µg/mL human recombinant IGF-1R extracellular domain (R&D systems, Gymea, NSW, Australia) solubilised in PBS. In each subsequent round a reduced concentration of IGF-1R was coated (round 2; 10 µg/mL and round 3; 5 µg/mL). The Immunotube was rotated overnight at 4 °C in order for the even coating of the entire surface. The coated immunotube was washed 3 times with PBS and blocked overnight with 5% (w/v) skim milk powder in PBS (MPBS) at 4 °C. An additional non-coated tube was also blocked. The tubes were again washed 3 times with PBS before 1012 library ‘I’ phage in 5% MPBS was added to the uncoated, blocked tube. This step was included to deplete any phage encoding for ScFv’s that are reactive to the immunotube, or skim milk proteins. The phage solution was transferred to the IGF-1R coated tube and rotated at room temperature for 1 h followed by no rotation for an additional h. The phage solution was discarded and a low stringency wash with PBST (PBS supplemented with 0.1% (v/v) Tween 20) was used to remove non-binders. The first round of selection employed minimal stringency by washing the tube 5 times using PBST followed by 2 times with PBS. Subsequent rounds increased washing stringency to 10 PBST/2 PBS and 20 PBST/2 PBS washes for rounds 2 and 3 respectively. The immobilised IGF-1R bound phage was eluted by incubating with trypsin solution (1 mg/mL trypsin in PBS) for 10 mins at room temperature with rotation and the enriched phage was rescued by re-infection. The E. coli solution was plated onto TYE AG and incubated overnight at 37 °C. The resulting ampicillin resistant colonies harbouring library phagemid were scraped off the agar plate for use in the next round of panning. In order to conserve time and maximise the chance of isolating binders, the whole panning process against

Chapter 3: Phage Display selection and screening 45

IGF-1R was repeated, but with both phage libraries I and J combined and panned against IGF-1R simultaneously.

3.2.8 Phage selection against αv integrin with library ‘I’ and ‘J’ The screening of ScFv binding integrin was conducted in a similar manner to the screening of IGF-1R outlined in section 3.2.7. In this instance, recombinant human αv integrin was coated onto an immunotube at a concentration of 5 µg/mL for the first round of screening, and was reduced to 2.5 µg/mL for the 2nd round. This was further reduced to 0.5 µg/mL in the 3rd and final round. For each concentration, 1 mL total volume was used. The entire panning process was conducted once against αv integrin

3.2.9 Polyclonal phage ELISA To determine the sensitivity and specificity of the panning process, a polyclonal phage ELISA was performed. The procedure is similar to the library selection process in section 3.2.7 but rather than coating the immunotube with IGF-1R, a Maxisorp® 96 well plate (Nunc) was used. Maxisorp® plate wells were coated with IGF-1R (5 μg/mL), BSA (100 μg/mL, Invitrogen) or ubiquitin (100 μg/mL, Sigma) overnight at 4 °C with rotation, washed with PBS and blocked with MPBS overnight. Phage from the selection rounds, along with BSA and ubiquitin reactive phage were diluted in MPBS and added to the wells and incubated for 1 h at room temperature followed by 3 PBST washes. Bound phage was detected with either an α-pIII primary antibody (New England Biolabs, Arundel, Qld, Australia) followed by an α-mouse-HRP secondary antibody for library ‘I’, or a more streamline α-pIII-HRP primary antibody (GE healthcare, Rydalmere, NSW, Australia) for library ‘J”. Subsequent to a wash step with PBST, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Millipore, Kilsyth, Vic, Australia) was added to all wells, which reacts with the antibody bound HRP to form a coloured compound. The development reaction was allowed to continue for 10 mins, was acidified with HCl and the resulting signal read at 450 nm. The data is expressed as a signal to noise ratio (S:N); determined by:

46 Chapter 3: Phage Display selection and screening

3.2.10 Monoclonal soluble ELISA Clones at the end of the panning rounds were picked into a 96 plate containing 200 μL of 2xYT AG and cultured at 37 °C with shaking at 250 RPM overnight. From the overnight culture, 5 μL was used as the inoculum in a new 96 well plate containing 200 μL of 2xYT AG. The new culture was maintained at 37 °C with shaking at 250 RPM for 3 h, at which point the plate was centrifuged at 3,200 g to pellet the E. coli and the supernatant was discarded. The pellets were resuspended with 2xYT AG supplemented with 1 mM IPTG and cultured at 30 °C with shaking at 250 RPM for 16 h. After which the plate was centrifuged at 3,200 g for 10 mins and 50 μl of the expressed ScFv containing supernatant was used in an ELISA as per the polyclonal phage ELISA (see section 3.2.9).

The threshold value for the further characterisation of binders was set as the total of average S:N of binders + the standard deviation of the binders.

3.2.11 Sequencing of positive binders E. coli clones determined as positive binders from ELISA analysis were cultured overnight in 5 mL of 2xYT medium overnight with shaking at 250 RPM at 37 °C. The overnight cultures were centrifuged at 3,000 g for 10 mins to obtain the pellet of E. coli cells. A miniprep procedure was performed using the QIAGEN QIAprep Spin Miniprep Kit to harvest the ScFv encoding phagemid according to the manufacturer’s instructions.

The purified phagemid was submitted to the Australian Genome Research Facility (AGRF) for sequencing using the purified DNA (PD) service. The sequencing primers used are listed in Table 3-2. The DNA sequences were analysed using CLC Main workbench 7.6.2.

Chapter 3: Phage Display selection and screening 47

Table 3-2 Sequencing primers for the phagemid vector pIT2

Primer Name Sequence 5’ to 3’ Binding location

LMB3 CAG GAA ACA GCT ATG AC upstream of the ribosome binding site

pHEN CTA TGC GGC CCC ATT CA 3’ Downstream of myc tag

3.3 RESULTS

3.3.1 rIGF-1R can be passively coated onto MaxiSorp® immunotubes To enable phage display selection, the target of interest must be immobilised to a solid support. Therefore, in order to determine if rIGF-1R can be passively adsorbed onto polystyrene MaxiSorp® immunotubes, 10 μg/mL of IGF-1R prepared in either 1 mL PBS or 1 mL NaHCO3 buffer was added to individual immunotubes and incubated overnight with rotation at 4 °C. Samples were taken prior to and at the conclusion of each period of incubation. In addition, a standard curve was constructed ranging from 0.325-10 μg/mL IGF-1R. The samples were separated on SDS-PAGE, transferred onto nitrocellulose, and probed with an anti-IGF-1R-α antibody.

The resulting blot showed that each rIGF-1R sample separated into two anti- IGF-1R reactive bands, at approximately 160 kDa and 125 kDa (Figure 3-3). The heavier molecular weight band corresponds to the un-cleaved single-chain form of IGF-1R containing both the α and β chains (truncated). The lighter band, which constitutes the majority of immune-reactivity, in each sample corresponds to the α chain alone.

Both IGF-1R-α antibody reactive bands were quantified and combined to form one signal value. The standard curve series signal values were graphed and the results show a high degree of linearity (R2=0.9977) between the concentration chosen and the near infrared signal. As demonstrated in Figure 3-3, IGF-1R was successfully coated

48 Chapter 3: Phage Display selection and screening

onto polystyrene based MaxiSorp® immunotubes. The binding capacity of the tubes was less than the 10 μg/mL of IGF-1R introduced into the tube, as seen by the residual uncoated IGF-1R at the end of day 1 and day 2 under both PBS and NaHCO3 coating conditions. The binding capacity of the immunotubes for rIGF-1R in PBS was calculated from the average of the amount adsorbed per tube per day and found to be 3.33±0.91 μg with a maximum adsorption of 5.141 μg per day. The average binding capacity for rIGF-1R diluted in NaHCO3 was 3.31±0.15 μg. This suggests that both binding conditions are similar and that the surface is being saturated with IGF-1R at 10 μg/mL by passive adsorption. Given this result, immunotubes used for phage display panning were incubated overnight with 10 μg/mL of IGF-1R in PBS to ensure saturation.

Chapter 3: Phage Display selection and screening 49

rIGF-1R STD curve 2000000 R square: 0.9977 )

m 1500000 n

0 0 6

( 1000000

l a n g i 500000 S

0 0 5 10 15 IGF-1R STD ( g/mL)

IGF-1R STD ( g/mL) Absorbed (g) Intensity (600 nm)

PBS Day1 7.339 2.661 1120000 PBS Day2 2.198 5.141 346000 PBS Day3 0.011 2.187 16700 NaHCO3 Day1 6.808 3.192 1040000 NaHCO3 Day2 3.188 3.620 495000 NaHCO3 Day3 0.047 3.141 22100

Figure 3-3: Efficiency of IGF-1R passive adsorption onto polystyrene. Recombinant IGF-1R was diluted to 10 μg/mL in PBS or NaHCO3 and added to individual Nunc MaxiSorp® immunotubes. The tubes were incubated with rotation for 24 h at 4 °C after which a small sample was taken (Day 1) with the remainder transferred into a new tube and put back into rotation. Samples were also taken after the 2nd day (Day 2) and the third day (Day 3). A standard curve of recombinant IGF- 1R was constructed ranging from 0.325 μg/mL to 10 μg/mL in order to quantify IGF- 1R adsorption. The standard curve and the post-coated IGF-1R samples were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with an anti-IGF-1R-α antibody. The blot was visualised on the Licor Odyssey system and the bands were quantified using Image Studio 4.0. The standard curve and the adsorbed samples were calculated using GraphPad Prism® version 6.0.

50 Chapter 3: Phage Display selection and screening

3.3.2 Panning and polyclonal phage ELISA of IGF-1R binders To assess the enrichment of positive IGF-1R binders, phage samples from each panning round were tested in a polyclonal phage ELISA against immobilised IGF-1R as per outlined in section 3.2.7. The results of the polyclonal phage ELISA with library ‘J’ are shown in Figure 3-4, with the signal:noise ratio calculated as the absorbance ratio between the IGF-1R coated wells and SMP blocked wells. It was expected that as the IGF-1R binders increase after each subsequent round of panning, the signal should also increase. The maximum signal:noise ratio to IGF-1R of 6.5 was achieved after 2 rounds of panning which decreased to 2.7 in round 3. This might be due to several factors: 1) enrichment of non-specific binders or; 2) The signal has reached the saturation point of the spectrophotometer, therefore unable to detect higher binding signal. Based on this data, 96 individual clones were selected from round 2 of selection rather than round 3 to minimise the selection of non-specific binders.

Polyclonal Phage IGF-1R ELISA Library 'J'

8 IGF-1R 20 g/mL IGF-1R 5 g/mL 6 IGF-1R 1 g/mL e s i

o BSA N

:

l 4 Ubiq a n g i S 2

0 0 1 2 1 2 d d d # # R R R 3 3 d d R R

Panning Round

Figure 3-4: Library ‘J’ polyclonal phage ELISA. IGF-1R (1, 5 and 20 μg/mL), BSA, and ubiquitin were coated onto a 96 well Maxisorp® immunoplates. Phage samples from before the panning selection (Round 0), and at each round of selection (Rd1, Rd2, Rd3) were added to the coated plate. Non binders were determined when signal:noise is at 0. The binding assay for round three was conducted in duplicate (Rd 3 #1, #2).

Chapter 3: Phage Display selection and screening 51

3.3.3 Soluble ScFv IGF-1R expression and Monoclonal ELISA To select for individual ScFv clones that encode IGF-1R binders, soluble ScFv expression was induced from individual clones from the 2nd round of panning. This allowed for selection of ScFv which can bind to IGF-1R. To express the soluble ScFv, IPTG was used to induce the lac promoter, driving the expression cassette housed within the phagemid. The resulting supernatant containing ScFv was used in an ELISA as per 3.2.9 but instead of detection with α-pIII antibody, bound ScFv to IGF-1R were detected using protein-L-HRP. The results of the 96 clones screened are shown in Figure 3-5 with the the signal to noise (S:N) threshold for further investigation set at 7 and non-binders at 1.0. Of the 96 clones screened, 7 returned a S:N above the threshold Table 3-3. Of the remaining 89 clones, 42 bind to IGF-1R and 47 did not bind.

52 Chapter 3: Phage Display selection and screening

Figure 3-5 Soluble IGF-1R ScFv ELISA. 96 clones from the 2nd selection round were induced to produce soluble ScFv. The expressed ScFvs were added to IGF-1R coated plate and bound ScFv to IGF-1R was detected using protein-L-HRP. The minimum threshold level (dotted line) is set based on 3.2.11. Non-binding is set at Signal:Noise = 1

Chapter 3: Phage Display selection and screening 53

Table 3-3: IGF-1R binding clones above threshold

Clone Signal to noise A4 7.6 A7 8.6 A11 9 D3 7.2 F1 8 F2 7.3 H3 9.6 H9 9.3

3.3.4 Panning and monoclonal ELISA of αv integrin ScFv binders In a similar manner to above, 96 clones at the end of the 3rd round of panning against αv integrin were chosen for soluble ScFv expression as per outlined in section 3.2.10. Given the limited availability of αv integrin, the polyclonal ELISA was replaced in favour of a monoclonal ELISA, allowing for the direct selection of individual ScFv. The resulting supernatant containing ScFv was used in a monoclonal ELISA with plates coated with αv integrin. The results of the 96 clones screened are shown in (Figure 3-6) with the signal to noise (S:N) threshold for non-binders set at 1.0. Of the 96 clones screened, 8 returned a S:N above the threshold (Table 3-4). Of remaining 88 clones, 52 bind to αv and 36 did not bind.

54 Chapter 3: Phage Display selection and screening

Figure 3-6 Selection of αv integrin binding ScFvs. 96 clones from the 3nd selection round were induced to produce soluble ScFv. The expressed ScFvs were added to αv integrin coated plate and bound ScFv was detected using protein-L-HRP. The minimum threshold level (dotted line) is set based on 3.2.11. Non-binding is set at Signal:Noise = ≤1

Chapter 3: Phage Display selection and screening 55

Table 3-4: αv integrin binding clones above threshold

Clone Signal to noise A2 2.46 B1 1.77 C1 2.33 E2 2.64 F3 3.04 F5 2.13 F8 2.42 G7 1.91 3.3.5 Sequencing of IGF-1R binding clones All 8 IGF-1R binding clones above the threshold of the phage ELISA, when panned with both Tomlinson libraries I and J, were sequenced. The sequencing primers used are listed in Table 2-3, with the forward sequencing LMB3 primer binding upstream of the VH-VL domains of the ScFv while the reverse primer pHEN bound downstream of the myc tag. The resulting sequencing chromatograms were analysed with CLC Main Workbench with all 8 clones sequenced returned a single unique sequence (Figure 3-7). This was not expected as the ELISA results demonstrated different binding capabilities between clones, which is indicative of different ScFv sequences. The full DNA sequencing chromatogram can be found in Appendix 3. To assess if the isolated IGF-1R ScFv was unique, the ScFv CDS was submitted to a Basic Local Alignment Search Tool (BLAST) search (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSe arch&LINK_LOC=blasthome). The result, listed in Appendix 4, showed that the top ranked result was an α-TNFα ScFv, which differs from the isolated ScFv by 16 amino acid residues. All residues were located within the CDRs, with identical framework region sequences. Based on the similarity in the framework regions, the α-TNFα ScFv was most likely isolated from the Tomlinson phage display library.

Analysis of the sequenced showed an amber stop codon sequence (TAG) at position 169-171 within the CDRH2 coding region. This extraneous stop codon was introduced as a remnant of the Tomlinson libraries generation process and would prematurely terminate the translation of the full length ScFv, requiring removal prior to expression. In suppression E. coli strains such as TG1, containing the genotype

56 Chapter 3: Phage Display selection and screening

supE, this amber stop codon is read-through as glutamic acid without termination. However, the expression E. coli strain used for this project HB2152 lacks the supE genotype and will terminate translation prematurely. Therefore, site-directed mutagenesis was required to change the DNA sequence from TAG to GAG, which encodes for glutamic acid.

Figure 3-7: Sequence map for IGF-1R binding clone. The ScFv DNA sequence encoding for the variable heavy complementarity determining region (CDRH2, CDRH3) and the variable light complementarity determining region (CDRL2, CDRL3) joined by a (Gly3Ser)3 polypeptide linker. The 6 histidine repeats (His tag) enable purification of the protein using affinity based chromatography. An amber stop

Chapter 3: Phage Display selection and screening 57

codon (TAG) was detected in the coding sequence of CDRH2. The DNA sequence was translated into amino acid sequence in one letter code using Main Workbench 7.0.

58 Chapter 3: Phage Display selection and screening

3.3.6 Sequencing of αv integrin binding ScFv clones Based on the results of the αv monoclonal phage ELISA (Figure 3-6), 8 clones (A2, B1, C1, E2, F3, F5, F8 and G7) were selected to be sequenced by the Sanger reaction. The resulting DNA sequences were compared with each other to determine unique ScFv construct sequences. From the 8 sequenced clones, 3 unique sequences were returned. The three clones that were unique are A2 clone B1 and clone F3 (

Figure 3-8), All three clones were verified for their sequence fidelity and all encode full-length ScFv. Both clone A2 and B1 encode a premature amber stop codon sequence (5’ TAG 3’) in the CDRH2 section, which needed to be removal before expression. The full DNA sequencing chromatogram can be found in Appendix 5-7.

Chapter 3: Phage Display selection and screening 59

Clone CDRH2 CDRH3 CDRL2 CDRL3 A2: DNA TCTATTACTT CGTCTGGGGA AAGGCATCCC CAACAGGGGC CGACGGGTGT CTTTTGACTA ATTTGCAAAG ATGTTCGGCC GAAGACATAG C T TCCGACG TACGCAGACT CCGTGAAGGG C

A2: Protein SITSTGVKT*YA RLGTFDY KASHLQS QQGHVRPPT DSVKG

B1: DNA ACTATTCCTC AGGGCTGCGC AATGCATCCA CAACAGGCTC GGTAGGGTCG GGTTTGACTA CTTTGCAAAG GGCTGAAGCC GAATACAAAT C T TACTACG TACGCAGACT CCGTGAAGGG C

B1: Protein TIPR*GRNTNYA RAARFDY NASTLQS QQARLKPTT DSVKG

F3: DNA GGTATTACGA ACTTCTCGGT CGTGCATCCC CAACAGGGGC AGCAGGGTGA CGTTTGACTA TGTTGCAAAG ATACGAGGCC TGGTACACAT C T TCCTACG TACGCAGACT CCGTGAAGGG C

F3: Protein GITKQGDGTHYA TSRSFDY RASLLQS QQGHTRPPT DSVKG

Figure 3-8: DNA and translated amino acid sequence for αv integrin clones A2, B1 and F3 ScFv.

The ScFv DNA sequence encoding for variable heavy complementarity determining region (CDRH2, CDRH3) and the variable light complementarity determining region (CDRL2, CDRL3) were determined using Sanger sequencing. Amber stop codon (TAG) was detected in the coding sequence of CDRH2. The DNA sequence was translated into amino acid sequence in one letter code using Main Workbench 7.0. Letters in bold indicates possible mutation sites engineered as part of the Tomlinson phage display libraries.

60 Chapter 3: Phage Display selection and screening

3.4 DISCUSSION

The aim of this chapter was the selection of IGF-1R and αv integrin binding ScFv using phage display technologies. The Tomlinson ScFv libraries I & J were chosen based on extensive use in the literature and the diverse applications for which they have been employed (Eteshola, 2010; Goletz, et al., 2002; Kim, Wang, et al., 2014; ten Haaf, et al., 2015; Wang, et al., 2012). In addition, as both libraries were created from human immunoglobulin sequences, the likelihood of an adverse immune reaction, such as the Human anti-mouse antibody (HAMA) reaction, is eliminated (Azinovic et al., 2006).

The phage display process is complex and many factors contribute to the successful selection of ScFv against the chosen target. The first factor to consider is the choice of immobilisation strategies onto a solid substrate. The two most common methods are passive adsorption onto polystyrene or an affinity based system such as biotinylating the protein and using streptavidin coated beads. The first method, adsorption onto polystyrene, is the simplest, requiring only the incubation of the antigen in a suitable buffer. However, in some cases the function of the protein can be affected (Butler et al., 1992). The use of streptavidin beads would require less target protein, since the protein coated beads could be reused in subsequent panning rounds. Burtrum, et al. (2003), described the coating of IGF-1R onto plastic for selection in phage display experiments, thus, this study served as a guide for the present undertaking. The MaxiSorp® surface chosen for this project reached saturation when incubated with recombinant human IGF-1R at a much lower concentration than expected. In the first round of panning against IGF-1R binders, 1 mL of IGF-1R at 20 μg/mL was added to a MaxiSorp® surface immunotube to be passively adsorbed. Subtractive concentration analysis demonstrated that a maximum of 5.141 μg was adsorbed to the surface when using either PBS or carbonate based buffers with average adsorption equalling 3.33±0.91 µg and 3.31±0.15 µg respectively. Initially, 20 µg/mL was thought to be low as the Burtrum et al. study used 50 µg/mL of IGF-1R as the immobilisation concentration for the first round of panning (Burtrum, et al., 2003). Practically speaking, significant difficulties were experienced endeavouring to evenly coat the immunotube with only 1 mL of liquid, it is thus most likely that >1 mL of 50 μg/mL IGF-1R was used by Burtrum and colleagues. It is unclear why such a high

Chapter 3: Phage Display selection and screening 61

amount of IGF-1R was used, given the saturation levels observed in the present study. Potentially the high levels of IGF-1R guarantees the surface reached saturation, allowing for the maximum number of rare IGF-1R reactive phage to bind during the first round of phage selection. Also, it is possible that multiple tubes were coated with IGF-1R, which would require the high IGF-1R concentration.

Another critical element of phage display is the selection of polystyrene blocking agents as the ScFv non-specifically binding to the Immuno tube may give false positive binders through the panning rounds. Furthermore, if a blocking agent such as skim milk powder (SMP) or bovine serum albumin (BSA) (Adey, Mataragnon, Rider, Carter, & Kay, 1995) is used, there may be ScFv selected for binding to this rather than the target, in this case IGF-1R and αv integrin. Therefore, to address this issue, a polystyrene and SMP depletion step was included at the start of each panning round. Similar depletion procedures have been employed in the literature (Eteshola, 2010).

The polyclonal and monoclonal ELISA procedures used for this project are routinely used as a screening tool for positive binding during phage display panning (Patil, et al., 2015; Petters, Sokolowska-Wedzina, & Otlewski, 2015; ten Haaf, et al., 2015). One problem was that identical sequence ScFv resulted in different ELISA signal levels, when in theory they should be identical. The most likely explanation is the different expression levels of clones grown in a 96 well plate, due to varied environmental conditions experienced across the plate. In addition, the levels of the initial bacterial inoculum was not quantified at the start of the culture, however, since an overnight culture was used, the concentration of E. coli would have reached saturation and be similar in numbers (Sezonov, Joseleau-Petit, & D'Ari, 2007). To maintain consistent ScFv input in ELISA, it is possible to perform a high throughput purification with 96 well plates coated with Ni-NTA (Qiagen Ni-NTA HisSorb Plates) or protein-L. The resulting purified ScFv could then be quantified normally using a standard protein quantification assay.

Due to the inherent random enrichment of clones resulting from the phage display process, the selection of clones harbouring identical ScFv CDS is possible.

62 Chapter 3: Phage Display selection and screening

Initially, in order to quickly discriminate among unique clones, a restriction fragment analysis using the enzyme BstNI on the ScFv CDS was conducted. However, it was unable to discriminate between unique clones fully (results not shown). The clones in the Tomlinson libraries are mutated in one of the possible 18 amino acid residues. The probability of BstN1 recognising its DNA cleavage sequence (CC’(A/T)GG) at one of these positions is low. However, BstN1 digestion has been successfully used in other phage display libraries that are more heterogeneous in nature, and potentially producing restriction fragments of different sizes (Pansri, Jaruseranee, Rangnoi, Kristensen, & Yamabhai, 2009b). Due to the failure of BstN1 discrimination, the slower and more expensive method of sequencing was used in order to determine unique clones.

Sequencing of the four unique clones which bind either IGF-1R or αv integrin, 3 (one IGF-1R and 2 αv integrin binders) had a premature amber stop codon sequence (TAG) within the CDHR2 encoding sequence. For both IGF-1R ScFv and also αv integrin B1, it was located in the sequence encoding the 5th amino acid residue, while for αv integrin A2, the amber stop codon was located in the 10th codon of the translation sequence. The amber stop codon is ineffective during phage display screening as the TG1 E. coli strain reads the codon as glutamic acid rather than a stop, due to repression of stop tRNA’s through the supE gene. Amber stop codons have been described in selected ScFv clones from the Tomlinson libraries used within previous studies (Eteshola, 2010; Wu, Ke, & Doudna, 2007). While the amber stop codon can be tolerated in the TG1 strain, when transferring the selected ScFv expressing plasmid into an expression E. coli strain, such as HB2151, the amber stop codon requires mutation. This can be achieved using site-directed mutagenesis to modify the DNA sequence TAG to GAG (Glutamic acid) (Wu, et al., 2007). The following chapter will detail the mutagenesis steps required. The enrichment of the desired phage depends on equal ScFv-phage expression and growth. If the displayed ScFv sequence has a growth disadvantage, such as using rare bacterial tRNAs, that sequence would propagate more slowly relative to others. Even if the ScFv has better binding characteristics, leading to the enrichment of sub-optimal binding clones among each panning round.

Chapter 3: Phage Display selection and screening 63

It is unclear why the panning of IGF-1R with both ScFv libraries only yielded one unique ScFv sequence. The screening process is complex, therefore, isolating the underlying cause is difficult. Since the process of IGF-1R immobilisation onto polystyrene and the subsequent panning was successful in the study by Burtrum, et al. (2003), potentially the problem stems from the Tomlinson libraries. In that study, the researchers used a library containing 3.7 x 1010 clones, which is ~100 times more diverse than the Tomlinson libraries I & J of 1.47 x 108 and 1.37 x 108 respectively. Even with a vastly more complex library, the phage panning process conducted by Burtrum et al was only able to yield 4 clones with only one clone demonstrating ligand blocking functionality. Other studies using the Tomlinson I & J libraries yielded 3 unique sequences from 96 characterised (Yan, Ko, & Qi, 2004), 14 from 44 clones (Eteshola, 2010) and 3 from 800 clones screened (Patil, et al., 2015). Taken together, there is evidence to suggest that the low number of unique binders (total of 4) obtained from phage panning could be related to the Tomlinson libraries used.

Therefore, it is likely that in using a smaller library for this project that only one IGF-1R binding sequence can be obtained. Since the commencement of this project, commercial phage display libraries, which contain a higher magnitude of complexity than used herein, have become available and can be sourced from Creative Labs (http://www.creative-biolabs.com/). For example, the HuScL-4(R): Human Single Chain (ScFv) Antibody Library has a complexity of 1.5x1016. The downside to obtaining these libraries is the substantial costs involved, upwards of $20,000. In addition to sourcing ScFv libraries with an increasing complexity, alternative libraries encoding for different antibody framework could also be investigated. In particular, single domain antibodies, which consist of one heavy chain domain (VHH) are gaining popularity due to its desirable characteristics such as being non-immunogenic in humans, small size, and aggregation resistance (Omidfar & Shirvani, 2012; Rozan et al., 2013).

In spite of the technical difficulties encountered, the panning process was able to yield 1 IGF-1R and 3 αv integrin unique binding sequences. The next step in fulfilling the aims of this project is the expression of these ScFv which is detailed in the following chapter.

64 Chapter 3: Phage Display selection and screening

Chapter 3: Phage Display selection and screening 65

ScFv expression and purification

Chapter 4: ScFv expression and purification 67

4.1 INTRODUCTION

The previous chapter described the selection of αv integrin and IGF-1R binding ScFv candidates using antibody phage display technologies. As a result of the panning procedure, the 4 unique ELISA confirmed clones were selected for expression in order to perform structural and functional characterisation studies.

The selected ScFv binding candidates described in the last chapter were chosen from a phage display library that has been widely used in the literature (Eteshola, 2010; Goletz, et al., 2002; Kim, Wang, et al., 2014; ten Haaf, et al., 2015; Wang, et al., 2012). Both Tomlinson libraries (I+J) are housed within the phagemid pIT2 vector (Figure 3-1) that has features which dictate both the expression and purification methods. The ScFv expression cassette is under the control of the lac promoter, which is induced by the addition of IPTG to cultures. Through the inclusion of an amber stop codon between the ScFv and pIII coding sequences, the construct can produce ScFv-pIII fusion for phage display, or alternatively, soluble ScFv when expressed in a non-amber suppression strain such as HB2151. Historically, the expression of ScFv using E. coli expression systems has been hampered by stability and yield problems, often resulting in misfolded proteins contained in inclusion bodies. The inclusion of the PelB signal peptide encoding sequence in the pIT2 vector directs the expressed ScFv to be exported to the bacterial periplasmic space, a more favourable environment for protein folding.

Various purification methods are commonly used for the purification of ScFv. The approaches can be broadly divided into two categories: 1) Classical chromatographic methods such as affinity, ion exchange, hydrophobic interactions and size exclusion, and 2) Alternative methods such as metal chelate affinity precipitation, and macroporous cryogels (Malpiedi, Díaz, Nerli, & Pessoa Jr, 2013). One method of antibody purification, immobilised metal affinity chromatography (IMAC), relies on the attraction between the engineered polyhistidine motif within the protein of interest, and immobilised metal ions, usually Ni2+ or Co2+. The main advantage of this approach is the ability to conduct the purification process under native or denaturing conditions. Advantageously, denaturing conditions can be utilised to recover misfolded proteins

68 Chapter 4: ScFv expression and purification

from inclusion bodies. There are several ways in which this could be achieved, usually the ScFv within the inclusion bodies are encouraged to refold naturally upon stepwise dialysis in a favourable environment (Sinacola & Robinson, 2002). However, this process is often time consuming and requires large volumes of buffer. Also, several parameters such as, high binding, low cost, and mild elution conditions makes IMAC attractive for ScFv purification (Block et al., 2009). However, bacterial proteins containing sequential histidines can also bind to the IMAC resin, resulting in low specificity within the IMAC system. Consequently, additional polishing steps are often used in conjunction to IMAC, such as size exclusion chromatography (SEC) or ion exchange chromatography. Another affinity purification method which can be employed exploits the fact that some bacteria express natural antibody binding proteins on their surface. Protein-L has a high affinity to the various classes of immunoglobins, including fragments such as ScFv (Björck, 1988). A major advantage of using protein- L in purification is that it does not require any additional purification motifs such as the polyhistidine tag, as in the case of Ni-NTA, as it binds through κ-light chain interaction (Nilson, Solomon, Björck, & Akerström, 1992). In addition, there is evidence to suggest that purification by protein-L results in a greater purity in comparison to Ni-NTA (Das, Allen, & Suresh, 2005; Malpiedi, et al., 2013).

Sequencing results from the last chapter have revealed that out of the 4 unique ScFv encoding sequences, 3 contain a premature amber stop codon (TAG) within the main ScFv (CDH2) sequence. This stop codon was suppressed during phage display using the E. coli strain TG1, allowing for the display of the full length ScFv-pIII fusion. However, for large scale periplasmic expression producing soluble ScFv lacking pIII a non-suppressive E. coli strain such as HB2151 is required. Therefore, site-directed mutagenesis was needed to mutate the amber stop codon (TAG) to one encoding glutamic acid (GAG) and enable expression of the full length ScFv.

The aims of this chapter are to:

 Mutate the extraneous amber stop codon interrupting the translation of the CDH2 domain of ScFv in selected clones to allow for expression in the non-amber suppressive E. coli strain HB2151.

Chapter 4: ScFv expression and purification 69

 Express the 4 ScFv using E. coli expression system and to purify ScFv for both structural and functional characterisations.

4.2 EXPERIMENTAL PROCEDURES

4.2.1 Materials The affinity chromatography resin nickel-nitrilotriacetic acid (Ni-NTA), QIAquick Gel Extraction Kit and QIAprep Spin Miniprep Kit were purchased from Qiagen (Chadstone, Australia). For protein purification and detection, protein-L agarose and HRP-conjugated protein-L were sourced from Thermo Fisher (Scoresby, Australia). SnakeSkin™ dialysis tubing (MWCO 3,500 Da), Slide-A-Lyzer™ dialysis cassettes (MWCO 10,000 Da) and Slide-A-Lyzer™ mini dialysis devices (MWCO 3,500 Da) were also sourced from Thermo-Fisher. The Q5® Site-Directed Mutagenesis Kit was acquired from NEB (Ipswich, US).

4.2.2 Site directed mutagenesis Extraneous amber stop codons in ScFv coding sequences were mutated using site- directed mutagenesis to alter the codon sequences from TAG to GAG, which encodes for glutamic acid. Clones which contained an interrupted CDS are: α-αv clone A2, α- αv B1 and α-IGF-1R. The Q5® Site-Directed Mutagenesis Kit was used (NEB). The workflow is summarised below in Figure 4-1.

Mutagenic primers were designed using NEBaseChanger (http://nebasechanger.neb.com/). The original and the desired DNA sequence were submitted into the program and the resulting primers and melting temperature outputs are listed in Table 4-1. Each mutagenesis reaction consisted of the DNA polymerase master mix, forward mutagenic primer, reverse mutagenic primer and plasmid DNA of α-αv-A2, α-αv-B1, or α-IGF-1R independently. The reaction mix and volumes are listed in 8. Each complete reaction mix was incubated in a thermocycler according to the conditions in

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Table 4-2. The post PCR samples were separated on a 1% agarose gel to visualise amplification of the ~5,000 bp pIT2 phagemid vector and check for non- specific amplification. The band corresponding to the correct size was excised and a gel extraction performed using the QIAquick® Gel Extraction Kit (Table 4-4). After PCR cycling, the linear PCR products contained a mixture of unmodified (TAG) and modified (GAG) DNA. In order to remove the template sequence, and to re-circularise the mutated product for transformation into E. coli, the PCR product is treated with a proprietary mixture of kinase, ligase and the restriction enzyme DpnI (KLD) as listed in (Appendix 8). The circularised vectors were transformed into competent HB2151 E. coli cells using the standard heat-shock protocol.

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Figure 4-1: Site-Directed Mutagenesis workflow. A) Exponential PCR amplification using the NEBaseChanger™ designed mutagenic primers with the mismatched based highlighted as a star. B) The inverse PCR yields a linear product with the mutated base in both DNA strands. C) The kinase, ligase and Dpn1 (KLD) reaction. The kinase phosphorylates the 5’ end of the PCR product (C.1), which allows the recirculation of the product by ligase (C.2). The Dpn1 restriction digest cleaves the template DNA which is methylated (C.3). D) The final product is listed. The forward primer is coloured blue while the reverse primer is in orange.

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Table 4-1: Mutagenic primers for Site-Directed Mutagenesis Target Action Sequence (5’ to 3’) Ta A2 A2-mut-TAG→GAG F TGTGAAGACAgAGTACGCAGAC 61 A2-mut-TAG→GAG R CCCGTCGAAGTAATAGATG °C

B1 B1-mut-TAG→GAG F TATTCCTCGGgAGGGTCGGAA 66 B1-mut-TAG→GAG R GTTGAGACCCACTCCAGC °C

IGF-1R IGF1R-mut-TAG→GAG GATTTCTCAGgAGGGTGATTATA 61 F C °C IGF1R-mut-TAG→GAG CTTGAGACCCACTCCAGC R

Table 4-2: Site-Directed Mutagenesis PCR cycling conditions Step Temp Time Initial Denaturation 98 °C 30 seconds 25 Cycles 98 °C 10 sec A2, IGF-1R, 61 °C 10-30 sec B1, 66 °C 72 °C 20-30 seconds/kb Final Extension 72 °C 2 minutes Hold 4-10 °C ∞

4.2.3 Preparation of competent HB2151 E. coli The E. coli strain HB2151 were made chemically competent using the calcium chloride method; briefly, single colonies were obtained by streaking a glycerol stock of HB2151 onto a TYE plate and incubating at 37 °C overnight. A single HB2151 colony was picked from the plate to initiate an overnight starter culture in 5 mL of 2YT medium and again cultured overnight at 37 °C but with shaking at 250 RPM. This overnight culture was diluted 1:100 in a total volume of 100 mL of 2YT and shaken at 250 RPM while being maintained at 37 °C for 3 hours to allow for exponential growth. The E. coli culture was chilled on ice and subsequently harvested in a 4 °C centrifuge at 3,000 g. The resulting pellet was resuspended in 10 mL of cold 100 mM CaCl2 and placed on ice for 20 mins. The HB2151 was again pelleted and resuspended as above and placed on ice for an additional 20 mins. The competent cells were harvested by a final centrifugation and resuspended in 5 mL of 100 mM CaCl2 supplemented with 15% (v/v) glycerol. The competent HB2151 were aliquoted and frozen at -80 °C until required.

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4.2.4 Transformation An aliquot consisting of 50 µL competent HB2151 cells was thawed on ice and 5 µL of post KLD treated DNA (see 4.2.2) from each clone was added. The samples were incubated on ice for 30 mins followed by a heat-shock step at 42 °C for 20 sec in a water bath. The tubes were chilled on ice for 2 mins and 500 mL of SOC medium added to each tube. The tubes were incubated at 37 °C with shaking at 250 RPM for 1 h. Subsequently, the samples were plated onto TYE AG plates and incubated overnight at 37 °C. Single colonies were picked from each plate, cultured overnight in 2xYT and plasmids extracted using the QIAGEN Mini-Prep Kit according to the manufacturers instructions. The purified DNA were sent for sequence verification by AGRF using LMB3 (5’ CAG GAA ACA GCT ATG AC 3’) and pHEN (5’ CTA TGC GGC CCC ATT CA 3’) sequencing primers.

4.2.5 Induction time pilot study An overnight starter culture was initiated from α-αv clone A2 glycerol stock. Five millilitres of 2xYT containing 100 µg/mL ampicillin and 4% glucose (2xYT AG) was inoculated from frozen E. coli stock using a sterile pipette tip and shaken at 250 RPM, 37 °C overnight. The resulting cultures were diluted 1:100 into 500 mL of 2xYT AG and placed in a 37 °C incubator and shaken at 250 RPM for an additional 2 h. The cell cultures were centrifuged at 3,200 g and the resulting pellet was resuspended in 500 mL of 2xYT AG with 1 mM IPTG. The incubation temperature was lowered to 30 °C and the culture shaken at 250 RPM. At 3 h intervals, a sample was taken for Western blot analysis to confirm the expression of ScFv, and to determine the ratio between periplasmic and supernatant located ScFv.

4.2.6 Detection of the expressed ScFv by Western blotting Samples collected at each step of the expression and purification processes were separated based on molecular weight using Nu-PAGE SDS-PAGE and immune- blotted as per section 2.7.1. The expressed ScFv was detected using HRP conjugated protein-L, which binds to the kappa light chain of immunoglobins, including truncated forms such as ScFv.

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4.2.7 Large scale ScFv expression A starter culture was made by inoculating 5 mL of 2xYT AG from glycerol stock and incubating overnight at 37 °C and 250 RPM. The next morning, the culture was diluted into 500 mL of pre-warmed 2xYT AG in Erlenmeyer baffled cell culture flasks and shaken at 37 °C, 250 RPM for 2.5 h. At this point, the culture was harvested and centrifuged at 3,300 g for 10 mins. The resulting pellet was resuspended in 2xYT A and supplemented with 1 mM IPTG. The culture was incubated at 30 °C with shaking at 250 RPM for an additional 9 hours. The 9 hours post inducting time point was chosen from the result of the induction time pilot (section 4.2.5).

4.2.8 Periplasmic extraction The phagemid vector pIT2 was engineered to encode the signal peptide PelB which allows for the expressed ScFv to be directed to the bacterial periplasmic space. To selectively lyse the outer membrane, a periplasmic extraction procedure was used, which is based on lysis by osmotic shock. The E. coli expression culture was centrifuged at 3,300 g for 10 mins. The supernatant was separated and the pellet resuspended in 25 mL of pre-chilled periplasmic extraction solution 1 (20 % (w/v) sucrose, 100 mM Tris-HCL [pH 8.0] and 1 mM EDTA). The suspension was slowly mixed using a magnetic stirrer at 4 °C for 30 mins. After the incubation, the solution was centrifuged at 10,000 g for 10 mins and the supernatant poured off and retained. The pellet was resuspended in 25 mL of periplasmic extraction solution 2 (5 mM

MgCl2) and stirred in the cold room for an additional 20 mins. The supernatant was obtained by an additional centrifugation at 10,000 g for 10 mins. The supernatant post extraction 1 and 2 were combined and stored at 4 °C for further processing.

4.2.9 Precipitation of ScFv from culture medium using PEG Protein was extracted from the culture supernatant using PEG 6000 as the precipitation agent. Culture medium containing expressed ScFv was incubated with an increasing concentration of PEG 6000 from 0 % to 20 % (w/v) with rotation at 4 °C for 1 h. The samples were centrifuged for 10 mins at 10,000 g to obtain the precipitated proteins. Samples taken throughout the procedure were separated by molecular weight

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on SDS-PAGE, immunoblotted onto nitrocellulose, and ScFv was detected using protein-L HRP as per section 4.2.6.

4.2.10 Purification using Ni-NTA affinity chromatography Approximately 50 mL of periplasmic extracts were dialysed using SnakeSkin™ Dialysis Tubing (Thermo-Fisher) against 10 L of Ni-NTA native binding buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) overnight at 4 °C. The dialysed ScFv extracts were added to 1 mL of Ni-NTA resin (equilibrated with binding buffer) and rotated at 4 °C for 1 h. The Ni-NTA was retained by gravity flow into a disposable chromatography column. The column was washed once with 10 mL of Ni-NTA wash buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). The bound ScFv were eluted from the column using the Ni-NTA elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) with 500 µL fractions collected.

4.2.11 Small scale purification using protein-L agarose Agarose conjugated with protein-L was employed as an alternative method of purifying ScFv. Rather than relying on an affinity tag such as the 6x histidine for Ni- NTA based purification, protein-L interacts with the kappa light chain of immunoglobulins, including ScFv. To determine if protein-L can improve yield or purity of the isolated ScFv, a test Ni-NTA eluate fraction from section 4.2.9 was dialysed in Slide-A-Lyzer™ mini dialysis device for 4 h at 4 °C against PBS. It was then applied to a protein-L agarose column equilibrated with PBS and allowed to flow by gravity. The column was washed once with 5 mL of PBS to remove loosely bound proteins. Remaining proteins were eluted with protein-L elution buffer (100 mM of glycine-HCl solution, pH 2.7) and 500 μL fractions were collected with tubes pre- filled with 50 μL of protein-L antibody neutralisation buffer (1 M Tris, pH 8.0). Samples were interrogated for purity with SDS-PAGE and Western blotting as per section 4.2.6.

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4.2.12 Large scale protein-L agarose expression and purification from culture medium The procedure for large scale protein-L agarose expression is similar to the previously described sections (4.2.7 – 4.2.11), with some modifications. Rather than harvesting the E. coli at 9 hours post induction with IPTG, the culture was maintained overnight at 30 °C with shaking at 250 RPM. The supernatant was harvested by centrifugation at 3,300 g for 10 mins and dialysed against 10 L of PBS overnight at 4 °C. The material was applied to a pre-equilibrated protein-L agarose column and allowed to flow by gravity. The column was washed with 5 mL of PBS and eluted with protein-L elution buffer in 500 μL fractions and immediately neutralised with 50 μL of protein-L antibody neutralisation buffer. At each step of the purification process, a sample was retained for assessment by silver stained SDS-PAGE for quality control.

4.2.13 Expression of αIGF-1R ScFv using EnPresso B EnPresso B is a proprietary expression medium system for E. coli claiming to guarantee an expression yield increase of at least 5-fold over traditional media such as LB medium. A starter culture was initiated by inoculating ScFv transformed E. coli into 5 mL of 2xYT A from α-IGF-1R ScFv glycerol stock and incubated at 37 °C with shaking at 250 RPM for 6 h. The EnPresso B medium was reconstituted by adding the contents of two satchels to 100 mL of sterile water into a 1 L Erlenmeyer baffled cell culture flask and supplemented with ampicillin to a concentration of 100 μg/mL. The starter culture and 50 μL of proprietary Reagent A was added to the shaker flask and incubated overnight at 30 °C with shaking at 250 RPM. Next, IPTG was added to a final concentration of 1 mM, along with 2 proprietary booster tablets and 50 μL of Reagent A. The culture was again incubated with shaking as previous for an additional 24 h. After the incubation period, the culture was harvested and centrifuged at 3,300 g for 10 mins. The supernatant was retained and subjected to the protein-L agarose purification regime as per section 4.2.12.

4.2.14 Protein Quantification The protein concentration of the purified ScFv was estimated using the Bicinchoninic Acid (BCA) protein assay kit (Pierce) as per the manufacturer's

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instruction. The protein BSA was used as a protein standard diluted in PBS to create a concentration range from 125 μg/mL to 2000 μg/mL. The BCA reaction plate was read at 562 nm using the Bio-rad benchmark plus microplate spectrophotometer system. The resulting data was curve fitted using the Microplate Manager Software version 6.2 (Bio-rad). BSA was chosen as the concentration standard rather than an IgG such as bovine gamma globulin (BGG) because ScFv lack the post-translational modifications of the constant region of an IgG (Huhn, Selman, Ruhaak, Deelder, & Wuhrer, 2009). Therefore, BSA is more representative of ScFv than an intact IgG for protein quantification.

4.3 RESULTS

4.3.1 Amber stop codon modification using Site-directed mutagenesis Of the 4 unique receptor binding ScFv candidates identified by ELISA (3.3.4 and 3.3.5), 3 had an amber stop codon within their CDS. Since the phage display selection was conducted in TG1 E. coli, a suppressive strain, this premature stop codon was suppressed. However, to facilitate the periplasmic expression of the ScFv in a non- suppressive strain such as HB2151, the sequence encoding for the extraneous amber stop codons (TAG) was mutated to one encoding glutamic acid (GAG) using site- directed mutagenesis. Candidates which underwent SDM were α-αv-A2 and B1 and α-IGF-1R. After the mutagenic PCR, each reaction was separated on an agarose gel to confirm amplification of the correct 5 kb plasmid product (Appendix ) and the band was subsequently excised using a gel extraction kit. Unexpectedly, an additional band of 750 bp was detected in the sample B1 reaction which was still present when the reaction was repeated. However, given that the 5 kb product was present, that band was excised and used in the KLD treatment to re-circularise the PCR product and also to remove the original template.

The DNA sequencing results of the procedure are shown in Figure 4-2. The sequencing data demonstrated that the SDM steps were successful in changing the selected thymine base to guanine and thus modifying the amber stop codon to encode a glutamic acid residue. The sequence verified clones were transformed into

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chemically competent HB2151 E. coli strains for stock generation and expression studies.

Before

After

Before

After

Before

After

Figure 4-2 Modification of amber stop codon sequence from ScFvs.

The sequencing data and the translated amino acid sequence from α-αv-A2 and B1 and α-IGF-1R clones pre and post site-directed mutagenesis (SDM). The highlighted region shows the location of the mutagenesis in which the thymine base, as part of the amber stop codon (TAG), was mutated to a guanine (GAG), encoding for glutamic acid.

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4.3.2 Post-induction ScFv expression time course In order to determine the optimum time for harvesting of the expression culture post induction with IPTG, an expression time course was conducted using α-αv clone A2. Samples were collected at 0, 3, 6 and 9 hours post IPTG addition to the culture media. The harvested E. coli were centrifuged to separate the pellet and supernatant fractions. The E. coli pellet was resuspended in PBS to the same starting volume of the original sample. The total, supernatant and pellet samples were resolved by SDS- PAGE, blotted onto a nitrocellulose membrane and probed using protein-L HRP to detect the presence of the expressed ScFv (Figure 4-3).

The resulting blot showed that at 3 hours post induction, the expressed ScFv could be detected in the total and pellet samples, most likely present within the periplasmic space. Such a result is to be expected due to the pIT-2 phagemid encoding the signal peptide PelB, which directs the translated protein into the periplasm. Over the period monitored (9 hours), the total yield of ScFv increased in a time depended manner. Though the expressed ScFv could be found in high concentration within the pellet, a greater total ScFv could be found in the culture medium, which could be recovered using a more involved purification approach.

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Figure 4-3: Time course trial of ScFv expression.

Clone α-αv A2 expressing E. coli were cultured and induced with IPTG. At time 0 and every 3 hours after, samples were taken (total) and centrifuged to separate the culture medium (supernatant) and E. coli (pellet). It was expected due to the PelB signal peptide that most of the α-αv A2 ScFv detected within pellet are in the periplasmic space with the remaining ScFv located in the cytosol. The samples were separated by SDS-PAGE and probed with protein-L conjugated HRP in a Western immunoblot.

4.3.3 Precipitation of proteins from culture medium using PEG formed insoluble products Even though the PelB signal peptide directs the expressed ScFv to the bacterial periplasm, some ScFv could be detected in the culture supernatant, as observed in Figure 4-3. To determine if the expressed ScFv in the culture medium could be recovered for further purification, a protein precipitation procedure using PEG 6000 was employed. Precipitation of the ScFv from culture medium has a distinct advantage because it minimises the volume of liquid for processing by downstream purification processes in addition to less protein complexity compared to a periplasmic extract.

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Test culture medium samples containing expressed ScFv were incubated with various increasing concentrations of PEG 6000 (0-20% (w/v)) and centrifuged. Samples were taken of the resulting supernatants to determine if the ScFv remains in solution and at what minimum PEG % the ScFv were no longer observed. Figure 4-4 (A) shows ScFv could still be detected at PEG concentrations of 5 and 10 % (w/v). When the PEG concentration increased to 15%, ScFv could not be detected in the supernatant and was thus inferred to have precipitated. Subsequently, expressed culture medium was incubated with 15% (w/v) PEG 6000, incubated at 4 °C with rotation for 1 h and centrifuged. The supernatant and pellet were separated and PBS added to the pellet for re-suspension but it remained insoluble, requiring the direct addition of SDS-PAGE sample buffer and heating in order to solubilise for SDS-PAGE analysis. In order to confirm the plausibility of the approach, 15% (w/v) PEG was added to a fresh batch of expression culture media, incubated, centrifuged and separated into supernatant and pellet samples and probed with protein-L HRP in an immunoblot as previously described (Figure 4-4 (B)). ScFv was detected in the pre- treatment culture media (-PEG) while a reactive band could not be detected in the supernatant sample (+PEG). Furthermore, the pellet sample (PPT) could be observed to contain a strong immuno-reactive band equivalent to the expected MW of ScFv. Together, these data demonstrate high efficiency of the precipitation reaction. The insoluble pellet suggests the proteins had aggregated and additional steps would be required if this precipitation approach is to be used.

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Figure 4-4 Precipitation of ScFv by PEG 6000.

(A) Culture medium containing expressed ScFv was incubated with increasing concentrations of PEG 6000, ranging from 5 to 20 % (w/v). The reactions were incubated at 4 °C with rotation and centrifuged. The expressed ScFv could not be detected at or above a PEG concentration of 15 % (w/v). (B) Samples of the expression culture media starting material (-PEG), supernatant (+PEG) and pellet (PPT) of 15% PEG treated expression media were subjected to SDS-PAGE and immunoblotted against protein-L HRP. Protein-L HRP-reactive ScFv is only observed in the starting material and PEG treated pellet.

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4.3.4 Optimisation of Ni-NTA purification with the addition of urea Initially, purification of expressed ScFv was undertaken by Ni-NTA-based affinity chromatography to selectively bind the 6xHis C-terminal tag encoded by the expression construct, a system widely used with success (Zhao, Chan, Lee, & Cheung, 2009). Unexpectedly, initial preliminary purification runs using periplasm extracts from an α-IGF-1R ScFv expression culture failed to produce pure protein in the Ni- NTA elution fractions (Figure 4.5 (A) and could be found in the flow-through sample. As ScFv was detected in the flow-through sample, it is plausible to assume the running and sample conditions are unfavourable for Ni-NTA binding. A common reason for proteins not to bind the Ni-NTA moiety is that the proteins adopted a folded conformation that hinders access to the polyhistidine tag. In an attempt to circumvent this problem, many optimisation steps were performed (Appendix 9). Finally, the addition of 1M urea to the starting periplasmic extract, in order to disrupt potential hydrophobic interactions among ScFv molecules, before addition to the Ni-NTA packed column was found to be successful in enabling efficient protein binding.

Samples were taken at each step of the purification process and interrogated using silver-stained SDS-PAGE and Western blot using protein-L HRP. In the process without urea (Figure 4-5 (A)), the silver-stained SDS-PAGE shows that no band (equivalent to the ScFv or otherwise) was observed in the elution fraction. This was confirmed by Western blot which revealed effectively equal amounts of ScFv (protein- L HRP-reactive band of ~28 kDa) could be found in the load and flow-through samples, suggesting the ScFv is unable to bind Ni-NTA under these conditions. On the other hand, with the addition of 1M urea (Figure 4-5 (B)), a single protein band of the predicted molecular weight could be detected in the elution fraction of the silver- stained SDS-PAGE and is reactive to protein-L HRP, suggesting that the band is the expressed ScFv. Unlike the purification procedure without urea, the absence of detectable ScFv in the flow-through shows that the binding process between the ScFv and the affinity resin is efficient. The results demonstrate that the addition of 1M urea to the starting load facilitates the binding of the ScFv to Ni-NTA. Based on this result, 1 M urea was included for ScFv purification using the Ni-NTA system.

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Figure 4-5: Improved Ni-NTA affinity purification of α-IGF-1R ScFv with the addition of 1 M urea.

Periplasmic extracts of an α-IGF-1R ScFv expressing E. coli culture were subjected to purification by Ni-NTA and interrogated with silver stained SDS-PAGE (left) and Western blotting with protein-L (right). The starting sample (load) without 1M urea (A) and with 1M urea (B) were passed through the Ni-NTA packed column with the unbound materials (Flow-through) retained. Non-specific binding proteins were removed by flowing binding buffer through the resin (Wash). The column was eluted by the addition of 250 mM imidazole in binding buffer (Elution).

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4.3.5 Large scale Ni-NTA purification of ScFv from periplasmic extracts Once the conditions for the Ni-NTA purification process were optimised, a large scale expression and purification was conducted with the 4 ScFv clones as follows; each expression culture, consisting of 500 mL total volume, was harvested 9 h post induction by IPTG and a cell pellet obtained by centrifugation. The expressed ScFv housed in the periplasmic space of the E. coli was harvested by osmotic shock and the resulting extract dialysed against Ni-NTA binding buffer. Urea was added to a final concentration of 1M before the addition of pre-equilibrated Ni-NTA resin for batch chromatography. Samples were taken at each stage of the process to determine the efficiency of the purification process and were subjected to silver-stained SDS-PAGE analysis.

The resulting gels (Figure 4-6) show all 4 ScFv could be purified to a high degree using Ni-NTA. In all 4 expressions, there was minimal protein found in the wash fraction, suggesting minimal non-specific interaction between the affinity resin and the crude sample, and also that any bound ScFv was strongly attached.

Each ScFv clone exhibited a slightly different elution profile, with most ScFv found in the 2nd or the 3rd elution fractions. The positive elution fractions for each ScFv (A2: E2-3, B1: E3, F3: E1-3, IGF-1R: E2-3) were pooled and underwent dialysis against PBS overnight followed by filtration with a 0.22 µm device to sterilise the sample. Each ScFv was aliquoted and stored at -20 °C.

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Figure 4-6: Purification of ScFv from E. coli periplasmic extracts using Ni-NTA.

E. coli clones expressing ScFv α-αv-A2 (top left), B1 (top right) and F3 (bottom left) and α-IGF-1R (bottom right) were harvested and the periplasmic extracts obtained by osmotic shock. The samples were dialysed against Ni-NTA binding buffer overnight and urea was added to a final concentration of 1M. Each sample was incubated with Ni-NTA in batch purification. The column was washed once with Ni-NTA binding buffer. The bound protein was released using Ni-NTA elution buffer. L= Load, F = Flow-through, W= Wash, E1-E5 = Elution fractions 1-5.

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4.3.6 Verification of the final Ni-NTA affinity chromatography purified ScFv The pooled, dialysed and filtered samples of the 4 purified ScFv were subjected to silver-stained SDS-PAGE and Western blot analysis to verify the identity and the purity of the final product. The results, shown in Figure 4-7, reveal all 4 ScFv were purified to a high degree (≥90%) when assessed using silver stained SDS-PAGE. Additional protein bands were detected in the α-IGF-1R ScFv sample, indicating the sample contained impurities. Western blot analysis using protein-L HRP demonstrated the protein bands observed in the silver-stained SDS-PAGE (~28 kDa) were reactive to protein-L, while the impurities found in the α-IGF-1R ScFv sample were not reactive, confirming their status as impurities. However, based on the staining intensity of the ScFv band in both SDS-PAGE and Western blot, the vast majority of the sample contained the α-IGF-1R ScFv.

Figure 4-7: Verification of the final Ni-NTA purified ScFv by silver-stained SDS- PAGE and Western blotting.

Elution fractions from the Ni-NTA purification process which were positive for ScFv were combined and dialysed overnight against PBS. The ScFv solutions were filtered through a 0.22 µm syringe filter and samples were subjected to silver-stained SDS- PAGE and Western blotting (protein-L HRP) analysis.

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4.3.7 Improved purification using protein-L agarose During the process of Ni-NTA optimisation, alternative purification methods were simultaneously investigated in the hope of increasing yield while also improving purity and robustness of the purification process. One such alternative is another affinity approach utilising protein-L conjugated to agarose beads. Much like protein- A, which has been used extensively in the purification of antibodies, protein-L binds to the immunoglobulin class of molecules, instead of a specific affinity tag. Specifically, protein-L binds to the kappa light chains of immunoglobins, which ScFv contain. It was not chosen as the primary purification method due to the considerable cost of obtaining the reagent. A 2 mL lot of protein-L agarose cost $478.40 AUD to purchase, which is considerably more than the Ni-NTA resin. Nevertheless, to test if the protein-L purification method would provide a significant improvement over Ni- NTA, α-BSA control ScFv was purified using the standardised Ni-NTA procedure followed by re-processing of the Ni-NTA elution fraction with the protein-L purification strategy (section 4.2.11). The results were electrophoresed onto SDS- PAGE and silver-stained (Figure 4-8).

The result showed the protein-L affinity purification method can improve the purity of ScFv to a greater degree in comparison to Ni-NTA. Ni-NTA elution fraction 2 is observed to contain two major protein bands. One is consistent with the molecular weight of ScFv (28 kDa), and another unknown band between 37 and 50 kDa. This elution fraction was reprocessed using protein-L affinity chromatography and the result showed that this higher molecular weight band was found in the flow-through and did not bind to protein-L. The protein-L elution fractions clearly contain a higher purity product as evident by the single band in lanes E2 and E3 (Figure 4-8). To further test the purity of the protein-L eluted fraction, the sample was concentrated using a microfuge protein concentrator with a molecular cut-off of 7.5 kDa. Any contaminating proteins that were below the detection limit of the silver stain in the elution fraction should also be enriched and become visible once the concentrated sample is interrogated. The resulting concentrated sample (lane ‘Conc elutions’ Figure 4-8) did not exhibit any additional bands, indicating a very high level of purity was achieved using the protein-L affinity chromatography process.

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Figure 4-8: Two step purification of α-BSA ScFv using Ni-NTA and protein-L conjugated agarose.

The periplasmic extract containing the expressed ScFv was processed using Ni-NTA affinity chromatography. The resulting eluate fraction E2 was further processed by protein-L agarose affinity chromatography. The protein-L agarose elution fractions E2 and E3 were combined and concentrated using a spin column concentrator (Conc elution).

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4.3.8 Large scale purification of ScFv from medium using protein-L agarose Previous purifications were conducted using the periplasmic extract. Given that protein-L has shown improved binding of ScFv over the Ni-NTA based system, in addition to yield improvements, all subsequent ScFv batches were purified from the supernatant containing the expressed ScFv using protein-L as per section 4.2.11. Samples were taken from each step of the purification process, and the resulting silver- stained SDS-PAGE (Figure 4-9) showed that a band corresponding to the correct molecular weight (28 kDa) was detected in the elution fractions, indicating that all three ScFv clones targeting the αv integrin (A2, B1, F3) were successfully purified.

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Figure 4-9: Large scale purification of ScFv using protein-L agarose.

E. coli supernatant from expression cultures of α-αv-A2, B1 and F3 ScFv were harvested by centrifugation. After overnight dialysis against PBS, samples were applied to protein-L agarose. The column was washed with 5 mL of PBS and eluted with protein-L elution buffer in 500 μL fractions and immediately neutralised with 50 μL of protein-L antibody neutralisation buffer. Samples were separated using SDS- PAGE and silver stained. L= load, F=Flow-through, W=wash, E1-E5 = Elution fraction 1-5.

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4.3.9 Expression and purification of αIGF-1R ScFv using EnPresso® medium The EnPresso® B expression medium is a proprietary medium from BioSilta which guarantees a minimum 5-fold increase in yield in comparison to an E. coli expression culture using traditional LB medium. According to the patent document, the key technology driving this medium was termed enzyme-based fed-batch (EnBase) (Neubauer, Neubauer, & Vasala, 2010) which consists of a controlled release of glucose into the medium by breaking down a glucose-polymer enzymatically.

To assess whether this medium can streamline the production of ScFv, an expression culture was conducted using EnPresso® B and the α-IGF-1R ScFv clone. The expression culture supernatant was harvested via centrifugation and passed through protein-L agarose. Samples were taken at each step of the purification process for analysis using silver-stained SDS-PAGE and Western blotting, with the result shown in Figure 4-10. The purification process was successful in purifying the ScFv in the 1st pass of the protein-L agarose with a single band of the expected MW (28 kDa) in elution fractions 2 and 3 (E2 and E3). There was only a small decrease in the intensity of bands between the load and the flowthrough in the Western blot of pass 1, suggesting either that the binding efficiency was low, or that the resin had reached binding capacity. Therefore, the flow-through from pass 1 was reprocessed onto regenerated protein-L agarose. Again, samples were taken at each step and both the silver stained gel and Western blot shows pure ScFv was isolated predominantly in elution fractions 2 and 3. When comparing the load and the flow-through, there is a substantial reduction in band intensity, suggesting that indeed the binding capacity was reached in pass 1 and that a 2nd pass was required to isolate the majority of expressed protein. The positive elution fractions from pass 1 (E2, E3) and pass 2 (E2, E3) were combined and dialysed against PBS. The total yield obtained, as deduced by BCA protein estimation, from 100 mL of Enpresso Medium was 2.2 mg, which translated to a recovered yield from the supernatant to ~22 mg/L. Given that additional ScFv within the E. coli remains (periplasm), further purification could be performed by conducting a periplasmic extraction on the E. coli pellet and combining it with the supernatant.

Chapter 4: ScFv expression and purification 93

Figure 4-10: Expression of α-IGF-1R ScFv using EnPresso® medium and purification using protein-L agarose

The supernatant from α-IGF-1R ScFv expressing E. coli cultured using EnPresso® medium were flowed onto protein-L agarose. Samples were taken at each stage of the purification process and assessed using silver-stained SDS-PAGE and Western blotting (protein-L HRP). Given the large quantity of ScFv detected in the flow- through of pass 1, it was flowed over protein-L agarose for an additional pass.

94 Chapter 4: ScFv expression and purification

4.4 DISCUSSION

The chapter herein described the successful expression and purification of the 4 ScFv generated through the phage panning process detailed in Chapter 3.

Carrying over from the selection of specific binding clones was the fact that 3 of the 4 contained an amber stop codon within the CDRH2 coding region. The presence of amber stop codons within the Tomlinson I & J libraries can be traced back to the methods of their mutagenic generation. The diversity of the Tomlinson phage display libraries (I+J) was generated by mutagenesis (Tomlinson & Winter, 2005). The CDRs of library ‘I’ have been generated using DVT triplets, targeting a total of 18 resides (D = adenine, guanine, thymine, V = adenine, guanine, cytosine, T = thymine). For library ‘J’ CDR variation was generated by NNK triplet mutations (N = any base, K = cytosine, guanine). As a direct consequence of the NNK mutation scheme, there is a 1/32 chance for insertion of an amber stop codon (TAG) into the CDR coding sequence (Cwirla, Peters, Barrett, & Dower, 1990), prematurely stopping the translation of the ScFv in non-suppression strains. Of the 4 ScFv candidates as a result of the phage display process, 3 contained an amber stop codon and each was successfully mutated by SDM to encode for glutamic acid in place of the amber stop codon (Section 4.3.1). Mutagenesis to glutamic acid was chosen to maintain homogeneity to the residue encoded by the amber stop codon in a suppressive E. coli strain as used during panning.

The signal peptide pelB was successful in the transport of the expressed ScFv into the periplasmic place as evident by the success of the periplasmic extraction process (Figure 4-3). However, some ScFv was found to have ‘leaked’ into the supernatant. This is consistent with other studies which observed that protein destined for the periplasm could be detected in the supernatant (Kipriyanov, Moldenhauer, & Little, 1997; Ukkonen, Veijola, Vasala, & Neubauer, 2013). In some studies, only the periplasmic fractions were used while in others the supernatant and the periplasmic extracts were combined in the downstream purification processes, depending on their yield requirements (Das, et al., 2005; Patil, et al., 2015; Rouet, et al., 2012; Wang, et al., 2012). The periplasmic extraction process required several incubation steps followed by centrifugation. In contrast, isolation from the supernatant only required

Chapter 4: ScFv expression and purification 95

the removal of the E. coli cells. In addition, there is also a reduction on E. coli host proteins using the supernatant as the starting material, minimising possible downstream contamination during purification steps. Given the potential in the total recoverable ScFv in the supernatant in addition to the reduced purification steps, the periplasmic extraction procedure was abandoned in favour of purification directly from the supernatant.

The Ni-NTA affinity purification process required extensive optimisation as the 6x histidine tagged ScFv were not binding to the resin. After numerous repeats, it was determined that the addition of 1M urea to the periplasmic extract containing the expressed ScFv was required for it to bind to the resin. A possible explanation is that since ScFv is prone to form aggregates, such a structural arrangement potentially makes the histidine tag inaccessible and therefore cannot bind to the Ni-NTA resin. Urea is a potent denaturant and 8 M urea is commonly used for Ni-NTA purification under denaturing conditions. The 1 M urea used should not completely denature the ScFv but allowed the histidine tag to be accessible. A study by Chen et al. (2006) purified ScFv from E. coli inclusion bodies under denaturing conditions with 8 M urea. The protein was refolded with buffer containing 1 M urea and subsequent dialysis against PBS. Their refolding protocol yielded a high proportion (70%) of soluble product. Given that 1 M urea was used instead of 8 M, it is likely that the ScFv was not structurally damaged by the use of 1M urea.

The formation of aggregates could be the reason why the PEG precipitation procedure was unsuccessful. To test for the presence of aggregates, various techniques could be employed, including size exclusion chromatography (SEC) using physiological buffers such as PBS or dynamic light scattering (DLS). Indeed, this will be undertaken in the next chapter.

Two affinity purification resins were used for this project, Ni-NTA and protein- L agarose. Even though the Ni-NTA purification system was able to purify ScFv to an acceptable level for additional characterisation, protein-L agarose was chosen in favour because it yielded a product with even greater purity, achieving a product at

96 Chapter 4: ScFv expression and purification

>90% purity. Initially, Ni-NTA was selected because of the previous experience of using this system and also because of the reduced cost in comparison to protein-L agarose. As an example, 25 mL of Ni-NTA cost 686 AUD while 2 mL of protein-L agarose from Thermo Fisher cost 478.40 AUD, an 8.7 fold increase in the cost per mL for resin of protein-L compared to Ni-NTA sourced from Qiagen. Similar to protein- L, another alternative that can be considered is protein-A, which can also bind to various immunoglobulin molecules, including ScFv (Forsgren & Sjöquist, 1966). Ultimately, protein-L was chosen as the immune-affinity purification method because of its higher binding strength to ScFv than protein-A (Sheng & Kong, 2012).

Improvements in protein yield and the simplification of the antibody fragments expression are sort after in both research and commercial settings. Examples of such attempts are the introduction of additives including L-arginine and sucrose to the culture medium which have been shown to increase expression yield (Kipriyanov, et al., 1997; Schäffner, Winter, Rudolph, & Schwarz, 2001). EnPresso® was recently introduced as a novel expression medium for bacterial and yeast expression. The results were promising, with between 2 and 5 fold increases in expression of single- domain antibodies (Ta et al., 2015; Zarschler, Witecy, Kapplusch, Foerster, & Stephan, 2013). Within the current study the EnPresso® expression medium was able to induce α-IGF-1R ScFv expression to a final yield of ~22 mg/mL, which is much higher than the yield of ~4.954 mg/L of an α-IGF-1R ScFv batch using LB medium previously conducted within this study (results not shown). The lower yield of ScFv using LB medium agrees with the literature values of 1-5 mg/L for ScFv from the Tomlinson libraries (Rouet, et al., 2012). However, given the relative higher price of the medium in comparison to the 2xYT used throughout this chapter, the use of the EnPresso® medium might be situational rather than for general purpose. A use could be during clonal screening of numerous binding candidates, where small volumes of concentrated ScFv are required.

Additional characterisation studies validating the expression process would be a valuable addition to the sample characterisation of the purified ScFv. For example, N- terminal protein sequencing can be performed to ascertain if the cleavage of the pelB signal peptide from the mature ScFv was both successful and complete. This is

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important as nonspecific signal peptide cleavage can occur at unexpected sites, generating potentially undesired ScFv heterogeneity (Kotia & Raghani, 2010). In addition to N-terminal sequencing, mass spectrometry can be included in the analysis workflow. Information that can be generated includes the molecular weight, the amino acid sequence and any degradation products (Mo, Tymiak, & Chen, 2013).

The successful expression and purification of the phage display selected binding candidates has prepared the way for critical structural analyses. Experiments will be undertaken to test the isolated ScFv and determine binding affinity to their target in addition to assessment of structural homogeneity.

98 Chapter 4: ScFv expression and purification

Biophysical characterisation

Chapter 5: Biophysical characterisation 99

5.1 INTRODUCTION

Numerous downstream quality control steps are performed on therapeutic monoclonal antibodies, including purity, identity, stability and structural integrity studies (Beck, Wagner-Rousset, Ayoub, Van Dorsselaer, & Sanglier-Cianférani, 2013). While the purity of the generated ScFv were assessed and deemed acceptable in the previous chapter, SDS-PAGE analysis in a denaturing environment does not give a true indication of the native structural integrity of the ScFv sample. Proteins including IgG and their smaller fragments such as ScFv can form aggregates, as indicated by their non-native quaternary structure (Narhi, Schmit, Bechtold-Peters, & Sharma, 2012). There is a diverse range of protein aggregates that can be classified based on particle size, reversible or non-reversible, status of the conformation, covalent modification or morphology. Aggregation has the potential to affect the function and stability of therapeutic proteins, particularly antibodies (Wang, 2005). Given the potential impact of protein aggregates on function, additional structural studies using analytical size exclusion chromatography (SEC) were conducted to assess the structural characteristics of the purified ScFv under physiologically similar conditions. The detection and quantification of dimers, trimers and higher ScFv aggregates forms the basis of the SEC experiments.

While there is no agreed maximum limit on product aggregation as some proteins might be safe, stable and functional with a certain percentage of aggregates, ScFv dimers/monomers will be monitored and quantified. Given the propensity of ScFv to form dimers (Nieba, Honegger, Krebber, & Plückthun, 1997; Rouet, et al., 2012), it is expected that all of the ScFv samples will contain various amounts of dimer. The formation of dimers can be attributed to many factors, with some intrinsic to the inter-domain amino acid sequences (Zhao et al., 2010), while others can arise from changes in environment experienced during expression and purification (Telikepalli et al., 2014).

In addition to SEC, another parameter which will be assessed is the elucidation of the binding affinity between the generated ScFv and its cognate target. The binding affinity constant (KD) is a function of on rate (ka) and off rate (kd) and confers particular

100 Chapter 5: Biophysical characterisation

characteristics in vivo (Pompliano, Meek, & Copeland, 2006). For example, the longer the Kd, the longer that the antibody resides bound to the target. For example, a kd value of 10-5/s translates to a dissociating time of 18 h, which is ideal for a therapeutic designed to have a long retention time (Weiner & Adams, 2000). Conversely, it might be desirable for an antibody conjugated with a toxin to have a fast kd in order to deliver the payload and quickly dissociate. Therefore, the affinity of the ScFv samples will be compared to the affinity of commercially available antibody-based inhibitors to estimate the affinity range needed for function.

The aims of this chapter are to:

 Detect and assess the presence of monomers and dimers in the ScFv preparations using analytical SEC.

 Determine the affinity of the ScFv to its target using the BIAcore system.

5.2 EXPERIMENTAL PROCEDURES

5.2.1 Materials A Superdex 75 10/300 GL size exclusion chromatography column was purchased from GE Healthcare. The molecular weight size standards BSA and carbonic anhydrase were obtained from Thermo-Fisher and Sigma-Aldrich respectively. All BIAcore reagents, including CM5 sensor chips, amine coupling kit and assay specific buffers were sourced from GE healthcare. All other reagents unless specified were purchased from Sigma-Aldrich and were of the highest grade.

5.2.2 Analytical size exclusion chromatography To assess the structural heterogeneity of the purified ScFv samples in a physiological condition, analytical size exclusion chromatography using PBS as the mobile phase was conducted. ScFv samples of 50 µL were aliquoted into glass vials with a polypropylene insert and injected into a Superdex 75 10/300 GL column running on a Shimadzu Prominence HPLC instrument, in order to observe the molecular size separation of the sample. Initial optimisation experiments were conducted using PBS as the running buffer at an isocratic flow rate of 0.5 mL/min. Optimisation of the protocol resulted in all subsequent separation cycles conducted in

Chapter 5: Biophysical characterisation 101

a high salt condition (PBS supplemented with additional NaCl [500 mM final]). The resolved proteins were monitored at both 214 nm and 280 nm wavelengths over a period of 40 minutes. To determine the molecular weight of the ScFv peaks, a calibration curve was generated by including the protein standard Bovine serum albumin (MW: 66 kDa) and carbonic anhydrase (MW: 29 kDa). The standards were separated in the same conditions as the samples and repeated three times. A blank sample consisting of low salt PBS was injected between each standard injection to monitor for protein retention by the column. The data collected was analysed using LabSolutions version 5.57 and exported to GraphPad Prism® version 6 and histograms generated. The portion of dimers and monomers were determined from the area under each peak expressed as a total percentage as calculated by the LabSolutions software.

5.2.3 BIAcore The affinity of the interaction between the generated α-IGF-1R ScFv to IGF-1R and α-αv integrin ScFv to αvβ3 integrin were measured by Surface Plasmon Resonance (SPR) on a BIAcore X-100 instrument. Recombinant human IGF-1R (rh- IGF-1R) or recombinant human integrin αvβ3 were coupled to the surface of individual CM5 sensor chips at pH 4.5 by amine coupling chemistry. In short, N- Hydroxysuccinimide (NHS, 11.5 mg/mL) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 75 mg/mL) were mixed at a 1:1 ratio and injected into both flow-cells at 10 µL/min for 420 s to activate the chip surface. The diluted rh- IGF-1R or αvβ3 in 10 mM sodium acetate buffer, pH 4.5 was injected into flow-cell two at 10 µL/min. Even though both flow-cells one and two on the sensor’s surface were activated by NHS-EDC amine coupling reagents, only flow cell two was immobilised with protein as flow cell one was used as a reference surface. Ethanolamine hydrochloride was lastly applied over both flow-cells to deactivate remaining active NHS-esters for 420 s at 10 µL/min. The immobilisation level target of 260 RU was set for IGF-1R while for αvβ3 the target was set at 450 RU. The relative low level of target immobilisations was chosen to minimise the effect of mass transfer limitations (GE Healthcare, 2012). Kinetic runs were then conducted in a single cycle manner with 5 increasing concentrations of ScFv or antibody control (α-IGF-1R) injected sequentially over the sensor’s surface at a flow rate of 30 µL/min (Table 5-1).

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Different concentration sets were used to assess if there were any concentration depended effects.

Table 5-1: Concentration series of analyte used in BIAcore affinity experiments

Analyte Ligand Concentration Concentration Concentration series 1 series 2 series 3

IGF-1R IGF-1R 1500, 500, 166.66, 1000, 333.33, ScFv 55.55, 18.51 nM 111.11, 37.03, 12.34 nM

IGF-1R IgG IGF-1R 50, 16.66, 5.55, 40, 13.33, 4.44, 30, 10, 3.33, 1.85, 0.61 nM 1.48, 0.49 nM 1.11, 0.37 nM

αv IgG αvβ3 70, 23.33, 7.77, 2.59, 0.86 nM

Each ScFv and control IgG were diluted in the BIAcore running buffer HBS- EP+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20), this buffer was also used for blank runs used for reference subtraction. The association time for each injection was set at 120 s with the final dissociation phase at 800 s. The resulting sensorgrams were double reference subtracted (fc 2-1 and blank injections) using the BIAevaluation software version 2.0.1. After each cycle, the surface was regenerated by flowing running buffer overnight to facilitate complete dissociation of the analyte. The affinity of antibody/antigen pair was calculated using the steady-state model within the BIA evaluation software.

5.3 RESULTS

5.3.1 Generation of a SEC molecular weight calibration curve To estimate the molecular weight of the ScFv peaks, protein standards were chosen based on their similarity in molecular weight to the calculated molecular weight of the ScFv (MW: 28 kDa) and its potential dimer (~56 kDa); Carbonic anhydrase (MW: 29 kDa) and BSA (MW: 66 kDa). The standards were resolved on the SEC

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column (Figure 5-1) and monitored at 214 nm (Figure 5-1: A) and 280 nm (Figure 5-1: B). The retention times at both wavelengths were identical, with the major peak at 17.25 ± 0.05 mins for BSA and two major peaks at 18.25 ± 0.04 and 21.49 ± 0.05 mins for carbonic anhydrase. The first retention peak of carbonic anhydrase at 18.25 ± 0.04 mins is similar to the peak of BSA at 17.25 ± 0.05, suggesting that this peak is the dimerised form (58 kDa) whereas the peak at 21.49 ± 0.05 mins corresponds to the expected size for the monomer form. Given the three data points, a linear regression model was applied and the resulting R squared value of 0.99 suggests a high degree of linearity at the molecular weight tested.

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Equation Y = -0.1134*X + 24.79

Figure 5-1: Size exclusion chromatography of reference proteins.

Bovine serum albumin (MW: 66 kDa) and carbonic anhydrase (MW: 29 kDa) were separated independently using HPLC on a Superdex 75 10/300 GL column. The mobile phase consisted of PBS at a flow rate of 0.5 mL/min. Each run was monitored simultaneously at A) 214 nm and B) 280 nm over 40 mins. The raw data from three experiments were exported from LabSolutions version 5.57 and graphed using GraphPad Prism® version 6.0. C) The BSA peak (17.25 ± 0.05 mins) and carbonic anhydrase peaks (apparent dimer = 18.25 ± 0.04 mins, monomer = 21.49 ± 0.05 mins) were plotted against their molecular weights and a linear regression model applied.

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5.3.2 Optimisation of SEC resolution of α-IGF-1R ScFv Once the molecular weight calibration curve was generated, a sample of the α- IGF-1R ScFv was separated to assess the molecular weight pattern of the purified protein sample (Figure 5-2). The initial running buffer was PBS (Figure 5-2: A) which showed a poor resolution of separation as can be seen by the lack of sharp peaks. In addition, the chromatogram did not regress back to baseline, suggesting some non- specific ionic-based interaction between the ScFv and the Superdex chromatography matrix (Arakawa, Ejima, Li, & Philo, 2010). Therefore, an additional run was performed where the PBS running buffer was supplemented with additional NaCl to a final concentration of 500 mM. The result (Figure 5-2: B) showed vastly improved peak separation and the ScFv separated into two distinct peaks at 17.79 ±0.031 and 20.70±0.039 mins. In conjunction with the molecular weight standard curve from section 5.3.1, the first peak was determined to be the dimer form of the ScFv with a calculated MW of 61.72 kDa based on a retention time of 17.79 ± 0.031 mins. The second peak, with a retention time of 20.70 ± 0.039 mins, was calculated to represent a MW of 36.07 kDa. Therefore, this second peak was inferred to be the ScFv monomer, based on the similarity when in comparison to the expected MW of 28.3 kDa.

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Retention Calculated Size Expected size Possible Time (mins) (kDa) (kDa) Identity Peak 1 17.79 ± 0.031 61.72 56.6 Dimer Peak 2 20.70 ± 0.039 36.07 28.3 Monomer

Figure 5-2: Size exclusion chromatography of α-IGF-1R ScFv.

Samples were separated on a Superdex 75 10/300 GL column using HPLC. The mobile phase consisted of A) PBS and B) PBS supplemented with high salt (total 500 mM NaCl) at a flow rate of 0.5 mL/min. The higher salt concentration in the mobile phase reduced the charge interaction between the ScFv and the Superdex stationary phase, allowing for separation between the monomers and dimers. Each run was monitored over 40 mins at both 214 nm (green) and 280 nm (black).

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5.3.3 Fractionation of α-IGF-1R ScFv To confirm the identity of the two major peaks during the separation of the IGF- 1R ScFv by SEC, fractions were collected over the course of the run (Figure 5-3). Four fractions were collected overall, with E1 and E2 representative of the larger dimer while E3 and E4 included the suspected monomer form of α-IGF-1R ScFv (Figure 5-3:A). The collected fractions (E1-E4) were interrogated by SDS-PAGE and silver stained. The resulting gel (Figure 5-3:B), run under denaturing conditions, demonstrated that there was no discernible difference in molecular weight (~28 kDa) between the monomer and dimer fractions. These data suggest that the observed difference in retention time in (Figure 5-3:A) is due to transient interactions among

ScFv between the VH and VL of two or more ScFv molecules rather than a mixed population of single molecule ScFv with different molecular weights. To confirm that the fractionation process was successful, the monomer fractions (E3-E4) were combined and re-analysed by SEC. The result (Figure 5-3:C) contained only one elution peak equivalent to the monomer derived previously, demonstrating that the SEC process was successful in obtaining the monomer fraction within the ScFv sample.

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E1 E2 E3 E4 E1 E2 E3 E4

200

150 100 75

kDa 50

37

25 20

15

10

Silver WB: protein-L

Figure 5-3: Fractionation of α-IGF-1R ScFv monomers and dimers using HPLC.

A) α-IGF-1R ScFv were separated using SEC and collected as four elution fractions (E1-E4). B) The dimer (E1-E2) and monomer population (E3-E4) were resolved on SDS-PAGE and silver stained. C) Fraction E3 and E4 were combined and analysed using SEC to confirm the fractionation of monomer within the sample. Each run was monitored over 40 mins at both 214 nm (green) and 280 nm (black).

Chapter 5: Biophysical characterisation 109

5.3.4 SEC characterisation of α-αv ScFv The three purified α-αv ScFv (A2, B1, F3) were resolved using SEC to assess the molecular weight pattern of the protein sample. The resulting chromatograms (Figure 5-4) demonstrate that all three ScFv were successfully resolved by SEC under pseudo-physiological conditions and were separated into two major peaks with similar retention times compared to α-IGF-1R. For ScFv sample A2, the two major peaks could be detected at a retention time of 17.53 and 20.475 mins, B1 at 17.9 and 20.233mins and for F3, 17.79 and 20.68 mins. Based on the MW calibration curve (Figure 5-1 C), the first major peak represents the dimer population (56 kDa) while the second major peak represents the monomer fraction (28 kDa). The area under both curves were determined using the LabSolutions software expressed as a % area, which directly correlates as the proportion of dimers and monomers. All three ScFv candidates contained >50 % monomer ScFv; with A2 containing 54.71%, B1 - 64.27% and the F3 clone containing the highest ratio of monomer at 71.13 %.

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(A) A2 200000 214 nm 280 nm

150000 s t i n U

y t

i 100000 s n e t n I 50000 39 % 54 %

0 0 10 20 30 40 Retention time (minutes)

(B) B1 200000 214 nm 280 nm

150000 s t i n U

y

t 100000 i s n e t n I 50000 29 % 64 %

0 0 10 20 30 40 Retention time (minutes)

(C) F3 300000 214 nm 280 nm s

t 200000 i n U

y t i s n e t n

I 100000 27 % 71 %

0 0 10 20 30 40 Retention time (minutes)

Figure 5-4: Characterisation of α-αv ScFv by SEC.

α-αv ScFv samples (A2, B1, F3) were separated on a Superdex 75 10/300 GL column using HPLC. The mobile phase consisted of PBS supplemented with higher salt (total 500 mM NaCl) at a flowrate of 0.5 mL/min. Each run was monitored over 40 mins at both 214 nm (green) and 280 nm (black). The integrative area for each peak is expressed as a percentage of the total area when observed at 280 nm.

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5.3.5 Affinity of α-IGF-1R ScFv to IGF-1R To determine the affinity between α-IGF-1R ScFv and its target, IGF-1R, BIAcore experiments were conducted. The recombinant IGF-1R was immobilised onto a CM5 sensor surface using amine coupling to a density of 452 RU. The affinity of a commercially available α-IGF-1R IgG antibody to IGF-1R was also measured in order to both act as a positive control and also as an affinity strength reference. For kinetic analyses, concentration series of ScFv or positive control IgG were flowed over the immobilised IGF-1R. The resulting double referenced sensorgram (Figure 5-5) showed that both the ScFv and IgG control can bind to IGF-1R. The response from each injection of α-IGF-1R IgG was plotted against concentration (Figure 5-5: A1) and a curve fit using a steady-state affinity model was applied. The calculated affinity -8 (KD) between α-IGF-1R IgG and IGF-1R from three experiments was 3.2x10 ± 1.31x10-8 M. The affinity of the α-IGF-1R ScFv (Figure 5-5: B1) was calculated in the same manner as the IgG control, with the resulting response from each injection plotted against concentration (Figure 5-5: B2). The calculated KD between α-IGF-1R ScFv and IGF-1R was 2.58x10-7 ± 3.236x108 M. There was an ~8 fold difference in affinity between the commercial IgG and the ScFv.

112 Chapter 5: Biophysical characterisation

ScFv IGF-1r (KD, M) IGF-1R IgG (KD, M) Mean 2.580e-007 3.200e-008

Figure 5-5: BIAcore affinity determination of ScFv and IgG targeting IGF-1R.

Representative sensorgrams of: A:1) Five increasing concentrations of a commercial α-IGF-1R IgG was sequentially flowed over IGF-1R A:2) The resulting response plotted against concentration and steady state affinity model was applied. B:1) Five increasing concentrations of α-IGF-1R ScFv was also sequentially flowed over IGF- 1R B:2) The resulting response plotted against concentration and steady state affinity model was applied. C) The KD values for three experiments were exported from Biaevaluation 2.0.2 and the mean ± SEM were graphed using GraphPad Prism® version 6.0.

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5.3.6 Affinity of α-αv integrin ScFv to αvβ3 integrin In a similar manner to the IGF-1R BIAcore experiments, the affinity of αv integrin targeting ScFv to αvβ3 was determined using BIAcore analysis. Recombinant αvβ3 was immobilised onto a CM5 sensor surface using amine coupling to a density of 260 RU. A concentration series of a commercial α-αv IgG antibody (Table 5-1) was flowed over the immobilised αvβ3 in a single cycle manner. The IgG antibody was chosen as the positive control to assess αvβ3-Ab binding response and also as an affinity reference to the generated ScFv. The resulting double referenced sensorgrams, (Figure 5-6A) showed that the control IgG did bind to αvβ3 and the response from each IgG injection was plotted against concentration (Figure 5-6B). From three independent experiments, the steady-state affinity model was applied to the binding -8 -9 responses and the calculated KD was 2.589 x 10 ± 4.866 x 10 M. The 3 generated ScFv targeting αv were also flowed over αvβ3. The initial affinity runs did not result in any response, therefore, to demonstrate non-binding, a high concentration of each ScFv (1 μM) was injected over αvβ3. Even at this higher concentration, there was a continued trend on non-binding for all three ScFv against αv integrin (Figure 5-6C- E).

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Figure 5-6: BIAcore analysis of αv targeting ScFv and IgG to αvβ3 integrin.

Representative sensorgrams of: A) Five increasing concentrations of a commercial αv targeting IgG was sequentially flowed over immobilised αvβ3. B) The resulting response plotted against concentration and steady state affinity model was applied with the resulting affinity (KD) from three experiments expressed as ±SEM. C, D and E) High concentration (1 μM) of each ScFv was flowed over αvβ3.

Chapter 5: Biophysical characterisation 115

5.4 DISCUSSION

This chapter set out to characterise the structural heterogeneity of ScFv expressed and purified in the preceding chapters. It is important to establish the samples structural state/s as the presence of aggregate has the potential to impact function. Dimerised ScFv could be detected in all ScFv samples, ranging from a low of 27% for sample α-αv integrin-F3, to a high of 39% for α-αv integrin-A2 (Figure 5-4). The propensity of ScFv to form dimers is widely known in the literature. ScFv have been engineered with a modified linker sequence length which favours the formation of ScFv heterodimers termed diabodies (Asano et al., 2008). In this case however, the dimerised ScFv were fully functional and shown to bind to their respective target with equivalent capacity as the monomeric form (Asano, et al., 2008).

Indeed, a study has revealed that the length of the linker sequence between the VH and the VL domains plays a key role in the formation of higher order structures (Völkel, Korn, Bach, Müller, & Kontermann, 2001). By reducing the length of the linker from 15 amino acid (aa) residues (as found in ScFv used within this study) higher order structures such as diabodies (9 aa), triabodies (5 aa) and tetrabodies (1 aa) can be generated. Given that the ScFv generated within this study contained a linker length of 15 aa, it is unlikely that the dimer detected are defined diabodies.

Therefore, the potential impact of dimers on function still needs to be determined and assessed. Obviously, if the dimerised ScFv were found to be non-functional this would have a significant impact on effective yield, require further processing (to remove or refold the dimers) and/or necessitate higher sample concentrations in functional applications. Ideally the ScFv should be fractionated using preparative SEC, however, the analytical HPLC instrument used has a maximum injection volume of 100 μL, which is problematic in processing larger volumes. Also, given that SEC considerably dilutes the recovered proteins in the elution fractions, the ScFv load solution should be at a high concentration, which has inherent problems since aggregation is concentration-dependent (Zhao et al., 2011)

-7 The affinity between the generated ScFv against IGF-1R (KD= 2.58 x 10 ± 3.236 x 10-8 M) was approximately 8 fold weaker than the commercially available IgG

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-8 -8 inhibitor (KD= 3.2. x 10 ± 1.31 x 10 M). However, an antibody with high affinity does not necessarily translate to an ideal tumour targeting agent, mainly because of their poor tumour penetration (Adams et al., 2001). The poor tumour penetration is due to a low dissociation (kd) rate, therefore limiting available antibody for binding once the initial antibodies have bound to the tumour’s surface and causing heterogeneous distribution. The term ‘binding site barrier’ effect has been termed to describe this process (Juweid et al., 1992). For example, a study by Adams, et al. (2001) performed a biodistribution study on mice with anti-HER-2/neu ScFv mutants, with various affinities ranging from 10-7 to 10-11 M, it was concluded that tumour retention did not increase with affinity beyond 10-9 M. It is therefore suggested that additional parameters such as avidity and specificity should also be considered in addition to affinity (Rudnick & Adams, 2009).

Even so, the affinity of the IGF-1R targeting ScFv could be improved by the process of affinity maturation, a process traditionally applied to B-cell antibody selection and enrichment (Tangye, Ma, Brink, & Deenick, 2013). Through somatic hypermutation of the antibody encoding variable regions, B-cells which express high affinity antibody to the target antigen are positively selected and enriched. The in vitro affinity maturation process using phage display aims to mimic this process; starting with a selected clone, mutations are introduced into the variable encoding regions of the antibody gene, followed by additional selection procedures. Mutagenesis methods such as error-prone PCR, E. coli mutant strains and site-directed mutagenesis have all been used to introduce mutations into the antibody coding sequence and therefore increasing antibody diversity for additional selections (Kim, Stojadinovic, & Izadjoo, 2014). Other non-mutagenic methods are based on the shuffling of the antibody coding sequences, such as chain shuffling, DNA shuffling, and CDR shuffling (Mandrup, Friis, Lykkemark, Just, & Kristensen, 2013; Rojas et al., 2004).

Antibodies which have undergone the affinity-maturation process can have a substantial increase in affinity (Ahmed et al., 2015; Petters, et al., 2015; Wang et al., 2011). Another study, using PCR based mutagenesis of clones generated from the same phage library (Tomlinson I + J) used in this project, described a modest 2 fold -2 increase (387 nM to 185 nM) in KD and 6 fold decrease of the off rate (7.63 x 10 1/s

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to 1.23 x 10-2 1/s) (Petters, et al., 2015). On the other hand, using a similar technique, the affinity of a ScFv targeting a tumour-inhibitory ligand was increased 160 fold to a

KD of 0.9 nM (from 144 nM) (Ahmed, et al., 2015).

The response between αvβ3 and the positive control IgG αv targeting antibody was low, suggesting the surface was not fully functionalised in an active state. From a theoretical perspective, the maximum response (Rmax) of any interaction can be calculated using the formula:

() = × ()

Given that ~260 RU of αvβ3 was immobilised, the theoretical Rmax:

(155 ) () = × 260 () = 210 αvβ3 (191.3 kDa)

Even though the calculated Rmax value of 210 RU is only theoretical, in practice when using amine coupling a response in the region of 70% of the Rmax can be achievable (GE Healthcare, 2012). The response reached between αv and the IgG antibody was much lower than 210 RU, suggesting that the αvβ3 immobilised to the sensor’s surface was not fully active, caused potentially by the amine coupling procedure, or by structural issues with the recombinant protein. Even though integrins in cells require bivalent cations such as Mg2+ or Mn2+ for structural stability and function, the addition of 1 mM Mg2+ and Mn2+ to the HBS running buffer did not increase binding response (Appenix 11).

The binding interaction model originally chosen to assess the binding of control IgG/ScFv to their cognate target using BIAcore was the 1:1 binding kinetics model. In contrast to the steady-state affinity model used for this project, the 1:1 binding model allows for the determination of the rate constants, association (ka) and dissociation (kd), from which the affinity constant (KD) could be calculated. Both the ka and kd allow for better assessment of binding than simply an end point value. Given that the kinetics model was the preferred option, the steady-state affinity option was chosen instead because the IGF-1R ScFv sensorgram (Figure 5-5:B1) did not fit the 1:1 model. Given that the binding of ScFv to IGF-1R was expected to be a 1:1 interaction, optimisation

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studies were conducted which ruled out two-state interaction (data not shown). Therefore, all BIAcore runs were analysed using the steady-state model. It is likely that given the presence of dimers in each ScFv sample, fractionation of the monomers using SEC could improve response fitting to the 1:1 model. However, since SEC dilutes the input sample considerably, this option was not investigated further for this project due to the substantial time investment required to produce sufficient quantities of ScFv for fractionation and subsequent BIAcore analyses.

The decision of using a steady-state affinity model could also have an impact -8 when comparing the calculated KD between the intact IgG/IGF-1R (KD= 3.2. x 10 ± -8 -7 -8 1.31 x 10 M) and the α-IGF-1R ScFv/IGF-1R (KD= 2.58 x 10 ± 3.236 x 10 M). The main factor to consider was avidity effects caused by the potential of a IgG antibody binding to two IGF-1R molecules simultaneously. Avidity effects will slow down the dissociation rate of the IgG/IGF-1R interaction, leading to an overestimation of the KD value (Klein et al., 2009). In the BIAcore assay format chosen, avidity will play a role with the full Ab but not with the ScFv. In an attempt to counteract the effect of avidity, experiments were conducted using low ligand immobilisation levels (IGF- 1R). However, potentially the difference in affinity between the full Ab and the ScFv is lower than the 8 fold as calculated.

The heterodimer αvβ3 was chosen for affinity analysis preferentially over the integrin subunit αv even though the phage panning process enriched for only αv binders. This was because integrins exist in nature as a heterodimer consisting of one α subunit and one β subunit. If panning were performed using αvβ3 as the target, ScFv binding the β3 may have been selected, something that would have been deleterious to the aim of generating pan-αv binding ScFv. In addition, αvβ3 was readily available and substantially cheaper to obtain and thus fit the requirements for the BIAcore analyses. Also, the binding affinity between αv targeting ScFv and αvβ3 is more physiologically relevant than between only the αv subunit and ScFv. However, it was soon discovered that even when injecting high concentrations of ScFv over αvβ3, there was a lack of response observed. One possible explanation was that the αv binding site for the ScFv was obscured by the β subunit. Therefore, given the lack of a response from the αv ScFv candidates against αvβ3, the next logical step was to assess if the

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ScFv can bind to the αv monomer. It was expected that the ScFv should bind because the phage panning process enriched for αv binders and this binding has been confirmed by ELISA. Surprisingly, the αv protein failed to immobilise onto the BIAcore sensor chip using amine coupling chemistry (data not shown). Up to this point there was no reason to doubt the integrity of the recombinant αv protein. The protein was sourced from a reputable company in which the protein was expressed using their proprietary technology, with their own quality control. In addition, antibody screening was listed as one of the applications for this protein (Appendix 11). Given the spurious results of the α-αv ScFv and the lack of immobilisation of the αv protein onto the BIAcore sensor chip, the protein was subjected to analysis by silver-stained SDS-PAGE and Western blotting (Appendix 12). The results showed that the αv protein contained a high portion of aggregates creating a high MW smear on the silver-stained SDS- PAGE. In addition, no immune-reactive bands could be detected when interrogated with an α-αv antibody in Western blotting (Appendix 12). Given the SDS-PAGE and Western blotting results, along with the amine coupling failure, there is a strong indication the recombinant αv integrin used for phage panning might not be representative of αv structure in vivo. The rationale for using αv monomer in phage display screening rather than the heterodimer was that it was hoped that a pan-αv inhibitor could be generated. Because the 3 ScFv generated against αv demonstrate no binding affinity against αvβ3 in BIAcore studies (Figure 5-6), it potentially points to lack of inhibitory function.

The chapter herein described the biophysical assessments of the isolated ScFv for their properties. All 4 ScFv contained a majority of monomers when interrogated using SEC. The α-IGF-1R ScFv showed the most potential, with a high portion of monomers (Figure 5-2) in addition to quantifyable binding affinity to IGF-1R (Figure 5-5). The next chapter will use cell-based assays to assess the inhibitory function of the ScFv in the relevant setting of tumour growth and metastasis.

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Functional characterisation

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6.1 INTRODUCTION

The tumour micro-environment (TME) and its interaction with growth factors (GFs) and the extracellular matrix (ECM) plays an important role in tumour cell biology. One such interaction is between the insulin like growth factor (IGF) and integrin axes. The insulin-like growth factor (IGF) system consists of ligands (IGF-I and IGF-II), receptors (IGF-1R, IGF-2R) and IGF binding proteins (IGFBP 1-6). Upon receptor ligand binding, the receptor is autophosphorylated leading to downstream signal transduction and activation of mitogenic pathways such as PI3K/AKT and MAPK/ERK. In breast cancer, several abnormalities in the IGF family can be found, including an increase in IGF-I and IGF-II ligands (Hankinson et al.), a decrease in IGFBP (Hoeflich & Russo, 2015), and altered IGF-1R expression. Recently, it was found through mRNA analysis that over 45% of breast cancer samples showed a molecular alteration in at least one member of the IGF system (Farabaugh, et al., 2015). Antibodies that are targeting the IGF-1R have been demonstrated in vitro to inhibit cancer cells lines (Burtrum, et al., 2003; Hollier, et al., 2008; Lu et al., 2004)

Cell migration is a key step in the progression of malignant cells from a primary localised tumour to an invasive metastatic phenotype (Vinci, Box, Zimmermann, & Eccles, 2013). This is mediated by the interaction of extracellular matrix proteins such as vitronectin with its cognate integrin receptor. It has been demonstrated that there is crosstalk between the IGF associated tyrosine phosphatase SHP-2 and the focal adhesion kinase which plays a role in cell migration, and survival (Beattie, McIntosh, & van der Walle, 2010; Mañes et al., 1999; Zheng et al., 2009).

The breast cancer cell line MCF-7 has been widely used as a cancer model for studying the role GFs and integrins in relevant cell processes (Hollier, et al., 2008; Kashyap, et al., 2011). Even though the MCF-7 cell line has been extensively used as a breast cancer model, it is particularly suited to application herein because it expresses both IGF-1R and the αv integrins αvβ3 and αvβ5. In addition, in vitro studies have revealed that MCF-7 cells in basal medium have a low propensity to migrate and attach to tissue culture plastic. Therefore, functional effects observed when investigating cell migration and attachment are confidently attributable to the treatment.

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Transwell® assays, or Boyden chamber assays (Chen, 2005), have been used to determine the migration response of MCF-7 upon simulation with VN+IGF-I, and subsequent blunting of VN+IGF-I response by blocking their cognate receptors with ScFv or control antibodies. Newer methods of measuring cell migration exist, such as the real time cell monitoring xCELLigence RTCA-DP instrument. Given the extensive literature on MCF-7 migration using the established Transwell® assays, this assay was opted as the method of choice for this project (Hollier, et al., 2008; Kashyap, et al., 2011; Van Lonkhuyzen, et al., 2007).

As described in the previous chapter, phage display screening yielded one IGF-1R and three αv binding candidates. These candidates were subsequently expressed and purified to a high degree of purity. The next step of this project was to assess the inhibitory function of the ScFv in a breast cancer cell line MCF-7 upon stimulation by IGF-I and VN. To do so, MCF-7 cells were treated with the generated ScFv along with commercially available receptor blocking monoclonal antibodies in proliferation, attachment, and migration assays.

The aim of this chapter was to:

 Assess the functionality of the ScFv in cell based assays to determine if it can inhibit the metabolic activity, attachment, and migration when induced with IGF-I and VN.

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6.2 EXPERIMENTAL PROCEDURES

6.2.1 Materials All reagents, unless stated, were purchased from Sigma-Aldrich and are of the highest quality. Transwell® plates containing 8.0 µm pore size insert for migration assays were obtained from Corning (Mount Martha, Vic, Australia). The CellTiter 96® AQueous One proliferation assay was sourced from Promega (Madison, USA). Human VN purified from serum was purchased from Promega while recombinant human IGF-I produced in E. coli was obtained from GroPep (Adelaide, SA, Australia). Commercially available receptor blocking monoclonal antibodies against IGF-1R (αIR3) and αv integrin (AV 1) were sourced from Merck Millipore. Isotype control mouse IgG antibody (G3A1) was bought from Cell signalling (Beverly, MA, USA).

6.2.2 Cell culture The human breast adenocarcinoma cell line MCF-7 (ATCC: HTB-22) was routinely cultured in growth media (GM) containing Dulbecco’s Modified Eagle Medium/F12 (1:1 DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 0.1 mg/mL streptomycin, 50 units/mL penicillin, 1 μg/mL gentamicin. The cell line was maintained in a humidified incubator at 37 °C with 95% air and 5% CO2 with a change of media every 3-4 days or upon 80% confluence, at which point cells were passaged at a ratio of 1:3. Cells were detached from the tissue culture flasks using TrypLE™ Express, a trypsin replacement dissociation agent. All functional assays were performed using serum free media (SFM) supplemented with 0.05% (w/v) bovine serum albumin (BSA).

6.2.3 Metabolic assay The CellTiter 96® AQueous One proliferation assay is based on the conversion by viable cells of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS into a coloured formazan product. This coloured product is measured spectrophotometrically and correlates to metabolically active cells. MCF-7 cells were passaged the day before the assay at a ratio of 1:1 and cultured in GM in a humidified incubator at 37 °C with

95% air and 5% CO2. The cells were then serum starved for 4 h by aspirating the GM,

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washing twice with PBS, before the addition of SFM. A series of treatments including a commercial IGF-1R blocking antibody, matched isotype control and increasing concentrations of IGF-1R targeting ScFv were set up in SFM+0.05% BSA. Serum starved MCF-7 cells were dissociated from the culture flask using TrypLE™, 5,000 cells added to each treatment within a microfuge tube and allowed to incubate at room temperature for 30 mins. After this incubation period, the treated cells were transferred into individual wells of a 96 well plate and stimulated with or without VN at a final contraction of 1 µg/mL and IGF-I at 30 ng/mL. The treated cells were incubated at 37

°C with 5% CO2 for 24 h, at which time 20 µL of CellTiter 96® AQueous One Solution Reagent was added into each well and a further 1.5 h incubation at 37 °C was performed for the formation of the coloured formazan product. The absorbance of each well was measured at 490 nm using the Benchmark Plus microplate spectrophotometer (Bio-rad).

6.2.4 Migration assay To investigate the effect of ScFv as inhibitors of VN and IGF-I dependent migration, a Transwell®-based assay was performed. MCF-7 cells were passaged the day before the assay to maintain cells in the proliferative phase. On the experiment day, cells were serum starved by aspirating the GM, twice washing with SFM and incubating with SFM for an additional 4 h. During the SFM incubation, the lower chambers of the Transwell® plates were incubated with both IGF-I (30 ng/mL) and VN (1 µg/mL) diluted in SFM+0.05% (w/v) BSA. The serum starved cells were harvested by washing the monolayer with PBS/EDTA followed by TrypLE™ application. In a similar approach to the metabolic assay, treatments of commercial IGF-1R or αv integrin function blocking antibody along with the unique ScFv clones generated from phage display were diluted in SFM+0.05% BSA to concentrations of 10 μg/mL for the IGF-1R IgG and 5 μg/mL for the αv integrin antibody. For the ScFv treatments, a concentration range from 10 - 100 μg/mL was generated. The serum starved MCF-7 cells were added at 60,000 cells per treatment, in microfuge tubes, and allowed to incubate with the antibody-based treatments for 0.5 h at room temperature before seeding into the upper chamber of a Transwell®. Plates were then incubated for

15 h at 37 °C with 5% CO2 within a humidified incubator. At the completion of the migration period, the non-migrated cells were physically removed from the upper

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surface of the semi-porous Transwell® membrane by cotton buds in a circular motion. Cells which had migrated to the lower surface of the membrane were fixed in ~3.7% formaldehyde and stained with 0.01% (w/v) crystal violet in PBS. To quantify migrated cells, the crystal violet was extracted from the stained cells using 10% (v/v) acetic acid and subsequently transferred into a 96 well plate. The absorbance was measured at 595 nm using the Benchmark Plus microplate spectrophotometer.

6.2.5 Attachment assay MCF-7 cells were passaged the day before the assay to synchronise the cell cycles before serum starving for 4 h by aspirating the GM, washing twice with SFM and finally incubating with SFM for the starvation period. Treatments targeting the αv integrin and antibody controls were diluted in SFM+BSA at various concentrations. Serum starved cells were harvested by aspirating the SFM, washing once with PBS+EDTA before the addition of TrypLE™. The detached MCF-7 cells were added at 20,000 cells per treatment and incubated for 30 mins at room temperature. During this incubation, a 10x solution of IGF-I and VN (300 ng/mL, 10 μg/mL) was made and added to individual 96 well plate wells. After the 30 mins incubation, the treated cells were seeded into the IGF-I+VN containing 96 well plate and incubated for 30 mins at

37 °C with 95% air and 5% CO2 to allow for cell attachment. Cells which have not attached after this period were aspirated, and washed twice with PBS. The cellular attachment was determined by the addition of 100 μL of SFM + 20 μL MTS reagent to each well and incubating for 1.5 h at 37 °C with 95% air and 5% CO2 for the conversion of MTS into a coloured product. This product was quantified at 490 nm using the Benchmark Plus microplate spectrophotometer.

6.2.6 Isolation of functional Insulin-like growth factor receptor MCF-7 Cell lysis MCF-7 cells were grown until 80% confluent in growth media (GM) containing Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 0.1 mg/mL streptomycin, 50 units/mL penicillin, 1 μg/mL gentamicin. The cultures were maintained in a humidified incubator with 5% CO2 and 95% atmospheric air at 37 °C. The cells were serum starved for 4 hours by firstly

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removing the old media, washing once with PBS, then adding SFM. After 4 hours, the media was aspirated and 2 mL of non-denaturing lysis buffer consisting of 50 mM Tris (pH 7.4), 100 mM NaCl, 1% (v/v) Triton X-100 and complete protease inhibitor (Roche) was added to each of the T-175 flasks. The cells were harvested from the cell culture flasks using cell scrapers and transferred into pre-chilled low protein binding microfuge tubes. To assist with cell lysis, the samples were immersed in a sonication water bath at 4 °C for 5 mins. The tubes were then centrifuged at 14,000 g for 20 mins and the supernatant was retained and stored at -80 °C for protein quantification and purification.

Cell lysate quantification of protein content The protein concentration of the cell lysate was estimated using the the Bicinchoninic Acid (BCA) protein assay kit (Pierce) as per the manufacturer's instruction. BSA was diluted in PBS to form a concentration range from 125 µg/mL to 2000 µg/mL and was used as the protein concentration reference. The BCA reaction plate was read at 562 nm using the Biorad benchmark plus microplate spectrophotometer system. The resulting data was curve fitted using the Microplate Manager Software version 6.2 (Biorad)

Wheat Germ Agglutinin purification Wheat Germ Agglutinin (WGA) conjugated to agarose beads were used to enrich for the glycoprotein IGF-1R from MCF-7 cell lysates. The beads were firstly washed in PBS to remove the N-acetyl-D-glucosamine (GlcNAc) from the storage buffer. MCF-7 cell lysates containing 24 mg of protein in 10 mL were added to the 1 mL of equilibrated beads and mixed overnight at 4 °C. The lysate/bead mixture was slowly transferred into a disposable column and beads allowed to settle. Once the sample passed through, 5 mL of wash buffer consisting of 50 mM Tris (pH 7.4), 100 mM NaCl, 0.1% Triton X-100 and complete protease inhibitor (Roche) was added to the top of the resin. To elute the bound proteins, elution buffer containing 50 mM Tris (pH 7.4), 100 mM NaCl, 0.1 % (v/v) Triton X-100, and 500 mM GlcNAc was added. Eluted fractions were collected in 0.5 mL increments and stored at -80 °C.

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6.2.7 SDS-PAGE and Western immunoblotting Samples collected throughout the purification process were prepared for electrophoresis by the addition of NuPAGE® LDS sample buffer and reducing agent to a final concentration of 141 mM Tris base, 2% LDS, 10% Glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G, 0.175 mM Phenol Red, 50 mM DTT, pH 8.5 and heated at 70 °C for 10 mins. The protein samples were separated based on molecular weight on a precast NuPAGE® 4-12% SDS-PAGE gel with NuPAGE® MES-SDS running buffer. The electrophoresis proceeded at a constant 200V for 35 mins. SDS- PAGE separated proteins were transferred onto a nitrocellulose membrane using the wet tank blotting system (Biorad) containing Towbin buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol) at a constant 200 mA for 90 mins. The blot was blocked for 1 h at room temperature with 5% (w/v) BSA in Tris buffered saline (10 mM Tris, 0.5 M NaCl, pH 7.6) with 0.1% Tween-20 (TBST). The blocked membrane was incubated with α-IGF-1R antibody (1:1000) diluted in blocking buffer and incubated overnight at 4 °C. The blot was then washed for 5 mins in TBST, repeating a further 2 times. After the washes, diluted α-rabbit horse radish peroxidase (HRP) conjugated antibody (1:5000) in blocking buffer was added and incubated for 1 h at room temperature. An additional 3 washes of 5 mins each in TBST were performed to remove unbound secondary antibody. The immune complex bound proteins were detected with the addition of ECL prime (GE) and visualised on an X-ray film processed using the AGFA CP 1000 film developer.

Quantitative Westerns were performed using the LI-COR Odyssey® system with a similar workflow to the chemiluminescence Westerns described above. After transfer, the blot was blocked with Odyssey® Blocking Buffer (OBB) at room temperature for one hour. The blocking buffer was removed and 1:1000 diluted rabbit α-phospho IGF-1R β antibody in OBB was added and incubated overnight at 4 °C. The blot was then washed for 5 mins in TBST 3 times. Anti-rabbit conjugated with DyLight 800 secondary antibody diluted at 1:10,000 in OBB were added. Three washes of 5 mins each of TBST and an addition wash of TBS for 5 mins was performed to remove any residual tween. The blot was scanned using a LI-COR® Biosciences Odyssey® Infrared Imaging System and data quantified using Image Studio software version 4.0. The blot was stripped by incubating the blot at room temperature for 15

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mins with Restore™ Western Blot Stripping Buffer. The blot was re-blocked and probed with a total IGF-1R antibody diluted at 1:1000 in OBB overnight at 4 °C. Anti- rabbit conjugated with DyLight 680 secondary antibody diluted at 1:10,000 in OBB was added. The resulting blot was imaged and processed in the same manner as the blot probing for phosphorylated IGF-1R.

6.2.8 Receptor in vitro phosphorylation assay Elution fractions from the Wheat-germ agglutinin purification determined to be IGF-1R positive were used to performed in vitro phosphorylation assays. A dose curve was set up with each reaction consisting of 2 µL of purified IGF-1R, 100 µM ATP, 5 mM MnCl2 and an increasing concentration of IGF-I (5-500 ng/mL). The reaction was incubated at room temperature for 10 mins before the addition of NuPAGE® LDS sample buffer and reducing agent as above. The resulting samples were subjected to SDS-PAGE and immunoblotted firstly with α-phopho-IGF-1R, stripped, and re- probed with total IGF-1R as per section 6.2.7.

To allow for the potential of high throughput screening, without the time consuming step of performing a SDS-PAGE and subsequent immunoblot transfer, a dot blot process was employed whereby each IGF-1R + IGF-I reaction was spotted directly onto nitrocellulose membrane and allowed to dry. The resulting dot blot was processed as per the above SDS-PAGE/Western blot protocol (6.2.7), post-transfer.

Quantification of the elution fraction Recombinant human IGF-1R consisting of the extracellular domain portion was used as the concentration standard in order to estimate the IGF-1R concentration in the WGA purification elution fractions. The recombinant IGF-1R was serially diluted 1:1 with PBS from 50 µg/mL to 97.65 ng/mL, yielding 10 dilution standards. Each standard concentration and WGA eluted fractions were separated by mass using SDS- PAGE and electro-transferred onto nitrocellulose. Quantitative Westerns were performed using an α-IGF-1R (total) antibody using the method in section 2.7.2. The resulting signal unit for each standard concentration band was plotted against protein concentration using GraphPad Prism® version 6.0.

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6.3 RESULTS

6.3.1 The effect of IGF-1R targeting ScFv on MCF-7 cell metabolic activity The ScFv developed in previous chapters was designed to inhibit cell activation and migration with the ultimate aim being to repress cancer cell metastasis and survival. To determine if the generated α-IGF-1R ScFv can inhibit the functional effect of IGF-I stimulation in a cell growth setting, MCF-7 cells were treated with various concentrations of α-IGF-1R ScFv, along with control antibodies, before inducing with either IGF-I or IGF-I combined with VN. The relative response of each treatment was measured in a 24 h cell metabolic assay (Figure 6-1). The raw absorbance data was shown in addition to normalised data where the Iso IgG response (uninhibited) was set at 100% and each treatment group expressed as a comparative percentage. When comparing the IgG isotype control absorbance values between IGF-I (0.36 ±0.02) and the VN+IGF-I (0.48 ±0.02) treated samples, the addition of VN induced a significant metabolic increase (p<0.05) in MCF-7 cells. Interestingly, a commercial IGF-1R targeting antibody did not reduce the effect of IGF-I induced metabolic activity. No significant difference was observed between the iso control samples (green bars) and IGF-1R targeting IgG (red bars) in response to either IGF-I or VN+IGF-I. Treatment with α-IGF-1R ScFv produced a dose dependent reduction in metabolic activity (blue shaded bars). Indeed, ScFv added at 40 µg/mL drove significant inhibition (IGF-I: 75.26 ± 2.34%, VN+IGF-I: 73.37 ± 1.77%) of cell metabolism compared to the isotype control (ρ <0.05).

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Figure 6-1: MCF-7 proliferation with IGF-1R inhibitors.

Serum starved MCF-7 cells were treated with a commercial antibody against IGF-1R (10 µg/mL), matched IgG isotype control (10 µg/mL), and three increasing concentrations of generated α-IGF-1R ScFv (10, 20, 40 µg/mL) before stimulation with either IGF-I (30 ng/mL) or VN (1 µg/mL) and IGF-I (30 ng/mL). Cells were cultured for 24 h before the addition of MTS reagent into each treatment. The conversation of MTS into coloured formazan product by metabolically active cells was quantified at 490 nm. The raw absorbance results (top) were normalised against iso IgG within each treatment group (Bottom) (IGF-I or VN+ IGF-I). Values shown are mean ± SEM (n=9). * ρ < 0.05.

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6.3.2 The effect of IGF-1R targeting ScFv on MCF-7 cell migration In addition to cell viability, another fundamental aspect of cancer cell metastasis is cell migration, thus the generated IGF-1R targeting ScFv were assessed for their ability to inhibit IGF-I+VN stimulated migration in a TranswellTM-based assay (Figure 6-2). The IgG isotype control, which is understood to have no effect on cell responses, was defined as 100% and treatments were assessed in terms of response relative to this uninhibited control (green bar). In contrast to the viability assay, the IGF-1R targeting commercial antibody demonstrated significant inhibition of MCF-7 cell migration when compared to the iso control (ρ <0.05), with migration reduced to 38.37 ± 6.51 %. Mirroring the proliferation assay, increasing concentrations of IGF-1R targeting ScFv showed a dose depended inhibitory trend. Although treatments of 10 μg/mL (purple bar, 99.23 ± 13.63 %), 20 μg/mL (orange bar, 97.29 ±7.99 %) and 40 μg/mL (black bar, 82.95 ± 2.779 %) did not inhibit migration to a significant extent, compared to the iso control, the highest ScFv treatment tested (100 µg/mL) reduced MCF-7 migration to 47.67 ± 14.18% which was a significant inhibition of MCF-7 cell migration (ρ <0.05).

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Figure 6-2: MCF-7 migration with IGF-1R inhibitors.

Serum starved MCF-7 cells were treated with a commercial antibody against IGF-1R (10 µg/mL), matched IgG isotype control (10 µg/mL), and four increasing concentrations of generated IGF-1R ScFv (10, 20, 40, 100 µg/mL) before seeding into an upper chamber of a Transwell®. The lower chamber was pre-filled with IGF-I (30 ng/mL) and VN (1 µg/mL). The cells were allowed to migrate for 15 h. The number of migrated cells in response to each treatment was expressed as a percentage relative to IgG isotype control. Values shown are mean ± SEM (n=6) from three independent experiments. * ρ < 0.05.

6.3.3 Effect of αv-integrin targeting ScFv on MCF-7 cell attachment Prior to cellular migration, MCF-7 cells have to firstly attach to a substrate, to which the activation of various integrin receptors is necessary. Therefore, the functional effect of αv-integrin targeting ScFv with respect to attachment was assessed (Figure 6-3). The IgG isotype control was defined as 100% and responses were measured relative to this control. Coating culture wells with VN+IGF-I facilitated significant levels of cell attachment (VN+IGF-I) in comparison to the non-coated controls (SFM, orange bar, -3.68 ± 5.63 %). The α-αv integrin commercial antibody was able to significantly inhibit cell attachment to IGF-I+VN (purple bar, 40.52 ± 8.48 %, ρ < 0.05). In terms of the 3 ScFv treatments, no significant functional effect relative to the isotype control was observed at any concentration tested.

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Figure 6-3: MCF-7 attachment with αv integrin inhibitors.

MCF-7 cells were treated with a commercial antibody against αv integrin (5 µg/mL), matched IgG isotype control (5 µg/mL), and three unique ScFv in increasing concentrations (20, 40, 100 µg/mL) before seeding each treated cells into individual wells in a 96 well plate containing IGF-I and VN (final concentration IGF-I: 30 ng/mL, VN 1 µg/mL). Cells were incubated at 37 °C to facilitate cell attachment at which point the unattached cells were removed. The attached cells were measured using the MTS reagent and quantified at 490 nm. Values shown are mean ± SEM (n=9) from three independent experiments. * ρ < 0.05.

6.3.4 The effect of αv-integrin targeting ScFv on MCF-7 cell migration As part of the dual targeting approach of this thesis, the functional effect of ScFv targeting the αv integrins was investigated in a similar migration assay to that described above. The IgG isotype control was again defined as 100% (green bar) and treatments were assessed in terms of response relative to the IgG isotype control (Figure 6-4). The commercial antibody against αv integrin was able to significantly inhibit the effect of IGF-I+VN (purple bar, 4.29 ± 2.32%). Indeed, this treatment was comparable to the SFM negative control (orange bar, 8.72 x 10-7 ± 1.86 %) demonstrating almost complete inhibition of the VN stimulation. In terms of MCF-7 cells treated with the various concentrations of three different ScFv, no significant functional effect relative to the isotype control was observed in any of the treatments. The largest inhibition response was clone B1 at 40 µg/mL (red bar, 40.28 ± 7.24 %), but no significant difference was observed.

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Figure 6-4: MCF-7 migration with αv integrin inhibitors

MCF-7 cells were treated with a commercial antibody against αv integrin (5 µg/mL), matched IgG isotype control (5 µg/mL), and three unique ScFv in increasing concentrations (20, 40, 100 µg/mL) before seeding into an upper chamber of a Transwell®. The lower chamber was pre-filled with IGF-I (30 ng/mL) and VN (1 µg/mL). The cells were allowed to migrate for 15 h. The number of migrated cells in response to each treatment was expressed as a percentage relative to IgG isotype control. Values shown are mean ± SEM (n=6) from three independent experiments. * ρ < 0.05, ** ρ < 0.01.

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6.3.5 Isolation of IGF-1R was successful using WGA agarose The recombinant human IGF-1R used for phage display screening contained only the extracellular domain. It lacks the kinase domain required for signal transduction post binding with its ligand, predominantly IGF-I. Both ELISAs to determine phage/ScFv binding only yielded information about if there is an interaction between the ScFv and the extracellular domain of IGF-1R. Therefore, a ScFv could potentially be binding strongly to the non-essential portion of the receptor that is needed for ligand-receptor activation. In other words, a positive binding candidate for the receptor might not necessary prevent ligand activation, which is the goal of this project. A more refined model for ScFv screening might be to determine if a ScFv candidate has the capability to prevent phosphorylation of the receptor upon ligand addition. Therefore, full length IGF-1R was needed containing both the extracellular domain, and also the tyrosine kinase domain to be suitable as the test model. The goal of this section was to develop a high through-put screening system using the isolated receptors including auto-phosphorylation studies when induced with the ligand IGF-I.

To isolate native IGF-1R, MCF-7 cells were lysed using a non-denaturing lysis buffer containing the non-ionic detergent Triton X-100. The silver stained SDS-PAGE in Figure 6-5 revealed that most proteins are non-reactive to WGA due to the similar protein staining found between the load and the unbound protein flow-through. This was expected as WGA can only bind to glycoproteins containing N-acetylglucosamine or chitobiose, such as IGF-1R. Washing of the resin resulted in the removal of the non- specifically bound proteins. Analysing the elution fractions, the majority of retained protein is eluted in fraction 2 from whence the intensity decreases across the subsequent fractions.

The same samples were interrogated by Western immunoblotting using an α- IGF-1R-α antibody. The difference in total intensity of the IGF-1R band between the load and the flow-through suggested that IGF-1R is being captured by the WGA with high efficiency. This is further evidenced as no IGF-1R could be detected in the wash sample. The majority of IGF-1R could be detected in elution fraction 2, with a smaller portion in fraction 3.

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Figure 6-5: Wheat Germ Agglutinin (WGA) purification of IGF-1R from MCF- 7 cell lysate.

MCF-7 cells were lysed using a non-denaturing lysis buffer (50 mM Tris (pH 7.4), 100 mM NaCl, 1% Triton X-100 and complete protease inhibitor) and batch affinity purified using agarose bound WGA. At each step of the purification process, a sample was taken for A) Silver stained SDS-PAGE and B) Western blot analysis with an IGF- 1R antibody. Load = Starting lysate, Flowthrough = unbound protein to WGA, Wash = loosely/unspecific bound protein, Elution= competitive removal of bound proteins to WGA by N-acetylglucosamine.

The yield of IGF-1R isolated in elution fraction 2 was determined using quantitative Western blotting on the LI-COR® Odyssey system. A standard curve was generated

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using known concentrations of recombinant IGF-1R. The standards and the elution fraction 2 were separated using SDS-PAGE as per section 2.7.1 and probed using an α-IGF-1R-α antibody using the procedure described in 2.7.2. The resulting blots are shown in Figure 6-6 A. The IGF-1R-α bands were quantified using Image Studio 4.0 and the concentration of the unknowns were determined in duplicate to be 0.899 and 0.862 µg/mL respectively. Given that the elution fractions were collected in 0.5 mL increments, the total IGF-1R recovered was calculated to be 0.440 ± 0.00925 µg from 10 confluent T-175 flasks. In addition, the WGA purified IGF-1R exhibit only one immune-reactive band to the IGF-1R-α antibody, unlike the recombinant IGF-1R with two. This suggests that the pro-IGF-1R in the isolated sample has been correctly cleaved into the α and β chains and would likely be functional.

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4000000

) 3000000 m n

0

0 R square 0.9978 6

( 2000000

l a n g i

S 1000000

0 0 10 20 30 IGF-1R STD (ug/mL)

IGF-1R STD (ug/mL) Signal (600 nm) (Interpolated) (Entered) E2.1 0.899 279000.000 E2.2 0.862 271000.000

Figure 6-6: Concentration determination of WGA purified fractions using quantitative Western immunoblotting. Known concentrations of recombinant human IGF-1R extracellular domain (50-0.097 µg/mL), and WGA purified elution fraction 2 underwent SDS-PAGE, transferred onto nitrocellulose membrane, and interrogated using α-IGF-1R-α antibody. The single chain form of IGF-1R has an approximate mass of 160 kDa while the alpha chain has a mass of 125 kDa. The blot was visualised on the LI-COR® Odyssey system and quantified using Image Studio 4.0. The standard curve was construction using the signal of the IGF-1R standards (0.097 - 25 µg/mL) on GraphPad Prism® version 6.0.

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6.3.6 Development of a functional screen for in vitro IGF-1R activation To achieve the aim of developing a screening tool for IGF-1R activation, it was required to determine if the IGF-1R isolated from cell lysates was functional. Samples of purified IGF-1R were stimulated with an increasing concentration of IGF-I (0, 5, 10, 25, 50, 100, 500 ng/mL). The samples were subsequently separated by SDS-PAGE and electro-blotted onto nitrocellulose. Blots were initially probed for receptor phosphorylation using an α-phospho-IGF-1R-β antibody, the blot was then stripped and re-probed with an α-IGF-1R (total) antibody. The results shown in Figure 6-7 demonstrate that isolated IGF-1R can be activated by IGF-I in a dose depended manner. Two different Western blotting system were utilised; A) traditional X-ray filmed based with the use of a HRP conjugated secondary antibody and B) Near infrared based Odyssey system using Dylight conjugated fluorescent secondaries. The IGF-1R antibodies have been optimised and tested in the traditional HRP based system, therefore it was used as a positive control to the Odyssey system, which has a higher linear dynamic range than X-Ray film, allowing for more accurate quantification of the detected proteins.

For both systems, both phospho-IGF-1R and IGF-1R antibodies exhibit sufficient specificity as minimal non-specific bands could be detected. In both systems, the addition of increasing IGF-I leads to an increase in IGF-1R activation in a dose dependent fashion as evident by the increasing intensity of the phospho-IGF-1R β band (95 kDa). This indicates at the isolated IGF-1R is functional and can be activated by the addition of IGF-I. The intensity of the total IGF-1R bands across all samples remains consistent which is an indicator of equal protein loading.

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kDa

Figure 6-7: IGF-I stimulation on WGA purified IGF-1R. An increasing concentration of IGF-I (0, 5, 10, 25, 50, 100, 500 ng/mL) was added to WGA purified IGF-1R receptor supplemented with ATP and MnCl2. The reactions were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Anti-phospho-IGF-1R-β was added (WB: p-IGF-1R) to the membrane, imaged, stripped and probed with a total IGF-1R antibody (WB:IGF-1R). A) Imaging using HRP conjugated secondary antibody and development onto X-Ray films using enhanced chemiluminescent reagent. B) LI-COR® Odyssey system was used to visualise blots probed with DyLight™ 680 (red) or DyLight™ 800 (green) conjugated antibodies.

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6.3.7 High throughput of IGF-1R stimulation assay Having established an IGF-1R activation screening process, in order to realise the aim of generating an assay capable of screening a large number of potential modulators, further optimisation was required. The process of protein separation by SDS-PAGE and the downstream immune-blotting is both time consuming and does not scale with large numbers of samples. Therefore, to determine if similar activation responses could be achieved by an alternative method, IGF-1R which has been activated by IGF-I in a dose curve fashion, was blotted directly onto nitrocellulose, in a dot blot fashion, and probed with phospho-IGF-1R, stripped, and reprobed with IGF- 1R total in a similar manner as above and according to section 6.27.

The resulting dot blots Figure 6-8:A were scanned and each protein spot quantitated using Image Studio 4.0. The signal for each phospho-IGF-1R spot (upper panel, green) was normalised against the total IGF-1R signal (lower panel, red). The fluorescence intensities were plotted with the normalised signal units against log IGF- I concentration using GraphPad Prism® version 6.0. The resulting graph, Figure 6-8:B, displays the classical sigmoidal dose-response curve.

This high throughput IGF-1R activation model has the potential to be used as a screening method of receptor inhibition for large sample numbers.

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Figure 6-8: IGF-I stimulation on WGA purified IGF-1R. An increasing concentration of IGF-I ranging from 0-500 ng/mL was added to WGA purified IGF- IR receptor. Each reaction was spotted onto nitrocellulose, and processed as per section 6.27. A) The dot blot was probed with an α-phospho-IGF-1R-β antibody, stripped, then re-probed with an α-IGF-1R (total) anybody. The image was visualised on the LI- COR® Odyssey system at 700/800 nm wavelengths. B) The signal from each spot was quantified using Image Studio 4.0 and the normalised values curve fitted using GraphPad Prism® version 6.0. An increase in p-IGF-1R fluorescence signal indicates an increase in receptor activation.

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6.4 DISCUSSION

The aim of this project was to generate ScFv which can inhibit two pathways which have been implicated in tumour progression, namely the IGF and integrin systems. Both systems have been implicated in the proliferation, attachment and migration of tumour cells and have as such been a target for drug development.

The IGF-1R ScFv was able to inhibit both IGF-I depended metabolic activity (Figure 6-1) and migration (Figure 6-2) in MCF-7 cells. In terms of metabolic activity, the IGF-1R targeting ScFv demonstrated a dose depended response and was able to significantly reduce stimulation to 75.26% ±2.34 % and 73.37 ± 1.77% when MCF-7 cells were induced with IGF-I or VN+IGF-I respectively. In addition, it was also able to significantly reduce migration to 47.67 ± 14.18% at 100 μg/mL of ScFv tested.

Even though this demonstrates that the ScFv was functionally active, the exact mechanism of inhibition was not determined. The control blocking antibody against IGF-1R (αIR3) used for this study, binds to IGF-1R as an allosteric inhibitor of IGF-I (Doern et al., 2009). Signalling studies will be required to confirm if the ScFv acts to block the binding of IGF-I and the subsequent activation of IGF-1R. This could be performed by interrogating the cell lysate in Western blots and probing for phosphorylated IGF-1R which is a measure of receptor activation. In addition, signalling molecules downstream of IGF-1R such as MEK/ERK and PI3K should be monitored for activation or inhibition as further evidence of IGF-1R modulation by the ScFv (Hollier, et al., 2008). Another method in which an antibody against IGF-1R could modulate the effects of IGF-I is through the down regulation of the receptors expression. For example, a monoclonal antibody currently in clinical trials, termed A12, has been shown to prevent ligand-dependent signalling, and also down- modulation of IGF-1R (Burtrum, et al., 2003). To determine if the isolated ScFv can act in a similar manner, MCF-7 cells could be incubated with or without ScFv and the relative amount of IGF-1R could be determined using fluorescence-activated cell sorting analysis.

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Given the BIAcore results of the αv ScFv in the previous chapter, there was a potential that the phage display derived ScFv would be non-functional in inhibiting integrin activation. The results in the chapter showed that all of the candidates were not effective in ablating the action of VN in both migration and attachments studies at the concentrations tested (Figure 6-3, Figure 6-4). Even higher concentrations of α- αv ScFv beyond 100 μg/mL could be tested, however, due to lack of any response trend observed so far, it was unlikely that a higher dose would produce a positive response. Given the amount of resources invested into the generation of the αv integrin ScFv, this was an extremely disappointing result.

The screening of positive binders used in this project only discriminated between binding or non-binding ScFv. There is a need for a functional screening process in which additional selection criteria such as preventing receptor activation are included in addition to binding. In order to address this need, partially purified IGF-1R was isolated from MCF-7 lysate using WGA agarose (Figure 6-5). Upon in vitro stimulation with IGF-I, the receptor was successfully autophosphorylated and quantified using a high throughput process (Figure 6-8). Despite the isolated IGF-1R being functional, further optimisation steps are required before it can be used as a screening tool. One is that WGA agarose has a high affinity to not only IGF-1R, but also to other glycoproteins such as the insulin receptor and epidermal growth factor receptor (Akiyama, Kadooka, & Ogawara, 1985; Burant, Treutelaar, & Buse, 1986). In order to obtain a pure IGF-1R sample free of other glycoproteins, an immunoprecipitation procedure using an IGF-1R specific antibody (Cell signalling, #3027) could be used. Upon the completion of further optimisations, this assay would allow for the functional screening of phage clones, which would give a better indication of a positive clone earlier in the characterisation process.

A total of 1 unique ScFv against IGF-1R and 3 targeting αv integrin were tested for their ability to inhibited VN and IGF-I depended activities on MCF-7 cells. Of particular interest was the α-IGF-1R ScFv, which demonstrated functionalities in inhibiting both cell metabolic activities and also migration, which are highly relevant to a potential breast cancer therapeutic.

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General discussion

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Breast cancer remains pervasive in the community, with 1 in 8 women developing the disease. Even with improved detection and treatments, it is still the second most common cause of cancer related death in Australian women (Australian Institute of Health and Welfare, 2014). The treatment of breast cancer can be broadly divided into two main groups; 1) Surgery and radiation therapy targets cancer locally and 2) systemic approaches such as chemotherapy, hormonal therapy, and targeted molecular therapy. In addition, the choice of treatments depends on various factors such as the cancer stage, status of hormone receptors (oestrogen, progesterone), and the status of human epidermal growth factor receptor 2 (HER2) (Gradishar, Anderson, et al., 2015). Breast cancer treatments such as surgery and chemotherapy can have severe side effects, including hair loss, nausea, fatigue, increased risk of heart failure, and physical and emotional stress (Love, et al., 1989; Maunsell, et al., 1993; Volkova & Russell, 2011). Also, treating metastatic breast cancer (MBC), or cancer which has spread from the confines of the primary tumour to secondary sites remains problematic, with an average five year survival rate of 24% (Siegel, et al., 2014). As a result of such side effects, coupled with limited success in treating MBC, researchers have been focused on generating therapeutic treatments with greater specificity and thereby minimal side effects. Targeted molecular therapy is an avenue of particular interest due to the potential specificity the process promises, targeting only malignant cancer cells. An example of a well-known targeted therapy is Herceptin® (trastuzumab), a monoclonal antibody for the treatment of breast cancer. Herceptin® binds to HER2 tyrosine kinase, preventing ligand activation and downstream signal transduction, leading to inhibition of tumour cell proliferation (Vu & Claret, 2012). A study conducted on 4,046 patients has compared the value of Herceptin® when used in conjunction with chemotherapy, compared to chemotherapy treatment alone. The study revealed that Herceptin® therapy increased the 10 year survival rate by 37% in addition to increasing the disease-free survival at 10 years by 40% (Perez et al., 2014). Given the need for improved therapeutics against MBC, and the proven success of targeted therapy, this project aims to generate novel single chain variable fragments (ScFv) which inhibit the Insulin-like growth factor (IGF) and the αv integrin systems, in order to impede the migration and survival of breast cancer cells.

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The interaction between tumour cells and the micro environment has a profound effect on the cells ability to metastasise into distal sites. Complex interactions between growth factors such as insulin-like growth factors (IGF-I) and extracellular matrix (ECM) proteins such as vitronectin (VN) have been shown to increase migration, survival and proliferation of cancer cells (Hollier, et al., 2008; Kashyap, et al., 2011; Noble, Towne, Chopin, Leavesley, & Upton, 2003; Van Lonkhuyzen, et al., 2007). With this knowledge, the study contained in this thesis described the production and functional assessment of ScFv targeting αv integrin and IGF-1R, with the goal of ablating the functional effect of IGF-I and VN on breast cancer cells. Ultimately, the project sought to combine the αv integrin and IGF-1R targeting ScFv through the creation of a dual-targeting or bi-specific antibody (BsAb). The rationale for generating a single BsAb compared to 2 individual ScFv molecules targeting IGF-1R and αv integrin are that BsAb 1) streamline production and quality control as one single molecule compared to a number of molecules, and 2) assuming preclinical success, require a single clinical trial compared to separated safety trials for each component followed by a combination efficacy trial. The total cost of assessing a single cancer therapeutic candidate in phase I to phase IV clinical trials was conservatively calculated to be US$78.6 million (Office of the assistant secretary for planning and evaluation, 2014). Clearly, there is potential cost savings in conducting a single clinical trial for a BsAb compared to a combination treatment. Furthermore, associated with cost is the lengthy timeline from the start of clinical testing through to approval, which has been estimated to be 7.5 years (DiMasi, Hansen, & Grabowski, 2003). Taken together, BsAb are an attractive strategy of inhibiting two known pathways which have been implicated in cancer progression.

To generate IGF-1R and αv integrin targeting ScFv, Phage display was chosen as the method of antibody selection for this project. It has advantages over alternative methods using laboratory animals including: 1) all selection steps are performed in vitro, therefore bypassing ethical considerations in animal maintenance and wellbeing; and 2) allows for the selection of human antibodies without cross-species reaction or resorting to transgenic mice encoding for human antibody genes. Of the present monoclonal cancer therapeutic antibodies approved for use in the United States, a large proportion were derived from a non-human host such as mice and were subsequently

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humanised (n=9/23) or are a chimeric entity (n=4/23). Indeed, an analysis of pre- clinical and under investigation antibodies listed in the Therapeutic Antibody Database ((Tabs), http://tabs.craic.com), demonstrated that the use of animal derived antibodies remains the most prevalent source for potential treatments. The Therapeutics Antibody Database was queried based on the discovery platforms and the clinical trial status (

Figure 7-1). The results revealed that antibodies generated by humanising rodent antibodies had the highest percentage at each stage of the approval process, ranging from a low of 32 % (n=140/434) in the preclinical stage, to a high of 54 % in phase II (n=68/124). Phage display derived antibodies ranked 2nd in the early approval stages, with both preclinical and phase I at 19% (n=83/434 and n=20/105 respectively). Given the advantages of phage display technologies mentioned previously, the reason for the relatively low percentage of antibodies derived from phage display technologies is unclear. It may be due to the well characterised and mature process of creating chimeric and humanised antibodies from animal sources (Jones, Dear, Foote, Neuberger, & Winter, 1986; Neuberger et al., 1984). In addition, phage display is dependent on the library used for antibody screening, which translates the ownership, patents and licencing to be of key commercial importance, and could limit use in industrial and/or commercial settings. Finally, because of the long development time between preclinical and approval stage, improvements in phage technologies maybe not have reached the therapeutic pipeline.

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Figure 7-1: Antibody discovery platform and their clinical trial status.

The total antibody candidates from preclinical to approval of the five major platforms (rodent, chimeric rodent, humanised rodent, phage display and transgenic mouse) was determined using data sourced from The Therapeutics Antibody Database (http://tabs.craic.com)

The phage display selection process is complex. Both the adsorption and the panning of IGF-1R and αv integrin was conducted in PBS without calcium or magnesium. This selection choice was made due to the previous studies involving the panning of IGF-1R. Of integral importance is that the substrate being panned against is in a structural state representative of the in vivo conformation. On the cell surface, integrins exist as heterodimers of one α and one β subunit. Critical to this structure are divalent cations which stabilise the receptor and allow ligand binding (Pesho, Bledzka, Michalec, Cierniewski, & Plow, 2006; Zhang & Chen, 2012). It was however undertaken in this study to pan against an αv monomer instead of an αv containing heterodimer. It was theorised that by using the αv monomer instead of a heterodimer such as αvβ3 in the phage selection process, a pan αv integrin ScFv binder could be selected and therefore be able to block all members of the αv integrin family. Rather than selectively targeting αvβ3 or αvβ5, a pan αv ScFv would inhibit both αv integrins which have been implicated in breast cancer progression (Goodman & Picard, 2012). It has been demonstrated that a pan αv inhibitor is more effective in inhibiting migration compared to a specific αvβ5 inhibitor (Hollier, et al., 2008), likely due to additional αv integrins targeted in addition to αvβ5. Should an αv dimer such as αvβ3 or αvβ5 have been used for panning, the addition of 1 mM calcium or 1 mM

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magnesium would have been employed during panning. In addition to the binding conditions, another critical aspect of the phage display selection process is the library itself. The complexity of the phage display library dictates the number of unique antibody fragments it encodes. Because the phage selection process is random, a more complex library has a greater chance of selecting high affinity binders. The library used in this project, the Tomlinson I & J libraries, has a complexity of 2.8 × 108, which when compared to newer libraries such as the Morphosys proprietary HuCAL® PLATINUM® with 4.5 × 1010 is ~160 times less diverse (de Wildt, et al., 2000; Prassler, et al., 2011). The reduced diversity of the libraries used may explain yielding only 1 unique binder for IGF-1R. Indeed such a trend has been observed in previous studies using the Tomlinson I & J libraries, Yan and colleagues described 3 unique sequences from 96 binders (Yan, et al., 2004), 14 from 44 clones were generated by Eteshola et al. (Eteshola, 2010) and a further study found 3 from 800 clones screened (Patil, et al., 2015). Historically, phage display libraries were generated and used in an academic setting. It was quickly recognised that phage libraries have commercialisation potential, leading to spinoff companies and the acquisition of libraries by commercial entities. Given the commercial importance of display libraries, publicly available libraries are limited. An alternative, which was considered at the start of this project, is the creation of a naïve phage display library, which would be valuable in future phage display projects, in addition to bypassing licencing and intellectual property issues. In brief, the library construction involves the amplification of variable heavy and variable light regions from donor B-cells, ligated into phagemid vectors, and subsequent transformation into an E. coli host (de Haard et al., 1999; Pansri, et al., 2009a; Sheets, et al., 1998). Donor B-cells can be sourced from the blood of volunteers or, after ethical clearance, from blood stocks with the Australian Red Cross. However, due to potential time constrictions, a commercial phage display library was believed to be a more prudent resource and was obtained instead. Recently, the antibody Portrazza™ (Necitumumab) derived from a naïve library described by de Haard, et al. (1999) was approved by the FDA, giving evidence supporting this method of library generation.

As part of the standard phage panning procedure, an ELISA was performed after each round to select clones that bind the target with high affinity. While identification

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of binders is important, in the setting of the current study ScFv that not only bound but also blocked receptor activation were the aim, something an ELISA could not select or test for. Thus, an additional screening tool to identify ScFv binding in such a way as to block receptor activation would have been of great benefit to the project. One such possible method is the inclusion of biotin labelled IGF-I or VN during the monoclonal ELISA screening process. In this competitive ELISA, ScFv which have high affinity to the target should displace the labelled ligand, which can be quantified and ranked. A similar assay was performed by Burtrum, et al. (2003), with the IGF-I labelled with a radioisotope instead of biotin. However, this method still does not assess the state of activation of the target receptor. In an attempt to address this short coming, a high throughput IGF-1R stimulation assay was partially developed in this project. IGF-1R was enriched from MCF-7 cell lysate using agarose bound wheat germ agglutinin (WGA). The isolated IGF-1R was observed to autophosphorylate upon in vitro stimulation with IGF-I, suggesting that the receptor is functional and can bind to IGF-I (Figure 6-8). Using this assay to improve the screening process would involve applying the ScFv clones to immobilised receptor followed by addition of IGF-I and analysing for receptor activation. An ScFv would be considered positive if it could inhibit the activation of IGF-1R upon stimulation with IGF-I. This approach is similar to the kinase assays used for compound screening for IGF-1R kinase inhibitors, in which only the kinase domains of IGF-1R are used (Moriev et al., 2013).

Full length antibodies are large glycoproteins which require complex folding and are post-transactionally modified, making expression in prokaryotic hosts such as E. coli generally unsuitable (Robinson et al., 2015). However, due to the reduced size and the lack of the glycosylated Fc region, ScFv are commonly expressed using an E. coli host (Frenzel, Hust, & Schirrmann, 2013). Furthermore, the panning process involved selection of E. coli expressed ScFv on the surface of the phage, therefore, an E. coli expression system was chosen to produce the ScFv in this project. Since the phage selection process relied on displayed proteins generated using an E. coli host, the large scale expression of the ScFv would have similar binding characteristics to the ScFv- pIII fusion in the phage display. A disadvantage of using E. coli is that Lipopolysaccharide (LPS) is a major component of the outer membrane and may contaminate the sample. LPS is a potent endotoxin and must be removed before any

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in vivo and therapeutic use (Schwarz, Schmittner, Duschl, & Horejs-Hoeck, 2014). Recently, a LPS free E. coli strain has been developed, which would minimise downstream steps and potentially reduce production costs (Mamat et al., 2015). This strain of E. coli, termed ClearColi®, is already commercially available and should be a target for use in future expressions of ScFv that are destined for in vivo studies.

In preparation for ScFv purification, literature was consulted in depth and the vast majority outlined simple, straightforward protocols for purifying ScFvs generated using the Tomlinson libraries (Eteshola, 2010; Kim, Wang, et al., 2014; Wang, et al., 2012; Yan, et al., 2004). Therefore, that the major obstacle encountered in this project was the difficulties in purifying the expressed ScFv using the Ni-NTA based system was an unexpected turn of events. Different binding and wash conditions for Ni-NTA were trialled, however, the expressed ScFv did not bind to the affinity resin. Since this purification process required the polyhistidine affinity tag to be accessible, potentially the tag was obstructed due to ScFv aggregating into higher order structures such as dimers. To test this hypothesis, ScFv samples were analysed using SEC with PBS as the running buffer. The resulting chromatogram revealed two major populations of ScFv containing the monomer and dimer form (Figure 5-2,Figure 5-4). In order for the ScFv to bind to Ni-NTA, 1M urea was added during the purification process. The goal of urea is to weaken hydrophobic interaction among ScFv molecules, without denaturing (Wörn & Plückthun, 1998; Zangi, Zhou, & Berne, 2009). The formation of aggregates can be difficult to eliminate. Therefore, there has been considerable effort in researching ways to produce aggregation resistant antibodies. Of particular interest is the identification of key residues within the antigen binding sites (VH: amino acids 28, 30-33, 35; VL 24, 49-53, 56) that when substituted for an aspartic acid or glutamic acid residue, improved biophysical properties including aggregation resistance were observed (Dudgeon, Rouet, & Christ, 2013; Dudgeon et al., 2012). Researchers have already ‘retro-fitted’ commercial therapeutic antibodies in this fashion to successfully reduce aggregation, thus, potentially the same technique can be applied to the ScFv isolated in this project. Alternative expression hosts such as mammalian or insect cell based systems could also be used to express ScFv and BsAb molecules. It has been shown that E. coli have difficulties in the expression of higher complexity antibodies

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such as BsAb (~56 kDa) and alternatives such as CHO may be a better choice (Mack, Riethmüller, & Kufer, 1995).

Given the inefficacy of Ni-NTA, alternative affinity based solutions were investigated. In the commercial production of recombinant therapeutic IgG, protein-A chromatography is commonly used in the purification process, enabling capture through highly specific interaction with the Fc region. The resulting product is of high purity and achieved in a single step (Liu, Ma, Winter, & Bayer, 2010). Because binding is via interactions with the Fc regions, the addition of extraneous amino acids residues such as polyhistidine are not required, therefore eliminating any immunogenicity concerns related to such tags. Furthermore, it has been demonstrated that the addition of the peptide affinity tag can adversely affect protein function (Schmeisser et al., 2006). Therefore, purification strategies which do not employ the use of an affinity tag have distinct advantages. However, protein-A binding requires the presence of VH3 domains, which most antibody fragments such as ScFv lack (Nilson, Lögdberg, Kastern, Björck, & Åkerström, 1993). Therefore, protein-L affinity chromatography was chosen for this project, which can bind with high affinity to antibody fragments via kappa light chain interactions (Nilson, et al., 1993). By using protein-L in the purification of ScFv from expression culture supernatant, an increase in purity was observed compared to Ni-NTA (Figure 4-8). This is consistent with other studies that used protein-L in comparison to Ni-NTA, concluding that protein-L was a more versatile and robust method (Das, et al., 2005; Rouet, et al., 2012). Furthermore, the use of protein-L enables utilisation of antibody phage display libraries which do not encode a purification tag.

In order to assess the binding affinity of ScFv to its target, the purified ScFv were interrogated using the BIAcore system. The goal was to compare to binding affinity between the generated ScFv and commercially available antibodies as a guide to potential functionality in cells. Recombinant IGF-1R or αvβ3 was successfully immobilised onto a BIAcore sensor chip using amine coupling chemistry. Corresponding ScFv was sequentially injected over the sensor surface and the interaction measured in real time. The calculated affinity (KD) between the commercial IGF-1R targeting antibody and IGF-1R was 3.2 ± 1.31 x10-8 M (Figure 5-5). In

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-9 comparison, another similar study returned a KD value of 1.3 ± 0.5 x10 M, which was ~25 times stronger to the results from this study (Mehrnaz Keyhanfar, 2005). This difference in binding affinity could be due to the immobilisation method used, which may have impacted binding. In this study, IGF-1R was covalently immobilised onto the sensor surface whereas the Mehrnaz Keyhanfar (2005) study used a capture system to immobilised the antibody and receptor was injected over. Potentially this would allow the receptor to be less structurally constrained due to it being assessed in a solution phase. In comparison to the commercial antibody, the calculated KD between the generated α-IGF-1R ScFv and IGF-1R was 2.58x10-7 ± 3.236x108 M, which was an ~8 fold difference in binding affinity. Given the confirmation of dimers in the ScFv samples upon SEC analysis, it is likely that a mixed population consisting of ScFv dimers/monomers would decrease binding affinity, compared to a homogeneous sample consisting only of monomers (Figure 5-2). A similar BIAcore analysis was performed on a commercially available IgG targeting αv integrin and 3 generated ScFv candidates. The heterodimer αvβ3 was chosen preferentially in BIAcore binding analysis over the αv monomer used during the panning process because integrin exists on cells as a heterodimer. The commercial antibody returned a calculated KD of 2.589 x 10-8 ± 4.866 x 10-9 M, however, none of the 3 ScFv candidates showed any binding response even at high concentrations (Figure 5-6). Given the lack of binding to αvβ3, the next logical step was to determine if the ScFv could bind to αv monomer, for which it was generated against. Surprisingly, the αv protein failed to immobilise onto the BIAcore sensor chip, raising questions about the integrity of the commercially sourced integrin protein, and the potential functional influences in cell assays.

The purified ScFv against IGF-1R and αv integrin were tested for their ability to inhibit key cancer progression mechanisms using MCF-7 breast cancer cells. This cell line has been extensively used as a test model for the study of VN and IGF-I interactions. The IGF-1R ScFv generated from this study was able to inhibit both IGF- I depended metabolic activity (Figure 6-1) and migration (Figure 6-2). In terms of metabolic activity, the IGF-1R targeting ScFv demonstrated a dose depended response and was able to significantly reduce stimulation by 24.74% ±2.34 % and 26.63 ± 1.77% when MCF-7 cells were induced with IGF-I or VN+IGF-I respectively. In addition, it was also able to significantly reduce migration by 52.33 ± 14.18% at a dose

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of 100 μg/mL. Direct comparison in potency among preclinical antibodies is difficult, due to differences in the cell line used, functional assays and the lack of studies generating ScFv molecules against IGF-1R or αv integrin (Tabs, http://tabs.craic.com/). In addition, because a dose response curve of the ScFv was not generated from this project, comparing half maximal inhibitory concentration (IC50) is not possible. However, 5 µg/mL of 4D5, the parental murine antibody of Herceptin®, was able to inhibit the proliferation of the SK-BR-3 breast cancer cell line by 56% (Hudziak et al., 1989). A recent patent filing on an antibody targeting IGF-1R detailed testing with doses ranging from 0.8 µg/mL to 100 µg/mL on the prostate cell line DU145 (Chang, Losman, & Goldenberg, 2015). Cells were stimulated with IGF- I at 10 ng/mL and metabolic inhibition was measured using MTS reagent in a manner similar to this project. Significant inhibition compared to the control was achieved at 20 µg/mL (p<0.01) and 100 µg/mL (p<0.001), reducing stimulation to ~66% and ~50% respectively. Given that ScFv molecules are smaller in size compared to IgG, 100 µg/mL of the IGF-1R ScFv is ~25 molar excess to 20 µg/mL of IgG. However, as discussed earlier, aggregates were detected in the ScFv samples and may have reduced the potency of the ScFv in binding to their targets. Ideally the ScFv preparations would have been fractionated to allow harvest of the monomer fraction for functional assessment. A small scale analytical HPLC fractionation study was performed in Section 5.3.3, however, it was clear a large amount of preparation would be required to produce enough monomer for assessment. Therefore, due to the time scale pressures of this study, additional optimisations are required for the large scale preparative separation of ScFv monomer. A comparative analysis of aggregate vs monomer in cell- based assays would be an intriguing investigation. If the results were to indicate that the aggregate fraction is non-functional the implication would be that the monomer fraction would show inhibition at much lower concentrations than those observed in the current undertaking. The 3 αv integrin ScFv candidates were also functionally assessed in attachment and migration assays. Given the lack of binding shown in BIAcore studies, it was likely that the isolated ScFv were also non-functional in cell- based assays. The results from the assays showed that this was indeed the case, with all the candidates showing a lack of inhibition at all concentrations tested (Figure 6-3,Figure 6-4). In contrast, the αv targeting IgG was able to significantly inhibit cell attachment by 59.48 ± 8.48 % and cell migration by 95.71 ± 2.32 % supporting the idea of αv integrin as a therapeutic target. Integrins, such as αvβ3 and αvβ5, belonging

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to the αv family have been linked to cancer metastasis. Given this knowledge, one aim of this project is to develop as part of a BsAb a pan-αv integrin inhibitor, which would inhibit both αvβ3 and αvβ5. By using the monomer αv instead of the heterodimer in the phage display screening process, it was hoped that the isolated ScFv would inhibit all αv integrins. However, BIAcore studies revealed that the isolated ScFv did not bind to αv integrin (Figure 5-6), furthermore, function assessment in MCF-7 cells also returned negative results (Figure 6-3,Figure 6-4).

Given the BIAcore and the functional results for the αv integrin ScFv candidates, there was doubt about the recombinant αv integrin protein used during the phage screening process. Therefore, the recombinant αv integrin was interrogated using SDS- PAGE in order to determine protein purity and integrity. The results showed that the commercially generated and sourced αv integrin protein was not in its native conformation (Appendix 12) potentially explaining why the generated ScFv against αv integrin was observed to be non-functional in assays. This suggests that there was a breakdown in the in vitro wheat germ expression system (Nozawa et al., 2011), purification, or quality control steps employed by the manufacturer. In contrast, a recombinant αvβ3 integrin sourced from another supplier demonstrated high purity in silver stained SDS-PAGE, with the staining revealing a distinct band corresponding to the calculated molecular weight for the αv and β3 monomers. In addition, the band corresponding to the suspected αv subunit was immune-reactive to an α-αv antibody, whereas no such immunoreactivity was observed with the commercial αv integrin protein. As an alternative, the αv integrin protein could have been expressed and purified in-house, using a mammalian expression system such as HEK293 cells. It was reported by Tartaglia et al. (2013) that functional heterodimer αvβ3 was produced in this system, and thus, an αv subunit generated in this way could be used in future experiments. Clearly, at the beginning of this study there was no reason to doubt the integrity of the purchased αv protein and by the time it was deduced to be unsuitable, there was no possibility of rerunning the panning procedure let alone expressing and purifying the αv monomer beforehand. Despite problems encountered in the αv integrin selection, it remains a variable therapeutic target. Currently, a total of 14 antibodies targeting αv integrins are under investigation, with half in preclinical assessment. The most developed is , which is currently at stage 2/3 (

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Appendix 2).

Due to experimental difficulties encountered throughout the project, a singular ScFv targeting IGF-1R was developed and demonstrated functionality. However, as a consequence of the selection of non-functional αv integrin ScFv, the ultimate goal of this project, the generation of a BsAb, was not able to be fulfilled. Nevertheless, the IGF-1R targeting ScFv demonstrated functionality in a 2D culture. The next logical step is to retest the ScFv with MCF-7 cells cultured in 3D. There is evidence that breast cancer cells cultured in 3D, as compared to a 2D monolayer, mimic more closely tumour cells in vivo and their interaction with the tumour microenvironment. Differences in cells cultured in 3D compared to 2D include altered drug responses (dit Faute et al., 2002), changes in gene expression (Kenny et al., 2007) and altered proliferation and morphology (Krause, Maffini, Soto, & Sonnenschein, 2010). In addition to providing a more representative ScFv-tumour response, this assay would also give evidence if the generated smaller ScFv (~28 kDa) confers any advantages over IgG (~150 kDa) given generally a smaller molecule can penetrate deeper into a tumour (Yokota, Milenic, Whitlow, & Schlom, 1992). To achieve this, the ScFv and control IgG conjugated with Quantum dots would be added to fluorescence labelled MCF-7 cells cultured in 3D. Both the size of the tumour in addition to the spatial distribution of the labelled ScFv would be monitored using fluorescence imaging. (Xu, et al., 2012).

Throughout the cell-based assay, it was evident, with the exception of α-IGF-1R in the MTS assay, that the control antibodies elicited significant inhibition. Indeed, these properties may be exploited as an alternative strategy for the development of a BsAb against IGF-1R and αv integrin. An overview of three possible alternative methods for BsAb production are detailed in Figure 7-2. The first method requires the de novo sequencing of intact commercially available IgG-based inhibitors to deduce the amino acid sequence in order to obtain the VH and VL coding sequences. From there, ScFv could be ‘reverse engineered’ based on the sequences obtained. Historically, Edman degradation has been the default choice for protein sequencing, but is time consuming and routinely returns only 15-20 residues per run (Pham, Tropea, Wong, Quach, & Henzel, 2003). Today, newer techniques based on tandem

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mass spectrometry called Comparative shotgun protein sequencing (CSPS) can be used to sequence large proteins such as antibodies (Bandeira, Pham, Pevzner, Arnott, & Lill, 2008). CSPS has advantages over Edman degradation including comparatively rapid turnaround and an increase in sequence coverage. The resulting amino acid sequence can be back-translated into nucleotide sequences using informatics tools such as EMBOSS Backtranseq (http://www.ebi.ac.uk/Tools/st/emboss_backtranseq/) which can take into account codon usage requirements. Once both VH and VL sequences are obtained, an expression vector encoding for the ScFv would be constructed, followed by the expression, purification and characterisation and subsequently, BsAb generation. De novo sequencing is particularly important in antibodies derived from an immunised host, in which sequence determination is essential for patent filing, FDA approval and for quality control purposes (Center for Biologics Evaluation and Research (CBER), 1996; Pham et al., 2006).

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Figure 7-2 Alternative workflows for the generation of BsAb against αv integrin and IGF-1R.

Methods of generation for BsAb by recombinant and chemically conjugation which can incorporate the α-IGF-1R ScFv sequence isolated from this project. Three main approaches can be employed: 1) Reverse engineering of commercially available antibodies against IGF-1R and αv integrin by de novo sequencing followed by the generation and recombinant expression of the BsAb (orange box). 2) Reselection of αv integrin binding ScFv by phage display and subsequent expression (blue box). 3) Redox method of BsAb generation using commercially available antibodies (green box.

The second alternative method is a hybrid approach which incorporates the IGF- 1R sequence obtained from this project, using additional sequence information for αv integrin ScFv sourced either from de novo sequencing in method one, or repeating the phage display panning process using recombinant αv integrin generated as per Tartaglia, et al. (2013). Once the VH and VL coding sequences are deduced, the workflow continues in an identical manner to method one.

The third alternative method of BsAb generation is the redox method, where each of the two parental antibodies are selectively reduced at the heavy inter chain bond using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA) (Carlring, De Leenheer, & Heath, 2011; Owais, Kazmi, Tufail, & Zubair,

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2014). The partially reduced antibodies are combined in an oxidising environment, allowing for the re-formation of the parental antibody, or the novel formation of a BsAb. The antibody mixture is put through two affinity based purification steps to remove the parental antibody, leaving only IgG-based BsAb. Out of the three methods discussed, the redox method has the potential to be the simplest to perform. However, this process is inefficient, with only 19% of BsAb recovered from 1 mg of each of the parental antibodies as a starting point (Carlring, et al., 2011). This would need to be scaled down due to cost considerations. In addition, while the generation of this IgG based BsAb would be useful as a proof of concept and as a control antibody in functional assays, its usefulness is limited, due to 1) requiring the purchase of both monospecific antibodies, and 2) the generated BsAb is at a structural endpoint, therefore, unable to improve the binding affinity using processes like affinity maturation. There is also likely to be intellectual property issues which will likely limit commercial usefulness.

In summary, this project aims to generate function blocking ScFv targeting IGF- 1R and αv integrin with the goal of inhibiting the growth and migration of tumour cells. Phage display technologies were used to screen for ScFv binders against IGF- 1R and αv integrin. The result of the panning process returned 1 unique IGF-1R and 3 αv integrin ScFv binders which were expressed and purified. Problems were encountered using the conventional Ni-NTA based affinity purification scheme, an alternative protein-L based system was used and yielded ScFv of high purity. The binding affinity for the purified ScFv against IGF-1R was comparable to the commercially available antibodies when assessed using BIAcore. However, none of the αv integrin candidates showed any binding activities. Subsequent cell-based functional assays revealed that the α-IGF-1R ScFv generated was able to functionally inhibit both the metabolic activity and migration of breast cancer cells to a significant degree. However, all three αv integrin binding candidates had no functional effects. Given that the α-IGF-1R ScFv demonstrated functionality, the next logical steps are to perform affinity maturation in order to increase its affinity towards IGF-1R along with optimisation of the ScFv sequence and production process to generate preparations with a very high proportion of monomers. Subsequently, the optimised ScFv should be assessed using MCF-7 3D culture based assays before progressing into a mouse

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xenograft model to determine if the in vitro results observed in this project can be translated into an in vivo animal setting.

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Rational design of IGF-1R/αv integrin bi-specific antibodies

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8.1 INTRODUCTION

The preceding chapters of this dissertation described studies conducted with the goal of generating bi-specific antibodies (BsAb) targeting pathways critical to tumour development and progression. The BsAb’s were designed to specifically inhibit both insulin-like growth factor (IGF) and the integrin systems, as previous studies have shown the activation of both pathways promote cancer cell growth and survival (Hollier, et al., 2008; Kricker et al., 2010; Van Lonkhuyzen, et al., 2007). The proposed recombinant BsAb construct was to consist of two arms, each made up of an independent IGF-1R and αv integrin binding single chain variable fragment (ScFv). To identify the monospecific ScFv binders, a phage display screening process was undertaken using a ScFv library. In respect to the IGF-1R arm, a single ScFv clone was found to bind the receptor with high affinity and was demonstrated to inhibit MCF-7 cell proliferation and migration (Figure 6-1, Figure 6-2). Although the phage enrichment process was successful, the resulting clones against αv integrin demonstrated no function in cell-based assays, as observed by the lack of significant difference in responses compared to controls in both metabolic and migrations assays. Additional receptor binding studies using surface plasmon resonance also yielded no response from each clone isolated against αv integrin. In order to ascertain the reason for the observed lack of function, it was discovered that the the αv integrin protein sample used as the target in ScFv selection was degraded and may not have been representative of the true αv integrin structure, therefore, the panning process would not have selected specific αv integrin binding ScFv. In spite of the difficulties experienced in this project, the results obtained with the IGF-1R targeting ScFv demonstrate that the rationale is sound and the ultimate aim of generating a function blocking BsAb would be possible with further investigation. In an effort to provide a fulsome body of work, this chapter sets out to describe a refined methodology for the further development and successful generation of an IGF-1R-av integrin targeting BsAb able to inhibit cancer cell survival and metastasis

As detailed in chapters 5 and 6 of this document, the major obstacle encountered was the lack of function demonstrated by the α-αv integrin ScFv clones resulting from the phage display screening process. Without the isolation of a ScFv targeting the αv integrin, the generation of the proposed BsAb targeting both IGF-1R and αv integrin

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was, therefore, not possible. To address this problem, the phage display panning process can be repeated using an alternative source of αv integrin. However, the commercial availability of recombinant αv integrin from other vendors is limited. Previous studies have shown that integrins can be expressed using insect or mammalian systems (Mathias, Galleno, & Nemerow, 1998; Tartaglia, et al., 2013). Furthermore, in these instances the expressed recombinant protein retained the ability to bind cognate ligands, such as vitronectin. This suggests that the expressed proteins are functional and retain their native confirmation. Even with the use of an accurate αv-integrin sample as the selection target, there remains the possibility that the newly isolated ScFv could still exhibit limited functionality, despite the input of additional time and resources. For example, the ScFv could bind to a non-critical portion of the target receptor uninvolved with signalling activation. Thus, although positive binding ScFv would be isolated, inhibitory function as required may not be exhibited. Thus, to provide adequate confidence and detail for a fulsome proposal within this chapter an alternative strategy is required.

A substitute process for selecting αv integrin targeting antibody sequences may be to search the literature and patent databases for proven antibodies effective at targeting and inhibiting the function of αv integrins. While such an approach would deliver a proven functional candidate without the rigours of selection, it would, however, be undesirable as the use of published sequences would significantly limit both the novelty of the project and the commercial potential/value of the final BsAb. A promising monoclonal antibody currently in clinical trials specifically targeting the αv integrin is abituzumab (EMD 525797, 17E6) (Élez et al., 2015; Mitjans et al., 1995; Uhl, Zühlsdorf, Koernicke, Forssmann, & Kovar, 2014; Wirth et al., 2014). It was modified from antibody clone (17E6) generated from a mouse immunised with placental-derived αvβ3 (Mitjans, et al., 1995). It has demonstrated the ability to inhibit cell attachment and tumour cell proliferation in both in vitro and in vivo studies. In preclinical studies, abituzumab was able to inhibit the attachment of melanoma cells to substrate bound vitronectin by ~75% (Mitjans, et al., 1995). To complement the in vitro studies conducted, an in vivo nude mouse model was used to examine the effect of abituzumab on tumour development. The results showed that not only was abituzumab able to suppress tumour growth, but also the frequency of metastasis. The

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preclinical data demonstrated the potential of abituzumab and subsequently phase I clinical trials were conducted to assess the pharmacokinetics and safety profile (Uhl, et al., 2014; Wirth, et al., 2014). An increasing single dose of abituzumab was administrated in both studies, ranging from 35 to 1500 mg. From the phase I trial results it was ascertained that abituzumab was well tolerated at all concentrations, and supported the advancement to phase II trials using 750 mg and 1500 mg doses. Recently, the results of a phase II study on patients with metastatic prostate cancer revealed that progression-free survival did not significantly increase in response to abituzumab treatments (Hussain et al., 2016). However, there was evidence demonstrating prostate cancer–associated bone metastasis inhibition, further strengthing the role of αv integrin in tumour metastatic progression. Taken together, abituzumab has demonstrated functionality that makes it an ideal candidate from which to base the αv integrin targeting arm of the BsAb. Given that abituzumab has shown activity in blocking αv integrin-dependent processes, a patent search was conducted to ascertain the amino acid sequence of the heavy and light chains which were found to be detailed within US patent US8562986. The heavy and light chain amino acid sequences from abituzumab can, therefore, form the basis of a theoretical ScFv construct through incorporation into an appropriate vector structure. Although there are numerous instances where ScFv sequences have been converted to IgG format for use as monoclonal Ab therapies (Bujak, Matasci, Neri, & Wulhfard, 2014; B. Liu et al., 2007), the reverse (which is required in this instance) has also proven successful (Asano et al., 2013). Using the abituzumab sequences in combination with the IGF-1R targeting ScFv described in this thesis would enable the generation of a functional BsAb fulfilling this projects aims of targeting the IGF-1R and αv integrins. Functional assessment of this newly generated BsAb has the potential to validate the BsAb generation approach, in which two independent targeting ScFv molecules are recombinantly expressed as one dual targeting entity through the use of a linker sequence. Furthermore, the functional data generated from cell-based assays would also increase our understanding of targeting both the IGF and integrin pathways as a possible tumour inhibiting mechanism.

The choice of the ScFv expression host and vector should also be investigated, given the problems encountered as previously described in chapter 4 of this thesis. Of

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the four ScFv clones isolated from the phage display screening process, three contained an amber codon within the coding sequence, requiring modification prior to large scale expression. The Tomlinson phage display libraries I+J were constructed on the pIT2 vector backbone and paired with the HB2151 E. coli strain for expression. As demonstrated from results presented in this thesis, there were some shortcomings in using this particular phage display library in combination with the HB2151 strain. Firstly, an inherent property of the Tomlinson libraries was the potential for the random generation of amber stop codon sequences (TAG) within the ScFv CDS. In a non-amber codon suppression strain such as HB2151, this would lead to premature termination of translation. Mutation of the amber stop codon (TAG) DNA sequence, through SDM, to encode for glutamic acid (GAG) resulted in maintained translation, which was equivalent to that observed during phage selection rounds. The SDM procedure, detailed in Section 4.2.2, required extensive additional time and resources: 1) Generation of a new mutagenic primer set for each ScFv clone; 2) The mutagenesis process can be lengthy with PCR, cloning, and transformation steps; and, 3) Verification of the SDM process can only be done by DNA sequencing, which is slow and resource intensive. A second shortcoming of the pIT2/HB2151 system was the observed low expression yield. When using a routine medium such as 2YT, the final recovered ScFv of a single candidate was ~4.95 mg/L of culture. The low yield was problematic as this required multiple expression and purification batches in order to produce enough ScFv for both the biophysical and function characterisation assays. In addition, the expression culture volume was limited by commonly available laboratory equipment. An attempt was made to counteract the yield problem by using the EnPresso B medium, which increased the ScFv yield to ~22 mg/L, however, it was still limited by the phage display focused pIT2 vector.

Recently, a new vector termed pET-ScFv (https://www.addgene.org/67843/) was designed in order to address the limitations of the pIT2 and HB2151 E. coli expression system (Ossysek et al., 2015). Specifically, the problem with amber codon generation within the ScFv coding sequence. During the phage display process using an amber suppression E. coli strain such as TG1, the amber codon has no negative effect. However, it prevented full length ScFv expression in a non-suppressor strain such as HB2151, which has been traditionally chosen as the expression strain in studies

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utilising the pIT2 vector in Thomlinson I & J libraries (Scott et al., 2008; Xiong, Li, Yang, Burgess, & Dynan, 2009; Xu et al., 2004). In addition to the problems associated with amber stop codons, the pIT2 vector was generated to include a lac promoter. This promotor has been demonstrated to be loosely regulated, causing basal level expression which can affect the growth rate of E. coli and subsequent protein yield (Rosano & Ceccarelli, 2014). To address the limitations outlined herein, a new vector was constructed based on the popular pET expression platform, under the control of a T7 promoter (Wurm et al., 2016). The T7 promoter has been demonstrated to facilitate higher expression yield, and also greater control when compared to the lac promoter found in pIT2 (Tegel, Ottosson, & Hober, 2011). In combination with RosettaBlue(DE3)pLysS E. coli, ScFv selected from the Tomlinson libraries were successfully expressed at high levels. Indeed, an almost tenfold increase in protein yield was achieved using pET-ScFv/RosettaBlue(DE3)pLysS compared to pIT2/HB2151 in LB medium. In addition to the increased expression yield, the RosettaBlue(DE3)pLysS strain suppresses the undesirable translation of amber stop codons to terminate protein synthesis. This would allow for large-scale protein expression post-phage panning without additional intermediate steps. Given the advantages of the new vector and E. coli combination even when using LB medium during expression, it would be expected that the ScFv yield would further increase when a specialised medium such as Enpresso B is utilised. Given the problems in obtaining sufficient ScFv for both functional and biophysical characterisations, an increase in yield would decrease to amount of time and resources required and allow the undertaking of additional assays, such as the fractionation of the ScFv monomer and dimer for comparative functional assessment.

As part of the biophysical characterisation process to assess the integrity of the purified ScFv, samples were separated based on molecular shape and size using size exclusion chromatography (SEC). The results demonstrated that each ScFv preparation contained populations of dimers (Figure 5-2, Figure 5-4). Heterogeneity presented within ScFv samples has the potential to impact downstream characterisations, such as in BIAcore and functional studies (Vázquez-Rey & Lang, 2011). Ideally, each ScFv preparation would have been fractionated into the monomer and dimer for comparative functional assessment. While improvements in the

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expression host and vector system previously outlined above would be expected to reduce the generation of dimers, another complementary strategy is for the minimisation of dimer formation through modification to the purification process. Of particular concern was the low pH buffer (100 mM of the glycine-HCl solution, pH 2.7) used to elute the bound ScFv from protein-L agarose. It has been shown that antibody aggregates can form from various stressors, including low pH conditions (Sahin, Grillo, Perkins, & Roberts, 2010). Although samples were immediately neutralised, the low pH environment of the elution process can be inferred to have contributed to dimer formation. To overcome this problem, it has been demonstrated that the addition of sodium chloride (0.1-1.0 M) or Ethylene glycol to elution buffers raised the pH required (pH 7.0) to recover bound proteins (Vázquez-Rey & Lang, 2011), thus minimising the generation of additional aggregates due to the elution process. The reduction in the generation of potential dimers in addition to fractionation with SEC would further increase the stringency of the final product leading to increased confidence in the data generated.

The binding affinities of the generated ScFv were compared to commercially available Ab’s using Surface Plasmon Resonance (SPR) on the BIAcore X-100 instrument. Originally, the Langmuir kinetics model (1:1) of binding was chosen as the model of interaction between ScFv and its target when evaluating BIAcore results. However, experimental results obtained did not fit this model, likely due to the presence of dimers increasing the complexity of the interaction. Therefore, an alternative model, the steady-state affinity model, was used for this project. Even though both models can calculate the KD of the interactions, the steady-state affinity model does not yield the association (ka) and dissociation (kd) rate constants. This detail has significant implications upon data interpretations as it has been demonstrated that two compounds with identical KD can have vastly different pharmacokinetics due to the differences in ka and kd (GE Healthcare, 2012) . In addition, the presence of ScFv dimers would exhibit avidity effects, resulting in the overestimation of the KD value. A ScFv sample containing a higher proportion of dimer would give results less reflective of the actual affinity value compared to a sample with lower dimer proportions. Given each ScFv sample contained varying amounts of dimers, comparison of relative affinity can be problematic. Ideally, a sample with reduced

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heterogeneity would allow for the improved fitting of the interaction to the Langmuir kinetics model, allowing for the determination of the unique ka and kd values. In particular, kd values alone can be used to rank antibody candiates in a non- concentration dependend manner, allowing for the screening of non-purified antibody samples (Rouet, et al., 2012). Taken together, improvements in the BIAcore assay can be achieved by the fractionation and/or greater control of ScFv heterogeneity.

Functional characterisation of the generated ScFv was performed using the breast cancer cell line MCF-7 as detailed in chapter 6. These functional studies were successful in demonstrating that the α-IGF-1R ScFv significantly inhibited IGF+VN dependent metabolic activity and migration. To build on these positive results, additional functional assays would give further insights into the ScFv’s potential behaviour in an in vivo situation, as it was the ultimate goal of this project to produce a concept molecule for a novel human therapeutic. Compared to the traditional two- dimensional (2D) monolayer culture used in this study (Hongisto et al., 2013; Weigelt & Bissell, 2008), there is increasing evidence that cancer cells grown in three dimensional (3D) cultures may be more physiologically representative of the in vivo situation. In particular, MCF-7 cells cultured in 3D form round spheroids that closely resemble in vivo primary breast tumours (Vantangoli, Madnick, Huse, Weston, & Boekelheide, 2015). The difference in morphology between cells cultured in 2D vs. 3D has been demonstrated to result in altered drug responses, differences in gene expression and signalling pathways (Dhiman, Ray, & Panda, 2005; Vantangoli, et al., 2015). Of particular relevance to the goals of this project was the assessment of the growth kinetics of MCF-7 cells in 3D by Vinci et al. (2012). In this approach, the size of fluorescently labelled MCF-7 spheroids is monitored in real-time using the IncuCyte® Live Cell Analysis platform in response to treatments. In addition to providing a more representative tumour physiology, the use of a 3D spheroid assay would also give an indication as to whether the generated smaller BsAb (~56 kDa) confers any advantages over larger IgG (~150 kDa) given smaller therapeutics have been shown to penetrate deeper into tumours (Yokota, et al., 1992). The increase in penetrative depth could potentially translate into more effective inhibition of tumour growth, as monitored by the IncuCyte® system. Given the advantages over the 2D

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cell-based assays, 3D cell-based assays will form part of the proposed workflow to be detailed in this chapter.

While the combination of 2D and 3D cell-based assays are the initial steps in the function assessment of the BsAb, in vivo testing of the BsAb in an animal model is critical in the pathway for regulatory approval as a human therapeutic. Both the 2D and 3D in vitro models are limited as they do not yield information about the pharmacokinetics, nor the biodistribution and uptake of the ScFv/BsAb. All three limitations imposed by the in vitro models can be addressed using a labelled BsAb and subsequent injection into an in vivo system. Currently, there are several methods in labelling antibodies and antibody fragments such as ScFv. These include radioisotope labelling, use of near-infrared (NIR) fluorescence probes and labelling with fluorophores such as green fluorescence protein (GFP) via a conjugation reaction, or co-expression as a fusion protein (Hilderbrand & Weissleder, 2010; Lu, Gong, Yu, & Li, 2005; Pavlinkova, Beresford, Booth, Batra, & Colcher, 1999). However, radioisotope labelling requires specialised handling and detection methods (Kaur et al., 2012). Also, the direct expression of a large GFP molecule similar in MW to the ScFv (26.9 kDa) has the potential to both perturb expression as well as alter the biological function of the labelled antibody. The large size of GFP can be partially overcome using a split-GFP system, in which only the CDS of the 11th strand of GFP (GFP11), encoding the 16 amino acid sequence RDHMVLHEYVNAAGIT, is fused into the CDS of the protein of interest, in this case the BsAb (Ferrara, Listwan, Waldo, & Bradbury, 2011; Kamiyama et al., 2016). It has been demonstrated since GFP11 consists only of 16 amino acid residues, it does not interfere with the proper function and expression of the attached protein (Kamiyama, et al., 2016). To restore fluorescence, a recombinant protein consisting of the 10 strands of GFP (GFP1-10) is incubated with the ScFv-GFP11 fusion, at which point the complex self-assembles into the complete fluorophore. The split GFP system has been demonstrated to be successful in imaging cells and lower order organisms such as C. elegans, however, the delivery of the GFP1-10 protein for live imaging in an animal such as a mouse has not been demonstrated. Potentially, this problem can be mitigated through the determination of spatial information for the ScFv after the animal has been sacrificed and the tissue harvested.

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Due to the potential shown by the α-IGF-1R ScFv, this chapter aims to extend the results obtained from this study and provide the theoretical framework for the generation of a BsAb construct, subsequent expression and purification, and functional assessment.

8.2 EXPERIMENTAL PROCEDURES

8.2.1 Selection of ScFv encoding sequences In order to facilitate the generation of the tandem BsAb constructs, the amino acid sequence of each component were determined (Table 8-1). The IGF-1R ScFv amino acid sequence was obtained from this project as a result of phage display screening. For the generation of the negative controls, preselected ScFv targeting BSA, derived from the Tomlinson phage library, will be used. It is assumed that the incorporation of BSA targeting ScFv will have negligible effect in cell-based functional assays and will thus serve as a control domain in the BsAb’s. However, in binding BSA, it may potentially increase the pharmacokinetic characteristics of the BsAb by increasing the molecular weight (Müller et al., 2007). The amino acid sequence of the heavy and light chains of abituzumab were obtained from US patent no US8562986 (appendix 14). However, since it is an IgG monoclonal antibody, in silico modifications were needed to convert from IgG to ScFv format. Therefore, the

VL and the VH regions were isolated, with a (GlySer)3 linker joining the two domains.

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Table 8-1: The amino acid sequence of the IGF-1R, BSA and αv integrin targeting ScFv.

ScFv Amino acid sequence

IGF-1R ScFv AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRISQ (Phage EGDYTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRGVFDYWGQG display) TLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLN WYQQKPGKAPKLLIYRASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRK SPPTFGQGTKVEIKRAAAHHHHHH BSA ScFv AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIYY (Phage AGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGYYVFDYWGQG display) TLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLN WYQQKPGKAPKLLIYYASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDT SPTTFGQGTKVEIKRAAAHHHHHH αv integrin QVQLQQSGGELAKPGASVKVSCKASGYTFSSFWMHWVRQAPGQGLEWIGYINPRS ScFv (EMD GYTEYNEIFRDKATMTTDTSTSTAYMELSSLRSEDTAVYYCASFLGRGAMDYWGQGT 525797) TVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLAW YQQKPGKAPKLLIYYTSKIHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTFP YTFGQGTKVEIK 8.2.2 Construction and synthesis of the BsAb constructs Four constructs based on the isolated CDS are here proposed (Figure 8-1). All four BsAb present in the tandem ScFv format consisting of two ScFv joined using a middle linker: A) α-IGF-1R:α-αv, B) α-IGF-1R:α-BSA, C) α-αv:α-BSA and D) α- BSA:α-BSA. Constructs B and C were produced to ensure that the binding activity of α-IGF-1R or α-αv ScFv is retained when converted into the BsAb format. The middle linker chosen will consist of the amino acid sequence STDGNT, an optimised linker for the expression of tandem format BsAb in E. coli (Korn, Nettelbeck, Völkel, Müller, & Kontermann, 2004).

Each complete expression construct will be synthesised using the GeneArt© Strings service (Thermo Fisher Scientific, Scoresby). The resulting double-stranded linear DNA fragments are much more economical compared to traditional gene synthesis service. During this process, the desired amino acid sequence for each BsAb construct will be submitted for codon optimisation for E. coli expression, and also the addition of the restriction sites NcoI to the 5’ and NotI to the 3’ ends. The full codon optimised CDS and the translated amino acid sequence for all constructs can be found in Appendices 15-18.

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(A) α-IGF-1R+α-αv BsAb

(B) α-IGF-1R+α-BSA BsAb

(C) α-αv+α-BSA BsAb

(D) α-BSA+α-BSA BsAb

Figure 8-1:Proposed tandem bispecific antibody constructs against IGF-1R and αv integrin.

Schematics of four tandem bispecific antibody DNA construct encoding for A) α-IGF- 1R:α-αv B) α-IGF-1R:α-BSA C) α-αv:α-BSA D) α-BSA:α-BSA. The middle linker encodes for the amino acid sequence STDGNT. The restriction sites NcoI and NotI have been added 5’ and 3’ of the CDS respectively to facilitate the cloning of the insert into an expression vector. All constructs encode the GFP11 tag at the 3’ end, allowing for the detection of the BsAb using the split GFP system.

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8.2.3 Cloning of BsAb constructs into the expression vector pET-ScFv-T To insert the BsAb CDS into the pET-ScFv-T, double restriction digests using NcoI and NotI will be performed on both the vector and the BsAb CDS containing the engineered restriction sites (Figure 8-2) . The linearised vector will be incubated with the digested fragment insert in T4 ligase, followed by transformation into chemically competent E. coli. Successful clones containing the insert will be screened using colony PCR using the 5′ sequencing primer TAATACGACTCACTATAGG and 3′ sequencing primer GCTAGTTATTGCTCAGCGG.

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Figure 8-2 Cloning workflow of BsAb encoding fragment into the pET-ScFv-T vector.

The BsAb encoding fragment synthesised by GeneArt will contain the restriction enzyme sites Nco1 and Not1 to match the cloning site of the pET-ScFv-T vector backbone.

8.2.4 Transformation of BsAb constructs A 20 µL aliquot of chemically competent RosettaBlue(DE3)pLysS cells will be thawed on ice. The expression vector samples will then be mixed with cells and incubated on ice for 5 mins followed by a heat-shock step at 42 °C for 30 sec in a water bath. The tubes are chilled on ice for 2 mins and 500 µL of SOC medium added to

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each tube. The tubes will be incubated at 37 °C with shaking at 250 RPM for 1 h. Subsequently, samples will be plated onto TYE + Kanamycin (50 µg/mL) plates and incubated overnight at 37 °C. Single colonies will be picked from each plate, cultured overnight in 2xYT and plasmids extracted using the QIAGEN Mini-Prep Kit according to the manufacturer’s instructions. The purified DNA will be sent for sequence verification by AGRF using GCTAGTTATTGCTCAGCGG (5’) and TAATACGACTCACTATAGG (3’) sequencing primers.

8.2.5 Expression of BsAb EnPresso B is a proprietary expression medium system for E. coli claiming to guarantee an expression yield increase of at least 5-fold over traditional media such as LB medium. A starter culture will be initiated by inoculating E.coli encoding the BsAb into 20 mL of 2xYT+ Kanamycin (50 µg/mL) and incubated at 37 °C with shaking at 250 RPM for 6 h. The EnPresso B medium will be reconstituted by adding the contents of one silver bag (20 tablets) to 500 mL sterile water in a 2.5 L Erlenmeyer baffled cell culture flask. The starter culture and 250 μL of proprietary Reagent A will be added to the shaker flask and incubated overnight at 30 °C with shaking at 250 RPM. Next, IPTG will be added to a final concentration of 0.6 mM, along with Booster 500 powder and 520 μL of Reagent A. The culture will again incubate with shaking as previous for an additional 24 h. After the incubation period, the culture will be harvested and centrifuged at 3,300 g for 10 mins. The supernatant will be retained and subjected to protein-L agarose purification.

8.2.6 Affinity purification of BsAb using protein-L agarose The Supernatant containing the expressed BsAb will be purified using protein- L agarose chromatography. Initially, the supernatant will be filtered through a 0.45 μm membrane before dialysis against PBS. The clarified supernatant will then be applied to a protein-L agarose column equilibrated with PBS and allowed to flow by gravity. The column will be washed once with 5 mL of PBS to remove loosely bound proteins. Bound ScFv will be eluted from the beads using elution buffer (100 mM NaCl, 100 mM of glycine-HCl, 20% v/v Ethylene glycol, pH 7.0) and collected in 500 µL

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fractions. Samples will be interrogated for purity by SDS-PAGE and Western blotting as per section 4.2.6.

8.2.7 Expression and purification of αv integrin Based on the difficulties encountered with obtaining a commercial source of recombinant αv integrin, the expression and purification will be conducted in-house. The expression and purification of the αv integrin protein will be used only for BIAcore characterisation experiments. A study by Tartaglia, et al. (2013) demonstrated the successful expression and purification of the extracellular domains of αvβ3 by co-transformation of two vectors encoding for either αv or β3 integrins. The expression and purification of only the αv integrin monomer will be based on that study with modifications. In short, the CDS of the extracellular domains of αv integrin will be synthesised using GeneArt© Strings service, with the addition of NcoI and XhoI restriction sites at the 5’ and 3’ end, respectively. In addition, CDS encoding for 6 histidine residues will be added to the 3’ end. The codon optimised CDS will be cloned into the pQE-TriSystem vector for expression using the HEK 293 cell line. The resulting recombinant αv integrin will be purified using Ni-NTA affinity chromatography.

8.2.8 Fractionation of BsAb monomers The affinity purified BsAb will be fractionated using SEC on an HPLC instrument. In short, samples will be separated on a HiLoad Superdex 75 prep grade column that has been calibrated using a Low Molecular Weight (LMW) Kit which contained 5 protein standards spanning from 6 500 to 75 000 Daltons. The running buffer used will consist of high salt PBS (500 mM NaCl) as the running buffer at an isocratic flow rate of 0.5 mL/min. The resolved proteins will be monitored at both 214 nm and 280 nm wavelengths with 0.5 mL elution fractions will be collected. Monomer and dimer BsAb fractions collected will be individually pooled for affinity analysis using the BIAcore instrument.

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8.2.9 N-Terminal sequencing Purified BsAb will be submitted for N-terminal sequencing by Edman degradation to assess the correct cleavage of the PelB signal peptide from the N- terminal of the mature BsAb protein.

8.2.10 Surface Plasmon Resonance (SPR)-based affinity determination The BIAcore X100 system will be used to determine the binding affinity of the generated BsAb (α-IGF-1R:α-αv) and controls to IGF-1R and αv integrin. IGF-1R or αv integrin will be immobilised using amine coupling chemistry to a BIAcore sensor chip surface. In order to reduce avidity effects, the immobilisations levels will be kept low for all reactions. Due to the high structural homology of the insulin receptor (IR) to IGF-1R (Ullrich et al., 1986), the binding of BsAb to immobilised IR will also be conducted to measure potential cross-reactivity. Subsequently, five sequential injections consisting of increasing concentrations of BsAb will be applied over the sensor surface at a flow rate of 30 µL/min. The interaction will be analysed using a 1:1 kinetics model. The experiment will be repeated immobilising the BsAb on the sensor’s surface and injecting either IGF-1R, IR, αv integrin monomer or αv integrin dimers such as αvβ3 as the analyte.

8.2.11 Metabolic assessment of the BsAb on MCF-7 cells The CellTiter 96® AQueous One proliferation assay is based on the conversion of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS into a coloured formazan product by viable cells. MCF-7 cells will be passaged the day before the assay. The cells will then undergo serum starvation for 4 h by aspirating the GM, washing twice with PBS, before the addition of SFM. A series of treatments including commercially available IGF-1R blocking antibody, αv blocking antibody, matched isotype controls and a concentration range of BsAb and BsAb controls will set up in SFM. A combination treatment consisting both α- αv integrin and α-IGF-1R IgG will be used to benchmark the generated BsAb. Serum starved MCF-7 cells will be dissociated from the culture flask using TrypLE™, 5,000 cells added to each treatment within a microfuge tube and allowed to incubate at room temperature for 30

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mins. After this incubation period, the treated cells are transferred into individual wells of a 96 well plate and stimulated with or without VN at a final contraction of 1 µg/mL and IGF-I at 30 ng/mL. The treated cells are then incubated at 37 °C with 5% CO2 for 24 h, after which 20 µL of CellTiter 96® AQueous One Solution Reagent is added into each well and a further 1.5 h incubation at 37 °C performed for the formation of the coloured formazan product. The absorbance of each well is then measured at 490 nm using the Benchmark Plus microplate spectrophotometer (Bio-rad).

8.2.12 Migration assay MCF-7 cells will be serum starved for 4 h. During the SFM incubation, the lower chambers of the Transwell® plates will be incubated with both IGF-I (30 ng/mL) and VN (1 µg/mL) diluted in SFM. Treatments consisting of the four generated BsAb (IGF-1R:αv integrin, IGF-1R:α-BSA, αv integrin:α-BSA, α-BSA:α-BSA) and a combined treatment consisting of two commercially available inhibitors of IGF-1R and αv integrin will be prepared in SFM at multiple concentrations. The serum-starved MCF-7 cells will be harvested and incubated at 60,000 cells per treatment, in microfuge tubes and allowed to incubate with the antibody-based treatments for 0.5 h at room temperature before seeding into the upper chamber of a Transwell®. Plates will then be incubated for 15 h at 37 °C with 5% CO2 within a humidified incubator. At the completion of the migration period, the non-migrated cells will be physically removed from the upper surface of the semi-porous Transwell® membrane. Cells which had migrated to the lower surface of the membrane will be fixed in ~3.7% formaldehyde and stained with 0.01% (w/v) crystal violet in PBS. To quantify migrated cells, the crystal violet will be extracted from the stained cells using 10% (v/v) acetic acid and 100 µL aliquots transferred into a 96 well plate. The absorbance will be measured at 595 nm using the Benchmark Plus microplate spectrophotometer.

8.2.13 Attachment assay MCF-7 cells will be passaged the day before the assay to synchronise the cell cycles before serum starving for 4 h.. Treatments consisting of the four BsAb, α-αv+α- IGF-1R IgG and control IgG treatments will be diluted in SFM+BSA at various concentrations. Serum-starved cells are harvested and are then added at 20,000 cells

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per treatment and incubated for 30 mins at room temperature in a microcentrifuge tube. During this incubation, a 10x solution of IGF-I and VN (300 ng/mL, 10 μg/mL) is prepared and added to individual 96 well plate wells. After the 30 mins incubation, the treated cells will be seeded into the IGF-I+VN containing 96 well plate, diluting the

IGF-I+VN to 1x, and incubated for 30 mins at 37 °C with 95% air and 5% CO2 to allow for cell attachment. Cells which have not attached after this period are aspirated and wells washed twice with PBS. The cellular attachment is determined by the addition of 100 μL of SFM + 20 μL MTS reagent to each well and incubating for 1.5 h at 37 °C with 95% air and 5% CO2 for the conversion of MTS into the coloured product. This product will be quantified at 490 nm using the Benchmark Plus microplate spectrophotometer.

8.2.14 Cell signalling A method to determine if the generated BsAb can prevent the activation of IGF- 1R or αv integrin through binding of IGF-I and VN respectively is to assess receptor activation using Western blotting. MCF-7 cells will be seeded onto six-well plates at 7.5x105 cells per well and cultured overnight in growth medium. The growth medium will be aspirated and cells serum starved for 4 hrs. Increasing concentrations of the BsAb and control treatments will be added to the cells and pre-incubated for 1 hr at 37 °C, before stimulation with the addition of IGF-I and VN. Cells will be harvested and lysed at different time points. The resulting cell lysates will be separated based on MW through SDS-PAGE and blotted onto a nitrocellulose membrane. The blot will be probed with Phospho-IGF-1R Ab to directly determine IGF-1R activation, or Phospho-FAK (Tyr397) Antibody for integrin activation. Levels of activated downstream signalling pathways such as AKT and ERK will also be assessed.

8.2.15 3D tumour spheroid assays Briefly, 5,000 MCF-7 expressing nuclear-restricted RFP (Essen BioScience) will be seeded into each well of a 96 well ultra-low attachment plate (Corning). A centrifugation step at 1000 rpm for 10 mins will pellet the cells onto the bottom of each well. The spheroid formation rate will be monitored over the next 96 hrs using the IncuCyte ZOOM® instrument at which point the spheroids will be pre-treated with

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BsAb against IGF-1R/αv integrin, IGF-1R/BSA, αv integrin/BSA or BSA/BSA and IgG against IGF-1R and αv integrin before stimulation with IGF-I and VN. The size of the spheroids as indicated by the Total Fluorescence Object Area will be monitored for an additional 10 days. In addition to the total size and volume of the MCF-7 spheroid, the metabolic activity will be determined using the CellTiter-Glo® 3D Cell Viability Assay (Promega).

8.2.16 Xenograft model and stability testing Xenograft models will be performed as previously reported using luciferase labelled MCF-7 breast cancer cells (Burtrum, et al., 2003). Mice will be implanted with estradiol pellets one week prior to injecting tumour cells. Two million MCF-7 cells will be injected into the 4th inguinal mammary fat pad of 5-6 week old NOD/SCID 3 mice. Tumours will be allowed to reach 150-300 mm before intraperitoneal administration of vehicle control (saline), generated BsAb or commercial antibodies.

Tumours will be measured twice a week using the formula pi/6 (w1 (biggest diameter) xw2xw2). Once per week we will also perform live animal bioluminescence imaging using the IVIS Spectrum system (Caliper, Australia) to monitor tumour growth and metastasis. The weight of the mice will also be recorded. Upon the end of the study, the mice will be sacrificed and tumour and organ tissue harvested for immunohistochemical analysis using H&E, Ki-67 and tunnel stains. To assess the biodistribution and uptake of the BsAb tagged with GFP11, the tumour and organ tissue harvested will also be incubated with recombinant GFP1-10 protein, allowing for the spontaneous formation of functional GFP fused to the BsAb. The quantitation and localisation of the BsAb will be performed using fluorescence microscopy.

An in vivo BsAb stability study will also be performed by injecting a single intravenous dose of 1 mg of BsAb. The subsequent levels of the BsAb will be measured in serum collected at 5 min, 30 mins, 2 h, 7 h, 24 h, 48 h and 72 h post injection as compared to serum collected prior to BsAb injection. The detection of intact BsAb in serum will be conducted using the split GFP tag.

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8.2.17 Statistical analyses Quantitative assays will be performed in triplicates and will be presented as means ± standard error of the mean (SEM). To compare among treatments and controls, analysis of variance (ANOVA) with tukey post hoc test will be conducted. Significance will be accepted at ρ<0.05.

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8.3 DISCUSSION

Breast cancer is a burden in Australia, with an estimate of over 16,000 (150 males and 15,934 females) newly diagnosed cases in 2016 (Australian Institute of Health and Welfare, 2014). Despite advances in both detection and treatment, metastatic breast cancer (MBC) remains problematic to treat, leading to a poor long- term prognosis. MBC or stage IV breast cancer is characterised by the presence of secondary tumours that have disseminated from the primary tumour site (National Cancer Institute, 2011). Common areas in which distal metastases can be found are the bone, brain, liver and lungs (Kennecke et al., 2010), commonly causing interference with the functional integrity of these organs. Previous studies have demonstrated the relationship between the role of the extracellular matrix and the growth factor system in cancer metastatic process and tumour development (Clemmons & Maile, 2005; Doerr & Jones, 1996; Kim, et al., 2011; Weigelt & Bissell, 2008; White & Muller, 2007; Zent & Pozzi, 2009). In particular, two systems which have been implicated are the Insulin-like growth factor axis and αv integrins (Hollier, et al., 2008; Kashyap, et al., 2011; Van Lonkhuyzen, et al., 2007). It has been demonstrated by these studies that the co-activation of IGF-1R and αv integrin stimulates tumour cell growth and migration, through the synergistic activation of both pathways. Currently, therapeutic candidates in clinical trials target either the IGF or the integrin system individually, with none currently targeting both systems simultaneously (Crudden, et al., 2015; Ley, Rivera-Nieves, Sandborn, & Shattil, 2016). Given that both pathways are synergistically linked, a single molecule able to inhibit both IGF and integrin systems may offer a novel MBC therapy and potential improvements in prognosis.

One method of dual pathway inhibition is through the use of a bispecific antibody (BsAb). Unlike naturally occurring antibodies, BsAb molecules are synthetically generated and designed to bind two pre-determined targets (Kontermann, 2012). In the preceding chapters of this dissertation, the proposed BsAb described would comprise of α-IGF-1R and α-αv integrin single-chain variable fragment (ScFv) domains joined by a peptide linker. To obtain the coding sequences (CDS) encoding the ScFv molecules making up the two arms of the BsAb, they were to be generated individually through the phage display selection process with immobilised IGF-1R or αv integrin. Once the selection process was complete, as determined by the successful

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characterisation of candidates in biophysical and cell-based functional assays, clones can then be sequenced and a BsAb generated. In respect to the IGF-1R ScFv isolated as part of this process, it has been demonstrated to bind with high affinity to IGF-1R, which translates to the functional inhibition of both the migration and metabolic activity of tumour cells. However, unlike the promising result shown by the α-IGF-1R ScFv candidate, the αv integrin process yielded clones that did not bind to αv integrin and did not show functionality in cell-based assays. Given the lack of functional ScFv targeting the αv integrin, it was therefore not possible to generate the original BsAb incorporating both α-IGF-1R and α-αv integrin domains to fulfil the original objective of this project.

In spite of the difficulties experienced in this project, the results obtained with the IGF-1R targeting ScFv demonstrate the rationale is sound and the ultimate aim of generating a function blocking BsAb would be possible with further investigation. In order to fulfil the original aims of this project, a total of four new BsAb constructs were proposed for future study by incorporating a promising αv integrin antibody sequence derived from the literature. The proposed constructs were: A) α-IGF-1R:α- αv, B) α-IGF-1R:α-BSA, C) α-αv:α-BSA and D) α-BSA:α-BSA. Of the four new BsAb constructs proposed, three were designed to function as control antibodies. Additional variations of BsAb could be constructed from a combination of the three binding ScFv domains, such as swapping the order from α-IGF-1R:α-αv to α-αv:α- IGF-1R. In addition, the variable heavy and the light chains of each ScFv-based domain can also be rearranged internally. Despite the possibility in additional configurations, the proposed BsAb constructs should be characterised prior to further optimisation experiments. In order to obtain the αv integrin antibody sequence, a literature search was performed. Abituzumab was chosen as the αv integrin antibody, based on availability of its sequence information via patent information (US patent US8562986), in addition to having human pharmacokinetic and toxicology data as a result of phase I clinical trials (Uhl, et al., 2014; Wirth, et al., 2014). Having clinical data available for the αv integrin arm of newly generated BsAb would likely strengthen its support for further clinical trial progression. It should be noted that since the α-αv sequence was derived from a commercial source, the commercial value of the BsAb may be limited. Potentially, given the format conversion of Abituzumab into a BsAb

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arm along with partnering to α-IGF-1R domains, such steps may be considered sufficiently inventive and classified as a novel product. With the procurement of sequence information for the α-αv arm of the BsAb, 4 new codon optimised for E. coli expression constructs were proposed (Figure 8-1).

Currently, there is no consensus on the correct usage of a BsAb negative control in functional and clinical experiments (Choi et al., 2013). It has been commonly accepted that in experiments conducted using an intact full-length antibody, that an isotype match control is included as part of the study (Hulspas, O'Gorman, Wood, Gratama, & Sutherland, 2009). The rationale of including a negative control is to assess if there are any non-specific reactions due to non-CDR elements, or problems generated during the antibody production process. Commonly, this is due to the Fc receptor-mediated interactions (Datta-Mannan et al., 2015). However, in studies involving the use of antibody fragments, such as ScFv or BsAb, due to their synthetic nature, the generation of an isotype matched control is problematic and their usage is not consistent in the literature. In one report, an antibody-based negative control was not included at all as part of their study with the excipient (commonly PBS) of the ScFv used instead as a negative control (Taki et al., 2015). In an attempt to generate a more biologically relevant negative control for the BsAb, three of the four proposed BsAb constructs contain α-BSA ScFv domains. The α-BSA ScFv have been incorporated as negative control domains, having shown in a previous study that phage displaying anti-BSA ScFv were biologically inert in a mouse model (Dong et al., 2016). The proposed negative BsAb controls would validate the tandem BsAb format, and also allow for the robust interrogation of the constructs biological activities.

Protein heterogeneity is a concern for the characterisation and validation of human protein therapeutic candidates (Dorai & Ganguly, 2014). When the purified ScFv samples generated within this study were interrogated with SEC chromatography, a mixed population consisting of monomer and dimer species was revealed (Figure 5-2, Figure 5-4). Indeed, the detection and monitoring of protein aggregation is a major quality control step for any therapeutic protein production (Roberts, 2014). Previous studies addressed this mixed population by performing a SEC fractionation step to isolate the monomeric form of the ScFv (Persic et al., 1999;

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Rouet, et al., 2012). Initial fractionation testing within the current study showed that the final eluate was low in concentration, due in part to the low concentration of the starting unfractionated sample. It is believed the detailed improvements outlined in this chapter would significantly increase the expected expression yield, thus achieving a higher concentration of starting material prior to fractionation. In addition to the SEC fractionation, to further monitor for protein heterogeneity, N-terminal protein sequencing of the purified BsAb molecule was also added to the newly proposed characterisation workflow. This is to ensure that the engineered PelB signal peptide has been correctly cleaved from the mature BsAb molecule, as differential cleavage of signal peptides has been previously observed in E. coli expression systems (Pechsrichuang et al., 2016).

Another area of concern for any potential receptor targeting therapeutic is cross- reactivity, caused by inhibition of an off-target pathway. In particular, due to the high degree of structural homology between the IGF-1R and IR (Ullrich, et al., 1986), antibodies designed to bind the IGF-1R potentially can also bind non-specifically to the IR. This non-specific inhibition of the IR pathway can cause adverse effects such as hyperglycaemia (Atzori et al., 2011; Haluska, et al., 2014). In studies involving the generation of an IGF-1R inhibitor, assays generally include measures to assess the potential cross-reactivity with the IR. Various methods can be employed to monitor this interaction, such as using a solid phase receptor binding assay (Burtrum, et al., 2003) and Western blotting to assess IR activation (Girnita, et al., 2004). The pre- existing BIAcore assays as described previously in this thesis can be modified to assess for IR cross-reactivity. In the new workflow described in this chapter, IR cross- reactivity will be quantitatively assessed by immobilising IR alone on a BIAcore sensor, and BsAb will be subsequently injected onto the surface. This affinity of interaction will be compared to the affinity obtained from the BsAb injected over the surface with immobilised IGF-1R. The subsequent results will definitively show if cross-reactivity exists. Should cross-reactivity be demonstrated, additional rounds of development will be needed, such as repeating the phage display process, or alternatively, incorporating literature derived α-IGF-1R antibody sequences.

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The ultimate goal of this project was to facilitate the development of an anti- cancer therapeutic for use in humans. To achieve this aim, the BsAb candidates generated from this project have to be approved by regulatory bodies within each host country; such as the Food and Drug Administration (FDA) in the USA, European Medicines Agency (EMA) in the European Union and the Therapeutic Goods Administration (TGA) in Australia. To initiate this approval process in the USA, an Investigational New Drug (IND) application is needed to be submitted to the FDA, signalling that a pharmaceutical agent will be used in an experimental way (Holbein, 2009). A similar process exists in Australia, requiring a submission via the Clinical Trial Notification (CTN) Scheme or the Clinical Trial Exemption (CTX) Scheme. The IND contains information in three broad areas, which are 1) Data demonstrating that the product is reasonably safe for testing in humans, which is most likely to be supported by animal pharmacokinetics data, 2) Information concerning the manufacturing process, and 3) Clinical Protocols and Investigator Information. To assess if sufficient data has been generated with the additional steps proposed within this chapter, the pathway to a clinical trial of a parental antibody to the BsAb abituzumab should be replicated as a guide to the approval process needed for the BsAb. In the key pre-clinical study published by Mitjans, et al. (1995), the antibody clone 17E6 (abituzumab, EMD 525797) was generated by mouse immunisation with

αvβ3. The isolated antibody was characterised to determine αv integrin binding using in vitro methods such as enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS) and immunoprecipitation. In a similar manner, the BIAcore assays proposed in this chapter allow not only for the confirmation of binding between the two BsAb arms to either IGF-1R or αv integrin, but can also determine if the conversion from abituzumab IgG to ScFv/BsAb containing the abituzumab CDR has affected binding kinetics. Mitjans, et al. (1995) subsequently performed cell-based functional assays to determine tumour response to antibody treatment. One such experiment, the attachment assay, will be performed as proposed using a similar principle. Furthermore, in vivo mouse experiments will also be performed in a similar manner to preclinical studies for abituzumab. The safety profile of the BsAb can be extrapolated based on the parental abituzumab. A maximum dose of 1500 mg of abituzumab has been tested in clinical trial phase I, without inducing significant adverse reactions (Wirth, et al., 2014). Based on this information, it is reasonable that the same molar dose of the BsAb most similar to abituzumab (α-αv:α-BSA) can be

192 Chapter 8: Rational design of IGF-1R/αv integrin bi-specific antibodies

calculated. A 1500 mg dose of abituzumab (~150 kDa) is equivalent to 10 µmol. The equivalent molar dose of a BsAb (56 kDa) would be 560 mg, and this should be the initial upper limit for any clinical trials. Taken together, the assays proposed follow a similar trajectory of a drug currently under clinical trial phase II review. Therefore, it is highly likely that the modified assays in this chapter would increase the likelihood of success and facilitate further clinical progression.

Should the BsAb demonstrate success in blocking IGF and integrin dependent tumour progression, alternative pathways that are synergistic should also be investigated. One potential combination pair is between IGF-1R and Epidermal growth factor receptor (EGFR). They are both examples of tyrosine kinase receptors, which play important roles in tumour development through their influence on cell proliferation and migration. It has also been demonstrated that cross-talk exists between both IGF and EGF signalling pathways, making the targeting with a BsAb inhibiting both pathways commercially and clinically attractive (Jin & Esteva, 2008). A recent study investigating an IgG-based BsAb targeting IGF-1R and EGFR demonstrated potent tumour inhibition activities in both in vitro and mouse models (Schanzer et al., 2016). Given promising results revealed by the IgG based BsAb, further investigations into ScFv based BsAb targeting both IGF-1R and EGFR should also be warranted.

In conclusion, four novel BsAb were proposed using an in silico approach by incorporating the functionally active α-IGF-1R ScFv domains obtained from within this study and a promising α-αv integrin antibody currently under clinical trial. In addition to the new constructs, improved expression methodology was proposed with the aim of improving yield. Furthermore, the results from the additional quality control steps and improved functional assays would likely meet the requirements to warrant future pre-clinical and clinical studies. The newly proposed BsAb constructs and the additional methodologies contained within this chapter could be completed in 1 year, due to the optimisations of prior processes contained in this study. Such a development will enable new methods to treat and potentially mitigate breast cancer tumour growth and metastasis, thus improving the lives of breast cancer sufferers.

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194 Chapter 8: Rational design of IGF-1R/αv integrin bi-specific antibodies

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Appendices

Appendix 1: Current IGF-1R targeting antibodies in development. Source: The therapeutic antibody database (http://tabs.craic.com/)

Name Internal Antigen Company Trials Patents Phase Name ▼ Cixutumumab IMC-A12, IGF-1R ImClone 43 11 Phase 2 NSC742460 Ganitumab AMG 479 IGF-1R Amgen, 24 19 Phase 3 Millennium CP-751871, IGF-1R Amgen, Pfizer, 11 34 Phase 3 CP-751, 871 Schering- Plough RG1507 R1507 IGF-1R Genmab, 10 Phase 2 Roche Dalotuzumab MK-0646, IGF-1R Merck & Co 5 17 Phase 2 MK0646, h7C10 SCH IGF-1R Merck & Co, 4 19 Phase 2 717454, Schering- 19D12 Plough AVE1642 EM164 IGF-1R ImmunoGen 3 11 Phase 1 BIIB022 == M14 IGF-1R Biogen 3 9 Phase 1 MM-141 HER3, IGF- Dyax, 2 8 Phase 2 1R Merrimack RO4858696- IGF-1R Roche 19 Preclinical >> 000, RO4858696

Immunomedics hR1 IGF-1R Immunomedics 7 Preclinical patent anti-IGF- 1R GSK patent anti- IGF-1R GSK 4 Preclinical IGF-1R Biogen EI-04 == EI-04 EGFR, IGF- Biogen Preclinical 1R Roche patent Angiopoietin Roche 5 Preclinical anti-IGF- 2, IGF-1R 1R/ANGPT2

Xencor patent IGF-1R Xencor 2 Preclinical anti-IGF-1R Genentech IGF-1R Genentech 1 Preclinical patent anti-IGF- 1R U.Adelaide IGF-1R U.Adelaide 1 Preclinical patent anti-IGF- 1R

Appendices 245

Pierre Fabre 12B1 IGF-1R Merck & Co, 17 Preclinical patent anti-IGF- Pierre Fabre 1R Roche Glycart EGFR, IGF- Glycart 5 Preclinical patent anti- 1R EGFR/IGF-1R BMS patent anti- EGFR, IGF- BMS 5 Preclinical EGFR/IGF-1R 1R U.Hong Kong H10 IGF-1R U.Hong Kong 3 Preclinical patent anti-IGF- 1R >> NIH patent anti- IGF-1R NIH 4 Preclinical IGF-1R City of Hope IGF-1R City of Hope 2 Preclinical patent anti-IGF- 1R Pharmacia IGF-1R Pfizer 1 Preclinical patent anti-IGF- 1R Roche patent IR18 IGF-1R Roche 1 Preclinical anti-IGF-1R << Sorrento patent IGF-1R Sorrento 2 Preclinical anti-IGF-1R R-Pharm patent IGF-1R R-Pharm 1 Preclinical anti-IGF-1R U.Hong Kong HER2/neu, U.Hong Kong 1 Preclinical patent anti-IGF- IGF-1R 1R / Her2 << NRC Canada IGF-1R NRC Canada 3 Preclinical patent anti-IGF- 1R

246 Appendices

Appendix 2: Current αv integrins targeting antibodies in development. Source: The therapeutic antibody database (http://tabs.craic.com/)

Name Interna Antige Compa Paten Pape Phase l Name n ny ts rs Abegri Integri AME, 8 10 Phase 1/2 n, n αVβ3 Medim MEDI- mune 522, hLM60 9, Vitaxin , LM609 abituzumab EMD52 Integri Merck 8 4 Phase 2/3 5797, n αV Serono EMD 525797 , DI- 17E6, DI17E6 intetumumab >> CNTO Integri Centoc 23 9 Phase 1/2 95 n αV or Ortho STX-100 BG000 Integri Biogen 11 1 Phase 2 11 n αvβ6 , Strom edix IMGN388 << Integri Immun 1 Phase 1 n αV oGen VPI-2690B Integri Janssen 1 Phase 2 n αVβ3 Biotech , Vascu lar U.California patent anti- Integri Biogen 3 Preclinical Integrin αvβ5 n αvβ5 , U.Cal ifornia 264RAD Integri AstraZ 7 2 Preclinical n αvβ6 eneca, Medim mune Macrogenics mBLA Integri Macro 1 Preclinical patent anti- 3 n αvβ6 Genics Integrin Alpha V B 6 U.California 37E1B Integri U.Calif 7 Preclinical patent anti- 5 n αvβ8 ornia Integrin αvβ8

Appendices 247

Seattle Genetics patent anti- Integri Seattle 2 Preclinical Integrin alpha V beta 6 n αvβ6 Genetic s U.North Carolina patent Integri UNC 6 Preclinical anti-Integrin alphaV beta3 n αVβ3 MFEVP1 Integri Cancer 4 Preclinical n αvβ6 Resear ch Techno logy U.California patent anti- Integri U.Calif 1 Preclinical Integrin αvβ6 n αvβ6 ornia

248 Appendices

Appendix 3: Sequencing trace of αIGF-1R ScFv (WT)

Appendices 249

Appendix 4: Blast search of IGF-1R ScFv

anti-TNF alpha single chain Fv antibody [synthetic construct] Sequence ID: gb|AEX15593.1|Length: 245Number of Matches: 1 Related Information Range 1: 1 to 245GenPeptGraphicsNext MatchPrevious Match

Alignment statistics fo r match #1

Score Expect Method Identities Positives Gaps

426 bits(1094) 9e-150 Compositional matrix adjust. 229/245(93%) 232/245(94%) 0/245(0%)

Query 2 MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRISQEGDYT 61 MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS IS G T Sbjct 1 MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSTGAST 60

Query 62 LYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRGVFDYWGQGTLVTVSSGG 121 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKG FDYWGQGTLVTVSSGG Sbjct 61 TYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGAAFDYWGQGTLVTVSSGG 120

Query 122 GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL 181 GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL Sbjct 121 GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL 180

Query 182 LIYRASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRKSPPTFGQGTKVEI 241 LIY AS+LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ+ +P TFGQGTKVEI Sbjct 181 LIYSASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANNAPTTFGQGTKVEI 240

Query 242 KRAAA 246 KRAAA Sbjct 241 KRAAA 245

250 Appendices

Appendix 5: Sequencing trace of α-αv A2 ScFv (WT)

Appendices 251

Appendix 6: Sequencing trace of α-αv B1 ScFv (WT)

252 Appendices

Appendix 7: Sequencing trace of α-αv F3 ScFv (WT)

Appendices 253

Appendix 8: SDM reactions, gel extraction, and enzymatic treatments.

Site-Directed Mutagenesis reaction.

Reaction Final con Q5 Hot Start High-Fidelity 2X Master Mix 12.5 μl 1X 10 μM Forward Primer 1.25 μl 0.5 μM 10 μM Reverse Primer 1.25 μl 0.5 μM Template DNA 1 μl 1-25 ng Nuclease-free water 9.0 μl

Overview of Qiagen gel extraction kit

Step Reagent name Buffer compositions Mechanism

1 QG 5.5 M guanidine thiocyanate (GuSCN) Solubilisation of the DNA containing 20 mM Tris HCl pH 6.6 agarose 2 Isopropanol Isopropanol 3 DNA Binding Binding of DNA to the silica column 4 QG 5.5 M guanidine thiocyanate (GuSCN) Wash 20 mM Tris HCl pH 6.6 5 PE 10 mM Tris-HCl pH 7.5 Removal of excess salts from column 80% ethanol 6 Elution Water Elution of pDNA from column

Kinase, Ligase & Dpn1 (KLD) treatment.

10 μl RXN

PCR Product 1 μl

2X KLD Reaction Buffer 5 μl

10X KLD Enzyme Mix 1 μl

Nuclease-free water 3 μl

254 Appendices

Appendix 9: Sequential optimisation conducted for the purification of ScFv using Ni- NTA affinity chromatography.

Material Affinity Conditions Outcome system Cultures Periplasmic Ni-NTA Binding buffer: Silver The expressed ScFv expressing extract 50 mM NaH2PO4 stained was found in the flow- different Supernantant 300 mM NaCl SDS- through and the wash ScFv pH 8.0 PAGE, fractions, indicating clones Western poor binding of the Wash buffer: blotting ScFv to the resin. 50 mM NaH2PO4 300 mM NaCl 20 mM imidazole pH 8.0 Cultures Periplasmic Ni-NTA Binding buffer: Silver Elimination of expressing extract 50 mM NaH2PO4 stained imidazole in the wash different Supernantant 300 mM NaCl SDS- step did not improve ScFv pH 8.0 PAGE, retention of ScFv clones Western Wash buffer: blotting 50 mM NaH2PO4 300 mM NaCl 0 mM imidazole pH 8.0 ScFv clone Supernantant Ni-NTA Precipitation of ScFv Western The precipitation of using PEG6000 blotting ScFv was followed by NI-NTA supernantant was sucessul. However, the resulting pellet was insoulable in Ni- NTA buffer

Appendices 255

Appendix 10: BIAcore response of αv antibody on immobilised αv with or with Mg/Mn

256 Appendices

Appendix 11: Recombinant αv integrin datasheet

Appendices 257

Appendix 12: Quality control on recombinant αv integrin. Sample of the recombinant αv integrin was centrifuged at 10,000 g for 10 mins. The supernatant and the insoluble fraction was separated and SDS-PAGE loading buffer was added to both. Each sample was further divided into two with one set heated at 72 °C for mins prior to separation on a SDS-PAGE. The same samples were also used in a Western blot and interrogated with an α-αv antibody. Recombinant αvβ3 was included as a positive control for the αv portion of the heterodimer.

258 Appendices

Appendix 13: SDM removal of the amber stop codon. Each reaction sample post mutagenic PCR were separated on an agarose gel. The mutated (TAG to GAG) linearised vector of the correct size of ~5 kb (red arrow) was excised and the DNA contents isolated using a gel extraction kit.

Appendices 259

Appendix 14

Engineered anti-alpha V-integrin hybrid antibodies EMD 525797 US Patent No 8,562,986 B2 Date: 22.10.13

Variable (underlined) and constant heavy chain sequences QVQLQQSGGELAKPGASVKVSCKASGYTFSSFWMHWVRQAPGQGLEWIGY INPRSGYTEYNEIFRDKATMTTDTSTSTAYMELSSLRSEDTAVYYCASFLGRG AMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKP SNTKVDKTVEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQAQSTFRVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Variable (underlined) and constant light chain sequences DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWYQQKPGKAPKLLIYYTSK IHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTFPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC

ScFv sequence generated from variable heavy and light chains with linker sequence QVQLQQSGGELAKPGASVKVSCKASGYTFSSFWMHWVRQAPGQGLEWIGY INPRSGYTEYNEIFRDKATMTTDTSTSTAYMELSSLRSEDTAVYYCASFLGRG AMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTI TCRASQDISNYLAWYQQKPGKAPKLLIYYTSKIHSGVPSRFSGSGSGTDYTFT ISSLQPEDIATYYCQQGNTFPYTFGQGTKVEIK

260 Appendices

Appendix 15

α-IGF-1R+BSA BsAb

Protein AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW VSRISQEGDYTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GRGVFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGD RVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASHLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSRKSPPTFGQGTKVEIKRAAASTDGNTAMAEV QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIYY AGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGYYVF DYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTIT CRASQSISSYLNWYQQKPGKAPKLLIYYASNLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSDTSPTTFGQGTKVEIKRAAARDHMVLHEYVNAAGIT HHHHHH

DNA TAAGCAGCCATGGCCGAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGG TTCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACC TTTAGCAGCTATGCAATGAGCTGGGTTCGTCAGGCACCTGGTAAAGGTCT GGAATGGGTTAGCCGTATTAGCCAAGAAGGTGATTACACCCTGTATGCA GATAGCGTTAAAGGTCGTTTTACCATCAGCCGTGATAATAGCAAAAATAC CCTGTACCTGCAGATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTATT ATTGTGCAAAAGGTCGTGGTGTGTTTGATTATTGGGGTCAGGGCACCCTG GTTACCGTGAGCAGCGGTGGCGGTGGTAGTGGTGGTGGCGGTAGCGGAG GTGGTGGTTCAACCGATATTCAGATGACCCAGAGTCCGAGCAGCCTGAG CGCAAGCGTTGGTGATCGTGTTACCATTACCTGTCGTGCAAGCCAGAGCA TTAGCAGTTATCTGAATTGGTATCAGCAGAAACCGGGTAAAGCACCGAA ACTGCTGATTTATCGTGCCAGCCATCTGCAGAGCGGTGTTCCGAGCCGTT TTAGCGGTAGTGGTAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTG CAGCCGGAAGATTTTGCAACCTATTATTGTCAGCAGAGCCGTAAAAGCCC TCCGACCTTTGGCCAGGGCACCAAAGTTGAAATTAAACGTGCAGCAGCA AGCACCGATGGTAATACCGCAATGGCAGAGGTGCAACTGCTGGAATCAG GCGGTGGCCTGGTACAGCCAGGCGGTTCACTGCGTCTGTCATGCGCAGCA TCAGGCTTTACCTTTTCAAGCTATGCCATGTCATGGGTGCGCCAGGCTCC AGGCAAAGGCTTAGAATGGGTGAGCACCATCTATTATGCAGGTAGCAAT ACCTATTATGCCGATTCAGTGAAAGGTCGCTTCACCATTTCACGCGATAA TTCAAAAAACACGCTGTATTTACAGATGAACTCACTGCGTGCCGAAGATA CAGCCGTTTATTATTGCGCCAAAGGCTATTATGTCTTTGACTACTGGGGA CAGGGTACACTGGTGACAGTTAGCTCAGGTGGCGGAGGTTCAGGCGGAG GCGGATCAGGTGGTGGTGGAAGCACAGATATCCAGATGACACAGTCACC GAGCAGTCTGTCAGCATCAGTGGGTGATCGCGTGACAATTACATGCCGTG CCAGTCAGAGTATTTCTAGCTACTTAAACTGGTATCAACAAAAGCCTGGC AAAGCCCCTAAACTGTTAATTTACTATGCAAGCAACCTGCAGTCTGGTGT TCCGTCACGTTTTTCAGGTAGCGGTTCAGGTACAGATTTCACACTGACAA TTAGTTCACTGCAGCCAGAGGATTTCGCCACATACTATTGCCAGCAGTCA GATACCAGTCCGACCACATTTGGTCAGGGAACAAAGGTGGAAATCAAGC GTGCAGCCGCACGTGACCACATGGTCCTTCATGAGTACGTAAATGCTGCT GGGATTacaCATCATCACCATCATCATTAAGCGGCCGCTAAGCA

Appendices 261

Appendix 16

α-αv+BSA BsAb

Protein MQVQLQQSGGELAKPGASVKVSCKASGYTFSSFWMHWVRQAPGQGLEWI GYINPRSGYTEYNEIFRDKATMTTDTSTSTAYMELSSLRSEDTAVYYCASFL GRGAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGD RVTITCRASQDISNYLAWYQQKPGKAPKLLIYYTSKIHSGVPSRFSGSGSGTD YTFTISSLQPEDIATYYCQQGNTFPYTFGQGTKVEIKSTDGNTAMAEVQLLES GGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIYYAGSNT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGYYVFDYWGQ GTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSI SSYLNWYQQKPGKAPKLLIYYASNLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQSDTSPTTFGQGTKVEIKRAAARDHMVLHEYVNAAGITHHHHHH

DNA TAAGCACCATGGATGCAGGTTCAGCTGCAGCAGAGCGGTGGTGAACTGG CAAAACCGGGTGCAAGCGTTAAAGTTAGCTGTAAAGCAAGCGGTTATAC CTTTAGCAGCTTTTGGATGCATTGGGTTCGTCAGGCACCTGGTCAAGGTC TGGAATGGATTGGTTATATCAATCCGCGTAGTGGCTATACCGAATATAAC GAAATTTTTCGCGATAAGGCAACCATGACCACCGATACCAGCACCAGCA CCGCATATATGGAACTGAGCAGCCTGCGTAGCGAAGATACCGCAGTGTA TTATTGTGCAAGCTTTTTAGGTCGTGGCGCAATGGATTATTGGGGTCAGG GCACCACCGTTACCGTTAGCTCAGGTGGTGGTGGTAGTGGTGGCGGTGGT TCAGGCGGTGGCGGTAGCGATATTCAGATGACCCAGAGTCCGAGCAGTC TGAGCGCAAGCGTTGGTGATCGTGTTACCATTACCTGTCGTGCAAGCCAG GATATTAGCAATTATCTGGCATGGTATCAGCAGAAACCGGGTAAAGCAC CGAAACTGCTGATCTATTATACCAGCAAAATTCATAGCGGTGTTCCGAGC CGTTTTAGCGGTAGCGGTAGTGGCACCGATTATACATTTACCATTAGCAG TCTGCAGCCGGAAGATATTGCAACCTATTATTGTCAGCAGGGTAACACCT TTCCGTATACCTTTGGTCAGGGTACAAAAGTGGAAATCAAAAGTACCGAT GGTAATACCGCAATGGCAGAAGTGCAGCTGCTGGAATCAGGTGGCGGTC TGGTGCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAGCCAGTGGTTTT ACCTTTTCAAGCTATGCAATGAGCTGGGTGCGCCAGGCACCAGGCAAAG GTTTAGAATGGGTTAGCACCATCTATTATGCAGGCAGCAATACCTATTAT GCCGATAGCGTGAAAGGTCGCTTTACCATTTCACGTGATAATAGCAAAAA CACCCTGTACCTGCAGATGAATAGTCTGCGTGCAGAGGATACAGCCGTCT ATTATTGCGCAAAAGGCTATTATGTGTTCGATTACTGGGGACAGGGTACA CTGGTTACCGTGAGCAGCGGAGGTGGTGGATCTGGTGGCGGAGGTAGCG GAGGCGGAGGCAGCACCGATATCCAAATGACACAGAGCCCGTCAAGCCT GAGCGCCTCAGTGGGTGATCGCGTGACAATTACATGCCGTGCCAGCCAG AGCATTAGCAGCTATCTGAATTGGTATCAACAAAAACCTGGCAAGGCTCC CAAACTGTTAATTTACTATGCAAGCAATCTGCAGAGTGGTGTTCCGTCAC GTTTTTCAGGTAGCGGTTCAGGTACAGATTTTACCCTGACAATTAGCTCA CTGCAGCCTGAGGATTTCGCCACATATTATTGCCAACAGAGCGATACCAG TCCGACCACATTTGGCCAGGGCACGAAAGTTGAGATTAAACGTGCAGCA GCACGTGACCACATGGTCCTTCATGAGTACGTAAATGCTGCTGGGATTaca CATCATCACCATCATCATTAAGCGGCCGCTAAGCA

262 Appendices

Appendix 17

α-IGF-1R+αv BsAb

Protein AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW VSRISQEGDYTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GRGVFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGD RVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASHLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSRKSPPTFGQGTKVEIKRAAASTDGNTMQVQL QQSGGELAKPGASVKVSCKASGYTFSSFWMHWVRQAPGQGLEWIGYINPRS GYTEYNEIFRDKATMTTDTSTSTAYMELSSLRSEDTAVYYCASFLGRGAMD YWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCR ASQDISNYLAWYQQKPGKAPKLLIYYTSKIHSGVPSRFSGSGSGTDYTFTISSL QPEDIATYYCQQGNTFPYTFGQGTKVEIKRDHMVLHEYVNAAGITHHHHHH

DNA TAAGCAGCCATGGCCGAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGG TTCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACC TTTAGCAGCTATGCAATGAGCTGGGTTCGTCAGGCACCTGGTAAAGGTCT GGAATGGGTTAGCCGTATTAGCCAAGAAGGTGATTACACCCTGTATGCA GATAGCGTTAAAGGTCGTTTTACCATCAGCCGTGATAATAGCAAAAATAC CCTGTACCTGCAGATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTATT ATTGTGCAAAAGGTCGTGGTGTGTTTGATTATTGGGGTCAGGGCACCCTG GTTACCGTGAGCAGCGGTGGCGGTGGTAGTGGTGGTGGCGGTAGCGGAG GTGGTGGTTCAACCGATATTCAGATGACCCAGAGTCCGAGCAGCCTGAG CGCAAGCGTTGGTGATCGTGTTACCATTACCTGTCGTGCAAGCCAGAGCA TTAGCAGTTATCTGAATTGGTATCAGCAGAAACCGGGTAAAGCACCGAA ACTGCTGATTTATCGTGCCAGCCATCTGCAGAGCGGTGTTCCGAGCCGTT TTAGCGGTAGTGGTAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTG CAGCCGGAAGATTTTGCAACCTATTATTGTCAGCAGAGCCGTAAAAGCCC TCCGACCTTTGGCCAGGGCACCAAAGTTGAAATTAAACGTGCAGCAGCA AGCACCGATGGTAATACCATGCAGGTCCAGCTGCAGCAGTCAGGTGGTG AACTGGCAAAACCGGGTGCAAGCGTGAAAGTTAGCTGTAAAGCCAGTGG TTATACCTTTTCAAGCTTTTGGATGCATTGGGTGCGCCAGGCACCAGGTC AGGGTTTAGAATGGATTGGTTATATCAATCCGCGTAGCGGTTATACCGAA TATAACGAAATTTTTCGCGACAAAGCAACCATGACCACCGATACCAGCA CCAGCACCGCATATATGGAACTGAGCAGTCTGCGTAGTGAAGATACAGC AGTTTATTACTGCGCAAGCTTTTTAGGTCGCGGTGCAATGGATTACTGGG GACAGGGTACAACCGTTACCGTTAGCTCAGGTGGCGGAGGTTCAGGCGG AGGCGGATCAGGTGGTGGTGGGAGCGATATCCAAATGACACAGAGCCCG TCAAGCCTGTCAGCATCAGTGGGTGATCGCGTGACAATTACATGCCGTGC CAGTCAGGATATTAGTAATTATCTGGCATGGTATCAACAAAAGCCTGGCA AAGCCCCTAAACTTCTGATCTATTATACCAGCAAAATCCATAGCGGTGTG CCGTCACGTTTTTCAGGTAGCGGTTCAGGTACAGATTATACGTTTACCATT AGTTCACTGCAGCCAGAGGATATCGCCACATATTATTGCCAACAGGGTAA TACCTTTCCGTATACATTTGGTCAGGGAACGAAGGTGGAAATCAAACGTG ACCACATGGTCCTTCATGAGTACGTAAATGCTGCTGGGATTacaCATCATC ACCACCATCACTAAGCGGCCGCTAAGCA

Appendices 263

Appendix 18

α-BSA+BSA BsAb

Protein AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW VSTIYYAGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GYYVFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGD RVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASNLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSDTSPTTFGQGTKVEIKRAAASTDGNTAMAEV QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIYY AGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGYYVF DYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTIT CRASQSISSYLNWYQQKPGKAPKLLIYYASNLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSDTSPTTFGQGTKVEIKRAAARDHMVLHEYVNAAGIT HHHHHH

DNA TAAGCAGCCATGGCCGAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGG TTCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACC TTTAGCAGCTATGCAATGAGCTGGGTTCGTCAGGCACCTGGTAAAGGTCT GGAATGGGTTAGCACCATCTATTATGCAGGTAGCAATACCTATTATGCCG ATAGCGTTAAAGGTCGCTTTACCATTAGCCGTGATAATAGCAAAAATACC CTGTACCTGCAGATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTATTA TTGTGCAAAAGGCTATTATGTGTTCGACTATTGGGGTCAGGGCACCCTGG TTACCGTGAGCAGCGGTGGCGGTGGTAGTGGTGGTGGCGGTAGCGGAGG TGGTGGTTCAACCGATATTCAGATGACCCAGAGTCCGAGCAGCCTGAGC GCAAGCGTTGGTGATCGTGTTACCATTACCTGTCGTGCAAGCCAGAGCAT TAGCAGTTATCTGAATTGGTATCAGCAGAAACCGGGTAAAGCACCGAAA CTGCTGATTTATTACGCAAGCAATCTGCAGAGCGGTGTTCCGAGCCGTTT TAGCGGTAGTGGTAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGC AGCCGGAAGATTTTGCAACCTATTATTGTCAGCAGAGCGATACCAGTCCG ACCACCTTTGGCCAGGGCACCAAAGTTGAAATTAAACGTGCAGCAGCAA GCACCGATGGTAATACCGCAATGGCAGAGGTGCAACTGCTGGAATCAGG CGGTGGCCTGGTACAGCCAGGCGGTTCACTGCGTCTGTCATGCGCAGCAT CAGGCTTTACCTTTTCAAGCTATGCCATGTCATGGGTGCGCCAGGCTCCA GGCAAAGGCTTAGAATGGGTGAGTACAATTTATTATGCCGGTTCCAACAC GTATTATGCAGATTCAGTGAAAGGCCGTTTTACCATCTCACGCGATAATT CAAAAAACACGCTGTATTTACAGATGAACTCACTGCGTGCCGAAGATAC AGCCGTTTATTATTGCGCCAAAGGTTACTACGTCTTTGATTATTGGGGAC AAGGTACACTGGTGACCGTTAGCTCAGGTGGCGGAGGTTCAGGCGGAGG CGGATCAGGTGGTGGTGGAAGCACAGATATCCAGATGACACAGTCACCG AGCAGTCTGTCAGCATCAGTGGGTGATCGCGTGACAATTACATGCCGTGC CAGCCAGTCAATTAGCTCATACTTAAACTGGTATCAACAAAAGCCTGGCA AAGCCCCTAAACTGTTAATTTACTATGCATCAAACCTGCAGTCTGGTGTT CCGTCACGTTTTTCAGGTAGCGGTTCAGGTACAGATTTCACACTGACAAT TAGTTCACTGCAGCCAGAGGATTTCGCCACATACTATTGCCAGCAGTCAG ATACCTCACCGACAACATTTGGTCAAGGTACAAAAGTGGAAATCAAGAG AGCAGCAGCACGTGACCACATGGTCCTTCATGAGTACGTAAATGCTGCTG GGATTacaCATCATCACCATCACCACTAAGCGGCCGCTAAGCA

264 Appendices

Appendix 19

Appendices 265