Isolation and characterisation of against human and canine EphA2_ targets for comparative studies in brain cancer Arghavan Golbaz Hagh Bachelor of Biology Master of Biology-Physiology

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2019 The Australian Institute for Bioengineering and Nanotechnology (AIBN)

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Abstract Glioblastoma multiforme (GBM) is one of the high-grade gliomas, with a patient median survival of around 15 months from the time of diagnosis. Current treatments for this brain cancer initially comprise of surgery and radiotherapy, followed by administration of Temozolomide. Despite many advances in preclinical studies of GBM in mice, the outcomes have not been translated to the clinical level for humans. This is because the mouse model of GBM does not recapitulate the human disease and is not a suitable model for comparative studies of spontaneous tumours in humans. The canine model represents a powerful, large animal model of gliomas, since canine brain tumours occur spontaneously, with an incidence rate and patient age profile quite similar to human populations. EphA2 is one of the multi-domain receptors of the most significant tyrosine kinase family of receptors, and is highly expressed in both human and canine glioblastoma. Overexpression of EphA2 has been shown to correlate with tumour stage, progression and patient survival. Since EphA2 is not expressed in normal brain tissue, but is over-expressed on GBM cells, it is potentially a highly useful receptor for -based therapy of brain cancer in both humans and dogs. Thus, monoclonal antibodies (mAbs) which cross-react with both human and canine EphA2 would be valuable molecular entities with diagnostic and therapeutic potential. To generate mAbs specific for EphA2 with cross-reactivity to both canine and human EphA2 receptors, technology was employed using various strategies for the presentation of the EphA2 antigen to the phage antibody library. This included biopanning against firstly recombinant whole extracellular domain (ECD) of both human and canine EphA2, secondly recombinant ligand- binding domain (LBD) of human and canine EphA2, and lastly against cells expressing the native EphA2. Biopanning against recombinant EphA2-ECD generated one promising antibody (mAb AGH001) which showed cross-reactivity to both recombinant human and canine EphA2-ECD. However, mAb AGH001 was shown to also bind to EphA2 Knock Down (KD) cells, suggesting mAb AGH001 may bind to a domain within EphA2-ECD, which is conserved among EphA family receptors, other than the unique LBD of EphA2. To isolate specific anti-EphA2 antibodies for the LBD on EphA2-ECD, the human and canine LBD was utilised as an antigen for a biopanning campaign. Utilising this strategy of biopanning against the LBD of EpahA2, mAb HuB1 was isolated and showed binding to both human and canine EphA2-LBD and EphA2-ECD as well; however, mAb HuB1 also showed binding to the negative EphA2 cell line. This could be due to partially overlapping on other Eph receptors. mAb AGH001 and a positive control antibody, murine mAb 4B3 did not bind to EphA2-ECD sub- domains. It can be concluded that the recombinant ECD sub-domains did not recapitulate the native conformation of EphA2. Furthermore, we demonstrated that mAb AGH001 has a conformational II , therefore relies on a structural conformation provided by the recombinant ECD. Cell-based biopanning was subsequently employed to isolate the EphA2 mAbs with specificity to native, membrane-bound EphA2. Initial biopanning against EphA2 KD cells was carried out as a depletion step, followed by biopanning of EphA2 positive cells; however, binding studies of phage antibody clones showed that the antibodies were not specific for EphA2. Although biopanning of a phage antibody library against both canine and human EphA2 was partially successful to the extent that antibodies were isolated, these antibodies were not specific for EphA2 and were not employed in further studies. In studies associated with demonstrating that antibodies against EphA2 and EphA3 may assist in the diagnosis of GBM, commercial antibodies were used, as well as a control mAb that binds EGFR. These antibody preparations were used to detect EphA2 and EphA3 receptors on characterised GBM patient-derived cell lines by conjugation to Raman reporters. Binding studies were consistent with the expected expression levels on these cell lines. Multiplexing of biomarkers can be used to detect GBM heterogeneity for diagnostic purposes, and the goal is to develop an that can be used to detect GBM circulating tumour cells (CTC) from blood samples. This thesis highlights the challenges associated with isolating specific antibodies for a particular member of a receptor family, where there are high levels of homology to other members of the same family or where there are shared domains with other receptor families. However, the potential benefits of such antibodies towards the diagnosis and therapy of cancers with a low life expectancy, such as GBM, warrants the development of innovative methods to isolate specific mAbs that bind EphA receptors. Encouragingly, this research has shown the utility of using antibodies that bind EphA2 and EphA3 receptors as new diagnostic tools that would facilitate the monitoring of treatment efficacy. This would be a major breakthrough, since serial biopsies to monitor treatment efficacy are challenging and also medical imaging can yield false negatives/positives, and cannot be relied upon to detect treatment response.

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Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis.

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Publications included in this thesis

No publications included.

Submitted manuscripts included in this thesis

No manuscripts submitted for publication.

Other publications during candidature

Oral Conference Presentation

Golbaz A.H., Jones M. L., Puttick S., Howard C. B., Rose S., Whittaker A., Mahler S. M. Generation of theranostic antibodies for brain cancers optimised for comparative oncology through phage display technique. International Conference BioNano Innovation, Brisbane, Queensland, Australia. 24-29 September, 2017.

Contributions by others to the thesis My Supervisors Dr. Martina Jones, Dr. Simon Puttick, Dr. Christopher Howard and Prof. Stephen Mahler made significant contribution to the project design throughout. Dr. Jones, Prof. Mahler and Dr. Puttick provided substantial advice regarding the thesis writing.

Acknowledgment is given to Miss Zhen Zhang for her contribution in performing the CTC experiments.

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Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis.

Research Involving Human or Animal Subjects No animal or human subjects were involved in this research.

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Acknowledgements

First and foremost, my greatest gratitude goes to my principal supervisor, Professor Stephen M. Mahler, for having me in his group as a PhD student and being highly supportive in all steps of this project. Also, I'd like to express my sincere appreciation to my mentor and co-supervisor, Dr. Martina L. Jones, for her unlimited support from the first step to the last one through my PhD. It was she who taught me so many skills, in particular antibody phage display. Also, I would like to say many thanks to other supervisors Dr. Simon Puttick and Dr. Christopher B. Howard for their brilliant ideas and being ready to assist in different stages of this project.

I would like to express my special thanks to UQ for offering me a full scholarship and giving me the opportunity to perform my PhD.

Many thanks to Prof. Bryan Day and Dr. Brett Stringer at QIMR for donating GBM cells. Thanks and appreciation to Dr Amanda Nouwens in Proteomics Facility at SCMB for mass spectrometry analysis. I greatly appreciate Zhen Zhang for her collaboration with CTC detection experiments. Thank you to Dr. Sumukh Kumble for his professional drawing in Figure 1.4.

Thank you to all past and present members of the Mahler group for their valuable advice and assistance on different occasions. In particular, thank you to Dr. Christian Fercher, Dr. Xuan Bui, Dr. Mohammed Alfaleh, Dr. Lucia Zacchi, Christopher J. de Bakker, Michael Yeh, Irene Reto, Andri Wardiana, Nadya Panagides and Craig Barry.

The biggest thanks go to my husband and my best friend, Afshin, for his endless support, encouragement and love. This journey would have been much harder without you. Thanks for listening to me and your precious advice and making my life full of happiness.

I would like to extend my appreciation to my wonderful family. Big thanks to my sister and brother, Eli and Amir, for their supports and encouragement from miles away. Finally, this work is dedicated to my beloved parents, Fati and Mahmoud, for all the sacrifices you have made for me. Love you and thank you for everything.

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Financial support This work was supported by the Cure Brain Cancer Foundation (R14/2173) and the CSIRO Probing Biosystems Future Science Platform.

Arghavan Golbaz Hagh is academically sponsored by The University of Queensland International Scholarship (UQI).

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Keywords

Glioblastoma, comparative studies, monoclonal antibody, phage display, EphA2.

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 110702, Applied Immunology, 80% ANZSRC code: 100499, Medical Biotechnology not elsewhere classified, 20%

Fields of Research (FoR) Classification FoR code: 1004, Medical Biotechnology, 90% FoR code: 1108, Medical Microbiology, 10%

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Table of Contents Abstract ...... II Declaration by author ...... IV Publications included in this thesis ...... V Submitted manuscripts included in this thesis ...... V Other publications during candidature ...... V Contributions by others to the thesis ...... V Statement of parts of the thesis submitted to qualify for the award of another degree ...... VI Research Involving Human or Animal Subjects ...... VI Acknowledgements ...... VII Financial support ...... VIII Keywords ...... IX Australian and New Zealand Standard Research Classifications (ANZSRC) ...... IX Fields of Research (FoR) Classification ...... IX Table of contents ...... X List of Figures ...... XVII List of tables ...... XIX List of abbreviation ...... XX

Chapter 1. Thesis Introduction ...... 1 1.1 Brain cancer ...... 2 1.1.1 Malignant gliomas ...... 2 1.1.2 Who classification ...... 2 1.1.3 Symptoms ...... 2 1.1.4 Epidemiology ...... 2 1.1.4.1 GBM incidence in the world ...... 2 1.1.4.2 GBM incidence in Australia and mortality rate ...... 3 1.1.5 Diagnostic tools in brain cancer ...... 3 1.1.5.1 Imaging and radiology ...... 3 1.1.5.2 Molecular diagnosis and DNA-methylation profiling ...... 3 1.1.6 Prognostic factors ...... 4 1.1.7 Current treatment for glioblastoma ...... 4 1.1.8 Blood-Brain Barrier ...... 5 1.1.9 Glioblastoma biomarkers ...... 5 1.2 Comparative oncology: Canine primary brain tumours ...... 5

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1.3 Eph receptors classification, structure and binding of Eph/ephrin ...... 6 1.3.1 EphA/ephrin activation and signalling ...... 7 1.3.2 EphA2/ephrinA1 expression ...... 10 1.4 Monoclonal antibodies (mAbs) ...... 11 1.5 Generation of antibodies using phage display ...... 13 1.5.1 Presentations of membrane for affinity selection ...... 15 1.6 Project hypothesis and Aims ...... 17

Chapter 2. General methods ...... 18 2.1 Introduction ...... 19 2.2 Standard solutions ...... 19 2.3 Standards methods ...... 19 2.3.1 Polymerase chain reaction (PCR) amplification ...... 19 2.3.2 of DNA ...... 20 2.3.3 Cloning of PCR product into pcDNA 3.1(+) ...... 20 2.3.4 Bacterial transformation ...... 21 2.3.5 Colony PCR ...... 21 2.3.6 Inoculation ...... 21 2.3.7 Plasmid DNA purification ...... 21 2.3.8 DNA sequencing set up ...... 22 2.3.9 Large scale DNA preparation ...... 22 2.3.10 Transfection and expression of desired into Chinese Hamster Ovary (CHO) cells ... 22 2.3.11 via metal affinity chromatography ...... 23 2.3.12 Purification of protein A using HiTrap MabSelect Sure column...... 23 2.3.13 Size Exclusion Chromatography (SEC) ...... 23 2.3.14 Preparation and enzymatic digestion of protein samples in solution for peptide mass fingerprinting ...... 23 2.3.15 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) ...... 24 2.3.16 Protein immunoblot () ...... 24 2.3.17 Biopanning library on purified antigen ...... 25 2.3.17.1 Coating immunotubes and binding ...... 25 2.3.17.2 Elution of bound phage ...... 25 2.3.17.3 Infection ...... 26 2.3.17.4 Determination of output titre ...... 26 2.3.17.5 Preparation of glycerol stock and phage rescue ...... 26

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2.3.17.6 Titration of rescued phage stocks ...... 27 2.3.18 Polyclonal phage ELISA ...... 27 2.3.19 Monoclonal phage ELISA ...... 28 2.3.20 High-throughput sequencing of positive clones ...... 29 2.3.21 Reformatting of scFv particles to whole human IgG1 ...... 30 2.3.22. -Linked Immunosorbent Assay (ELISA) ...... 31 2.3.23 Competitive ELISA test ...... 31 2.3.24 Cell culture ...... 32 2.3.25 for reformatted scFv-derived antibodies ...... 32

Chapter 3. Isolation of canine and human monoclonal antibodies against recombinant canine and human EphA2 Extracellular domains (ECDs) ...... 34 3.1 Introduction ...... 35 3.2 Materials and methods ...... 36 3.2.1 Cloning of canine and human EphA2 Extracellular Domains (ECDs) into mammalian expression vector...... 36 3.2.2 Producing recombinant canine and human EphA2-ECD ...... 36 3.2.3 Biopanning on recombinant canine and human EphA2-ECD ...... 37 3.2.4 Sequences alignment and identification of the unique clones ...... 37 3.2.5 Reformatting of anti-canine and anti-human EphA2-ECD scFvs into whole human IgG1 .... 37 3.2.6 Production and purification of anti-canine and anti-human EphA2-ECD mAbs ...... 39 3.2.7 ELISA assay ...... 39 3.2.8 Kinetic analysis using Surface Plasmon Resonance (SRP) ...... 39 3.2.9 Analysis of antibody binding via flow cytometry ...... 39 3.3 Results ...... 40 3.3.1 Production of recombinant canine and human EphA2 Extracellular Domains ...... 41 3.3.2 Identification of canine and human EphA2-Extracellular Domains ...... 42 3.3.3 Generation of scFv phage binder against canine and human EphA2-ECD through affinity selection using phage display ...... 42 3.3.4 Enrichment of scFv phage binders to canine and human EphA2-ECD ...... 43 3.3.5 Screening individual phage positive clones specificity towards canine and human EphA2- ECD ...... 44 3.3.6 Sequence alignment and identification of unique clones to canine and human EphA2- ECD ...... 45

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3.3.7 Cross-Reactivity analysis of anti-human and anti-canine EphA2-ECD scFvs against human and canine EphA2 ...... 47 3.3.8 Reformatting of anti-EphA2 scFv phage binders to human Immunoglobulin G1 (IgG1) ...... 47 3.3.9 Expression of reformatted phage display-derived scFv into human IgG ...... 48 3.3.10 Binding of reformatted antibodies against recombinant canine and human EphA2-ECD .... 49 3.3.11 Affinity assessment of mAb AGH001 to recombinant human and canine EphA2-ECD ...... 50 3.3.12 Binding of mAb AGH001 to native EphA2 through flow cytometry ...... 51

3.3.13 Binding and competitive immunoassay ...... 54 3.4 Discussion ...... 55 3.5 Conclusion ...... 58

Chapter 4. Isolation of canine and human monoclonal antibodies against recombinant canine and human EphA2-Ligand Binding Domain (LBD) ...... 59 4.1 Introduction ...... 60 4.2 Materials and methods ...... 62 4.2.1 Cloning of canine and human EphA2- LBDs into mammalian expression vector ...... 62 4.2.2 Producing recombinant canine and human EphA2-LBD ...... 62 4.2.3 Preparation of human and canine EphA2-LBD in a solution for peptide mass fingerprinting analysis ...... 63 4.2.4 Biopanning against recombinant canine and human EphA2-LBD ...... 63 4.2.5 Sequence alignment and identification of the unique scFv phage clones to human and canine EphA2-LBDs ...... 63 4.2.6 scFv-phage production ...... 64 4.2.7 Reformatting of anti-canine and anti-human EphA2-LBD scFv particles into human IgG1 constant regions ...... 64 4.2.8 Expression and purification of the reformatted anti-human and anti-canine EphA2-LBD into CHO-XL 99 mammalian cells ...... 65 4.2.9 Analysis of anti EphA2-LBD antibodies binding via flow cytometry ...... 65 4.2.10 Binding ELISA assay for mAb 4B3 against recombinant canine and human EphA2-LBDs 65 4.3 Results ...... 65 4.3.1 Creation of truncated canine and human EphA2-LBD expression vector ...... 65 4.3.2 Production of recombinant canine and human EphA2-LBD ...... 66 4.3.3 Enrichment of scFv phage binders to canine, human EphA2-LBDs ...... 67 4.3.4 Screening individual phage clones for specificity to canine and human EphA2-LBD ...... 69 4.3.5 Sequence alignment and identification of unique clones to canine and human EphA2-LBD . 71

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4.3.6 Binding of scFv phage particles to canine and human EphA2-LBDs ...... 73 4.3.7 Reformatting of anti EphA2-LBD scFv phage binders to human Immunoglobulin G1 (IgG1) ...... 74 4.3.8 Production of reformatted CrossA11 and HuB1 into human IgG1 constant regions ...... 75 4.3.9 Binding and specificity of reformatted antibodies to recombinant human and canine EphA2- LBD and ECD ...... 75 4.3.10 Binding specificity of anti EphA2-LBD mAbs to native EphA2 receptors through flow cytometry analysis ...... 78 4.4 Discussion ...... 79

4.5 Conclusion ...... 81

Chapter 5. Identifying the epitope of mAb AGH001 within Extracellular Domain of the EphA2 receptor ...... 83 5.1 Introduction ...... 84 5.2 Materials and methods ...... 85 5.2.1 Cloning different domains of canine and human EphA2-ECD into a mammalian expression vector ...... 85 5.2.2 Expression of canine and human EphA2-extracellular domains ...... 87 5.2.3 ELISA assay against different domains of EphA2 ectodomain ...... 87 5.2.4 Protein immunoblot ...... 87 5.3 Results ...... 87 5.3.1 Creation expression vector for different domains of canine and human EphA2-ectodomain 87 5.3.2 Expression and production of recombinant canine and human EphA2 domains (CRD, LBD&CRD and FnI&II) ...... 89 5.3.3 Binding analysis of mAb AGH001 to different extracellular domains of human and canine EphA2 by ELISA ...... 90 5.3.4 Binding analysis of mAb AGH001 to different domains of human EphA2-ECD by western blot ...... 90 5.4 Discussion ...... 91

5.5 Conclusion ...... 92

Chapter 6. Isolation of human monoclonal antibodies against native EphA2 through cell- based biopanning ...... 94

6.1 Introduction ...... 95 6.2 Materials and methods ...... 97

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6.2.1 Cell culture ...... 97 6.2.2 Biopanning on U87 positive and negative EphA2 cell lines ...... 97 6.2.2.1 Binding and elution of phage ...... 97 6.2.2.2 Infection and amplification of eluted phage ...... 98 6.2.2.3 Titration of eluted phage ...... 98 6.2.2.4 Rescue of phage-infected E. coli ...... 98 6.2.2.5 Titration of rescued phage ...... 98 6.2.2.6 Analysis of enriched phage pools by flow cytometry ...... 98 6.2.2.7 Analysis of single phage clones by monoclonal phage ELISA ...... 99 6.3 Results ...... 100 6.3.1 Phage pool enrichment analysis by flow cytometry using EphA2 positive and KD U87 cells ...... 100 6.3.2 Monoclonal phage ELISA for human EphA2 scFv-phage binders ...... 101 6.4 Discussion ...... 106

6.5 Conclusion ...... 107

Chapter 7. Application of antibodies to detect Circulating Tumour cells in brain cancer .... 109 7.1 Introduction ...... 110 7.2 Materials and methods ...... 111 7.2.1 Antibodies ...... 111 7.2.2 Cell lines ...... 112 7.2.3 Adherent cell culture ...... 112 7.2.4 Single neurosphere cell culture ...... 112 7.2.5 Flow cytometry ...... 113 7.2.6 Gold nanoparticles synthesis ...... 113 7.2.7 Raman reporter preparation ...... 113 7.2.8 SERS-NPs functionalisation ...... 114 7.2.9 SERS-NPs labelling on single neurosphere primary glioblastoma cell lines ...... 114 7.2.10 SERS measurement ...... 114 7.3 Results ...... 115 7.3.1 Non-adherent sphere cell culture ...... 115 7.3.2 Binding specificity of commercial antibodies to the EphA2, EphA3 and EGFR on the surface of glioblastoma primary cells ...... 115 7.3.3 The specificity test of each Ab-SERS for EphA2, EphA3 and EGFR surface markers ...... 117 7.3.4 Heterogeneity profiling of multiple biomarkers for each glioblastoma primary single cell .. 118

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7.4 Discussion ...... 119 7.5 Conclusion ...... 121

Chapter 8. General discussion and conclusion ...... 122 8.1 General discussion ...... 123 8.2 General conclusion ...... 126

References ...... 127 Appendix 1: Full-length sequences of human and canine EphA2 receptors ...... 143 Appendix 2: Multiple sequence alignment of EphA-ECDs ...... 144 Appendix 3: The percentage of homology between EphA2-ECD and other EphA-ECDs ...... 146 Appendix 4: Screen capture of the MS-MS results for human and canine EphA2-ECD ...... 147 Appendix 5: Multiple sequence alignment of EphA-LBDs ...... 148 Appendix 6: The percentage of homology between EphA2-LBD and other EphA-ligand binding domains ...... 149 Appendix 7: Screen capture of the MS-MS results for human and canine EphA2-LBD ...... 149 Appendix 8: Alignment of cysteine-rich domain among EphA family receptors ...... 150 Appendix 9: The percentage of homology between EphA2-CRD and other EphA-Cysteine-rich domains ...... 151

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List of Figures

Figure 1.1 Schematic representation of ephrins and Eph structures…………………………………7 Figure 1.2 Schematic representation of Eph-ephrin signalling pathways……………………….…...8 Figure 1.3 Schematic illustration of scFv (A), and monoclonal antibody or IgG structure (B)….....12 Figure 1.4 Schematic illustration of a round of biopanning against immobilised recombinant protein………...... ………...... ………...... ………...... 15 Figure 3.1 pcDNA 3.1 (+)-Human EphA2-ECD-His plasmid map……………………………….. 41 Figure 3.2 SDS-PAGE analysis of both canine and human EphA2-Extracellular Domains ………42 Figure 3.3 Polyclonal phage ELISA for canine and human EphA2-ECD………………………….43 Figure 3.4 Monoclonal phage ELISA to screen for individual clones binding to canine EphA2- ECD…………………………………………………………………………………………………45 Figure 3.5 Colony PCR to amplify phagemid scFv of anti-human EphA2-ECD and anti-canine EphA2-ECD………………………………………………………………………………………...46 Figure 3.6 Cross-reactivity of human scFv-phage particle against canine EphA2-ECD, and three different canine scFv-phage particles against human EphA2-ECD………………………………...47 Figure 3.7 Coomassie blue staining of reformatted antibodies on SDS-PAGE…………………….48 Figure 3.8 Indirect ELISA to test for specificity of reformatted mAbs to recombinant canine and human EphA2-ECD………………………………………………………………………………...49 Figure 3.9 SPR sensorgrams (Biacore T200) for the interaction between ligand (AGH001) and human EphA2-ECD and canine EphA2-ECD analytes…………………………………………………… 51 Figure 3.10 Flow cytometric analysis of PC3 and LNCaP exposed to mAbs AGH001 and 4B3…..52 Figure 3.11 Flow cytometry analysis of positive and EphA2 knock down human glioblastoma cell line…………………………………………………………………………………………………..54 Figure 3.12 Inhibition ELISA for competitive binding to recombinant human EphA2-ECD between mAb AGH001 and mAb 4B3……………………………………………………………………… 55 Figure 4.1 Structure of the EphA2-LBD……………………………………………………………61 Figure 4.2. pcDNA 3.1 (+)-Human EphA2-LBD-His plasmid map………………………………..66 Figure 4.3 SDS-PAGE analysis of both canine and human EphA2-LBDs…………………………67 Figure 4.4 Polyclonal phage ELISA to test the enrichment of phage pools to canine and human EphA2-LBDs ……………………………………………………………………………………….68 Figure 4.5 Monoclonal phage ELISA to screen individual clones binding to canine and human EphA2-LBDs………………………………………………………………………………………..69 Figure 4.6 Colony PCR to amplify phagemid scFv of anti-canine EphA2-LBD…………………...72 XVII

Figure 4.7 ELISA assay to test the binding of scFv phage particles towards recombinant canine, human EphA2-LBDs………………………………………………………………………………..74 Figure 4.8 SDS-PAGE analysis of reformatted CrossA11 and HuB1 mAbs into human IgG1 in reduced and non-reduced conditions………………………………………………………………. 75 Figure 4.9 ELISA on purified canine and human EphA2-LBD and EphA2-ECD………………… 76 Figure 4.10 Binding analysis of HuB1 and CrossA11 mAbs to EphA2-LBD by western blot…... 77 Figure 4.11 ELISA assay to test the mAb 4B3 to recombinant canine and human EphA2- LBD…………………………………………………………………………………………………77 Figure 4.12 Flow cytometric analysis of U87 and EphA2 KD U87 cells exposed to HuB1, CrossA11 and 4B3 antibodies……………………………………………………………………… 78 Figure 5.1 pcDNA 3.1 (+)-Human EphA2-CRD-His plasmid map……………………………...…88 Figure 5.2 SDS-PAGE analysis of different domains of human and canine EphA2-ECD after SEC purification…………………………………………………………………………………………. 89 Figure 5.3 ELISA assay to test the binding activity of mAb AGH001 to different sub-domains of recombinant canine and human EphA2-ECD ………………………………………………………90 Figure 5.4 Binding analysis of A) mAb AGH001, B) mAb 4B3 on different domains of human EphA2 ………………………………………………………………………………………………91 Figure 6.1: Flow cytometry analysis of phage pools enrichment after three rounds of biopanning using positive EphA2 U87 cell line, EphA2 knock down U87 human glioblastoma cell line…….101 Figure 6.2 Monoclonal phage ELISA to screen individual phage clones binding to EphA2……..103 Figure 7.1 Binding analysis of commercial antibodies to GBM markers…………………………116 Figure 7.2 Specificity test of Ab-SERS labels on single neurosphere primary glioblastoma cells..118 Figure 7.3 Multiple biomarker heterogeneity profiling…………………………………………….119

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List of tables

Table 2.1 Oligonucleotide primers used for DNA sequencing of pcDNA 3.1……………………...21 Table 2.2 Oligo primers used for PCR and DNA sequencing of scFv-Phage clones……………… 30 Table 3.1 Oligonucleotide primers used for amplification of human and canine EphA2- ECD from full length human and canine EphA2 constructs……………………………………………………36 Table 3.2 Oligos names used for PCR of heavy and light variable scFv domains against human and canine EphA2-ECD………………………………………………………………………………... 38 Table 3.3 mAbXpress Vectors containing human constant backbone regions……………………..38 Table 3.4 Sequence of the primers used for amplifying heavy and light variable regions…………38 Table 3.5 Primers for screening colonies………………………………………………………...... 39 Table 3.6. The alignment of human and canine EphA2-ECD sequences…………………………..40 Table 3.7 Representative the CDR regions of the heavy and light chain variable regions…………46 Table 3.8 Affinity values for the interaction between mAb AGH001 and recombinant human and canine EphA2-ECD determined via SPR…………………………………..……………………….51 Table 3.9 Comparison of mAbs 4B3 and AGH001 CDRs……………………….…………………55 Table 4.1 Oligonucleotide primers used for PCR of human and canine EphA2-LBD……………..62 Table 4.2 Oligo primers used for PCR and DNA sequencing of scFv-Phage clones ………………63 Table 4.3 Oligos names used for PCR of anti EphA2-LBD heavy and light variable scFv domains……………………………………………………………………………………...………64 Table 4.4 Primer sequences used for amplifying anti EphA2-LBD heavy and light variable regions……………………………………………………………………………………...……….64 Table 4.5 Primers for screening anti EphA2-LBD colonies…………………...……………………65 Table 4.6 The alignment of human and canine EphA2-LBD sequences……………..……………..66 Table 4.7 Sequence alignment of complementary-determining regions of the heavy and light chain variable regions towards canine and human EphA2-LBDs………………………………….……..72 Table 5.1 Oligonucleotide primers used for PCR of human and canine EphA2 different domains 86 Table 5.2 The sequence of human EphA2 extracellular domains…………………...…88

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List of abbreviations

2YT 2× Yeast Extract Tryptone Ab Antibody ACA Anti-clumping agent ACN acetonitrile AFBP Anterior fat body protein AGRF Australian Genome Research Facility ANARC Antigen receptor Numbering And Receptor Classification CDR Complementarity-determining regions CRD Cysteine-rich Domain CTC Circulating Tumour Cell DTT Dithiothreitol ECD Extracellular domain ELISA Enzyme-Linked Immunosorbent Assay EGF Epidermal Growth Factor Eph Erythropoietin-producing hepatocellular Ephrins Eph receptor interacting protein FBS Fetal Bovine Serum FDA U.S. Food and Drug Administration KD Knock Down GBM Glioblastoma multiforme HRP horseradish peroxidase IAA Iodoacetamide Ig G immunoglobulin G IMAC immobilized metal ion affinity chromatography IMGT the international ImMunoGeneTics database LB Luria-Bertani medium LBD Ligand Binding Domain mAb Monoclonal antibody MAPK mitogen-activated protein kinase MW Molecular Weight MWCO Molecular Weight Cut-Off

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NBF National Biologics Facility PBS Phosphate-buffered saline PCR Polymerase chain reaction PMF Peptide mass fingerprinting RE Restriction Enzyme RTK Receptor Tyrosine Kinase SDS-PAGE Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis scFv Single-chain variable domain SEC Size Exclusion Chromatography sgRNA single guide RNA SPR Surface Plasmon Resonance SCMB School of Chemistry and Molecular Biosciences TMB 3, 3´, 5, 5´-Tetramethylbenzidine TMZ Temozolomide TFA Trifluoroacetic acid TTFields Tumour treating fields VH Variable domain of the heavy chain VL Variable domain of the light chain

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Chapter 1. Introduction

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1.1 Brain cancer 1.1.1 Malignant gliomas

Glioma is general terminology for primary brain tumours, with subdivision based on the origin of the cells. Subdivisions include oligodendrogliomas, astrocytic tumours (astrocytoma, anaplastic astrocytoma and glioblastoma), ependymomas, and mixture of gliomas [1, 2]. Glioma is reported to be around 80% of all malignant primary brain tumours [1, 3]. The most common form of gliomas is glioblastoma multiforme (GBM) with around 82% of all malignant gliomas [4]. Glioblastoma multiform is named after its characteristic cells being of different size and shape. The heterogeneity is a hallmark of GBM with wide diversity among patients [5] and also within the tumour [6]. Glioblastoma with astrocytic origin is an aggressive type of central nervous system (CNS) tumours with very poor prognosis. The median survival of patients is reported to be nearly 15 months from the time of diagnosis [7, 8].

1.1.2 Who classification

According to the World Health Organization (WHO), malignant gliomas are classified into four subclasses based on the histopathological standards (anaplasia, undifferentiation and aggressiveness) [9]. Grade I is benign and can be treated by surgery while grade II to IV are quite invasive. Grade II tumours include diffuse astrocytoma, oligodendroglioma and oligoastrocytomas. Grade III tumours, include anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic oligoastrocytomas; and Glioblastoma and variants are graded class IV by WHO [10, 11].

1.1.3 Symptoms

The symptoms of brain tumours can be varied based on the size, grade and locality of the tumour. Common symptoms are comprised of headache, nausea and vomiting, neurocognitive impairment, personality disorders, lack of balance in movement, epileptic seizure and focal neurological deficits, loss of urinary control and visual impairment [12].

1.1.4 Epidemiology 1.1.4.1 GBM incidence in the world

Glioblastoma is not a common brain tumour, with less than 10 per 100,000 incidences of brain tumour in the world, but the short median survival rate of fewer than two years after diagnosis makes it a critical public health problem [7, 13]. There is a gender male to female GBM ratio of 3:2 [7, 14]. The developed countries have a higher incidence rate than undeveloped countries [7] probably due to less diagnosed case reports, lack of access to the health care and diagnosis in the latter [15, 16]. It may appear in persons of any age [17], but it affects adults preferentially, with a peak incidence at the age 2 of 55-60 years old [14]. Several studies have reported a higher rate in white compared black people [13].

1.1.4.2 GBM incidence in Australia and mortality rate The brain cancer rate has gradually increased in Australia over time. In 2018 there was 1935 newly diagnosed brain cancer, ranking 18th most common cancer in Australia which has been in rise. The estimated rate of brain cancer in 2018 reflect that the chance of someone in Australia having brain tumour by their mid 80’s could be 1 in 124 [18], with a slightly higher chance of 1 in 112 for Queenslanders predicted by the Queensland Cancer Statistics Online (QCSOL). Statistics show that in 2016 brain cancer was the 10th most common cause of cancer death in Australia. It was predicted to be the 13th cause of cancer death in 2018 and estimated the highest cause of cancer deaths under the age of 24 [19].

1.1.5 Diagnostic tools in brain cancer

1.1.5.1 Imaging and radiology

Diagnosis and classification of GBM is most commonly achieved through magnetic resonance imaging (MRI) as the soft tissue contrast is superior to computed tomography [20]. While the standard

MRI acquisition series of T2 weighted, T1 weighted pre and post contrast and fluid attenuated inversion recovery (FLAIR) can be used to grossly define surgical resection margins and radiotherapy fields, these images provide non-specific contrast, are inaccurate when detecting invasive tumour with an intact blood-brain-barrier and cannot distinguish disease recurrence from treatment damage [21].

Nuclear medicine techniques such as PET (positron emission tomography) [20] and SPECT (single photon emission computed tomography) [20] have the potential to overcome many of the limitations of MRI and can differentiate between disease recurrence and treatment damage. Radiolabelled glucose and amino acid tracers are now widely used in PET scanning to provide information about the biology and metabolism of the tumour [22, 23].

1.1.5.2. Molecular diagnosis and DNA-methylation profiling

The data bank achieved from Next Generation Sequencing (NGS) and also DNA-methylation studies of patient glioma samples, facilitates the molecular diagnosis of gliomas based on their molecular profile [24, 25]. Molecular diagnostic biomarkers used for GBM include: Isocitrate dehydrogenase 1/2 (IDH1/2), NF1, Tumour suppressor protein p53 (TP53), PDGFRA, telomerase messenger expression [26].

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1.1.6 Prognostic factors

Several molecular and metabolic alterations have been reported for glioblastoma. They have reasonable potential to be used as prognostic markers, such as CDK4 and EGFR amplification, CDKN2A deletion, ATRX and TERT mutations [26]. The MGMT promoter methylation technique in GBM patient can be used as another prognostic factor, and it has been confirmed that the MGMT promoter methylation is correlated with more prolonged survival [27, 28]. MicroRNAs (miRNA) can also be applied as a GBM prognostic biomarker. The accuracy of prognosis can be improved by considering the regulation of miRNAs alongside other factors such as age [29]. There are still ongoing debates regarding the use of biomarkers such as nestin and CD133 as a prognostic marker [30] while in fact those markers have been involved in brain tumour development [31].

1.1.7 Current treatment for glioblastoma

The treatment of GBM is challenging due to the heterogeneity and location of the tumour [32]. Current treatments for brain cancer comprises surgery and radiotherapy, followed by adjuvant chemotherapy [33]. Despite many recent advances in conventional therapy for GBM, the treatment target and treatment response still remain poor with the average survival period between 10 and 15 months from the time of diagnosis [34]. Surgery is the first and foremost recommended treatment for glioblastoma [8]. After relevant neuroimaging, a neurosurgery treatment provides the most effective tumour resection, followed by adjuvant chemotherapy. Carmustine and temozolomide (TMZ) are two drugs which are particularly used for GBM treatment [35, 36]. Chemotherapy is usually employed with aim of targeting the DNA to hinder DNA replication.

High energy beams such as protons or X-ray are used to eliminate the cancer cells. The Optune device is widely used as a novel electric-field therapy in GBM tumours that applies low to intermediate frequencies, delivering to the tumour-treating fields (TTFields) by disrupting the electric fields of the tumour proliferating cells. Their effects are through disturbance of the mitotic spindle creation. Optune has been employed for recurrent or advanced GBM tumour after standard treatment since 2011. Later on, in 2015, the combination use of TMZ chemotherapy and Optune were officially accepted by the U.S. Food and Drug Administration (FDA) as the first-line of GBM therapy approach [37].

1.1.8 Blood-brain barrier

It is known that the blood-brain barrier (BBB) and the lack of lymphatic system isolate the brain from the rest of the body and makes studying of this organ difficult. Consequently, BBB avoids the penetration of antibodies and other immune cells to the brain, and there are specific area of research that has been dedicated to the disruption of the BBB to mediate the administration of anti-cancer 4 drugs to this region. Focused ultrasound (FUS) along with synthetic microbubble injection has been employed in an early clinical trial, and its safety for use in cancer treatments has been studied and approved without unfavourable influences [38]. However, latest research in this field has clarified that the immune cells can penetrate into BBB through circulation [39-41] and this finding sheds light to the misunderstanding that the CNS is an immune-isolated area.

1.1.9 Glioblastoma biomarkers

There have been many cell surface targets recognised for glioblastoma such as interleukin-13 receptor alpha chain variant 2 (IL13Rα2) [42, 43], PTPRZ1 (Protein Tyrosine Phosphatase Receptor Type Z1)[44], EGFR (Epidermal Growth Factor Receptor) [45], EGFR variant III [46, 47], c-Met (tyrosine- protein kinase Met) [48, 49] and PDGF (Platelet-derived growth factor) receptor [50], VEGF (vascular endothelial growth factor) [51], CD15[52], CD 133[53], BMI1[54], CD44, ITGA6 (Integrin Subunit Alpha 6), CD36 [55]. Additionally, Eph tyrosine kinase families have been correlated with poor GBM prognosis and recognised as a driver of tumorigenesis. Several receptor families have been identified as GBM markers including EphA2 [56], EphA3 [57], EphA7[58], EphB4 [59].

1.1.10 Comparative oncology: Canine primary brain tumours

Comparative oncology is a fast growing field in cancer research that applies to spontaneously occurring cancers in animals, and the comparison of those cancers to its human counterparts to identify treatments that can benefit both humans and animals [60-62]. This involves the cancer- correlated genes and studies of the novel therapeutic options for cancer management [63-65]. Although the number of veterinary species are high, the majority of research has been focused on pet dogs due to the similar physiologic, anatomic, and genomic comparisons, shared environment with human, and its similarity to the incidence of diagnosed spontaneous cancers in humans [66].

It is known that many features of cancers between dog and human are alike, including the basic biology of cancer, histological appearance, tumour genetics, molecular targets, biological behaviour, and response to conventional therapies. In addition, tumour commencement and progress are determined by the similar features in both species, such as age, sex, nutrition, environmental exposure and reproduction status [67].

Dogs like humans naturally develop primary central nervous system neoplasms that contain a wide range of tumours, including glial tumours (oligodendrogliomas, astrocytomas), meningiomas, and pituitary gland tumours [68], and various glioblastoma have been reported in dogs [69]. Mouse models of cancer have revealed many biochemical and biological pathways to be involved with cancer development and progress. However, they are restricted in disclosing all features involved in 5 human cancers, including genomic instability, treatment over long phases of time and also remarkable heterogeneity in both tumour cells and tumour microenvironment and stroma [66]. Domestic dogs with naturally occurring primary brain tumours are without these deficiencies and can be used as an ideal transitional model between human and mouse [70]. Therefore, there are many reasonable factors that make canine cancer models quite attractive for developing drugs against spontaneous cancers in both canine and human.

The prognosis of a brain tumour in canine with any histology seems poor with a median survival time of fewer than two months [71]. Comparative studies could be highly efficient for both human and dog oncology as having similar incidence rate in definite tumour formation [72], and eventually it can lead to effectively reducing the morbidity and mortality rate of these tumours in both species.

1.3 Eph receptors classification, structure and binding of Eph/ephrin

The Eph (Erythropoietin-producing hepatocellular) receptor family, are the largest of the tyrosine kinase (RTK) receptors. The first Eph gene was discovered in 1987 from an erythropoietin-producing hepatocellular carcinoma cell line [73]. The Eph family is classified into two subgroups EphA (EphA1-10) and EphB (EphB1-6) based on their sequence homologies and binding affinity to two different types of membrane-anchored ligands called “ephrins” (Eph receptor interacting protein) [59, 74-76].

The Eph family receptors (Figure 1.1, B) are type I transmembrane proteins consisting of highly conserved extracellular regions, including an NH2-terminal Ligand Binding Domain (LBD) required for ephrin recognition and binding [77] followed by a cysteine-rich domain (CRD) which contains a sushi domain as well as an Epidermal Growth Factor like motif and two fibronectin type III repeats at the carboxyl terminal of the extracellular domain that might be involved in receptor-receptor dimerization interaction [78]. The ectodomain is divided by a helix membrane-spanning section from the intracellular region, which encompasses a juxtamembrane segment, a tyrosine kinase domain and the sterile α-Motif (SAM) and a COOH-terminal PDZ binding motif (Postsynaptic density protein, Disc large, Zona occludens tight junction protein) [79] that mediate in receptor clustering and also signal transduction at a sub-cellular level [80, 81]. The SAM and PDZ binding motifs are involved in the communication with proteins located in cytosols [82, 83]. The ligand binding site is unique for each Eph sub-type; however, a similar folding structure is identified in some carbohydrate-binding proteins [84] such as lectin [85].

Ephrins known as Eph family receptors interacting proteins (Figure 1.1, A) are membrane-bound proteins and grouped into two sub-classes of ephrin-A and ephrin-B based on their structure and binding to the Eph family receptors. The class A-ephrin ligands (A1-6) are linked to the plasma 6 membrane by a glycosylphosphatidylinositol (GPI)-anchor and lack the transmembrane and cytoplasmic domains. The class B-ephrins (B1-3) possess a transmembrane-spanning segment [80, 86] and a cytoplasmic PDZ-binding motif [87].

The ephrins’ N-terminal receptor-binding domain (RBD) is formed from ´Greek key´ eight β strands, whereas the Eph receptor LBD contains a ´jellyroll´ fold of twelve β strands [88]. The nine different EphA (1-9) receptors, bind to and are activated by six ephrin-A (1-6) and the EphB (1-6) receptors. In some exceptional interactions, EphA4 and EphB2 can preferably bind to ephrin B (1-3) and ephrin A5, respectively, and EphB4 preferentially binds to ephrin A5 [59]. The membrane interaction of Ephs and ephrins together trigger a mechanism that causes multimerization of both structures in their membrane, resulting in signalling induction. Clustering of ephrinA1/EphA2 activates the tyrosine phosphorylation, followed by internalization and degradation of EphA2 [89-91].

Figure 1.1 Schematic representation of ephrins and Eph structures. (A) The ephrinA and ephrinB ligands structures. (B) The Eph receptor structure.

1.3.1 EphA/ephrin activation and signalling

Eph receptors and ephrins interaction are required for initiating bidirectional cell-to-cell communication through signal transduction [75, 92, 93]. Signalling triggered by the Eph receptors is termed “forward signalling”, followed by autophosphorylation of Eph cytoplasmic domain and

7 stimulation of ephrin ligand is referred to as “reverse signalling”. The signal is regulated by changing the forward and reverse signal degree. In forward signalling, the ephrin-Eph complex involved in activation of many kinase-dependent/independent signalling pathways [94]. EphA2 signalling is complicated and both hinder and stimulate oncogenic features, such as cell migration and invasion [95].

Figure 1.2 Schematic representation of Eph-ephrin signalling pathways. Signalling triggered by the Eph receptors is termed “forward signalling”, followed by autophosphorylation of Eph cytoplasmic domain. Stimulation of ephrin ligand is referred to as “reverse signalling”.

Activation of EphA2 by ephrin-A leads to tumour suppression signalling, which inhibits proliferation and migration [96]. Ephrin-A1 ligand acts like a tumour suppressor by attaching to EphA2 receptor, resulting in decreased proliferation and invasion [97, 98]. It has been reported that EphA2 can act as either a tumour suppressor (activated by ephrin-A1) named ligand-dependent signalling, or tumour promoter (phosphorylated by Akt) termed ligand-independent signalling [99]. In ligand-dependent signalling, the EphA2 prevents cell proliferation and invasion by Akt and Ras/MAPK (mitogen- activated protein kinase) inhibition [95, 100]. There is a possibility that due to ephrin-A1 deficiency in GBM, ligand-dependent signalling does not happen and alternatively occurs through phosphorylation of overexpressed EphA2 without ephrin-A1 motivation [99]. Despite the fact that ephrin-A1 can initiate signalling by cell to cell contact, it can trigger paracrine signalling by being released from the GBM cell surface as a soluble monomer [91].

Each of Eph and ephrin can act as a ligand and receptor; thus, each one can initiate bidirectional signalling. Physical interaction of adjacent Eph and ephrin cause the dimerization of both which is

8 mediated by high affinity binding sites. This dimerization results in a heterotetrameric complex by association of a second Eph/ephrin complex. Subsequently clusters of tetramers assemble for efficient forward signalling [101, 102]. This signalling leads to phosphorylation of many tyrosine residues in the cytoplasmic segment through either juxtamembrane or kinase domain. The consequence of this ligand-dependent signalling impacts many downstream signalling proteins located in the cytoplasmic region such as the small GTPases of the Rho and Ras family, focal adhesion kinase (FAK), the Janus kinase/signal transducers and activators of transcription (Jak/Stat) and phosphatidylinositol 3-kinase (P13K) pathways [103].

Activation of EphA receptor regulates the formation and movement of a cell by controlling the Rho GTPases including RhoA, Cdc42, Rac and Ras. This activation occurs by direct binding of individual Rho GTPases to the Rho proteins of EphA2 located in the cytoplasmic domain and effect on actin dynamics, which results in regulating the cell formation and motility.

Eph receptors particularly down-regulate the Ras/MAPK pathway, whereas they activate the Ras family of GTPases (H- and RRas) in a normal range, resulting in migration and proliferation [104]. Eph mediates the cell migration and adhesion by adjusting the molecules involved in integrin signalling comprising FAK and paxillin [105]. Their effect can be through stimulation or suppression of integrin cell signalling pathway. EphA receptor activation regulates the Jak-Stat signalling pathway, that plays a role in cell division and longevity [106]. The EphB receptors facilitate the cell migration and propagation through P13K/Akt pathway. The Abl and p53-family of tumour suppressor proteins are also regulated by Eph/ephrin [103].

The Eph receptors can also motivate “reverse” signalling in the ephrin-expressing cells after Eph/ephrin complex interaction [59, 80, 103, 107]. Since ephrin-expressing cells are deficient in the enzymatic domain, the reverse signalling is mediated by phosphorylation of cytoplasmic associated tyrosine kinases particularly Src family kinase members including Src, Fyn, Lyn, Yes. Following this, the phosphorylated ephrin B provides the SH2 binding domain site on itself which mediates the binding through SH2 and SH3 or PDZ domains to cytosolic adaptor proteins, for example, the adaptor Grb4 [108-110]. It has been reported that ephrinB reverse signalling suppression occurs through the binding of protein tyrosine phosphatase basophil like (PTP-BL) to ephrin B. This binding happens in ephrin B at the C-terminal of PDZ binding motif. PTP-BL terminate the reverse signalling by dephosphorylation of ephrinB and subsequently deactivation of Src family kinases [109]. The PDZ motif has a crucial role in assembling the signalling molecules.

The mechanism of reverse signalling in ephrinA ligand is not clear. Presumably like other GPI anchored proteins, the clustering of ephrinA ligands is initiated by activity of Fyn, a member of Src 9 family kinases where transmembrane adaptor molecules, coupling with the lipid raft proteins, transmit the intracellular signal [111-113]. EphA2 can be activated without the presence of ephrinA1 or kinase activity and can initiate the signalling as an oncoprotein through EphA2 S897 phosphorylation [95]. This independent activation of EphA2 (non-canonical signalling) induces phosphorylation of EphA2 on Serine897. The Serine897 residue is positioned in the linker loop between the sterile-a motif (SAM) and kinase domains. It has been reported that Akt, PKA and RSK have a critical role in the phosphorylation of S-897 [114, 115], due to their serine/threonine kinase activities [59, 95, 113, 116]. It has also been reported that the ephrinA1 attached to the EphA2 reduce the serine897 phosphorylation [114]. EphA2 extracellular domain with other neighbouring EphA2 interacts through two interfaces, so- called the heterodimerisation and clustering interfaces [117, 118]. The residues, including L223, L254, and V255 of CRD are involved in the clustering interface while the receptor-ligand and receptor-receptor contacts are involved in the heterodimerisation interface via residue G131 of LBD.

High-level EphA2 expression correlates to many factors occurring in tumour cells including DNA damage, as well as high expression of H-Ras oncogene, and E-cadherin, activation of MAP kinase pathway, and the loss of c-Myc and estrogen receptors [119-121].

1.3.2 EphA2/ephrinA1 expression

The expression level of Eph and ephrin are different between normal and tumour tissue, suggesting the importance of their contribution in tumorigenic properties, including metastasis, angiogenesis and invasion [80, 107, 122]. The EphA2 receptor tyrosine kinase is highly expressed in different cancers including melanoma, ovarian [123], gastric, pancreatic [124], breast [125-127], prostate [128, 129], bladder, lung, renal, cervical, colon [126, 130, 131], oesophageal, gastric cell carcinomas [132] and gliomas as well, suggesting the role of EphA2 as a common oncoprotein in many cancerous tumours [96]. Many studies have reported the importance of EphA2 and ephrin-A1 in glioma biology [91, 96, 133]. EphA2 is highly expressed in glioblastoma tumour tissue, whereas its ligand, ephrinA1, is expressed at a very low level. The cell-cell contacts are unstable in cancer tissue, and therefore the highly expressed EphA2 cannot interface to the neighbouring low expressed ephrinA1 cell [134], resulting in the inactivation of highly expressed EphA2 in GBM.

Activation of EphA2 by ephrinA1 in normal tissue negatively regulates the function of integrin, cell growth [134], proliferation [135], neovascularization and angiogenesis. Deregulation of Eph receptor expression correlates with the attainment of tumorigenic properties, tumour growth, metastasis and angiogenesis in different human cancers, such as gliomas which are associated with the level of

10 malignancy [59, 91, 136]. High levels of EphA2 expression correlates with tumour stage, progression, and patient survival [96, 97, 114, 137, 138] and also poor prognosis in patients suffering an invasive mesenchymal subtype of GBM [139]. Binda et al [56] found EphA2 is the most upregulated receptor amongst the Eph receptors. This has formerly been described in human grade IV gliomas, which associate with poor outcome [96, 140- 142], suggesting the importance of EphA2 in the pathogenesis of human GBMs. The natural function of Eph receptors impacts on adult nervous system[143], axon fasciculation [144], and stem cell niches [59, 145].

EphA2 has an essential role in signal transduction in normal physiology and is primarily found in adult human epithelial cells [146, 147], and is largely expressed in epithelial embryonic [139]. EphA2 mRNA expression has been detected in skin, spleen, colon, brain, liver [148]. There are some other EphA receptors involved in glioma malignancy such as EphA4 for which their high expression correlates with high-grade tumour [149]. Since EphA2 is not present in normal brain tissue, but is overexpressed in GBM cells [126], it is potentially a highly useful molecule for diagnosis and therapy approaches against brain cancer.

1.4 Monoclonal antibodies (mAbs)

The first monoclonal antibody (mAb) was generated through mouse immunisation and hybridoma technology in 1975 [150], and the first FDA approved therapeutic mAb was introduced in 1986. Hybridoma technology was discovered by Kohler and Milstein in 1975 [151]. This technology is based on fusing a myeloma cells with a B cells to produce immortalised hybridoma cells, with each hybridoma expressing one specific antibody. Since the mAbs produced through hybridoma technology were originally of murine origin, they are capable of provoking immunogenicity in humans, and this considered as a major hindrance in employing hybridoma technology for producing therapeutic antibodies [152]. To reduce immunogenicity, chimeric antibodies were generated by fusing the variable regions of one species such as a mouse with constant regions of human origin. Following this, CDR-grafted antibodies were produced by grafting the complementarity determining regions (CDRs) of a mouse antibody onto the framework regions of a human antibody [153, 154]. Fully human mAbs are now produced through either phage display technology or transgenic mouse technology, and the first fully human mAb, adalimumab (Humira), was approved for rheumatoid arthritis treatment in 2002 [155]. Nowadays mAbs are widely used for the treatment of various disease indications, predominantly cancer and inflammatory diseases. Around 84 mAbs has been approved by FDA as of Jun 2019 [156].

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A basic structure of IgG consists of two identical heavy (⁓ 50 kDa each) and two identical light (⁓ 25 kDa each) chains. Each chain consists of both constant and variable regions (Figure 1.3, B). Each heavy chain contains a variable region (VH) connected to the CH1, which is the first constant region of the heavy chain. A hinge region joins the CH1 to the Fc domain which consists of CH2 and CH3 regions. Each light chain consists of a variable region (VL), which can be either Kappa (VK) or Lambda (Vλ), followed by a light constant region (CL). Each variable region (VH, VK and Vλ) contain three CDRs which determine the specificity of each mAb binding to an antigen. The peptide configuration of heavy and light variable regions of CDRs have a critical role in antigen-binding activity determination [157, 158].

Engineered different versions of mAbs can create smaller antibody fragments. An antigen binding fragment (Fab) consists of the VL and CL domains of the light chain and the VH and CH1 domain of the heavy chain, and is created by enzymatic treatment. Recombinant DNA technology allows an even smaller fragment to be created, consisting of a peptide linker that fuses the heavy (VH) and light (VL) chain variable regions to create a single chain variable fragment (scFv) (Figure 1.3, A).

Figure 1.3 Schematic illustration of scFv (A), and monoclonal antibody or IgG structure (B). The variable regions of heavy and light chains are connected with a short linker peptide (A). The structure of an IgG protein consists of four polypeptide chains. The two heavy chains are connected by disulphide bonds and each heavy chain is linked to a light chain by one disulphide bond. Abbreviations: scFv; single chain variable fragment; VL: light chain variable; VH: heavy chain variable; Fv: variable fragment; CL: light constant region; CH: heavy constant region; Fab: antigen binding fragment; FC: fragment crystallisable region.

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1.5 Generation of antibodies using phage display

Antibodies and antibody fragments are arguably the most promising candidates to be deployed as targeted theranostics due to their high specificity, high affinity and robust manufacturing techniques. Hybridoma and phage display are the most common techniques used to produce mAbs. Despite many advantages of hybridoma methodology, including the high specificity and sensitivity of the resulting mAbs, there are some limitations in hybridoma technology, including it being time-consuming, and the immunogenicity of murine-derived mAbs. Unlike hybridoma, phage display technology is a very fast, controllable procedure and easy to screen a large diverse clones, without immunogenicity for naïve libraries. These advantages make phage display more desirable method for many applications [159]. The introduction of phage display was primarily marked in 1985 by George P. Smith who demonstrated that foreign (poly) peptides could be displayed on the surface of filamentous M13 phage particles [160], and later on it was further developed by Winter and McCafferty [161] to allow the display and isolation of specific antibodies. Notably and testament to the importance of the discoveries, Smith and Winter were awarded the Chemistry Nobel Prize in 2018 due to their pioneering works in the production of novel antibodies. Phage display technology for antibody isolation includes two main phases: construction of the antibody display library and biopanning of the library to isolate specific phage binders. Briefly, to prepare a phage library, cDNA is synthesised from the mRNA of antibody-producing B- lymphocytes, and then the variable region genes of antibodies are amplified, assembled as Fab or scFv genes, then ligated into a phagemid vector in-frame with a phage coat protein gene. In this process, the foreign gene is expressed as a fusion with the phage coat protein during bacterial infection. Helper phage is needed to provide essential genes for proteins involved in phage replication and assembly within bacterial cells. A variety of helper phages can be used, but M13 phages are more applicable since they do not destroy the host (E. coli) during amplification [162]. As a result, a unique fusion protein comprising a Fab or scFv antibody binder is exposed on the surface of a phage particle with the corresponding phagemid DNA enclosed within the phage, forming a good connection between phenotype and genotype [160, 163, 164]. Antibody fragments are used since E. coli are unable to express full length antibodies.

The diversity and quality of the library are critical for successful isolation of specific antibodies during phage display biopanning. Phage libraries usually consist of billions of different phage particles, with potentially as high as 1013 unique phage particles in a library. Phage display, among several various display methods, is the most common technique used for the first time by Lerner’s and Winter’s groups for isolating antibody [165, 166]. Libraries are classified as either ‘naïve’, where 13 blood samples are taken from random individuals with no bias towards any disease states, or ‘immunised’, where samples are collected from individuals with a particular disease of interest. Naïve libraries use V-gene sequences extracted from IgM mRNA, whereas the V-genes from IgG mRNA are used when creating immunised libraries [167, 168]. The naïve library can be employed for a wide range of antigens to isolate a variety of antibody specificities; however, the selected binders have lower affinities compared with isolated binders from an immunised library [169, 170].

In addition to naïve and immunised libraries, libraries can also be synthetic or semi-synthetic. Synthetic libraries are created by full DNA synthesis of antibody genes with randomisation in the CDRs, whilst semi-synthetic libraries are based on the replacement of the amino acids in the CDRs that creates a highly diverse library [171].

The second phase of phage display technology is a screening of phage libraries (biopanning) in which phage particles are isolated against a target antigen based on affinity and specificity selection. The biopanning process is as important as the construction of a library, and it generally consists of four key steps (Figure 1.4). Firstly, the phage library is incubated with the target protein, resulting in the binding of phage binder to the epitope of the antigen. Secondly, the non-adherent or weakly bound phage binders are removed by a washing step. Thirdly, the specific bound phages are eluted through the use of low pH or high pH buffers and infected into E. coli, and fourthly, the phage are amplified by concurrent infection with helper phage. These resulting enriched phage binders are purified and used for the next round of biopanning as a sub-library. Overall, 3-5 iterative rounds of selection are performed to reduce the number of non-specific binders and enrich the captured specific binders [172, 173].

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Figure 1.4 Schematic illustration of a round of biopanning against immobilised recombinant protein. The phage library is incubated with the target antigen (Step 1), followed by washing to remove the unbound phages (Step 2). The bound phage are eluted (Step 3) and amplified via bacterial infection (Step 4) to create sub-library for next round of biopanning. This cycle is repeated 3-5 times to enrich the specific phage binders, then the selected phage clones are identified by sequencing.

1.5.1 Presentations of membrane proteins for affinity selection

The presentation of a target antigen to a library is critical to the outcome. Different methods are employed to display antigens. The protein of interest can be presented either directly or indirectly on the solid surface. Direct immobilisation can occur on different types of solid supports such as microtiter plates [174], polystyrene immuno tubes, cellulose sheets [175], monolithic cryogel column [176] and BIAcore sensor chips [177]; or through chemical cross-linking on the solid phase. The indirect immobilization can be performed by fusion of target antigen to another recombinant antibody chimera, such as Fc region of immunoglobulin G (IgG) and capture on Protein A/G beads after phage library incubation [178]; by biotinylation of the target antigen and capture of bound phage using avidin- or streptavidin-covered magnetic beads [179]. Indirect immobilization of target protein on a solid surface offers some clear advantages over direct immobilization on a solid support such as requiring a lower concentration of target protein for the enrichment of high-affinity clone selection that avoids the avidity effect [180]. In addition, it increases the accessibility of the target ligand

15 binding site to the phage library, helping to maintain the correct folding of protein target [181], resulting in more efficient biopanning, and also bringing less epitope denaturation especially for small antigens. Direct immobilisation of antigens on surfaces can result in denaturation or inaccessibility of epitopes [182].

Alternatively, the target protein can be displayed on whole cells to screen binders in a phage library. This allows for isolating phage particles against membrane proteins in their correct physiological and conformational relevant state including correct glycosylation and other post-translational modifications [183, 184]. The biopanning selection can be conducted on suspension cells in media [185], confluent monolayer adherent cells in a culture flask [178], or cells fixed by formaldehyde [186]. Cancer cell lines can be used [159, 187-193] to isolate tumour specific binders for therapeutic purposes. Different selection strategies to present antigen in biopanning procedures are reviewed in detail by Molek et al. [178].

Beside filamentous phage display as a predominant, quick and economical method for identifying mAbs, there are other types of display libraries that have been reported and utilised including a VLP library [194], in vitro mRNA/cDNA display [195], [196], , mRNA display, cell surface display, covalent DNA display, library display, cis-activity based (CIS) display [197], in vitro RNA display, bacterial display and finally two-hybrid system. These techniques have been widely used in many different areas of bioscience, such as cancer research, drug discovery, neurobiology and immunology, and for all of these techniques, correct display of the target antigen is a necessary pre-requisite for obtaining high quality antibodies or other binding entities.

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1.6 Project hypothesis and aims

The EphA2 receptor is highly expressed in glioblastoma tumour cells compared to normal brain tissue. GBM naturally occurs in both humans and dogs, therefore it makes EphA2 a highly attractive therapeutic target for comparative oncology studies. We hypothesise that an antibody which cross- reacts with both canine and human EphA2 will be an ideal marker which can empower studies on canine EphA2 for direct translation into human studies. We hypothesise that antibodies which bind both human and canine EphA2 will be useful diagnostic and therapeutic tools to benefit canine as well as human patients. Furthermore, we presume that generated antibodies can be used to detect the EphA2 biomarker on circulating tumour cells (CTC). This application might be useful as a predictive prognostic tool to assist with better management strategies in clinical decision making.

In order to generate antibodies that are bind specifically to the EphA2 receptor, and not to other receptors containing similar domains, we hypothesize that the generated antibodies should target the ligand binding domain of EphA2, as this domain is the most unique for EphA2.

The work presented in this thesis aims to discover antibodies specific for EphA2 with cross-reactivity to both canine and human EphA2 receptors. To achieve this goal, the phage display technology was employed using different strategies for the presentation of the EphA2 antigen to the phage library, including biopanning against recombinant whole extracellular domain of human and canine EphA2, biopanning against recombinant ligand-binding domain only of human and canine EphA2, and finally biopanning against the native cell surface EphA2. The final goal was to characterise isolated antibodies after reformatting to whole human IgG.

To demonstrate binding of generated antibodies to the EphA2 ligand-binding domain, the different domains of canine and human EphA2 were expressed separately. The goal was to show the binding of generated antibodies to only EphA2-ligand binding domain and no other area on the EphA2 extracellular domain.

In order to demonstrate the feasibility of employing mAbs to detect CTCs, mAbs will be applied with Raman reporters using GBM patient-derived cell lines. The purpose was to monitor the binding of mAbs to cell surface biomarkers and to investigate the heterogeneity of GBM by multiplexing the antibodies for each cell line.

The work described in this thesis demonstrates the difficulty in isolating antibodies that are specific for particular cellular receptors that belong to a family of receptors with similar sequence and structure.

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Chapter 2: General methods

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2.1 Introduction

This chapter describes methods and materials which are generally applicable in multiple chapters and are mentioned frequently throughout this thesis.

2.2 Standard solutions

The following solutions were prepared in Milli-Q water and sterilised in an autoclave at 120 °C for 20 min. 2YT (2×Yeast Extract Tryptone Media): 10 g/L Yeast Extract, 16 g/L Tryptone or Peptone, 5 g/L NaCl The required antibiotic was added as required at 1/1000 dilution before use, from a 1000x stock of either Ampicillin (100 mg/mL), Tetracycline (3 mg/mL), or Kanamycin (30 mg/mL). When required the media was supplemented with 40% glucose (w/v) to a final concentration of 2%. 2YT Agar: 15 g/L Agar in 2YT Media. The required antibiotic was added after the autoclaved media had cooled but still warm. The prepared agar was poured into 90 mm or 150 mm petri dishes. Glucose-40% (w/v):

200 g glucose in 500 mL H2O, autoclaved MPBS (2% or 10%) (w/v): Skim milk powder dissolved in PBS at 1 g/50 mL or 1 g/10 mL PBS

PBS tablets dissolved in H2O, autoclaved PBS-Glycerol: PBS with 20% glycerol (v/v) PBS-T: PBS containing 0.1% Tween-20 (v/v) PEG-NaCl: 20% Polyethylene glycol-6000 (v/v), 2.5 M NaCl, autoclaved LB agar (Sigma):

14 g LB agar in 400 mL H2O, autoclaved

2.3 Standards methods 2.3.1 Polymerase chain reaction (PCR) amplification

19

The insert was amplified by PCR. The lyophilised DNA oligo was ordered from Sigma and resuspended in MilliQ water (Final concentration of 100 µM). PCR mixture contained 2 µL of each forward and reverse primers, less than 200 ng template, 25 µL 2x PrimeStar Max DNA polymerase (TaKaRa) and MilliQ water to reach the total volume of 50 µL reaction. A Thermal Cycler (Applied Biosystems) was used for PCR, and the thermal cycles were as following: 95 °C for 1 min, then 25 cycles of 98 °C for 10 s, 55 °C for 15 s, 72 °C for 15 s, then a final extension of 72 °C for 1 min.

2.3.2 Gel electrophoresis of DNA

The PCR product was mixed with Gel Loading Dye, purple (6X) (BioLabs) and electrophoresed at 90 V through 1% agarose gel (w/v) in TAE Buffer (Bio-Rad) containing RedSafe (iNtRON Biotechnology) for 20 min. The Gel was visualised by Gel Doc (Bio-Rad).

2.3.3 Cloning of PCR product into pcDNA 3.1(+)

PCR products were cloned into the expression vector pcDNA3.1 (+) (Invitrogen). The PCR product and vector were digested using the appropriate Restriction (REs) (NEB) (1 µL of each), 1 µg DNA, 5 µL 10× Cutsmart Buffer (NEB) to reach the final volume of 50 µL with Nuclease-free Water (Invitrogen) for each reaction. Digestion was performed by incubation at 37 °C for 3 h. The total volume of each reaction was electrophoresed on 1% agarose gel (w/v) (section 2.3.2), then visualised by E-Gel Safe imager (Invitrogen). The linearized vector pcDNA3.1 and insert band were extracted from the gel using scalpel blade and purified using ISOLATE II PCR and GEL KIT (Bioline) as per the manufacturer’s instruction. Insert DNA (75 ng), and linearized vector (25 ng) were ligated in a 20 µL reaction containing 1 µL Quick Ligase (NEB), 10 µL 2× Quick Ligation Reaction buffer (NEB) at RT for 10 min. Then bacterial transformation was done for the recombinant DNA as described in section 2.3.4. The transformation mixture was added to a LB-agar plate containing 100 µg/mL ampicillin and incubated overnight at 37 °C. A number of colonies were screened for successful ligation through colony PCR (section 2.3.5). Positive clones were selected, amplified (section 2.3.6) and purified using QIAprep Spin Miniprep Kit as per manufacturer’s instruction (section 2.3.7) then sent to the AGRF for sequencing (section 2.3.8). They were sequenced in duplicate in both directions using the BGH (Bovine growth hormone terminator) reverse primer and T7 promotor forward primer (Invitrogen) (Table 2.1).

20

Table 2.1 Oligonucleotide primers used for DNA sequencing of pcDNA 3.1.

Primer Name Sequence 5´ to 3´

T7 Promoter, Forward Primer TAATACGACTCACTATAGGG

BGH Reverse primer TAGAAGGCACAGTCGAGG

2.3.4 Bacterial transformation

One shot Top 10 chemically Competent E. coli (Invitrogen) were used to amplify the recombinant DNA as following: A vial of competent cells (40 µL) was thawed on ice then 1 µL of the ligation reaction was added to the competent cells and incubated on ice for 30min. The cells were heat shocked at 42 °C in water bath for 45 sec, followed by 2 min incubation on ice. Afterwards, sterile LB (Luria- Bertani) medium (250 µL) was added to the cells and incubated shaking at 37 °C for 1hr. The transformation mixture (100 µL) was used to spread on a LB-agar plate containing 100 µg/mL Ampicillin. The plate was incubated at 37 °C incubator overnight.

2.3.5 Colony PCR

Half of the grown cells from each colony were taken by a sterile micropipette tip and added to a PCR tube containing 1 µL of each forward and reverse primers (10 µM) (Table 2.1), 5 µL GoTaq Green Master Mix (Promega) and 4 µL MilliQ water. Then resuspend the cells in tube components completely. A PCR thermal cycler was used for colony PCR with the following thermal conditions: 95 °C for 5 min, followed by 25 cycles of 95 °C for 15 s, 55 °C for 15 s, 72 °C for 2 min, then a final extension of 72 °C for 1 min. Afterwards, 5 µL of each reaction was directly loaded on 1% agarose gel (w/v) (section 2.3.2) for screening the plasmids containing the desired insert.

2.3.6 Inoculation

A sterile pipette tip was used to transfer the selected single-positive colony from LB-agar plate. The tip was dropped into a 50 mL falcon tube and inoculated into 5 mL LB media containing the appropriate selective antibiotic; Ampicillin was used for recombinant pcDNA 3.1. The culture was grown overnight with 200 rpm shaking at 37 °C.

2.3.7 Plasmid DNA purification

The 5 mL overnight bacterial culture was harvested and centrifuged at 5000 rpm, for 15min at RT. Plasmid DNA was extracted from the overnight culture pellet using the QIA Prep Spin Miniprep kit 21

(QIAGEN) as recommended by the manufacturer. The concentration of DNA was determined using a NanoDrop spectrophotometer.

2.3.8 DNA sequencing set up

Two sequencing reactions were set up per purified DNA (PD), one in the forward direction and one in the reverse direction. Each sample included 600-1500 ng purified plasmid, 1 µL of 10 µM forward or reverse primer. The final volume was reached to 12 µL by adding Ultra-Pure water. Prepared samples were submitted to the Australian Genome Research Facility (AGRF), Brisbane node for PD sequencing. Results from the AGRF were analysed using Geneious version 9.0.5 software, and positive confirmation of the plasmid quality led to midiprep (section 2.3.9) being completed to prepare high-quality amounts of DNA template for sterile transient transfection.

2.3.9 Large scale DNA preparation

An Erlenmeyer flask containing 400 mL sterile LB with 400 µL ampicillin was inoculated with 50 µL of Top 10 cells transformed with the pcDNA3.1 expression vector containing the desired gene insert (section 2.3.4). The culture was incubated overnight at 37 °C, 200 rpm shaking. Next day, the culture was harvested and centrifuged at 5000 rpm, 4 ºC for 15 min. The supernatant was discarded, and plasmid DNA was extracted from the cell pellet using the NucleoBond® Xtra Midi kit as recommended by the manufacturer (Macherey-Nagel).

2.3.10 Transfection and expression of desired protein into Chinese Hamster Ovary (CHO) cells

The construct was expressed by PEI mediated transfection in suspension-adapted CHO-XL99 cells. The CHO-XL99 cells were maintained in CD-CHO medium (Invitrogen) supplemented with 8 mM GlutaMax (Invitrogen) and 0.4% anti-clumping agent (v/v) (Invitrogen). A day prior to transfection, the cells were passaged to remove anti-clumping agent that inhibits DNA/PEI complex formation and also to obtain cells at half the desired transfection concentration (1.8 × 106 cells/mL). The cells should double in 24 h, and therefore be at the right concentration on the day of transfection (3.0 × 106 cells/mL).Total DNA was 2 µg per mL of initial transfection volume and this for antibody transfection was 1 µg/mL of each heavy and light chains (for a total of 2 µg/mL of DNA). Briefly, the DNA and PEI-Max (Polysciences) were diluted separately in OptiPro SFM (Invitrogen), and after 30-60 second incubation, diluted DNA was mixed with diluted PEI-Max by gently pipetting, followed by 15 min incubation at RT without disturbing the mixture. Then the mixture was added to the cell flask and placed in a humidified incubator at 37 °C, 7.5% CO2, 130 rpm shaking. After 4-6 h of incubation, cells were diluted by doubling the total volume with CD-CHO medium and other feeds including Efficient Feed A (Invitrogen), Efficient Feed B (Invitrogen) and anti-clumping agent (Invitrogen). 22

The flask was then incubated at 32 °C, 7.5% CO2, 130 rpm for 10-13 d. The supernatant of culture was harvested post-transfection by centrifugation at 5000 ×g for 15 min at 4 °C and passed through a 0.22 µm filter membrane then it was stored at -20 °C until purification could be completed.

2.3.11 Protein purification via metal affinity chromatography

Proteins of interest with introduced 6xHis tags were purified using two sequential immobilized metal ion affinity chromatography (IMAC) steps. First using 1 mL HisTrap Excel column (GE Healthcare) on AKTA explorer (GE Healthcare) and followed by 1 mL HisTrap Fast Flow column (GE Healthcare) on AKTA to remove additional impurities. First, the Excel column was equilibrated with 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4. Then, filtered supernatant was loaded onto the column and washed with wash buffer containing 20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4. The protein of interest then was eluted using Elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, and pH 7.4). The eluted protein was buffer exchanged into PBS using a HiPrep 26/10 desalting column (GE Healthcare). Subsequently, the second purification step was performed using HisTrap Fast Flow column with the same buffers used previously. The concentration of eluted protein was determined by reading the absorbance at 280 nm via a Nanodrop 1000 spectrophotometer and using the absorbance extinction coefficient obtained through ProtParam tool (https://web.expasy.org/protparam/). The purity of the eluted protein was examined using SDS-PAGE (NuPAGE system, Invitrogen).

2.3.12 Purification of protein A using HiTrap MabSelect Sure column

Protein A Purification was performed using a 1 mL HiTrap MabSelect Sure column (GE Healthcare). PBS was used as a loading buffer and 0.1 M Glycine, pH 3.0 as an elution buffer. The eluted fraction was buffer exchanged into PBS using a HiPrep 26/10 Desalting column (GE Healthcare). The purity of the eluted protein was analysed using SDS-PAGE (NuPAGE system, Invitrogen).

2.3.13 Size Exclusion Chromatography (SEC)

The recombinant purified protein was concentrated to 400 µL by using Amicon Ultra-15 with 10,000 MWCO (Merck). The Superdex 200 Increase 10/300 GL column (GE Healthcare) was equilibrated with PBS wash buffer and then the concentrated protein was loaded to the column. The elution fractions were collected based on the protein molecular weight.

2.3.14 Preparation and enzymatic digestion of protein samples in solution for peptide mass fingerprinting

23

To reduce and alkylate the sample, Dithiothreitol (DTT) was added to the protein sample to give 5 mM final concentration. The sample was incubated at 56 °C for 30 min, then cooled to RT, followed by addition of Iodoacetamide (IAA) to 25 mM final concentration. Then the tube was incubated for 30 min at RT in the dark. DTT (5 mM final concentration) was added to the tube to quench excess iodoacetamide. To digest the protein trypsin (1:100 enzyme: protein ratio) was added to the tube and incubated at 37 °C overnight. Next day, the digested sample was centrifuged, and the supernatant was transferred to a new tube. To zip-tip (Merck) clean-up, zip-tip was hydrated with 100% acetonitrile (ACN, w/v) (3×10 µL) and tip was equilibrated with 5% ACN (w/v) / 0.1% TFA (v/v) (3×10 µL) (v/v). The sample was loaded (10 µg) and then tip was washed with 5% ACN (w/v) / 0.1% TFA (v/v) (3×10 µL). After that the sample was eluted with 80% ACN (w/v) / 0.1% TFA (v/v) (10 µL) into a new tube before dried by vacuum centrifuge at V-AQ mode for 20 min. Samples were submitted to the Proteomics Facility at the School of Chemistry and Molecular Biosciences (SCMB), UQ and analysis was completed using Protein Pilot Peptide mass fingerprinting (PMF) Spectrometry. 2.3.15 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The molecular weight of the protein of interest was determined using SDS-PAGE. The protein samples were prepared as below and heated at 95 °C for 5 min, then were ran on NuPAGE 4-12% Bris-Tris Protein Gel (Thermo Fisher) in the electrophoresis apparatus filled with MES buffer at 200 V for 35 min. A SeeBlue Plus2 Prestained Standard molecular weight (Invitrogen) was used as a protein marker.

1) Non-reduced condition: 10 µg of protein solution + NuPAGE LDS Sample Buffer (4×) 2) Reduced condition: 10 µg of protein solution + NuPAGE LDS Sample Buffer (4×) + NuPAGE Sample Reducing Agent (10×)

The gel was rinsed three times for 5 min with water, on a shaker. Then stained for 1 h on a shaker with enough SimplyBlue Safe Stain (Life technologies) to cover the gel, followed washing with water and imaged using ChemiDoc (Bio-Rad).

2.3.16 Protein immunoblot (Western Blot)

Western blot was applied to detect a specific protein by using antibodies. After SDS electrophoresis, the gel was placed on top of the Trans-Blot Turbo PVDF membrane (Bio-Rad) in transfer cassette (Bio-Rad) and ran at 200 V for 7 min. The membrane was washed once in 1× PBS-T and blocked with 1% MPBS-T for an hour with gentle shaking, followed by incubation with the diluted primary antibody in 1% blocking solution for an hour on the shaker. Then the membrane was washed three times with PBS-T for 10 min with gentle shaking. The HRP-conjugated secondary antibody was 24 diluted in blocking solution and added to the membrane for an hour at RT. The washing step was repeated three times. ECL chemiluminescent Substrate Reagent kit (Invitrogen) was used according to manufacturer’s instructions. The membrane was incubated in the substrate in the dark for less than five min. The luminescent signal was detected using Gel Doc (Bio-Rad). Multiple exposure time was used to identify the most optimal exposure time.

2.3.17 Biopanning library on purified antigen 2.3.17.1 Coating immunotubes and binding

Purified proteins were used to isolate binders from the naïve human scFv phage library [198], kindly provided by Dr. Martina Jones at National Biologics Facility (NBF, AIBN-UQ). This library compromised filamentous M13 phage clones expressing approximately 5×109 unique scFvs. On day one of biopanning, two immunotubes were coated each with 10 µg/mL of an unrelated His-tagged protein (AFBP-His) diluted in PBS and one immunotube was coated with the target protein as a positive antigen with 10 µg/mL concentration. This concentration decreased in subsequent rounds to 5 µg/mL. A 50 mL falcon tube containing 5 mL 2YT with Tetracycline was inoculated overnight with a single colony of E. coli XL1-Blue (Stratagene) as a starter culture at 37 °C with shaking. As a negative control, another single XL1-Blue was inoculated into 2YT media containing either Ampicillin or Kanamycin. On day two, all immunotubes were washed thrice with PBS and then blocked with 2% MPBS (w/v). For the first round of biopanning, 1 mL of phage scFv library stock containing 1013 phage particles were blocked with 2% MPBS in a 2 mL tube for 1hr at RT rotating. After 1 h incubation, the blocking solution was discarded and the blocked phage scFv library was added to the first negative immunotube and incubated for 1 h at RT with shaking. The blocking solution of the second negative tube was then discarded after 1 h, and the content of the first negative tube was added to the second emptied immunotube. After another one hour incubation, the blocking solution was removed from the positive tube, and phage particles were transferred to the positive tube and incubated for 1 h at RT with rotating. Meanwhile, 150 µL of overnight XL-Blue culture was inoculated into 15 mL 2YT with Tetracycline, and grown for approximately 2-3 h to reach the OD600 0.6-0.8.

2.3.17.2 Elution of bound phage

Phage suspension was removed from the positive tube to waste phage bottle and the tube washed three times with PBS-T, followed by three times with PBS for the first round. The washing step was implemented three times for round two and seven times for round three. Afterwards, 1 mL 200 mM glycine, pH 2.5 was added to the immunotube and incubated shaking for 8 min at RT to elute bound

25 phages. The eluted phage was transferred to two 2 mL tubes, each containing 500 µL 1 M Tris, pH 7.4. For storage, 500 µL of 50% glycerol (v/v) was added to one tube and kept at -80 °C.

2.3.17.3 Infection

10 µL of the eluted phage from the second tube took aside for output titration, and the rest was mixed with 10 mL XL1-Blue cells (OD600: 0.6-0.8) and incubated non-shaking for 30 min at 37 °C. Then, the cells were centrifuged at 2000 ×g for 10 min, and the supernatant was discarded. The cell pellet was resuspended in 0.5 mL 2YT media and spread evenly on two 150 mm 2YT plates containing 2% Glucose (v/v) and Ampicillin (1:1000). The plates were incubated overnight at 30 °C.

2.3.17.4 Determination of output titre

For phage titration, 10 µL of eluted phage was added to 90 µL PBS and serially diluted to 10-4 dilution. Then, 1 µL of each dilution transferred to 100 µL log phage E. coli XL-Blue cells and incubated non- shaking for 30 min at 37 °C. One 150 mm 2YT Agar plate containing 2% Glucose (v/v) and Ampicillin (1:1000) was used to draw quadrant and labelled for each dilution. 20 µL of phage- infected E. coli spread onto the plate corresponding to each dilution of eluted phage. The plate was incubated overnight at 37 °C. Next day, the number of colonies were counted for each quadrant to calculate the output titre in colony forming units (cfu) as follows:

Output titre (cfu) = Number of colonies × (100 µL/20 µL) × 1000 µL/mL × 2 mL

2.3.17.5 Preparation of glycerol stock and phage rescue

To prepare glycerol stock, 2.4 mL 50% glycerol (v/v) and 3.6 mL 2YT was added to the first overnight large plate and the plate was scraped until all bacteria were detached from the plate. Then the slurry was transferred to the second large plate and the plate scraped to detach all the bacteria from the second plate to prepare a uniform slurry. 1 mL aliquots of the slurry were prepared and stored at -80 °C.

Glycerol stock was added into 50 mL 2YT containing 2% Glucose (v/v) and Ampicillin (1:1000) to reach the starting OD600 to 0.05-0.1, in a 250 mL Erlenmeyer flask. The flask was incubated shaking 11 at 220 rpm at 37 °C to reach the OD600 0.4-0.6. To rescue the phage infected E. coli, 10 M13K07 helper phage particles were added. The flask was incubated at 37 °C for 30 min non-shaking, followed by 30 min shaking. The culture was centrifuged at 2000 ×g for 10 min, and the supernatant was discarded. The pellet was resuspended in 100 mL 2YT media containing ampicillin and kanamycin in 500 mL flask, and incubated overnight at 30 °C with shaking at 220 rpm. Next day, phage rescued

26 culture was centrifuged at 3200 ×g for 10 min, and 40 mL of supernatant was equally transferred to 2× 50 mL tubes. For phage precipitation, 8 mL of PEG/NaCl (1/5 vol) was added to each tube. The mixture was mixed thoroughly and placed on ice for at least 1 h. Next, the mixture was centrifuged 10,000 ×g at 4 °C for 15 min. The supernatant was discarded to bacterial waste. All phage pellets were resuspended in a total of 10 mL cold PBS, and combined into one one 50 mL tube. The precipitation step was repeated with addition of 2 mL PEG-NaCl and incubated on ice for 1 h. Afterward, the mixture was centrifuged at 10,000 ×g, 4 °C for 15 min. The pellet was resuspended in 3 mL ice-cold PBS-Glycerol. The aliquots of 3× 1 mL was prepared. 10 µL of that was kept aside for titration of rescued phage and the rest stored at -80 °C for subsequent rounds of biopanning. The prepared phage stock was then used for subsequent rounds of biopanning, and for polyclonal ELISA.

2.3.17.6 Titration of rescued phage stocks

On day one, a single XL1-Blue E. coli colony was inoculated into 5 mL 2YT media containing Tetracycline and the same inoculation was prepared with either Ampicillin or kanamycin antibiotic as a negative control. The cultures were incubated shaking at 37 °C overnight. Next day, 50 µL of overnight culture was inoculated into 5 mL 2YT containing Tetracycline. The culture was incubated shaking at 37 °C to reach the OD600 reading 0.6-0.8. Serial 1/10 dilutions of library phage were prepared down to 10-10 (10 µL + 90 µL PBS). Tips were changed between each dilution as phage particles are very sticky. 1 µL of 10-7 to 10-10 dilutions were transferred to new 1.5 mL tubes, and 100 µL of log-phase E. coli culture with the OD 0.6-0.8 was added to each dilution and mixed briefly, then they were incubated without shaking at 37 °C for 30 min. One 2YT plate containing 2% Glucose (v/v) and Ampicillin (1:1000) was divided into 4 quadrants and labelled for each dilution, and 20 µL of culture was spread on relevant sections. The plate was incubated at 37 °C overnight. The following day, the number of colonies were counted and the phage concentration in cfu/mL calculated for the countable dilution according to the following formula. Cfu/mL (stock) = # colonies on plate × 100/20 × 1000 × dilution factor

2.3.18 Polyclonal phage ELISA

On day one, a 96-well Nunc Maxisorp plate was coated with positive and negative antigens to 10 µg/mL in 10 mL PBS. 200 µL of negative antigen was added to all columns 2-6 wells and 200 µL positive antigen to all columns 8-12 wells. PBS (200 µL) was added to columns 1 and 7. The ELISA plate was incubated at room temperature overnight. Next day, the plate was washed 3 times with PBS. 400 µL PBS containing 2% MPBS (w/v) was added to each well. In separate 2 mL tubes, 90 µL of phage particles from library, round 1, round 2 and round 3 was added to 1800 µL 2% MPBS (w/v), 27 followed by 1hr incubation at RT. The plate’s content was discarded and 180 µL 2% MPBS (w/v) was added to all wells in column 3-5, and columns 9-11. Column 6 and 12 were filled with 200 µL 2% MPBS (w/v). 200 µL of blocked phage particles were added as below: Library Phage: A1, A2, B1, B2, A7, A8, B7, B8. Round 1 Phage: C1, C2, D1, D2, C7, C8, D7, D8. Round 2 Phage: E1, E2, F1, F2, E7, E8, F7, F8. Round 3 Phage: G1, G2, H1, H2, G7, G8, H7, H8.

Then 20 µL of blocked phage particles were transferred across the plate to obtain serially 1/10 dilution from column 2 to 5, and from column 8 to 11. Tips were changed between each dilution. Columns 1 and 7 were negative controls for no antigen coated and columns 6 and 12 were negative controls for no added phage. The immunoplate was incubated for 1 h at RT. Afterwards, the plate was washed thrice with PBS-T, and each well was incubated with 200 µL prepared Mouse anti-M13 monoclonal antibody (GE Healthcare Life Sciences) diluted 1/5000 in 2% MPBS (w/v) for 1hr at RT. The plate was washed with PBS-T three times and incubated with 200 µL Goat Anti-Mouse IgG (H+L)-HRP- conjugated (Bio-Rad) diluted 1/5000 in 2% MPBS (w/v) for 1 h at RT. The washing step was repeated. 3, 3´, 5, 5´-Tetramethylbenzidine (TMB) substrate solution (BioLegend) was prepared as manufacturer’s instructions (1:1), and 100 µL was added to each well for 10 min at RT. 100 µL sulphuric acid 1 M was added to each well to stop the reaction. The absorbance was measured at 450 nm using the microplate reader (BioTek PowerWave XS2).

2.3.19 Monoclonal phage ELISA To obtain individual colonies, 40 µL of last round glycerol library stock was spread over a small part on the edge of 150 mm 2YT Agar plate containing 2% Glucose (v/v) and Ampicillin (1:1000) using Streak plate technique. This process was repeated three times on 2YT Amp-Glu plates to obtain at least 96 individual colonies. The plates were incubated at 37 °C overnight. Next day, 150 µL 2YT containing 2% Glucose (v/v) and Ampicillin (1:1000) was distributed to each well of a new 96-well round-bottom sterile plate. Each well was inoculated with a single colony obtained from overnight plate except row H wells that act as a negative control. The 96-well plate was covered by parafilm and incubated shaking at 37 °C overnight. On day three, each well of a new 96-well round-bottom culture plate supplemented by 150 µL 2YT containing 2% Glucose (v/v) and Ampicillin (1:1000) was inoculated by 5 µL of corresponding wells of overnight culture and incubated shaking at 220 rpm at 37 °C for 3 h. 60 µL of 50% glycerol (v/v) was added to each well of the overnight culture plate to prepare a glycerol stock of each clone. Then, it was covered by parafilm and stored at -80 °C. To induce phage production in the new culture plate, 50 µL of 4×108 helper phage was added to each well after the 3 h incubation. The plate was incubated without shaking for 30 min following 30 min 28 with shaking at 37 °C. The plate was centrifuged at 3200 ×g for 10 min at RT. The supernatant was discarded to phage waste, and the pellet was re-suspended in 200 µL 2YT containing Ampicillin and Kanamycin. The plate was covered by parafilm and incubated overnight with shaking at 30 °C. To prepare ELISA plate, positive and negative antigens were diluted to 3 µg/mL in PBS, and 200 µL of each antigen was separately added to all the wells of one Maxisorp plate, except wells H7 to H12 as a negative antigen control. Plates were incubated at RT overnight. On day four, the overnight cultured plate was centrifuged at 3200 ×g for 10 min at RT and 150 µL of supernatant was transferred to corresponding wells of a new 96-well round bottom plate. 150 µL of 2% MPBS (w/v) was added to each well for blocking the phage particles. The plate was kept at RT until needed for ELISA. The overnight MaxiSorp plates were washed three times with PBS, and 400 µL of 2% MPBS (w/v) was added to each well then incubated at RT for 1 h. Wells were emptied by inversion. 100 µL of 2% MPBS (w/v) was added to each well and an additional 100 µL to wells H7-H12. 100 µL of blocked phage particles were added from corresponding wells to both the positive and negative ELISA plates. Randomly, 100 µL of the remaining blocked phage were used to add to wells H7 to H12 and incubated for 1hr at RT. The plates were washed thrice with PBS-T and each well incubated with 200µL prepared Anti-M13 monoclonal antibody (GE Healthcare Life Sciences) diluted 1/5000 in 2% MPBS (w/v) for 1 h at RT. The plate was washed with PBS-T three times and incubated with 200 µL Goat Anti-Mouse IgG (H+L)-HRP-conjugated (Bio-Rad) diluted 1/5000 in 2% MPBS (w/v) at RT for 1hr. The washing step was performed. The TMB substrate solution was prepared as manufacturer’s instructions (1:1), and 100 µL was added to each well and incubated at RT for 10 min. 100 µL 1 M Sulphuric acid was added to each well to stop the reaction. The absorbance was measured at 450 nm using the microplate reader. The positive signals were considered as an arbitrary cut-off value of A450 ≥ 1.0.

2.3.20 High-throughput sequencing of positive clones

High-throughput sequencing method was used for more than 45 positive clones achieved in monoclonal phage ELISA. Sequencing was performed in a 96-well plate format from PCR products, utilising AGRF’s PCR clean-up and sequencing service. PCR mastermix (100×) was prepared containing 1000 µL Bioline 2x MyTaq, 100 µL of 10 µM pPhNBF-PCR_For, 100 µL of 10 µM pPhNBF-PCR_Rev (Table 2.2) and 800 µL Milli-Q water. 20 µL of the mastermix was added to each well of a 96-well PCR plate. A multi-channel pipette was used to scrape the surface of each well of the frozen glycerol plate from monoclonal EISA plate to obtain a small amount of glycerol stock on the tip. The glycerol stocks were transferred to corresponding wells of PCR plate. PCR plate was sealed by Adhesive PCR film sealer (ThermoFisher) and placed in the thermal cycler. PCR was run with the following thermal conditions: 95 °C for 10 min, followed by 30 cycles of 95 °C for 30 s, 55 29

°C for 30 s, 72 °C for 1.5 min, then a final extension of 72 °C for 10min. 5 µL of PCR product was loaded on 2% 96-well E-Gel (ThermoFisher) to check whether PCR has worked. Expected product size is around 2400 bp. Afterward, 10 µL of PCR product was added to a new standard 96-well plate except for H1 well as a control, with water added to a total volume of 20 µL. The primers for sequencing were prepared separately in 1.5 mL tubes. Each tube contained either 200 µL 3.2 µM pPhNBF_Seq_For or 200 µL 3.2 µM pPhNBF_Seq_Rev (Table 2.2). Then, the plate and primers were sent to AGRF for PCR clean-up and sequencing service.

Table 2.2 Oligo primers used for PCR and DNA sequencing of scFv-Phage clones

Oligonucleotide Primers Sequences (5´to 3´)

pPhNBF-PCR_For TCACACAGGAAACAGCTATGACC

pPhNBF-PCR_Rev CCTTCATAATTTGCATAGCGATCCAGGG

pPhNBF_Seq_For TGATTACGCCAAGCGCGCC

pPhNBF_Seq_Rev TTTCAACGGTCTATGCGGCACC

For less than 45 positive clones in monoclonal phage ELISA, the phagemid DNA from corresponding clones were isolated and sequenced using primers flanking the scFv sequences (Table 2.2). On day one, A 150 mm 2YT Agar plate containing 2% Glucose (v/v) and Ampicillin (1:1000) was divided equally into eight sections and inoculated by selected well of glycerol stock for only one clone and incubated to grow at 37 °C overnight. On day two, for expanding culture, a single colony from each clone was picked and inoculated into 5 mL 2YT containing 2% Glucose (v/v) and Ampicillin (1:1000) with shaking at 37 °C overnight. On day three, to prepare glycerol stock, 300 µL of each culture was transferred to sterile tubes containing 200 µL of 50% glycerol (v/v) and stored at -80 °C. 2 mL of remaining overnight culture was used to extract phagemid DNA as described in section 2.2.7. Samples were prepared (section 2.3.8) for Purified DNA (PD) sequencing with both forward and reverse primers separately. Unique clones were determined by sequence alignment.

2.3.21 Reformatting of scFv particles to whole human IgG1

Phage clones of interest were reformatted to whole human IgG1 as published previously by our group [199]. Briefly, heavy and light mAbXpress vectors (1µg), containing human antibody constant regions, were digested in a reaction containing 0.5 unit of SacI (NEB) and 2.5 µL CutSmart buffer

(NEB) and H2O to reach the final volume of 25 µL, for 2 h at 37 °C. The digestion reaction was

30 cleaned up using QiaEX II kit, and the final product was eluted in 20 µL EB buffer. 3 µL of purified digestion product was run on 1% agarose gel (w/v) at 90 V for 20 min to check the full linearization. PCR was performed on the isolated phagemids to amplify separately the variable heavy (VH) and (VL) light regions using relevant primers with the following thermal conditions: 95 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, then a final extension of 72 °C for 1min. Afterwards, 5 µL of PCR product was run on 1% agarose gel (w/v) at 90 V for 20 min, to check for a single product. Subsequently, 5 µL PCR product was treated with 2 µL Cloning Enhancer (TaKaRa) and incubated at 37 °C for 15 min, followed by incubation at 80 °C for 15 min. Then, infusion reaction was set up using 2 µL 5× In-Fusion HD Enzyme Premix (TaKaRa), 1 µL linearized vector, 4 µL treated PCR product and 3 µL H2O, followed by incubation at 50 °C for 15 min. The reaction tube was placed on ice and transformation was performed as described previously in section 2.3.4. 100 µL of transformation mixture was spread on LB-agar plate containing kanamycin and incubated at 37 °C overnight. The following day, colonies were picked and inoculated into PCR mixture containing relevant primers for heavy and light chains (either Kappa or Lambda). Positive clones had around 600 bp, and negative clones had 350 bp bands on 1% agarose gel (w/v).

2.3.22. Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA was used to detect and quantify the desired protein. Briefly, 200 µL of positive and negative antigens diluted to 3 µg/mL in PBS were immobilised on positive and negative wells of Nunc MaxiSorp flat-bottom ELISA plate and incubated at RT overnight. Next day, the plate was washed 3× with PBS and blocked with 400 µL 2% MPBS (m/v) per well for 1 h at RT. The wells were emptied by inversion and incubated with 200 µL blocked primary antibody diluted in 2% MPBS (w/v) then incubated for 1 h, followed by 3× washing with PBS-T. 200 µL HRP-conjugated secondary antibody (diluted 1/5000 in MPBS) was added to each well and incubated for 1 h at RT. The washing step was repeated before adding 100 µL per well TMB solution to initiate the reaction. The reaction was stopped after 10 min by adding 100 µL per well 1 M sulphuric acid (H2SO4) and the absorbance was read at 450 nm by a plate reader.

2.3.23 Competitive ELISA test

Initially, the optimum dilution and concentration of the reformatted phage-derived antibody was determined against the recombinant antigen. This was conducted by ELISA assay as previously described in section 2.3.20 by making serial dilutions of reformatted antibody. For performing the competitive ELISA, an ELISA plate was coated with 3 µg/mL of recombinant antigen and incubated at RT overnight. The following day, the anti-EphA2 4B3 antibody was serially diluted 1 in 3 in 200

31

µL 2% MPBS (w/v) in a 96-well round bottom plate. Alongside, the reformatted antibody was blocked at a constant concentration in 200 µL 2% MPBS (w/v) in another 96-well round bottom plate. Both plates were incubated at RT for 1hr. Next, the overnight ELISA plate was washed three times with PBS. The antibodies were mixed (100 µL of reformatted antibody at constant concentration and 100 µL of serially diluted anti-EphA2 4B3 antibody) and then added to ELISA plate immobilised with recombinant antigen and incubated at RT for 1 hr. The plate was washed 3× with PBS-T and 200 µL anti-human HRP secondary antibody was added to each well and incubated for 1 h. The washing step was repeated for 3×, following adding 100 µL of TMB solution to each well and 10 min incubation. The reaction was stopped by adding 100 µL/well 1 M H2SO4. The absorbance was measured at 450 nm.

2.3.24 Cell culture

PC3, LNCaP and U87 were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (v/v) (Sigma), and 4 mM Glutamine (Gibco) at 37 °C with 5% CO2/ 95% humidified air atmosphere. At 90% confluency, the prostate cancer cell lines (PC3 and LnCap) and glioblastoma cell line (U87) were passaged using Cell Dissociation Solution (Non-enzymatic 1x, Sigma) and TrypLE Select Enzyme (1X, Gibco), respectively.

2.3.25 Flow cytometry for reformatted scFv-derived antibodies

Adherent cells were cultured to reach the 90% confluency. To resuspend the cells from adherent form, the media was aspirated from cell culture flask, washed with PBS and the cell dissociating solution was added to the flask based on cell types. The flask then incubated at 37 °C for less than 5 min. Media containing FBS was added to stop the activity of dissociation buffer and cells were resuspended gently by pipetting. Cell counting and viability assessment was performed using Automated Cell Counter (TC 20, Bio-Rad). The cell suspension was centrifuged at 400 ×g for 4 min. At least 200 µL of 1×106 cells/mL was aliquoted to each tube and blocked with PBS containing 2% FBS (v/v) for 30 min on ice. Cells were centrifuged and resuspended with 100 µL blocked primary antibody at 1/200 concentration and incubated for 1 h on ice. Some tubes were used as controls and weren’t treated with either primary antibody or secondary antibody or none of them and incubated only with blocking buffer. Cells were washed with ice-cold PBS containing 2% FBS (v/v) by centrifugation three times. Then cells were resuspended with 100 µL blocked secondary antibody at 1/200 concentration and incubated for 1 h on ice. The washing step was performed for three times and cells were resuspended in 1 mL cold PBS and flow cytometry was done using either Sysmex

32

Partec Cube 6 or BD Accuri C6 and results were analysed using FCS Express 4 Flow Research Editing.

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Chapter 3. Isolation of canine and human monoclonal antibodies against recombinant canine and human EphA2 Extracellular Domains (ECDs)

34

3.1 Introduction

EphA2 is a member of the largest family of receptor tyrosine kinases. The interaction between EphA2 and its membrane-bound ligand, ephrinA1, occurs by cell-to-cell contact. This interface induces downstream activation of tyrosine phosphorylation signalling and clustering EphA2-EphA2 receptors and result in adjusting cell migration and proliferation during development [59, 76]. Several studies have reported that activation of EphA2 without ligand leads to tumorigenic properties [114, 200-202]. EphA2 is one of the receptors that has been overexpressed in quite a few malignancies such as prostate, colorectal, bladder, melanoma, ovarian, lung, and brain cancer, including glioblastoma [58, 91, 96, 123, 136, 203, 204]. Glioblastoma is the most aggressive form of a brain tumour [205], and the survival rate of patients bearing glioblastoma highly depends on the level of EphA2 expression [91, 96, 136, 203]. EphA2 is overexpressed in around 90% of human and canine glioblastoma cells [206]; to the contrary, EphA2 receptors are not present in the normal brain [96]. This suggests that the EphA2 receptor is an attractive target for the diagnosis of glioblastoma as well as therapeutic trials. mAbs that cross react with both human and canine EphA2 could be used in comparative oncology models of glioblastoma in dogs, and could potentially be used as therapeutic or diagnostic agents for glioblastoma, and other tumours, in humans.

The goal of the work described in this chapter was to isolate mAbs that bind to the extracellular domain (ECD) of both canine and human EphA2. To achieve this goal the ECDs of canine and human EphA2 were expressed as recombinant proteins, and used to screen a naïve human scFv phage display library to isolate specific mAbs. To isolate antibodies against EphA2 through phage display, sufficient purity of the protein was required and the success in isolation of specific binders was dependent on the purity of EphA2 utilised in the biopanning campaign.

The strategy of targeting the extracellular domains of the receptors has been adopted due to difficulties on isolation and expression of the full-length membrane receptor due to the extensive hydrophobic domains embedded within the membrane [207-209]. The purified recombinant protein or peptide can be provided through expression in yeast [210], bacteria [211], insect cells [212] or mammalian cells [213]. However, the production of recombinant extracellular protein domains in bacteria may not be suitable due to the lack of posttranslational modifications and codon bias [214]. Purification of proteins away from other protein contaminants with similar physicochemical properties presents challenges and in some cases, when used in biopanning experiments, can lead to isolation of irrelevant antibody binders.

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This chapter describes the expression and purification of recombinant canine and human EphA2 ECDs, followed by the biopanning of a naïve human scFv phage library (generated and supplied by the National Biologics Facility-NBF) to isolate specific binders towards the human and canine EphA2-ECD. The phage binders were reformatted to whole human IgG1, and further characterization and validation was performed on generated mAbs.

3.2 Materials and methods

3.2.1 Cloning of canine and human EphA2 Extracellular Domains (ECDs) into mammalian expression vector

The DNA sequences for the full-length canine (Accession Number XP_544546) and human (Accession Number P29317) EphA2 receptor were synthesised by GeneArt (ThermoFisher Scientific, Germany) (Appendix 1). The extracellular domains (ECD) of canine (residues 1-534) and human (residues 1-534) EphA2 were PCR-amplified from the full-length sequences. Forward primers were the same in both canine and human EphA2 but reverse primers were different due to differing sequences. A hexahistidine tag and stop codon were added to the 3´ end of reverse primers. The primers are shown in Table 3.1 and the PCR conditions are as described in section 2.3.1. The cloning protocol into pcDNA3.1 expression vector was previously outlined in section 2.3.3. The BamHI and Nhel restriction enzymes were used to digest both PCR products and pcDNA 3.1 (+) vector. Sequencing was performed for recombinant constructs using Table 2.1 primers.

Table 3.1 Oligonucleotide primers used for amplification of human and canine EphA2- ECD from full length human and canine EphA2 constructs

Oligonucleotide Name Sequence (5´ to 3´)

EphA2_NheI_For AAGCTGGCTAGCCCACCATGGAAC Ca_EphA2_ECDHis_Ba AACCGTGGATCCCTAGTGATGGTGATGGTGGTGATTGCCGC mHI_Rev TGGCCTCGGTGGAC Hu_EphA2_ECDHis_Ba AACCGTGGATCCCTAGTGATGGTGATGGTGGTGGTTGCCA mHI_Rev GAGCCCTCAGGAGAC

3.2.2 Producing recombinant canine and human EphA2-ECD

The expression vectors containing canine and human EphA2-ECD genes were amplified and purified through DNA Midiprep protocol (section 2.3.9), followed by transfection of constructs into CHO-

36

XL99 cells for mammalian expression (part 2.3.10). The secreted proteins were harvested from the culture supernatant and purified using HisTrap and FF excel columns by immobilized metal ion affinity chromatography as described previously in section 2.3.11.

3.2.3 Biopanning on recombinant canine and human EphA2-ECD

The naïve human scFv phage library with a diversity of 5×109 unique scFvs was provided by the National Biologics Facility (mAbLab library, AIBN, UQ) and used in the biopanning procedure. Three rounds and four rounds of biopanning were accomplished for purified recombinant canine and human EphA2-ECD respectively (section 2.3.17). Hexahistidine-tagged anterior fat body protein (AFBP) was used in a subtractive step as a negative control to remove non-specific histidine binders. Our group at UQ has previously provided AFBP (MW 34 kDa). The depleted phage library was then biopanned against the extracellular domain of canine and human EphA2 immobilised on immunotubes through adsorption. The biopanning procedure was followed by polyclonal phage ELISA (section 2.3.18) and monoclonal phage ELISA (section 2.3.19).

3.2.4 Sequences alignment and identification of the unique clones

The positive phage clones based on the monoclonal phage ELISA results were selected for Colony PCR (section 2.3.5) and sequenced through a High-throughput sequencing method (section 2.3.21) by using primers listed in Table 2.2. Sequences were analysed using the Geneious version 9.0.5 software program, and then multiple amino acid sequences were aligned using Clustal Omega program (EMBL-EBI, https://www.ebi.ac.uk/Tools/msa/clustalo/).

3.2.5 Reformatting of anti-canine and anti-human EphA2-ECD scFvs into whole human IgG1

Reformatting was performed to ligate heavy and light chain variable regions of the identified scFv candidates onto human heavy and light chain constant regions by using the ligation-free method mentioned in section 2.3.21. Primers were designed (Table 3.2 and 3.4) to amplify the heavy and light variable regions with compatibility to cloning into mAbXpress vectors (Table 3.3). The amplified heavy (VH) and light variable (VL) regions of scFv candidates were cloned into heavy (NBF-305) and light (NBF either 304 or 326) human constant regions, respectively.

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Table 3.2 Oligos names used for PCR of heavy and light variable scFv domains against human and canine EphA2-ECD Clone Name Heavy F Heavy R Light F Light R (10 µM) (10 µM) (Lambda or (Lambda or kappa) (10 µM) Kappa)(10µM)

Hu78 mAbX_VhF7 mAbX_VhR6 mAbX_VhF3 mAbX_VLR4 Ca 38 mAbX_VhF7 mAbX_VhR7 mAbX_VkF12 mAbX_VKR4 Ca 53 mAbX_VhF6 mAbX_VhR1 mAbX_VKF9 mAbX_VKR1 Ca 64 mAbX_VhF7 mAbX_VhR6 mAbX_VLF3 mAbX_VLR4

Table 3.3 mAbXpress Vectors containing human constant backbone regions Region of human constant backbone Name of vector Heavy constant region hG1- NBF305 Lamda light constant region hLambda- NBF326 Kappa light constant region hKappa- NBF304

Table 3.4 Sequence of the primers used for amplifying heavy and light variable regions Primer Name Sequence (5´ to 3´) mAbX_VhF7 CAGGTGTCCACTCCGAGGTACAGCTGCAGCAG mAbX_VhF6 CAGGTGTCCACTCCGAGGTGCAGCTGGTGCAGTC mAbX_VhR6 GCGGAGGACACGGTGAGCATTGTCCCTTGGCC mAbX_VhR7 GCGGAGGACACGGTGAGCGTGGTCCCTTTGCCC mAbX_VhR1 GCGGAGGACACGGTGAGCGTGGTCCCTTGGCCC mAbX_VhF3 CAGGTGTCCACTCCGAGGTGCAGCTGCAGGAG mAbX_VkF12 CCGGCGTGCACTCCGAGATCTGGATGACCCAG mAbX_VKF9 CCGGCGTGCACTCCGAGACGACACTCACGCAG mAbX_VLF3 CCGGCGTGCACTCCGAGTCTGTGTTGACGCAGCCG mAbX_VLR4 GCCTTAGGCTGGCCGAGGACGGTCAGCTTGGTCC mAbX_VKR1 GGCCACGGTCCGCTTGAGTTCCAACTTTGTCCC mAbX_VKR4 GCCACGGTCCGCTTGAGCTCCAGCTTGGTCCC

After Infusion cloning, colony PCR was run for screening the colonies as described in section 2.3.5 by using Table 3.5 primers.

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Table 3.5 Primers for screening colonies Name of primers Type of chain Sequence (5´to 3´) mAbX_SeqF Forward primer TAACACCGCCCCGGTTTTCC Seq_hG1_R Reverse Heavy TCACCAGGCAGCCCAGAGCG Seq_hL_R Reverse Lambda CAGGCAGACCAGGGTGGCC Seq_hK_R Reverse kappa GTTCAGCAGGCACACCACGG

3.2.6 Production and purification of anti-canine and anti-human EphA2-ECD mAbs The reformatted mAbs were expressed in CHO-XL99 cells using the transfection protocol described in section 2.3.10. The secreted proteins were harvested from the cell culture supernatant and Protein A purification was performed using HiTrap MabSelect Sure columns. The purification procedure is detailed in section 2.3.12. The concentration of eluted soluble proteins was reported using the following formula: Protein concentration C (mg/mL) = 280 nm UV light Absorbance measured by NanoDrop / Absorption Coefficient calculated through ProtParam program in ExPASy online.

3.2.7 ELISA assay

The ELISA was conducted to examine the binding of reformatted antibodies to recombinant canine and human EphA2-ECD (section 2.3.20).

3.2.8 Kinetic analysis using Surface Plasmon Resonance (SRP) The kinetics-of-binding assessment on the most promising mAb was made using SPR, conducted on a Biacore T200 instrument (GE Healthcare). An assay design employed the reformatted mAb as the ligand and recombinant human and canine EphA2-ECDs as analytes. Amine coupling kit (GE Healthcare) was used to immobilise the mAb to the surface of a CM5 sensor chip aiming for 500 RU (Response Units). This was followed by injecting increasing concentrations (12.5, 25, 50,100 µg/mL) of human and canine EphA2-ECD (Analyte) over the surface at a flow rate of 30 µL/min. Single- cycle kinetics model was performed using 1:1 binding. Blank subtraction was applied against the reference surface and using a cycle containing buffer (HBS-EP+) in place of analytes. Sensorgrams were analysed using T200 BiaEvaluation version 3.1 software (GE Healthcare).

3.2.9 Analysis of antibody binding via flow cytometry

The binding of anti-canine and anti-human EphA2-ECD reformatted mAbs to native EphA2 was assessed using flow cytometry (2.3.25) on prostate and glioblastoma cell lines expressing EphA2 (2.3.24).

39

3.3 Results

Expression vectors containing canine and human EphA2-ECD were created for transient expression of the ECDs in CHO-XL99 cells. Figure 3.1 represents the map of recombinant human EphA2-ECD into pcDNA 3.1. Confirmation of sequences was achieved through analysis via Geneious version 9.0.5 software. The alignment of the canine and human sequences is shown in Table 3.6, representing 93% identical matching sequence.

Table 3.6. The alignment of human and canine EphA2-ECD amino acid sequences, showing identity 498/534 (93%)1. Human MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMN Canine MERPGPRACLALLWGCVLASAAAAQGKEVVLLDFAAAKGELGWLTHPYGKGWDLMQNIMD

Human DMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFN Canine DMPIYMYSVCNVVAGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFN

Human LYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYL Canine LYYAESDVDYGTNFQKRQFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLSRKGFYL

Human AFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGG Canine AFQDIGACVALLSVRVYYKKCPELLQGLARFPETIAGSDAPSLATVAGTCVDHAVVPPGG

Human EEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPS Canine EEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKSEASESPCLECPVHTVLS

Human PEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDI Canine SEGATFCDCEEGYFRAPQDLLSMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGRDDI

Human VYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSG Canine VYSVTCEQCWPESGECGPCEASVRYSEPPHALTRTSVTVSDLEPHMNYTFAVEARNGVSE

Human LVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSN Canine LVASRSFRTASVSINQTEPPKVKLEGRTTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSN

Human SYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGN Canine SYNVRRTEGFSVTLDDLAPDTTYLIQVQALTQEGQGAGSKIHEFQTLSTEASGN

1 The highlighted yellow residues indicate the non-aligned amino acids between human and canine EphA2-ECD. 40

Figure 3.1 pcDNA 3.1 (+)-Human EphA2-ECD-His plasmid map. The pcDNATM 3.1 contains ampicillin resistance gene (amp marker), ampicillin promotor (amp prom), T7 promoter (T7 prom), lac promoter (lac prom), SV40 promoter (SV40 prom), Neomycin phosphotransferase II selectable marker (NTP_II marker), BGH polyadenylation signal sequence (bGH_PA) and human EphA2-ECD between BamHI and Nhel restriction sites. The pcDNATM 3.1(+)-canine EphA2-ECD-His plasmid map is similar to the above mentioned map and is not depicted.

3.3.1 Production of recombinant canine and human EphA2 Extracellular Domains The canine and human EphA2-ECD constructs were expressed in CHO-XL99 cells, and the expressed proteins were secreted to the supernatant, followed by two consecutive IMAC purification, first by using HisTrap Excel column and second by His-trap FF column affinity chromatography to achieve a greater purity. SDS-PAGE analysis showed the canine and human EphA2-ECD with an apparent molecular weight of 56 kDa. The purity of the expressed recombinant protein after each purification step is shown by SDS-PAGE gel in both reduced and non-reduced conditions. SDS-PAGE gel analysis (Figure 3.2) showed first and second purification of EphA2-ECD by HisTrap excel (Figure 3.2 c and d) and HisTrap FF columns (Figure 3.2 a, b, e and f). Results showed that reduced EphA2 (Figure 3.2 b and f) had a higher apparent molecular weight compared to that of the non-reduced EphA2 (Figure 3.2 a, c, d and e). This is a consequence of the drag forces of the predominantly beta-sheet structure of the reduced EphA2.

41

Figure 3.2 SDS-PAGE analysis of both canine and human EphA2-Extracellular Domains. Image shows the EphA2-ECD with an apparent molecular weight of 56 kDa after purification by HisTrap excel and HisTrap FF column chromatography. Band shows in lane a) purified canine EphA2-ECD after HisTrap FF in non-reduced condition b) purified canine EphA2-ECD after HisTrap FF in reduced condition c) purified canine EphA2-ECD after His-Trap excel in non-reduced condition d) purified human EphA2-ECD after HisTrap excel in non-reduced condition e) purified human EphA2-ECD after HisTrap FF in non-reduced condition f) purified human EphA2-ECD after HisTrap FF in reduced condition. Molecular weight of marker (SeeBlue® Plus2 Pre-Stained Standard) is illustrated in the left lane.

3.3.2 Identification of canine and human EphA2-Extracellular Domains

Mass spectrometry analysis was conducted for enzymatically digested recombinant canine and human EphA2-ECD in solution.The ProteinPilot Peptide mass fingerprinting (PMF) program was employed and the results confirmed the sequence coverage for identification of generated recombinant EphA2-ECD for both canine and human (Appendix 4).

3.3.3 Generation of scFv phage binder against canine and human EphA2-ECD through affinity selection using phage display

The purity of the protein used in biopanning as an antigen is a key success factor in biopanning outcomes. In this regard, two consecutive purifications were performed for both canine, and human EphA2-ECD and their identity was confirmed through mass spectrometry analysis. The naïve human scFv phage library was kindly provided by NBF. This library compromised filamentous M13 phage clones with a diversity of approximately 5×109 binders. Stringent washing (×5) in round two led to a decrease in output titration, thus washing step was completed by ×3 for round two, ×7 for rounds three and four. 42

3.3.4 Enrichment of scFv phage binders to canine and human EphA2-ECD

The enhancement of sub-libraries for specific phage binders after each round of selection was verified by polyclonal phage ELISA using recombinant canine and human EphA2-ECD. The phage pools obtained after each round of biopanning and the source naïve human scFv phage library were employed to determine the enhancement of specific scFv-phage binders towards the immobilised canine and human EphA2-ECD, separately. This was performed with two replicates, and sub-libraries were prepared in 1/10 serial dilutions. Bound phages were detected using anti-M13 phage HRP antibody, and absorbance was measured at 450 nm. The phage pool analysis showed the enrichment of phage binders to the EphA2-ECD through increasing rounds selection. This was evident from round 2 for canine EphA2-ECD (Figure 3.3, A) and round 3 for human EphA2-ECD (Figure 3.3, C). The highest enrichment was achieved by round 3 and round 4 for canine and human EphA2-ECD, respectively. In addition, all the phage pools were tested against immobilised anti-AFBP as a negative control for nonspecific binding. There was no binding towards AFBP for either of the phage pools (Figure 3.3 B&D). These results suggest that the enrichment of specific phage binders was become higher round by round.

Figure 3.3 Polyclonal Phage ELISA for canine and human EphA2-ECD. Phage pools generated during biopanning against purified canine EphA2-ECD (A and B) and human EphA2-ECD (C and 43

D) were tested against immobilised EphA2-ECD (A and C) and AFBP (B and D). Results indicated the average of two replicates for each sample and grouped through two-way ANOVA. The positive signals were considered as an arbitrary cut-off value of A450 ≥ 1.0.

3.3.5 Screening individual phage positive clones specificity towards canine and human EphA2- ECD

Cell glycerol stock from Round 3 of canine and round 4 of human EphA2-ECD were used for monoclonal phage ELISA to screen individual phage clones. Single colonies were grown in 96-well plates. Each well contained a potentially different clone; helper phage then was added to induce production of phage particles. The phage particles from each clone were tested by phage ELISA for specific binding to the EphA2-ECD. Phage–EphA2 binding was detected using anti-M13 phage HRP antibody. Monoclonal phage ELISA towards canine EphA2-ECD indicated 79 out of 84 positive phage clones (Figure 3.4), and this was 74 for human EphA2-ECD (Figure not shown).

44

Canine EphA2-ECD and AFBP vs phage scFv binders

2.0

Canine EphA2 ECD-his tag AFBP-his tag

1.5

)

m

n

0

5

4

(

e

c 1.0

n

a

b

r

o

s

b A

0.5

0.0 A1 A6 A11 B4 B9 C2 C7 C12 D5 D10 E3 E8 F1 F6 F11 G4 G9 H2 H7 H12 Number of phage clone

Figure 3.4 Monoclonal Phage ELISA to screen for individual clones binding to canine EphA2- ECD. The phage particles from each clone were tested by phage ELISA for specific binding to the canine EphA2-ECD and AFBP-his tag. Phage–EphA2 binding was detected using anti-M13 phage HRP antibody. The positive signals were considered as an arbitrary cut-off value of A450 ≥ 1.0.

3.3.6 Sequence alignment and identification of unique clones to canine and human EphA2-ECD

The DNA sequences encoding the VH and VL regions of the positive clones were amplified by PCR (Figure 3.5; protocol described in section 2.3.5) and sequenced (described in sections 2.3.8 and 2.3.20) using Table 2.2 primers. Multiple sequence alignment revealed that all 74 positive clones isolated from biopanning against human EphA2 were the same unique sequence (designated as hu78- scFv). Four unique sequences were identified from the 79 positive clones isolated from biopanning against canine EphA2 (designated as ca71-scFv, ca53-scFv, ca38-scFv, and ca64-scFv). Among those, the unique anti-human binder (hu78-scFv) had 100% identical sequence with one of the anti- canine unique phage binder (ca71-scFv), suggesting that this is a canine-human cross-reactive binder. The protein sequences corresponding to the CDR sequences of the clones obtained are presented in Table 3.7. 45

Figure 3.5 Colony PCR to amplify phagemid scFv of A) anti-human EphA2-ECD B) anti-canine EphA2-ECD. Lane M contains the Marker for each row. Rows A to H (lanes 1 to 12) contains the achieved positive clones against human (A) and canine (B) EphA2-ECD. DNA from positive clones migrated to around 1000 bp confirming the presence of anti-EphA2 scFvs. Products were obtained for all clones except for six from the canine EphA2 screening (shown by red circles). The plasmids for these clones were extracted separately by using Quick Plasmid Miniprep Kit. pPHNBF_Seq_For and pPHNBF_Seq_Rev primers were used for sequencing.

Table 3.7 Representative the CDR regions of the heavy and light chain variable regions. Represent CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR- CDR-L3 ative clone L2 hu78-scFv GGSISSG IYYSG FGVATKGDAF SGSSSNIGNNYV DNDK GTWDFSR (AGH001- GYY DI S RPS GEV scFv = ca71-scFv) ca64-scFv GYEFNIY IIYPSDSDTRYSP PDLGGKMTSD RASQSVAGNYL GASSR QHYGSSS WIG SFQG WFFDI A AT AT ca53-scFv GDTLISY GIIPILGTANYAQ AFSSGSPARFY RSSQSLLHSNG LGSNR MQALQTL AIS KFQG GMDV YNYLD AS T ca38-scFv GGSFSGY EINHSGSTNYNP GRNIAAPYYY RASQSISSYLN AASSL QQSYSTP YWI SLKSRI MDV QS WT

46

3.3.7 Cross-Reactivity analysis of anti-human and anti-canine EphA2-ECD scFvs against human and canine EphA2

Since the human and canine EphA2-ECDs have 93% homology (Table 3.6), the cross-reactivity of unique anti-human EphA2-ECD scFv (hu78-scFv) was assessed by ELISA against recombinant canine EphA2-ECD. Results indicated the binding of hu78-scFv phage to canine EphA2-ECD (Figure 3.6, A). This result was expected since the same scFv sequence was also isolated during biopanning against canine EphA2-ECD.

The cross-reactivity of three different anti-canine EphA2-ECD scFv binders was tested for binding to immobilised human EphA2-ECD (Figure 3.6, B). ELISA outcomes showed that among anti-canine EphA2-ECD scFvs, ca64-scFv and ca38-scFv had affinity to human EphA2-ECD whereas ca53-scFv had no positive reaction to human EphA2-ECD, suggesting the specificity of ca53-scFv to only canine EphA2-ECD. These experimental outcomes were consistent with their serial dilutions as well.

Figure 3.6 Cross-Reactivity of (A) human scFv-phage particle against canine EphA2-ECD, and (B) three different canine scFv-phage particles against human EphA2-ECD, measured by ELISA. Recombinant canine EphA2-ECD (panel A) was immobilised on ELISA plate and incubated with hu78-scFv (neat) and three 1/10 serial dilutions. In panel B, the Recombinant human EphA2- ECD was immobilised and incubated with ca38-scFv, ca53-scFv and ca64-scFv and three 1/10 serial dilutions of relevant anti-canine EphA2-ECD scFvs. The absorbance was measured at 450 nm and positive signals were considered as an arbitrary cut-off value of A450 ≥ 1.0 for neat concentrations.

3.3.8 Reformatting of anti-EphA2 scFv phage binders to human Immunoglobulin G1 (IgG1)

The scFv phage particles were reformatted to full human immunoglobulin G1 (IgG1) for further characterization. The In Fusion™ system [118] was used for ligation-independent In Fusion™ 47 cloning (Clontech). The sequencing results were matched to reference sequences constructed in Geneious version 9.0.5 software, representing the successful Infusion of scFvs into mAbXpress vectors encoding the heavy and light chains of human constant backbones. The identical scFv phage binder (hu78-scFv), with cross reactivity for both human and canine EphA2-ECD, was named mAb AGH001 after reformatting to fully human IgG1.

3.3.9 Expression of reformatted phage display-derived scFv into human IgG

The reformatted mAbs including AGH001, ca38, ca53 and ca64 were transiently expressed in CHO- XL99 cells and purified by Protein A affinity chromatography. The SDS-PAGE gel results (Figure 3.7) showed a 150 kDa band of a fully assembled antibody in the non-reduced sample lane. The 150 kDa band was split into two distinct bands in the reduced lane. This is characteristic of purified mAbs; the 50 kDa and 25 kDa bands represents the heavy and light chains, respectively.

Figure 3.7 Coomassie blue staining of reformatted antibodies on SDS-PAGE: A) AGH001, B) ca38 anti-canine EphA2-ECD mAb. SDS-PAGE gel results after purification using Protein A Column Chromatography shows the whole IgG with 150 KD band in the non-reduced lane and the characteristic heavy chain (50 KD) and light chain (25KD) bands in the reduced lane. Other bands in the non-reduced lane present different isoforms of reformatted mAbs. Markers in A and B are SeeBlue® Plus2 Pre-Stained Standard (Thermo Fisher Scientific) and Precision Plus Protein Unstained Standards (Bio-Rad), respectively. (Images are not shown for ca53-IgG and ca64-IgG mAbs, but these showed a similar profile).

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3.3.10 Binding of reformatted antibodies against recombinant canine and human EphA2-ECD

An indirect ELISA binding assay were set up to assess the binding of reformatted antibodies to purified recombinant human and canine EphA2-ECD immobilized on a microtitre plate. The recombinant canine and human EphA2-ECD antigens were coated at a concentration of 3 µg/mL and incubated with three fold serially diluted concentration of antibodies beginning at a concentration of 10 µg/mL (Figure 3.8). Absorbance results showed that mAb AGH001 demonstrated significant binding to both canine and human EphA2-ECD (Figure 3.8, A), whereas none of the anti-canine EphA2-ECD mAbs including ca38, ca53 and ca64 mAbs, bound to either recombinant canine or human EphA2-ECDs (Figure 3.8, B and C). The ELISA results for anti-canine mAbs are not consistent when compared to anti-canine scFvs binding, specifically for ca38-scFv and ca64-scFv. These two scFvs showed different binding specificity when reformatted to whole IgG. It was expected to have a better binding when scFvs are reformatted to full mAb as they are changed to two binding sites, resulting in increased avidity. This experiment was repeated, and the similar results were achieved. It is possible that the heavy and light chain variable regions did not assemble into the correct configuration when they were not constrained in a single polypeptide.

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Figure 3.8 Indirect ELISA to test for specificity of reformatted mAbs to recombinant canine and human EphA2-ECD. A) Recombinant human and canine EphA2-ECD were separately immobilised on ELISA plates at 3 µg/mL concentration and incubated with serially diluted mAb AGH001 starting with 10 µg/mL concentration with serially dilution. B) Recombinant human EphA2-ECD was immobilised at 3 µg/mL concentration, and the binding of three different reformatted anti-canine EphA2-ECD mAbs were tested. C) To test the binding of the different generated anti-canine mAbs, recombinant canine EphA2-ECD were coated on the plate. The absorbance (Y-axis) at 450 nm was plotted against the logarithm of the mAbs dilutions (X-axis), and results were analysed using GraphPad Prism Version 8.0.2 software.

3.3.11 Affinity assessment of mAb AGH001 to recombinant human and canine EphA2-ECD

Surface Plasmon Resonance (SPR, Biacore T200) was used to determine the real-time rate of association (binding) and disassociation (unbinding) of mAb AGH001 to human and canine EphA2- ECD. Single-cycle kinetic analysis was performed by injecting increasing concentrations of the analyte (EphA2-ECD) to flow cells with immobilized AGH001. 500 RU of mAb AGH001 was immobilised on activated CM5 biosensor chip. The sensorgrams were fitted with a 1:1 binding model to show the interaction between EphA2-ECD and mAb AGH001 according to the equation A+ B = AB. The sensorgrams are illustrated for the single cycle kinetics for recombinant human EphA2-ECD (Figure 3.9, A), and recombinant canine EphA2-ECD (Figure 3.9, B) and the measured affinity values are summarized in Table 3.7. Evaluation of the affinity parameters revealed that mAb AGH001 had an Equilibrium Dissociation Constant (KD) of 13.8 nM to human EphA2-ECD (A), and 12.7 nM to canine EphA2-ECD (B). Most antibodies isolated using phage display have a KD value between micromolar to nanomolar. The lower nanomolar value, the higher the affinity antibody for its ligand would be considered [215]. The Biacore results suggest that AGH001 is a moderately good antibody. SPR results were consistent with ELISA results.

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Figure 3.9 SPR sensorgrams (Biacore T200) for the interaction between ligand (mAb AGH001) and a) human EphA2-ECD b) canine EphA2-ECD analytes. 500RU of mAb AGH001 was immobilized directly on the surface of the CM5 sensor chip. This was followed by injecting at various concentrations (12.5, 25, 50,100 µg/mL) of recombinant EphA2-ECD (Analyte) over the surface at a flow rate of 30 µL/min. The green line (raw data) and black line (binding fit) are illustrated.

Table 3.8 Affinity values for the interaction between mAb AGH001 and recombinant human and canine EphA2-ECD determined via SPR. Association rate constant (ka), Dissociation rate constant (kd), Equilibrium dissociation constant (KD), Relative response (RU), Chi-square and U- value are presented in below table.

2 2 Analytes ka (1/Ms) kd(1/s) KD(M) Rmax(RU) Chi (RU ) U-value

Hu EphA2-ECD 7720 1.06E-04 1.38E-08 48.8 4.13 9

Ca EphA2- ECD 7416 9.42E-05 1.27E-08 48.97 4.16 9

3.3.12 Binding of mAb AGH001 to native EphA2 through flow cytometry At the beginning of this experiment, several available human prostate cancer cell lines were tested including PC3 and LnCap via flow cytometry to examine the specificity of mAb AGH001 to the native EphA2 expressed on the cancer cells membrane. Since EphA2 receptor is overexpressed in human prostate cancer cells [128] a number of available prostate cell lines were first used to test the binding of mAb AGH001 to EphA2. Additionally, mAb 4B3 was used as a positive control. mAb 4B3 is a murine antibody that binds human EphA2-Fc [216], generated by standard hybridoma 51 technique in the laboratory of Andrew Boyd at QIMR Berghofer Medical Research Institute. Goat pAb to Mouse IgG (DyLight 594) and Goat pAb to Human IgG (DyLight 594) secondary antibodies were used to detect mAb 4B3 and mAb AGH001, respectively.

Flow cytometry results showed that AGH001 bound to the above-mentioned prostate cancer cell lines expressing EphA2 (Figure 3.10, A and B). The results were consistent with flow cytometry analysis of mAb 4B3 (Figure 3.10, C and D). However, the comparison of 4B3 and AGH001 histogram intensities was not possible, since different secondary antibodies conjugated to the fluorescent dyes were used according to the primary antibody species. It is interesting to note that by increasing the mAb AGH001 concentration, the more shift was seen to the right. Though the different histogram intensities were not observed for the increased concentrations of 4B3, and almost all the histograms of different concentration were overlaid. This suggests that the mAb 4B3 has a high affinity, and that even at the lowest concentration used in the experiment, the binding is saturated.

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Figure 3.10 Flow cytometric analysis of PC3 and LNCaP exposed to mAbs AGH001 and 4B3. A) PC3, and B) LNCaP cells were exposed to three concentrations of mAb AGH001 (3, 10, 30 µg/mL). Goat pAb to HuIgG (DyLight 594) was used as a secondary antibody. C) PC3 and D) LNCaP were incubated with mAb 4B3 at three concentrations (3, 10, 30 µg/mL). The Goat pAb to Ms IgG (DyLight 594) was used as a secondary antibody. Secondary fluorescence antibodies (X-axis) were measured using 561 nm laser and 630/22 nm filter and the 10.000 events were counted on Y-axis.

Despite these findings, a negative cell line that does not express EphA2 was required to test the specificity of mAb AGH001. Since this project was based on the EphA2 receptor overexpressing in glioblastoma cell lines, accordingly the characterization was completed on the glioblastoma cell line. U87-MG is a human glioblastoma cell line; both positive and negative EphA2 cell lines were kindly provided by Dr Brett Stringer (QIMR Berghofer Medical Research Institute, Queensland, Australia). U87 EphA2 KnockDown (KD) cell was generated using CRISPR/Cas9 genome editing utilising a single guide RNA (sgRNA) technique. Analysis of antibody binding via flow cytometry showed that mAb AGH001 bound to both positive and negative EphA2 cell lines, as demonstrated by a positive shift in fluorescence (Figure 3.11, A), indicating that mAb AGH001 non-specifically bound to the EphA2 KD cell line, compared with no right shift for the positive control antibody, 4B3, in the negative EphA2 cell line (Figure 3.11, B).

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Figure 3.11 Flow cytometry analysis of positive and EphA2 knock down human glioblastoma cell line. A) The mAb AGH001 was detected by anti-human IgG DyLight 594 in both positive EphA2 U87 cells (pink histogram) and EphA2 KD U87 cells (light green). Both histograms were overlaid to each other, representing the non-specificity of mAb AGH001 to EphA2. Overlapping of anti-human IgG DyLight 594 histograms were showed in both U87 cells (black) and EphA2 KD U87 cells (grey). B) The mAb 4B3 was specifically detected by anti-mouse IgG DyLight 594 antibody histogram in positive EphA2 U87 cells (red), and no binding was seen for mAb 4B3 in EphA2 KD U87 cell (dark green), indicating the specificity of mAb 4B3. Secondary anti-mouse IgG DyLight 594 histograms has been overlapped both in positive (dark blue) and in negative (light blue) EphA2 U87 cells.

3.3.13 Binding and competitive immunoassay

Competitive ELISA was conducted to test the competition between mAbs 4B3 and AGH001 towards recombinant human EphA2-ECD. Firstly, to test the binding of mAb AGH001 against human EphA2- ECD, 1:3 serial dilutions of mAb AGH001 (green curve) were tested on 3 µg/mL of human EphA2- ECD (Figure 3.12, A). Based on the resulting curve, 3 µg/mL of mAb AGH001 was chosen as a constant concentration for the inhibition assay (showed by arrow). The competition assay (Figure 3.12, B) showed no competition between mAb 4B3 and mAb AGH001 towards recombinant human EphA2-ECD, since there was no significant difference observed between the constant concentration of mAB AGH001 (green curve) and blue curve (serial dilutions of mAb 4B3 pre-mixed with constant

54 concentration of mAb AGH001). The amino acid sequence alignment of mAbs AGH001 and 4B3 CDRs showed that they have different CDRs (Table 3.9)

Figure 3.12 Inhibition ELISA for competitive binding to recombinant human EphA2-ECD between mAb AGH001 and mAb 4B3. A) The ELISA plate coated with human EphA2-ECD at 3 µg/mL was incubated with 1:3 serial dilutions of mAb AGH001 to determine the optimum dilution for constant concentration in a competition assay. Bound mAbs were probed using anti-human IgG HRP secondary antibody. B) The recombinant human EphA2-ECD was immobilised on an ELISA plate, then incubated with serial dilutions of mAb 4B3 pre-mixed with 3 µg/mL of mAb AGH001 (blue curve). The absorbance (Y-axis) at 450 nm was plotted against the logarithm of the mAb dilutions (X-axis), and the results were analysed using GraphPad Prism Version 8.0.2 software.

Table 3.9 Comparison of mAbs 4B3 and AGH001 CDRs.

mAbs CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3

4B3 GYTFTDYD INPNNGG ARRSTMTYFD SSVSY RTS QQYHSYPLT A Y AGH00 GGSISSGGY IYYSG FGVATKGDAF SGSSSNIGNNYV DNDKRP GTWDFSRGE 1 Y DI S S V

3.4 Discussion

Although mouse models have been widely used in preclinical studies, histological features have been poorly characterised in rodents bearing brain cancer [217]. Xenograft models in mice and rats exhibit limited genetic heterogeneity and reduced invasiveness, as they are immune-deficient animals; these 55 animal models are not good comparisons to spontaneous tumours in human patients. The domestic dog represents a powerful, large animal model of glioma, as canine brain tumours occur spontaneously with an incidence rate and patient age profile similar to that of human populations [218-221]. Furthermore, it has been shown that many histological [222, 223] and genomic [37] characteristics are conserved between canine and human gliomas. These are some of the factors that have led to introduction of comparative oncology programs, by institutions such as the US NIH National Cancer Institute [224], as highly innovative and proven strategies towards the translation of therapeutic candidates. To facilitate comparative oncology studies, investigating particular overexpressed biomarkers, it is useful to have an antibody that cross-reacts with both the canine and human forms of the biomarker. In this chapter, the goal was to use phage display to isolate antibodies capable of binding canine and human EphA2.

As a target for biopanning, we cloned and produced the extracellular domain of canine and human EphA2 only. The full-length EphA2 receptor is of significant size (130 kDa) [225] to be easily expressed and purified, and also its transmembrane domain is hydrophobic, therefore cannot be stabilised in soluble form in biopanning procedure and might lose its native conformation. In total, four unique scFvs were isolated through separately biopanning against canine and human EphA2- ECD. Among these, one was isolated from both biopanning strategies, against both EphA2-ECD species. This scFv, after reformatting to whole IgG, was designated mAb AGH001, and showed cross-reactivity with both recombinant canine and human EphA2-ECD. The other scFvs reformatted to IgG did not show specific binding to either recombinant human or canine EphA2-ECD, whereas they had bound in their scFv phage format towards recombinant antigens. When a scFv is reformatted to an IgG, it is expected to result in improved binding to recombinant antigens, as opposed to the phage display-derived scFv format, due to the presence of two binding sites and increased avidity effect [226-228]. However, this was not observed and is inconsistent with our ELISA results. Reports have also indicated loss of affinity after switching of scFv fragment into IgG format. The conversion from monovalent to bivalent antigen binding format can in some cases result in inappropriate conformational arrangement of the variable regions that leads to a decrease in affinity, which may not be recompensed by an avidity effect [229].

The affinity of mAb AGH001, measured by SPR, was in the nanomolar range, consistent with other antibodies isolated from a naïve library with a range of 10-8 to 10-9 M [217, 230, 231]. Flow cytometry results showed that mAb AGH001 bound to U87 cells expressing EphA2 on the cell surface, though it also bound to another unknown entity on EphA2 KD U87 cells. In contrast, the mAb 4B3 bound

56 specifically to EphA2 on U87 cells, while no binding was observed for mAb 4B3 on EphA2 KD cells, suggesting the specificity of mAb 4B3 to EphA2.

There was no competition occurred between mAb 4B3 and mAb AGH001 towards binding to EphA2- ECD in competitive ELISA. This outcome suggests that these two antibodies bind to a different epitope of EphA2, potentially even a different domain. The sequence alignment of their CDRs revealed that they bind to different epitopes as well. We assumed that mAb 4B3 binds specifically to EphA2-ligand binding domain (LBD), since it has been demonstrated to have potential as a theranostic agent [232] and possibly mAb AGH001 binds to another area on EphA2-ECD. Since the EphA2 is a multi-domain receptor (Figure 1.1, B) and the biopanning procedure was accomplished on the extracellular domain of EphA2, it is not surprising to isolate an antibody which binds to another domain of EphA2-ECD. This binding could be within the cysteine-rich domain (CRD), two fibronectin type III domains (Fn III) or even part of both LBD and CRD. This antibody could be functional if it binds to Fn III regions. This ubiquitous domain plays key roles in the cell-cell connection and ECM (extracellular matrix) formation, and is present in approximately 2% of all animal proteins [233].

It has been reported [234, 235] that the ECD of EphA2 can be cleaved by membrane type I matrix metalloproteinase (MT1-MMP) on tumour cells and truncated EphA2 without the existence of ephrinA1 can initiate oncogenic signalling pathway. The cleavage sites are located near the N- terminal of Fn III domain starting with either amino acid sequences Y385, T395, V432 and N435. It has been reported that there is an abundance of this scaffold in human blood and also extracellular matrix, more specifically as a component of cytokine receptors, cell surface hormones and cell adhesion molecules [236, 237], and represents a natural, common scaffold [238]. In one study [235], it was demonstrated that N-terminally cleaved EphA2 could promote ligand-independent oncogenic signalling by switching from the ligand-dependent pathway. In addition, the phosphorylation pathway can be activated by N-terminally truncated EphA2 in tumour tissue. According to the above-mentioned studies, we can hypothesize that mAb AGH001 may bind to the Fn III domain of EphA2 ECD and may recognise other Fn domains that naturally exist on EphA2 KD cells. Therefore, this may suggest that mAb AGH001 can bind to multiple targets. An alternative hypothesis is that mAb AGH001 can block other Eph family receptors as well. All the Eph receptor ectodomains comprise a highly conserved N-terminal LBD, followed by a CRD, and two Fn III repeats. Since the extracellular segment is highly conserved in all Eph families [239] and Eph receptors are co-expressed [89] then we can subsequently assume that mAb AGH001 may also binds to another Eph receptor on EphA2 KD cells.

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3.5 Conclusion

We conclude that mAb AGH001 is unlikely to bind to the receptor-binding site of EphA2 according to the competitive assay result using the positive control mAb 4B3 antibody. We were not sure exactly where on the EphA2 molecule mAb AGH001 binds, therefore epitope binding analysis of mAb AGH001 is required to clarify the AGH001 binding site (Chapter 5). By proving that the mAb AGH001 binds to the Fn III domains, we can use this antibody for suppressing EphA2 oncogenic signalling pathway in tissues with a lower level of ephrinA1. It has been documented that ephrinA1 is present at considerable low levels in GBM while EphA2 is overexpressed [96, 98]. Isolation of an antibody by selectively targeting EphA2 is challenging since this receptor belongs to a large family with closely related proteins [240]. Then, it would be a good strategy for targeting the EphA2-LBD domain in both canine and human to specifically isolate anti-EphA2 antibodies.

The following chapter is dedicated to isolating anti-EphA2 antibodies that bind canine and human EphA2-LBD domains through phage display to screen specific binders for this specific region.

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Chapter 4 Isolation of canine and human monoclonal antibodies against recombinant canine and human EphA2 Ligand- Binding Domain (LBD)

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4.1 Introduction

Many studies have reported the generation of novel antibodies for membrane proteins. In such studies, either native cells or the ectodomain of the target receptor were employed as an antigen [241-246]. There has been significant successes for antibody isolation through these kinds of approaches. Although these strategies can yield antibodies for the desired epitope, they may also result in the generation of antibodies that bind to domains or epitopes other than the one of interest, especially for multi-domain receptors. An alternative strategy to target particular domains of multi-domain receptors is to use only a small, discrete structural domain of the receptor in the antibody selection procedure [247]. However, it is important to keep in mind that in some cases, such domains may fail to recapitulate the correct 3-D structure of the native domain, and subsequently can result in loss of native epitopes. This strategy could be employed for screening antibodies to cell surface receptor sub- domains, where antibody domain-selectivity is required. One study targeted the individual domains of multi-pass transmembrane GluA4 receptor, and an antibody was successfully isolated against the GluA4-ligand binding domain [247]. Consequently, this strategy could be an ideal way to target the selection and generation of antibodies against the specific epitope.

Isolating antibodies through phage display by biopanning against only the ligand binding domain (LBD) of a given receptor is a viable strategy from an antibody specificity perspective. Several peptides have been isolated from peptide phage libraries that bind explicitly to the LBD of the targeted receptors [248-250] and were actually able to mimic the native receptor-binding motifs of corresponding ligands [251, 252]. A number of membrane proteins, including the Eph family tyrosine kinase receptors, are differentiated by their binding domains. Members of such family receptors are identifiable by their receptor binding domain, which is different from one member to another [77, 240, 253].

Binding of ligand to the extracellular-LBD of EphA2 activates the kinase domain and consequently triggers a signal transduction cascade within the EphA2-expressing cell, resulting in endocytosis of EphA2 bound to its ligand. Therefore, generating antibodies by targeting the EphA2-LBD can mediate the transport of cytotoxic agents into cancer cells, or inhibit or promote downstream effects of ligand binding. The LBD is a conserved domain in Ephs ectodomain, and an adjacent cysteine-rich domain appears to contribute to LBD for oligomerization [78]. The EphA2-LBD forms a compact circular feature (Figure 4.1) with a β-sandwich jellyroll folding configuration. It has been shown [93] that the EphA2- LBD is a monomer when at low concentration (10 µM) but assembles as a dimer asymmetrically at high concentration (Figure 4.1). Dimerization occurs by the connection of D-E and J-K loop residues.

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The DE loop has significant importance for target specificity but not in target binding compared with the other two loops [254]. Crystal structure study of EphA2-ephrinA1 complex has shown that the binding affinity of ephrinA1 to EphA2 occurs through the G-H loop of EphA2 comprising 15 conserved amino acids connecting the G to H β sheets [255]. This domain has contributed to the understanding of EphA2 activation. Targeting a smaller part of a receptor (i.e. domain) for antibody discovery may increase the specificity in the highly conserved receptor family. However, a binder to this particular region of the receptor might not be able to recognise the receptor in its physiological microenvironment [164]. The most crucial difference between EphA and EphB families of receptors is the length of the H-I loop. The H-I loop in EphA2 receptors has four less amino acid residues in comparison with that of the EphB receptors [256].

Figure 4.1 Structure of the EphA2-LBD. Secondary structure elements are displayed. Dimer structure of EphA2‐LBD in the asymmetric unit. Monomers are illustrated in different colours (Adapted from [93]).

The work described in this chapter aimed to isolate mAbs that bind to the (LBD) of both canine and human EphA2. To work towards this goal, the LBDs of canine and human EphA2 were expressed as recombinant proteins and used to screen a naïve human scFv phage display library to isolate specific mAbs. Simultaneously, three separate soluble antigen biopannings were conducted: first against canine EphA2-LBD, second against human EphA2-LBD and third by alternating canine and human EphA2-LBD in each round. Totally four rounds were applied for each biopanning campaign, to

61 isolate phage binders that bound selectively to canine and human LBD of EphA2. Isolated scFv phages were reformatted to a fully human IgG1, and characterised for binding to EphA2.

4.2 Materials and methods 4.2.1 Cloning of canine and human EphA2- LBDs into mammalian expression vector

Start and end sequences of both canine and human EphA2-LBDs have been selected based on reported methodology of Seiradake et al. [118]. The LBD of canine and human EphA2 were PCR- amplified from the full length sequences of human (Accession Number P29317) and canine EphA2 (Accession Number XP_544546) synthesised by GeneArt (ThermoFisher Scientific, Germany). A pcDNA3.1 (+) (Invitrogen) expression vector already containing an insert for DENV-NS1 protein with a mammalian secretion signal was obtained from the National Biologics Facility (NBF, Brisbane, Australia). AgeI and XbaI restriction enzymes were used to digest the vector to remove the existing insert but leaving the secretion signal. The same enzymes were used to digest the PCR products. A hexahistidine tag and stop codon were added to the C-terminal of reverse primers. The primers are shown in Table 4.1.

The cloning protocol into pcDNA3.1 expression vector and DNA preparation for sequencing were previously described in section 2.3.3 and section 2.3.8, respectively. Sequencing was performed for recombinant constructs using Table 2.1 primers.

Table 4.1 Oligonucleotide primers used for PCR of human and canine EphA2-LBD

Oligo Name Sequence (5´ to 3´) HuEphA2_LBD_Agel_F TGCAGCTACCGGTGTCCACTCCCAGGGCAAAGAA HuEphA2_LBD_Xbal_R TCTAGACTAGTGATGGTGATGGTGGTGGGGGCACTTCTTG TAGTAGACC Ca_EphA2_LBD_ GCTACCGGTGTCCACTCCCAGGGCAAAGAAGTGGT Agel_F Ca_EphA2_LBD_His_X TCTAGACTAGTGATGGTGATGGTGGTGGGGGCACTTCTTG bal_R TAGTACACC

4.2.2 Producing recombinant canine and human EphA2-LBD

The expression vector constructs including pcDNA 3.1(+)-hu-EphA2-LBD-His and pcDNA 3.1(+)- ca-EphA2-LBD-His were transfected separately into CHO-XL99 cells for mammalian expression as previously described in section 2.3.10. The secreted proteins were purified using HisTrap excel column on AKTA explorer, followed by Size Exclusion Chromatography (SEC) Superdex TM 200 62

10/300 GL column. The SEC was applied (section 2.3.13) to collect the highest purity of the recombinant proteins based on their molecular weight for using in biopanning procedure.

4.2.3 Preparation of human and canine EphA2-LBD in a solution for peptide mass fingerprinting analysis

Described previously in section 2.3.14.

4.2.4 Biopanning on recombinant canine and human EphA2-LBD

The naïve human scFv phage library with a diversity of 109 unique scFvs was kindly provided by NBF (MJ-SM scFv library). This library became available after work in chapter 3 was done and employed in biopanning campaigns to isolate phage binders against canine and human EphA2-LBDs. Hexahistidine-tagged anterior fat body protein (AFBP) was used in a subtractive biopanning step to remove any histidine-tag binders, before the depleted phage library was deployed against the LBD of canine and human EphA2 immobilised on immunotubes through adsorption. Separately, three soluble antigen biopanning procedures were conducted (sections 2.3.17) using: 1) canine EphA2-LBD, 2) human EphA2-LBD and 3) alternating canine and human EphA2-LBD in each round. Overall, four rounds of were carried out for each biopanning campaign, to isolate phage binding selectively to LBD of EphA2. The biopanning procedure was followed by polyclonal phage ELISA (section 2.3.18) and monoclonal phage ELISA (section 2.3.19).

4.2.5 Sequence alignment and identification of the unique scFv phage clones to human and canine EphA2-LBDs

The positive phage clone binders based on the monoclonal phage ELISA results were selected for Colony PCR (section 2.3.5) and sequenced through a High-throughput sequencing method (section 2.3.21) by using Table 4.2 primers. Sequences were analysed using the Geneious version 9.0.5 software program, and then multiple amino acid sequences were aligned using Clustal Omega program (EMBL-EBI, https://www.ebi.ac.uk/Tools/msa/clustalo/).

Table 4.2 Oligo primers used for PCR and DNA sequencing of scFv-Phage clones

Primers name Sequences (5´ to 3´) pPhNBF2_PCR_For TTGTGTGGAATTGTGAGCGG pPhNBF2_PCR_Rev CCTTCATAATTTGCATAGCGATCCAGGG pPhNBF2_Seq_For CAGGAAACAGCTATGACC pPhNBF2_Seq_Rev TTTCAACGGTCTATGCGGCACC

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4.2.6 scFv-Phage production

The 96-well glycerol stock plate was thawed on ice, and the selected individual phage clones were scraped using a pipette tip from the surface of the corresponding wells of the glycerol plate, and inoculated into 5 mL 2YT containing 2% Glucose (v/v) and Ampicillin (1:1000). The rest of the procedure for phage rescue and precipitation was as described in section 2.3.17.5 but in a smaller scale.

4.2.7 Reformatting of anti-canine and anti-human EphA2-LBD scFv particles into human IgG1 constant regions

Reformatting was performed to ligate heavy and light chain variable regions of the identified scFv candidates onto human heavy and light chain constant regions by using the ligation-free method mentioned in section 2.3.21. Primers were designed (Table 4.3 and 4.4) to amplify the heavy and light variable regions with compatibility to cloning into mAbXpress vectors (Table 3.3). The amplified heavy (VH) and light variable (VL) regions of scFv candidates were cloned into heavy (NBF-305) and light (NBF-326) human constant regions, respectively.

Table 4.3 Oligos names used for PCR of anti EphA2-LBD heavy and light variable scFv domains

Template VhFor VhRev VLFor VLRev HuB1-scFv mAbX_mVhF6 mAbX_mVhR1 mAbX_mVLF3 mAbX_mVLR4 Cross A11-scFv mAbX_mVhF3 mAbX_mVhR2 mAbX_mVLF3 mAbX_mVLR4

Table 4.4 Primer sequences used for amplifying anti EphA2-LBD heavy and light variable regions. Underlined sequences display the specific scFv regions altered from one clone to another

Primers Sequences (5´ to 3´) mAbX_VhF6 CAGGTGTCCACTCCGAGGTGCAGCTGGTGCAGTC mAbX_VhR1 GCGGAGGACACGGTGAGCGTGGTCCCTTGGCCC mAbX_VhF3 CAGGTGTCCACTCCGAGGTGCAGCTGCAGGAG mAbX_VLF3 CCGGCGTGCACTCCGAGTCTGTGTTGACGCAGCCG mAbX_VLR4 GCCTTAGGCTGGCCGAGGACGGTCAGCTTGGTCC mAbX_VhR2 GCGGAGGACACGGTGAGCAGGGTTCCCTGGCC

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After Infusion cloning, colony PCR was carried out for screening the colonies as described in section 2.3.5 by using primers listed in Table 4.5.

Table 4.5 Primers for screening anti EphA2-LBD colonies

Name of primers Type of chain Sequence (5´to 3´) mAbX_SeqF Forward primer TAACACCGCCCCGGTTTTCC Seq_hG1_R Reverse Heavy TCACCAGGCAGCCCAGAGCG Seq_hL_R Reverse Lambda CAGGCAGACCAGGGTGGCC

4.2.8 Expression and purification of the reformatted anti-human and anti-canine EphA2-LBD into CHO-XL 99 mammalian cells

Described previously in section 2.3.12.

4.2.9 Analysis of anti EphA2-LBD antibodies binding via flow cytometry

The binding of anti-canine and anti-human EphA2-LBD reformatted mAbs to native EphA2 was assessed using flow cytometry (2.3.25) on glioblastoma cell lines expressing EphA2 (2.3.24).

4.2.10 Binding ELISA assay for mAb 4B3 against recombinant canine and human EphA2-LBDs

Described in section 2.3.22

4.3 Results

4.3.1 Creation of truncated canine and human EphA2-LBD expression vector Expression vectors containing canine and human EphA2-LBD were created for transient expression of the LBDs in CHO-XL99 cells. Figure 4.2 represents the map of recombinant human EphA2-LBD into pcDNA 3.1. The confirmation of their sequence was achieved by analyses via Geneious version 9.0.5 software. The sequencing results showed a 100% match with the reference sequences. The alignment of the canine and human EphA2-LBD sequences is shown in Table 4.4, representing 96.1% identity in 178 residues overlap.

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Figure 4.2. pcDNA 3.1 (+)-Human EphA2-LBD-His plasmid map. The pcDNATM 3.1 contains Amp selection marker and human EphA2-LBD between AgeI and Xbal restriction sites. The pcDNATM 3.1(+)-canine EphA2-LBD-His plasmid map is similar to the above-mentioned map and is not depicted here.

Table 4.6 The alignment of human and canine EphA2-LBD sequences

Human 1 QGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTN Canine 1 QGKEVVLLDFAAAKGELGWLTHPYGKGWDLMQNIMDDMPIYMYSVCNVVAGDQDNWLRTN

Human 61 WVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDT Canine 61 WVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDVDYGTNFQKRQFTKIDT

Human 121 IAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCP Canine 121 IAPDEITVSSDFEARHVKLNVEERSVGPLSRKGFYLAFQDIGACVALLSVRVYYKKCP

4.3.2 Production of recombinant canine and human EphA2-LBD

To reach the highest purity of soluble canine and human EphA2-LBDs, two consecutive purifications were performed; firstly with HisTrap Excel column and secondly with Superdex 200 Increase 10/300 GL column. The latter purification allowed the removal of unrelated proteins and the collection of recombinant canine and human EphA2-LBDs, based on their molecular weight. Transfected CHO cells yielded 37.5 mg canine EphA2-LBD and 13.12 mg human EphA2-LBD proteins per volume of 187.5 mL transfected culture. SDS-PAGE analysis (Figure 4.3) showed the purity of the canine and human EphA2-LBD after HisTrap and SEC purifications in non-reducing conditions with an apparent molecular weight of 20 kDa. The recombinant canine and human EphA2-LBDs after SEC purification

66 were enzymatically digested in solution and Protein Pilot Peptide Mass Fingerprinting (PMF) spectrometry analysis confirmed the sequence coverage for identification of generated recombinant EphA2-LBD for both canine and human (Appendix 7).

Figure 4.3 SDS-PAGE analysis of both canine and human EphA2-LBDs. A) SeeBlue Plus2 pre- stained standard protein marker (Invitrogen) was used. B) Recombinant human EphA2-LBD elution after HisTrap purification. C) Recombinant human EphA2-LBD elution after SEC purification, showing the highest purity at ~ 20 kDa. D) Recombinant canine EphA2-LBD elution after Histrap purification. E) Canine EphA2- LBD elution after SEC purification, showing the highest purity at ~ 20 kDa

4.3.3 Enrichment of scFv phage binders to canine, human EphA2-LBDs

The enrichment of sub-libraries for specific phage binders was monitored after four consecutive rounds of selection, by the execution of a polyclonal phage ELISA (Figure 4.4). The naïve human scFv phage library (MJ-SM library) and phage pools collected after each round of biopanning were used, followed by 1 to 10 serial dilutions of each phage pool. The enrichment was observed to increase against canine EphA2-LBD from round 3 (Figure 4.4, A) whereas the enrichment was observed to increase for the two other biopannings from round 2 (Figure 4.4, B and C). Concurrently, all the phage pools were tested against immobilised AFBP as a negative control for nonspecific binding. There was no binding observed towards AFBP for any of the phage pools (Figure 4.4, A, B and C). The purpose of alternating the target proteins in a third biopanning strategy was to increase the possibility of finding phage binders that cross-react with both canine and human EphA2-LBDs. For this purpose, four rounds of biopanning were undertaken, with two rounds devoted to the purified canine EphA2- LBD and two rounds to the purified human EphA2-LBD. These results suggest that the enrichment 67 of specific phage binders to canine and human EphA2-LBD was increasing with iterative rounds of biopanning.

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Figure 4.4 Polyclonal phage ELISA to test the enrichment of phage pools to canine and human EphA2-LBDs. A) Biopanning rounds performed on canine EphA2-LBD tested against canine EphA2-LBD B) Biopanning rounds performed on human EphA2-LBD tested against human EphA2- LBD C) Biopanning rounds performed alternating between human and canine EphA2-LBD tested against human EphA2-LBD.

4.3.4 Screening individual phage clones for specificity to canine and human EphA2-LBD

Cell glycerol stock from Round 4 of canine and human EphA2-LBD were used for monoclonal phage ELISA to screen individual phage clones. Single colonies were grown in a 96-well plate’s format. Each well contained a potentially unique clone. Helper phage was added to induce production of phage particles. The phage particles from each clone were tested by phage ELISA for specific binding to the recombinant canine and human EphA2-LBD proteins, separately. The single phage clones obtained from biopanning by alternating recombinant canine and human EphA2-LBDs were used twice; once against canine EphA2-LBD and once for human EphA2-LBD. Monoclonal phage ELISA against canine EphA2-LBD indicated 37 positive clones (Figure 4.5, A), and 19 positive clones were isolated for the human EphA2-LBD (Figure 4.5, C). The phage binders alternated between purified canine and human EphA2-LBD showed 10 positive clones against canine EphA2-LBD (Figure 4.5, B), and 12 for human EphA2- LBD (Figure 4.5, D), of which 8 clones were common between two antigens. Clones with an arbitrary cut-off value of A450 ≥ 1.0 were selected as positive clones for further characterisation (Figure 4.5).

Ca EphA2-LBD and AFBP vs phage scFv binders

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Figure 4.5 Monoclonal phage ELISA to screen individual clones binding to canine and human EphA2-LBDs. A) Binding of phage clones obtained by biopanning against canine EphA2–LBD to canine EphA2-LBD, B) Binding of phage clones obtained by alternating the recombinant proteins to canine EphA2-LBD, C) Binding of phage clones obtained against human EphA2–LBD to human EphA2-LBD, D) Binding of phage clones obtained by alternating the recombinant proteins to human EphA2-LBD.

4.3.5 Sequence alignment and identification of unique clones to canine and human EphA2-LBD

The positive (binding) clones as determined by monoclonal phage ELISA were PCR amplified (Figure 4.6) to identify unique clones. The sequencing results were assembled and aligned using Geneious version 9.0.5 software, Virtual Ribosome version 2.0 (http://www.cbs.dtu.dk/services/VirtualRibosome/ ) and Clustal Omega online software (EMBL- EBI, https://www.ebi.ac.uk/Tools/msa/clustalo/). Sequence analysis showed 4 unique clones for canine EphA2-LBD, 6 for human EphA2-LBD and 4 unique clones for both canine and human EphA2-LBDs (Table 4.7). The international ImMunoGeneTics (IMGT)[257] database was used through Antigen receptor Numbering And Receptor Classification (ANARC)[258] to cluster the CDRs.

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Figure 4.6 Colony PCR to amplify phagemid scFv of anti-canine EphA2-LBD. Lane M contains the Marker for each row. Rows A to H (lanes 1 to12) contains the positive clones against canine EphA2-ECD. DNA from positive clones migrated to around 1000 bp confirming the presence of scFv amplicons. This was repeated for the remaining three plates (Gels are not shown).

Table 4.7 Sequence alignment of complementary-determining regions of the heavy and light chain variable regions towards canine and human EphA2-LBDs. The number of positive phage clones with an identical sequence is indicated in each cluster group. Four unique scFv binders obtained against canine EphA2-LBD (written in blue), Four unique scFv binders by alternating canine and human EphA2-LBD (written in red) and six unique scFv binders against human EphA2-LBD (written in green).

Panned Ag CDR CDR CDRH3 CDRV1 CDR CDRV3 Number Clone Name H1 H2 V2 of clones Ca EphA2- GYSF IYPG ARHRGEDA QNISS AAS QQNYIT 1 Not LBD TSY DSDT FDI Y PLT Characterised W GFT ISWN ARAGYSSG QDISR AAS QQGNSS 1 CaA6 FDDY SGSI WYAFNYYY W PWT A GMDV GFT ISWN AKSNSGRS SSDVG DVS SSYTSSS 1 Not FDDY SGSI AFDI GYNY TLYV Characterised A GFT ISYD AKDYRTGD SSNIG GNS QSYDSS 53 CaA1 FSRY GSNK FWSGASDY AGYD LSGVV G

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Alternating GYSF IYPG ARRMVGYC SSNIG DNN GTWDSS 2 CrossA12 Ca and Hu TSY DSDT TSTSCEGTR NNY LSAWV EphA2- W FDP LBD GFTF ISWN ARAGYSSG QDISR AAS QQGNSS 4 CrossH10 DDY SGSI WYAFNYYY W PWT A GMDV

GFTF ISWN AKAGGGRG SNDIG DVS SSYTTG 2 CrossH12 DDY SGSI YFDY TYNY NTPWV

A FTFS ISYD AKDYRTGD SSNIG GNS QSYDSS 17 CrossA11 RYG GSNK FWSGASDY AGYD LSGVV Hu EphA2- GFIFS SSSS AKVGTGWG KLGNK QDA QAWDS 11 HuF9 LBD DYY GRTI NWFDP Y NTAV GYTF ISAY NO CDR3 ------YAAS QELKSF 1 Not TSYG NGN present -- PRIT Characterised T GFTF ISWN AKSTLHCS SSNIG RNN QSYDSS 5 HuE7

DDY SGSI GGSCYSGA NNY LGGWI A FDI GGTF IIPIL ARDAYSYG SSNIA END GTWDSS 16 HuB1 SSYA GIA YFVSGYYY NNY LVSWV YGMD GYSF IYPG NO CDR3 SSNIG DNN GTWDSS 3 Not TSY DSDT present KNY LSAVV Characterised W GYSF IYPG NO CDR3 SSNIG DNN GTWDSS 2 Not TSY DSDT present NNY LSAWV Characterised W

Lack of CDRH3 framework was observed in three groups against human EphA2-LBD clustering results (Table 4.6). According to the importance of the CDR-H3 loop in antigen recognition and a significant contributor to binding strength and antibody specificity [259-262], the three groups against human EphA2-LBD including G2, G5 and G6 were removed from further study. First, the phage clones with higher frequency were selected for further study. CaA1-scFv and CaA6-scFv against canine EphA2-LBD, HuB1-scFv and HuF9-scFv against human EphA2-LBD, and CrossA11-scFv, CrH10-scFv, CrH12-scFv and CrA12-scFv for both canine and human EphA2-LBDs were selected for further characterization.

4.3.6 Binding of scFv phage particles to canine and human EphA2-LBDs

Indirect ELISA was performed to examine the binding of selected scFv phage clones to the recombinant canine and human EphA2-LBDs (3 µg/mL). The selected scFv phage binders were set up at 10 µg/mL (neat) concentration with serially diluted to 10-3 dilutions. 73

Results showed the HuB1-scFv binding to human EphA2-LBD in neat concentration as well as canine EphA2-LBD (Figure 4.7). No binding was observed for HuF9-scFv phage binder to either recombinant canine or human EphA2-LBD (Figure 4.7). CaA1-scFv bound to both canine and human EphA2-LBD whereas CaA6-scFv bound only to canine EphA2-LBD. Although CrossA11-scFv indicated cross-reactivity to both canine and human EphA2-LBD, the two remaining scFv phage binders including CrossH12, CrossH10 did not show any binding either to canine or human EphA2- LBDs. Since CrossA11-scFv and HuB1-scFv had a better absorbance, they were selected for reformatting into human IgG1 constant regions.

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Figure 4.7 ELISA assay to test the binding of scFv phage particles towards recombinant canine, human EphA2-LBDs. ELISA plate was coated with PBS, AFBP, human EphA2-LBD and canine EphA2-LBD indicated in the bar graph legends. Seven scFv phage binders as indicated (X-axis) were added to AFBP, human and canine EphA2-LBDs. Bound phages were detected using anti-M13 phage HRP antibody and the absorbance was measured at 450 nm (Y-axis). Positive signals were determined by using an arbitrary cut-off value of A450 ≥ 1.0.

4.3.7 Reformatting of anti EphA2-LBD scFv phage binders to human Immunoglobulin G1 (IgG1)

The CrossA11 and HuB1 scFv phage particles were reformatted to fully human IgG1 for further characterization. The In Fusion™ system [118] was used for ligation-independent In Fusion™ cloning (Clontech). The sequencing results were matched to reference sequences constructed in

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Geneious version 9.0.5 software, representing the successful Infusion of scFvs into mAbXpress vectors encoding the heavy and light chains of human constant regions.

4.3.8 Production of reformatted CrossA11 and HuB1 into human IgG1 constant regions

The reformatted mAbs including CrossA11 and HuB1 were transiently expressed in CHO-XL99 cells and purified by Protein A affinity chromatography. The SDS-PAGE gel image (Figure 4.8) showed a 150 kDa band of a complete antibody in the non-reduced sample lane. The 150 kDa band was split into two distinct bands in the reduced lane. This is characteristic of purified mAbs; the 50 kDa and 25 kDa bands represents the heavy and light chains, respectively.

Figure 4.8 SDS-PAGE analysis of reformatted CrossA11 and HuB1 mAbs into human IgG1 in reduced and non-reduced conditions. SeeBlue Plus2 pre-Stained protein (Invitrogen) was used as a marker to estimate the molecular weight in MES running buffer. SimplyBlue SafeStain (Life Technologies) was used for staining the PAGE gel. The image shows the full IgG MW in non- reducing condition at approximately 150 kDa for both CrossA11 mAb (A), and HuB1 mAb (C). Other bands in the non-reduced lane present different isoforms of reformatted mAbs. The antibodies heavy and light chains can be observed for CrossA11 mAb (B) and HuB1 mAb (D) in reduced lanes at approximately 50 and 25 KDa, respectively.

4.3.9 Binding and specificity of reformatted antibodies to recombinant human and canine EphA2-LBD and ECD

The activity and binding of HuB1 and CrossA11 mAbs were determined on immobilised canine and human EphA2-LBD by indirect ELISA assay. As shown in Figure 4.9, both antibodies bound to human and canine EphA2-LBD. These results were consistent with the phage particles ELISA (Figure 4.7). Since the LBD is a part of the EphA2-ECD, the binding of these mAbs were examined on 75 recombinant human and canine EphA2-ECD (Figure 4.9). The ELISA results indicated the binding of HuB1 mAb to both canine and human EphA2-ECDs. In contrast, CrossA11 mAb did not bind to either human or canine EphA2-ECD, but only to the purified EphA2-LBD (human and canine). This suggests that CrossA11 mAb is binding to an epitope in the purified LBD protein which is not exposed or present in the whole purified ECD. The binding of mAb AGH001 was tested on both canine and human LBD and ECD of EphA2. It was interesting to observe there was no binding for mAb AGH001 to either human or canine EphA2-LBD and this finding was consistent with the competition ELISA result between mAb 4B3 and mAb AGH001 (Section 3.3.13). Therefore, it is more likely that mAb AGH001 binds to domains of EphA2-ECD other than the LBD.

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Figure 4.9 ELISA on purified canine and human EphA2-LBD and EphA2-ECD. PBS and purified antigens (3 µg/mL) including human and canine EphA2-LBD and human and canine EphA2- ECD were immobilised on ELISA plate, and binding of HuB1, CrossA11 and AGH001 mAbs (10 µg/mL) were tested (X-axis) using anti-human HRP secondary antibody. The absorbance was measured at 450 nm (Y-axis) and positive signals were determined by using an arbitrary cut-off value of A450 ≥ 1.0.

The binding of both HuB1 and CrossA11 mAbs were tested against reduced and non-reduced recombinant EphA2-LBDs by Western Blot (Figure 4.10). Results showed HuB1 mAb specifically detects non-reduced human EphA2-LBD but not it’s reduced form (Figure 4.10, A). The detection of both non-reduced canine and human EphA2-LBD by CrossA11 mAb was observed albeit weakly, and there was also no binding observed in the reduced conditions (Figure 4.10, B).

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Figure 4.10 Binding analysis of (A) HuB1 mAb to human and canine EphA2-LBD in both reduced and non-reduced conditions, and (B) CrossA11 mAb to human and canine EphA2- LBD in both reduced and non-reduced conditions by western blot. An approximately 20 kDa band (purple arrow) represents the interaction of A) HuB1 mAb with non-reduced recombinant human and canine EphA2-LBD, and B) CrossA11 with non-reduced recombinant canine and human EphA2-LBD.

Binding of mAb 4B3, the positive control antibody, was tested on human and canine EphA2-LBD using ELISA. The first aim was considering mAb 4B3 binding to LBD, and then making a plan for competition ELISA between 4B3 and HuB1 mAbs against the LBD. The ELISA results indicated there is no binding for 4B3 against the EphA2-LBD, suggesting there is no competition between 4B3 and HuB1 on LBD (Figure 4.11).

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Figure 4.11 ELISA assay to test the mAb 4B3 to recombinant canine and human EphA2-LBD. PBS and anti-mouse IgG HRP and purified human and canine EphA2-LBD were immobilised on 77

ELISA plate and binding of mAb 4B3 was tested (X-axis) using anti-mouse HRP secondary antibody. The absorbance was measured at 450 nm (Y-axis).

4.3.10 Binding specificity of anti EphA2-LBD mAbs to native EphA2 receptors through flow cytometry analysis

The binding capability of generated HuB1 and CrossA11 mAbs was tested using EphA2 positive and KD U87 cells. Results achieved from flow cytometry showed a small shift for both HuB1 mAb (Figure 4.12, A) and CrossA11 mAb (Figure 4.12, B); however, they also bound (albeit to a lower level) to EphA2 KD U87 cells, suggesting the non-specificity of isolated antibodies to the native EphA2. The mAb 4B3, as a positive control, demonstrated a histogram shift for EphA2 positive U87 cells and a complete absence of binding to EphA2 KD U87cells (Figure 4.12, C).

Since different secondary antibodies conjugated to fluorescence dye were used for our generated and control antibodies, the comparison based on their histogram intensities is not valid.

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Figure 4.12 Flow cytometric analysis of U87 and EphA2 KD U87 cells exposed to HuB1, CrossA11 and 4B3 antibodies. U87 and EphA2 KD U87 cells were incubated with A) HuB1 mAb, B) CrossA11 mAb, and C) 4B3. Antibody binding was detected using DyLight 594 anti-human (A and B) and anti-mouse (C) IgG secondary antibodies. Secondary antibody fluorescence (X-axis) was measured using 561 nm laser and 630/22 nm filter and the number of events were counted on Y-axis.

4.4 Discussion

The interaction of EphA2 interface by ephrinA1 ligand can induce EphA2 dependent-ligand or forwarding signalling [113, 263, 264]. The ligand can be either the cell surface-anchored ephrinA1, soluble form of ephrinA1 which could be cleaved from the cell membrane by , or engineered short peptides[76]. The forward signalling leads to EphA2 receptor tyrosine phosphorylation and consequently, stimulation of various downstream signalling molecules. Since the LBD has a key role in interaction with its ligand (ephrinA1), it makes this region critical for initiating the downstream signalling. In addition, Eph tyrosine kinase family receptors are characterised by their LBD, and they are differentiated from one member to another by their LBDs conserved sequence and their binding affinities, which is necessary for ephrin ligand recognition [77, 240, 253]. Targeting this specific region of EphA2 structure as an antigen could provide the opportunity to isolate specific antibodies against this particular region. To employ this strategy in biopanning, we cloned and produced only the LBD of human and canine EphA2, based on the sequence data reported by Seiradake et al., [118]. Their identity was confirmed by mass spectrometry analysis through matching the amino acid sequence and their molecular weight size by SDS-PAGE gel. Once identity was confirmed, LBDs were used as antigen for the biopanning campaign. Since human EphA2-LBD has 96.1% homology with canine EphA2-LBD (Table 4.5), we expected to isolate several cross- reactive binders to both antigens. Subsequently, the biopanning campaign resulted in the isolation of a number of phage binders specific for the recombinant human EphA2-LBD, canine EphA2-LBD and cross-reactive to human and canine EphA2-LBDs. Sequence alignment revealed a number of phage binders that lacked the heavy chain CDR3 loop. The CDR-H3 has an extensive variety of shape and length [265, 266] and improvement to affinity and stability of antibodies is performed by manipulating this domain [267-270]. It has also been reported that the CDR3 loop is a significant contributor to binding strength and antibody specificity [271, 272]. Thus clones that lacked the CDR3 loop were not further characterised nor studied. Isolated HuB1-scFv and CrossA11-scFv showed higher binding among other scFv phage binders shown in Figure 4.7 to canine, and human EphA2-LBD and both scFvs displayed cross-reactivity to both antigens. The reformatted phage-derived HuB1 and CrossA11 mAbs conserved their affinity

79 and cross-reactivity to recombinant canine and human EphA2-LBDs. Furhermore, ELISA data for reformatted antibodies was consistent with the scFv phage ELISA. In addition, the binding of reformatted HuB1 and CrossA11 mAbs against the ECD of EphA2 was assessed by ELISA. Since these antibodies were isolated by biopanning on EphA2-LBD and this domain is a constituent of EphA2-ECD, it was anticipated that the ELISA would indicate HuB1 mAb binding to both human and canine EphA2-ECD.

In contrast, CrossA11 mAb bound to neither human nor canine EphA2-ECD. This could be due to the different conformation of the LBD in recombinant EphA2-LBD and recombinant EphA2-ECD. Therefore, CrossA11 mAb can only detect the segment or domain upon which it was originally biopanned against. HuB1 mAb could only detect the non-reduced human EphA2-LBD but not the reduced form of the protein. Similarly, the CrossA11 mAb bound to both non-reduced human and canine EphA2-LBD, but not the reduced. It is possible that the intramolecular disulphide bonds of LBD may have been disrupted by reducing agents and reformatted mAbs were not able to detect the reduced protein. There are five cysteine residues in the recombinant LBD, so there could be two disulphide bonds in each monomer. These disulphide bonds will create conformational epitopes that are destroyed in reducing conditions. Potentially, the disulphide bonds in the recombinant LBD are not paired up in the same manner as in the recombinant ECD, potentially explaining why some of the isolated mAbs can bind to the LBD but not the ECD.

Since mAb 4B3 did not bind to the purified EphA2-LBD, this suggests that our newly created recombinant LBDs used as an antigen, may have different conformation compared with the LBD present within the native EphA2. This can be due to an inappropriate, non-functional folding of LBD; alternatively there may be correct folding, but immobilisation on the solid surface resulted in altered orientation during biopanning [273], shielding the epitope of interest. Therefore, the selected phage binders may not recognise and bind to the naturally folded receptor on the cell surface.

Another hypothesis is that our recombinant LBD conformation is correct and HuB1 mAb can recognise the LBD. However, this small region, based on its structure, may not be exposed to detection, or even might be masked by other molecules on the cell surface; thus, it would not be easily detectable by our isolated antibody. Crystal structure studies of Eph-ephrin complex [209, 274, 275] reveal that the interaction of receptor-ligand is through an ephrin loop inserted into a hydrophobic region at the surface of Eph through its extended G-H loop.

Several peptides have been produced through phage display that specifically bind to the Eph receptor’s hydrophobic cavity and compete with cognate ligands [241, 276, 277]. The protein crystal study of these peptides [278, 279] has confirmed the penetration of these peptides into the 80 hydrophobic ligand-binding channel of Eph. It is feasible that the HuB1 mAb may not be able to penetrate into EphA2 cavity. However, flow cytometry results showed a small shift in both EphA2 positive and negative cells, suggesting HuB1 might bind to this channel at EphA2 and also have partially overlapped with other Eph receptors binding site. Since there is more than 50% identity between LBD in EphA2 and other LBD in EphA family receptors, it is feasible that there are many shared epitopes (Appendix 6).

In several studies, the selection of a protein fragment or domain as antigen for biopanning against a phage antibody library has been based on evaluation of the similarity between a truncated and native sequence, to identify the potentially immunogenic epitopes [280, 281]. Furthermore, a study has suggested for generating an antigen for isolating a potent antibody; it is better to select a protein as an antigen based on structure similarity to the native protein and put the sequence similarity in the next priorities [212].

4.5 Conclusion

In conclusion, there are many factors involved in successful antibody isolation through phage display, including the quality of phage library, the purity and structure of the antigen and the biopanning strategy. One of the most significant factors, especially when a truncated protein is used as an antigen in phage display, is the cloning and expression of appropriate target protein fragments or domains. The antibodies isolated from the phage libraries must be capable of detecting the intact, native protein. This is of particular importance in our studies where we are targeting the G-H loop-binding channel of Eph receptors.

Additionally, along with considering the importance of antigen sequence similarity with the native receptor, it is critical that the structure of the antigen employed for a biopanning procedure is identical to the native structure, thus conserving epitopes of the target receptor. However, the exact nature of the folding and conformation of EphA2-ephrinA1 domains, compared to the folding and conformation of the native EphA2-ephrinA1, has remained elusive and requires further study.

As previously discussed, immobilisation of protein antigens on solid surfaces may result in loss of conformation and/or altered orientation of epitopes. Thus to preserve native structure and ensure epitopes are accessible, liquid phase biopanning using soluble, biotinylated protein antigens can be utilised, using Streptavidin-magnetic bead pull-downs. However, this strategy may yield non-specific binders to streptavidin and so negative selection biopanning strategies against streptavidin are required.

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In conclusion, selectively targeting a small segment of EphA2 structure based on its sequence did not lead to the isolation of EphA2 receptor-specific antibodies, and understanding of EphA2 crystal structure is helpful to properly target this receptor for mAb isolation. However, this strategy was successful for isolation of antibody against LBD of GluA4 ionotropic glutamate receptor, it was not prosperous for generating mAbs against EphA2-LBD [247]. Another strategy is targeting native EphA2 through cell-based biopanning, in the quest to isolate high affinity antibodies against native EphA2 receptor. Chapter 6 is dedicated to isolating anti-EphA2 mAbs through cell-based biopanning, after epitope binding analysis of mAb AGH001, and mAb 4B3 to identify their binding site against different domains of EphA2 structure.

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Chapter 5. Identifying the epitope of mAb AGH001 within Extracellular Domain of the EphA2 receptor

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5.1 Introduction

Investigating the antibody binding site on its target antigen greatly assists in the ongoing development of diagnostic and therapeutic antibodies, especially considering the target epitope allows an understanding of the interaction between antigen and antibody. For example, antibodies that are used in flow cytometry and ELISA assays, may not be always be suitable in Western blot and analysis, due to the loss of conformational epitopes upon denaturation under reducing conditions. The epitope, also called antigenic determinant, is the part of the antigen that is recognised by an antibody, and conversely, the paratope is the part of the antibody which binds to the antigen. Epitopes may be either continuous or discontinuous; a continuous (or linear) epitope is where the antibody binds to a linear amino acid sequence; a discontinuous (or conformational) epitope is where the antibody binds amino acids in a region determined by the folding and native, three dimensional conformation of the protein antigen.

The EphA2-ECD has several sub-domains (Figure 1.1), including the ligand-binding domain (LBD), a cysteine-rich domain (CRD) and two repeats of Fn type III (FnI&II) domains. The amino terminus of EphA2-LBD is a key component, involved in the creation of a complex of receptor/ligand [93, 282], indicating the importance of this domain for the discovery of functional anti-EphA2 antibodies that may disrupt binding. It seems that EphA2/ephrinA1 interacts through two separate Eph/Eph interfaces; LBD and CRD [102, 283]. Furthermore, the crystal structure of non-ligated EphA2 shows that the CRD is involved in EphA2 ectodomain clustering [78] and its dimerization is stabilised by leucine zipper-like interactions in the CRD involving Leu-223, Leu-254 and Val-225 [284]. The CRD is located immediately after the C-terminus of the LBD. It consists of an evolutionarily conserved sushi domain preceding an epidermal growth factor (EGF)-like domain. Random coils and β sheets extend the structure of this domain. Five antiparallel β sheets form a β-sandwich in the N-terminus of CRD with one disulphide bond, whereas the C-terminus of CRD includes two β sheets plus six random coils wrapped firmly, with four S-S bonds. The N and C-termini of CRD are tightly compacted against each other. Both repeats of the FnIII domains have characteristic IgG-like folds [88]. The separate domains within the extracellular domain are organized linearly to facilitate inter- domain interfaces resulting in a rigid ectodomain.

FnIII domains conform to a β-fold and provide a wide binding interface at two poles, including non- adjacent loops [285]. Crystallographic studies of 50 natural FnIII domains reveal a high degree of similarity in the seven β-sheets, whereas the six loops are identified with a diversity of conformation. In contrast to immunoglobulins and T-cells in which binding occurs only on the poles having CDRs,

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FnIII binding can occur throughout loops at any pole [286, 287]. It has been shown that FnIII domains are present in approximately 2% of all human proteins and have been noted in other developed organisms [288].

The mAb AGH001 was isolated by biopanning against the whole EphA2-ECD (Chapter 3). Flow cytometry experiments showed that it bound EphA2-expressing U87 cells, however it also bound EphA2 KD U87 cells. We therefore assumed that mAb AGH001 binds to a domain of EphA2-ECD which is abundant on other receptors on the cell surface. Based on this, we hypothesised that mAb AGH001 might bind to the FnI&II domain due to the presence of this domain in many other proteins. Alternatively, since the CRD has an essential role in dimerization of EphA2 even when the receptor is free of ligand [289] and has more than 46% homology among other EphA tyrosine kinase family receptors (Appendix 9), it is also possible that mAb AGH001 binds to the CRD. To demonstrate the binding site of mAb AGH001 within the EphA2-ECD, the expression of each domain as recombinant proteins were required to test the binding of mAb AGH001.

The work described in this chapter aimed to construct expression vectors for the various domains of human and canine EphA2-ECD, including recombinant LBD (detailed in chapter 4), CRD, the LBD and CRD together and FnI&II. To work towards this goal, different domains of canine and human EphA2-ECD were expressed as recombinant proteins, and binding site analysis was performed in an attempt to elucidate the epitope of mAb AGH001.

5.2 Materials and methods 5.2.1 Cloning different domains of canine and human EphA2-ECD into a mammalian expression vector

Start and end sequences of both canine and human EphA2 domains including the LBD (detailed in chapter 4), the CRD, the LBD&CRD together, and FnI&II were selected, based on sequence data reported by Seiradake et al. [118]. The above-mentioned domains of canine and human EphA2 were PCR-amplified from the full-length sequences of human (Accession Number P29317) and canine EphA2 (Accession Number XP_544546), and synthesised by GeneArt (ThermoFisher Scientific, Germany). A pcDNA3.1 (+) (Invitrogen) expression vector, already containing an insert for DENV- NS1 protein with a mammalian secretion signal, was obtained from the National Biologics Facility (NBF, Brisbane, Australia). AgeI and XbaI restriction enzymes were used to digest the vector to remove the existing insert but leaving the secretion signal. The same enzymes were used to digest the PCR products. A hexahistidine tag and stop codon were added to the reverse primers for incorporation into the C-terminus of the expressed proteins. The primers are listed in Table 5.1.

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The cloning protocol into pcDNA3.1 expression vector and DNA preparation for sequencing were previously outlined in section 2.3.3 and section 2.3.8, respectively. Sequencing was performed for recombinant constructs using the primers shown in Table 2.1.

Table 5.1 Oligonucleotide primers used for PCR of human and canine EphA2 different domains

Oligo Name Sequence (5´ to 3´) Hu-EphA2-CRD- GAAGTGCCCCACCGGTGTCCACTCCGAACTGCTGCAGGGC Agel-For Hu_EphA2_CRD- GAGCCTGCCTCTATGCCCTGTCACCACCATCACCATCACTAGTCTAGA His_Xbal_Rev Hu-EphA2- TGCAGCTACCGGTGTCCACTCCCAGGGCAAAGAA LBD&CRD-Agel- For Hu_EphA2_ GAGCCTGCCTCTATGCCCTGTCACCACCATCACCATCACTAGTCTAGA LBD&CRD- His_Xbal_Rev Hu-EphA2-FnI&II- TCTATGCCCTGTACCGGTGTCCACTCCACCAGACCT Agel-For Hu_EphA2_FnI&II TCTCCTGAGGGCTCTGGCAACCACCACCATCACCATCACTAGTCTAGA -His_Xbal_Rev

Ca-EphA2-CRD- AGTGCCCCACCGGTGTCCACTCCGAGCTGCTGCAGGGCCTGG Agel-For Ca_EphA2_CRD- GACCTGCTGTCCATGCCCTGTCACCACCATCACCATCACTAGTCTAGA His_Xbal_Rev Ca-EphA2- GCTACCGGTGTCCACTCCCAGGGCAAAGAAGTGGT LBD&CRD-Agel- For Ca_EphA2_ GACCTGCTGTCCATGCCCTGTCACCACCATCACCATCACTAGTCTAGA LBD&CRD- His_Xbal_Rev Ca-EphA2-FnI&II- CTGTACCGGTGTCCACTCCACCAGACCCCCTTCCGC Agel-For Ca_EphA2_FnI&II TGTCCACCGAGGCCAGCGGCAATCACCACCATCACCATCACTAGTCTA -His_Xbal_Rev GA

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5.2.2 Expression of canine and human EphA2-extracellular domains

The expression vector constructs including pcDNA3.1(+)-hu-EphA2-CRD-His, pcDNA3.1(+)-hu- EphA2-LBD&CRD-His, pcDNA3.1(+)-hu-EphA2-FnI&II-His, pcDNA3.1(+)-ca-EphA2-CRD-His, pcDNA3.1(+)-ca-EphA2-LBD&CRD-His, and pcDNA3.1(+)-ca-EphA2-FnI&II-His were transfected separately into CHO-XL99 cells for mammalian expression as previously described in section 2.3.10. The secreted proteins were purified using HisTrap excel column on AKTA explorer, followed by Size Exclusion Chromatography (SEC) Superdex TM 200 10/300 GL column. The SEC was employed (section 2.3.13) to collect the highest purity of the recombinant proteins based on their molecular weight.

5.2.3 ELISA assay against different domains of EphA2 ectodomain

Described previously in section 2.3.22.

5.2.4 Protein immunoblot

Described in section 2.3.16.

5.3 Results

5.3.1 Creation expression vector for different domains of canine and human EphA2-ectodomain

Expression vectors containing canine and human EphA2-CRD, EphA2-LBD&CRD, and EphA2- FnI&II were created (Figure 5.1) for transient expression in CHO-XL99 cells. The confirmation of their sequences was achieved via Geneious version 9.0.5 software. The sequencing results were 100% matched with the reference sequences. The amino acid sequence of human EphA2-ECD, with the sub-domains highlighted, is shown in Table 5.2.

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Figure 5.1 pcDNA 3.1 (+)-Human EphA2-CRD-His plasmid map. The pcDNATM 3.1 contains ampicillin resistance gene (amp marker), ampicillin promotor (amp prom), T7 promoter (T7 prom), lac promoter (lac prom), SV40 promoter (SV40 prom), Neomycin phosphotransferase II selectable marker (NTP_II marker), BGH polyadenylation signal sequence (bGH_PA) and human EphA2-CRD between BamHI and Nhel restriction sites. The canine and human pcDNATM 3.1(+)-His plasmid map including EphA2-CRD, LBD& CRD and FnI&II are similar to the above mentioned map and is not depicted.

Table 5.2 The amino acid sequence of human EphA2 extracellular domains. Each colour indicates a different sub-domain. The LBD is highlighted by turquoise, CRD by yellow, FnII by green and FnI by magenta colour.

QGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDN WLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRL FTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYK KCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIG QCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQ DPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGEC GPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQ TEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDD LAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGV GFFIHR

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5.3.2 Expression and production of recombinant canine and human EphA2 domains (CRD, LBD&CRD and FnI&II)

To achieve maximal purity for each domain post expression, two consecutive purifications were performed; firstly with HisTrap Excel column and secondly with Superdex 200 Increase 10/300 GL column. The latter purification removed host cell proteins and yielded purified recombinant canine and human EphA2 domains based on their molecular weight. Recombinant EphA2-CRD did not express (both human and canine versions), in either CHO or HEK (Human Embryonic Kidney) cells. SDS-PAGE with Coomassie blue staining (Figure 5.2) showed the high purity of the canine and human EphA2 domains after SEC purifications in non-reduced and reduced conditions, with theoretical predicted MWs of 20, 32, 22, and 56 kDa for LBD, LBD&CRD, FnI&II, and ECD domains, respectively. It was expected that the heavier MW band on SDS-PAGE would correspond to the combination of LBD&CRD domains, compared with FnI&II domains. This difference of apparent MW with actual MW on SDS-PAGE can be due to post-translational modifications (PTMs) especially by glycosylation, increasing the protein mass, and methylation; PTMs of proteins affect the mobility of the protein and may cause a significant shift on SDS-PAGE [290]. Furthermore, some proteins are very stable and may not fully denature, retaining one or more disulphide bonds prior to electrophoresis.

Figure 5.2 SDS-PAGE analysis of different domains of human (A) and canine (B) EphA2-ECD after SEC purification. SeeBlue Plus2 pre-stained standard (Invitrogen) was used as a protein marker. A) Human EphA2 different extracellular domains, and B) Canine EphA2 different extracellular domains. 1) Non-reduced LBD, 2) Reduced LBD, 3) Non-reduced LBD&CRD, 4) Reduced LBD&CRD, 5) Non-reduced FnI&II, 6) Reduced FnI&II, 7) Non-reduced ECD, and 8) Reduced ECD

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5.3.3 Binding analysis of mAb AGH001 to different extracellular domains of human and canine EphA2 by ELISA

ELISA was performed to determine if mAb AGH001 binds to different domains of human and canine EphA2-ECD (Figure 5.3). Results showed that mAb AGH001 could only bind to the whole EphA2- ECD in both human and canine, whereas no binding was observed for the sub-domains of EphA2- ECD including LBD, LBD&CRD and FnI&II. Mouse-derived mAb 4B3 was used as a positive control against different domains of human EphA2-ECD. The binding results indicated that mAb 4B3 could only bind to whole human EphA2-ECD also.

) 2.0 m

n Hu EphA2-LBD&CRD

0

5 Ca EphA2-LBD&CRD

1.5

4 (

Hu EphA2-FnI&II e

c Ca EphA2-FnI&II

n 1.0 a

b Hu EphA2-ECD r

o Ca EphA2-ECD

s 0.5 b

A Hu EphA2-LBD 0.0 Ca EphA2-LBD mAb AGH001 mAb 4B3 mAbs(10 µg/mL)

Figure 5.3 ELISA assay to test the binding activity of mAb AGH001 to different sub-domains of recombinant canine and human EphA2-ECD. ELISA plate was coated with different domains of human and canine EphA2-ECD. mAb AGH001 was added to both human and canine EphA2-ECD sub-domains, whereas mAb 4B3 was added to only human EphA2-ECD sub-domains (X-axis). Bound mAb AGH001 and mAb 4B3 were detected using anti-human and anti-mouse HRP antibodies, respectively and the absorbance was measured at 450 nm (Y-axis).

5.3.4 Binding analysis of mAb AGH001 to different domains of human EphA2-ECD by western blot

The binding of mAb AGH001 to different domains within human EphaA2-ECD was tested through Western blotting (Figure 5.4, A). Results show that mAb AGH001 cannot bind to any of the domains, including the whole ECD. In contrast, mAb 4B3 bound to the ECD (Figure 5.4, B). This suggests that mAb AGH001 has a conformational epitope, whereas mAb 4B3 has a linear epitope, since the proteins are denatured when used in Western blot.

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Figure 5.4 Binding analysis of A) mAb AGH001, B) mAb 4B3 on different domains of human EphA2-ECD. Lanes 1 contain SeeBlue Plus2 Prestained Standard molecular weight (Invitrogen) as a protein marker. Lanes 2 contain human LBD. Lanes 3, 4 and 5 contain human LBD and CRD, Fn I&II and ECD, respectively. Panel (A) shows that mAb AGH001 did not bind to any of human EphA2-ECD sub-domains, whereas mAb 4B3 on panel (B) bound to only human EphA2-ECD.

5.4 Discussion

Antibodies bind to an antigen via specific epitopes, and since antigens contain multiple epitopes, then many different antibodies can interact with the same antigen. The position of the epitope can affect the functionality of the antibody; for example whether it blocks a ligand, induces internalisation or stimulates or inhibits the protein activity. For example, there are several therapeutic antibodies that target EGFR (epidermal growth factor receptor) with different epitope specificity, such as cetuximab, matuzumab and panitumumab [291].

The LBD in EphA2 is known as a key domain where the cell-to-cell interaction occurs [77]. The Fab 1C1 is an anti-human EphA2 mAb reported to induce phosphorylation, and is internalized and degraded. It therefore has potential for the delivery of conjugated toxic drugs to EphA2 expressing cells [292]. To characterise the epitope for Fab 1C1 mAb, X-ray crystallography and a mutational approach have been applied. Results suggest that the epitope (Fab 1C1 mAb binds with a KD of 810 pM) is located in the EphA2-LBD. The crystal structure recognised the third heavy-chain complementarity-determining region of Fab 1C1 mAb as the paratope merely involved in direct ephrin interface and the extended CDRH3 loop of Fab 1C1 mAb penetrated the EphA2 surface channel [138].

However, other sites on EphA2 not involved in ligand binding can also be useful epitopes for therapeutic targeting. For example, it has been reported that N-terminally truncated EphA2 by membrane type I matrix metalloproteinase (MT1-MMP) without binding to ephrinA1 can initiate 91 oncogenic signalling pathway [234, 235]. Therefore, identifying the binding site on different domains of EphA2-ECD would assist in inhibiting the oncogenic signalling pathways through generating antibodies.

Since mAb AGH001 binds to EphA2 KD U87 cells (chapter 3), we assumed that it binds to domains on EphA2 other than the LBD, since domains outside the LBD have high homology to other receptor proteins. Since the extracellular domain of EphA family receptors is conserved amongst the EphA family, and the homology of CRD and FnIII domains are high among this family receptors, mAb AGH001 may block any of these domains on EphA2 KD U87 cells.

Potential epitopes may be within the CRD, FnI&II domains or even part of both LBD and CRD junction. To demonstrate this, these recombinant domains were transiently expressed in CHO cells. All the recombinant domains were successfully expressed, except the CRD. This could be due to the disulphide bonds formed by 16 cysteine residues in this domain. The CRD structure is extended by random coils and β sheets [88]. It would appear that the difficulty of CRD expression, even using different host cells (CHO and HEK) is related to its complex structure that makes secretion in culture medium difficult.

There was no binding observed for mAb AGH001 against the different sub-domains of canine and human EphA2-ECD. An explanation for the lack of binding could be altered conformation of these recombinant purified domains compared with the natural folding within the native EphA2. Another possibility could be that the conformation of purified domains is correct, but the mAb AGH001 can only detect an epitope that exists within CRD, or between CRD and FnI&II domains. Also, this result was consistent for positive control antibody, mAb 4B3.

Antibodies may bind to either linear or conformational epitopes. Comparing the Western blot analysis between mAbs AGH001 and 4B3 in non-reduced conditions against the EphA2-ECD indicated that mAb AGH001 has a conformational epitope and the epitope was lost through denaturation due to heating during the sample preparation and the presence of denaturant SDS in the PAGE gel. These factors cause unfolding of the protein resulting in modification of conformational epitopes [293], resulting in the ability of mAb AGH001 to bind to the denatured ECD. Since mAb 4B3 binds the ECD in Western blot, it must bind to a linear epitope which can persist under denaturing conditions.

5.5 Conclusion

EphA2 is a multi-domain receptor, and each domain folds correctly only when in the context of the cell membrane. mAb AGH001 targets a conformational epitope, which can make identifying the epitope binding site challenging when using recombinant sub-domains for biopanning of phage

92 display peptide library. There are a variety of methods to map the interaction between antigen and antibody and no one method is appropriate for all the cases. If applied, x-ray crystallography could be the best choice, however the crystallisation of the AGH001-EphA2 complex was beyond the scope of this study. It is not still clear which domain of EphA2 the mAb AGH001 can bind from the studies presented here.

The extracellular domain of EphA receptors is evolutionarily conserved. To test whether mAb AGH001 may bind to other EphA tyrosine kinase receptors, a number of recombinant EphA receptor family ECDs can be utilised by analysing the mAb AGH001 binding against different EphA receptors. However, the availability of all the recombinant extracellular domain of EphA is limited, and it is not feasible to make an overall conclusion. An alternative strategy would be to employ co- (Co-IP) of mAb AGH001 with membrane extracts of cells. In this order, mAb AGH001 can pull-down the ligands to which it binds, and these can subsequently be identified using mass spectrometry.

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Chapter 6: Isolation of human monoclonal antibodies against native EphA2 through cell-based biopanning

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6.1 Introduction

Cell membrane proteins are attractive targets for monoclonal antibody (mAb) discovery. mAbs are important molecular entities, utilised in the diagnosis and treatment of many disease indications, principally cancer and inflammatory diseases. The main methodologies used for mAb discovery and generation are transgenic mice with human humoral immune system, and antibody phage display technology. Numerous studies have reported the isolation of mAbs using phage display by biopanning against an antigen of interest [242-244].

The previous chapters have described phage antibody biopanning campaigns, in the quest to isolate antibodies specific for the EphA2 receptor, through the use of a recombinant extracellular domain or the ligand-binding domain of EphA2. Although these strategies isolated mAbs capable of binding to recombinant EphA2 and to native cells expressing EphA2, the mAbs also bound to cells which had the EphA2 receptor knocked-down through CRISPR/Cas9 engineering. This suggests that the mAbs were not binding exclusively to EphA2, but to epitopes common to other cellular receptors.

Despite extensive research on the generation of mAbs through soluble antigen biopanning, the recombinant expression of membrane proteins and consequent purification are challenging. The quality of the antigen being employed through the antigen biopanning campaign is an essential factor in isolating native antigen-specific antibodies successfully [198]; however, the native conformational structure of a purified, recombinant antigen used may be compromised during expression and purification. Furthermore, it may denature when immobilised on the solid surface. This may lead to the use of an antigen, which is structurally different from the native form on the cell membrane [294, 295]. As a consequence, it may lead to the isolation of antibodies that cannot recognise the native epitopes of the target protein [296, 297].

Cell-based biopanning can be used as an alternative approach to eliminate these difficulties and allow generation of antibodies against membrane proteins in their native conformation. However, the success in this strategy depends on accessibility of the ectodomain of the target membrane protein, with the associated antigenic determinants [208, 298, 299]. In this case, the cell surface proteins are in their physiological and conformational relevant state with native glycosylation and other post- translational modifications [183, 184].

However, the protein-of-interest may be of low abundance on the cell membrane or might be masked by the presence of carbohydrates and other various endogenous membrane proteins that may be present in high abundance.

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Many studies have described the isolation of antibody fragments against cell-surface proteins by cell- based biopanning using antibody phage display with an associated immunoglobulin gene library. A number of different procedures have been applied to cell-based biopanning for presenting the target protein, including suspension cells [185], adherent cells attached to the surface of the culture flask [178], or cells fixed by formaldehyde [186]. One such study resuspends adherent cells to expose cells, resulting in a greater surface area of a target receptor being presented to the phage antibody library [300] and to decrease nonspecific phage antibody binders [301, 302]. Various cancer cell lines have been used in phage display, [159, 187-193] to obtain tumour-specific binders for diagnostic agents and therapeutic purposes. A depletion step using the same host cells but not expressing the protein- of-interest is commonly performed for isolation of known target proteins in phage display [183, 198, 245, 303]. This step allows the removal of phage antibodies that bind common antigens and epitopes, before selection of phage antibodies to the target antigen on antigen-positive cells. Cells with the gene-of-interest knocked out through CRISPR/Cas9 can be used as a subtractive step and cells expressing a high level of target receptor are then utilised for target antigen selection [184]. This depletion step is especially useful for depleting binders to epitopes that may be shared between the protein-of-interest and other related receptor proteins.

EphA2 tyrosine kinase receptor is a cell surface biomarker [56], and its over-expression is correlated to poor prognosis and tumorigenesis properties in many cancers [123-125, 127-129, 131], EphA2 is, therefore, a desirable target for mAb discovery using phage display technology and subsequent antibody therapy. The isolation of antibodies against EphA2 through biopanning against purified antigen was challenging for a number of reasons; firstly there is the possibility of receptor structure alteration during purification and immobilisation, resulting in the isolation of antibodies which do not bind to the receptor in their native conformation; secondly, there are domains that are shared with other receptors, so that antibodies will not be specific for one receptor. Some of the antibodies isolated using recombinant antigen could bind to cells, but also bound to EphA2 knock-out cells suggesting that the mAbs were binding to epitopes that are common on many receptors.

The work described in this chapter aimed to isolate mAbs that bind specifically to the native human EphA2 ectodomain, with no cross-reactivity to related Eph receptors. To work towards this goal, the adherent positive and negative EphA2 U87 cells were used for biopanning. Three rounds of biopanning were completed in the campaign, to isolate specific binders towards human EphA2 ectodomain.

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6.2 Materials and methods

6.2.1 Cell culture

EphA2 positive and knock down (KD) U87 cells with a lower number passage were cultured, as outlined in section 2.3.24.

6.2.2 Biopanning on U87 positive and negative EphA2 cell lines

EphA2 KD U87 and positive EphA2 U87 cells were used during biopanning for subtraction and positive selections during biopanning, respectively. The specific methodologies for cell-based biopanning and recombinant protein antigen biopanning are similar. The following sections explain the details of whole cell-based biopanning.

6.2.2.1 Binding and elution of phage

A day before starting biopanning, a 50 mL tube containing 10 mL 2YT with Tetracycline was inoculated overnight with a single colony of E. coli XL1-Blue (Stratagene) as a starter culture at 37 °C with shaking. As a negative control, another single colony was inoculated into 2YT media containing either Ampicillin or Kanamycin.

On day one, the adherent EphA2 KD U87 cells were detached from one confluent T75 flask to use as suspension form. The cells were centrifuged at 300 ×g for 3 min at 4 °C, and the cell pellet was resuspended in 10 mL of ice-cold PBS. The washing step was repeated two more times. The pellet was resuspended in 5 mL 2% MPBS (w/v), followed by 30 min incubation at 4 °C. Along with this, a vial of MJ-SM scFv library (NBF) with a diversity of 109 unique scFvs was blocked with 5 mL 2% MPBS (w/v) with rotation for 30 min at 4 °C. Following this step, the blocked EphA2 KD U87 cells were mixed with blocked phage library and incubated with rotation for 1 h at 4 °C. This step was to deplete any phage binding to proteins of the non-target cell by using EphA2 KD U87 cells. This step was performed two times to discard any non-specific phage binders. The first incubation of the mixed phage library and blocked negative cells were spun at 10,000 ×g for 5 min at 4 °C. The high-speed rotation was performed since the cells were no longer needed for the next step. The supernatant was taken and added to the second blocked negative cells tube and incubated for 1 h at 4 °C with rotation. Centrifugation was done again at high speed. The depleted phage library supernatant was taken to the blocked positive EphA2 U87 cells and incubated with rotation for 1 hr at 4 °C. The mixture was spun at 400 ×g for 4 min at 4 °C. The supernatant was discarded, and the cell pellet was washed three times by centrifuging the cells and resuspending with 10 mL of ice-cold PBS-citrate buffer, pH 5.0 containing 0.1% Tween 20 (v/v). This step was to eliminate any non-specifically adsorbed phage

97 particles. Additional washing steps were then performed three times with 10 mL ice-cold PBS as described above, and finally, the cell pellet was resuspended in 5 mL ice-cold PBS containing 1mM EDTA. Cells were centrifuged at 400 × g for 4 min. The supernatant was discarded, and the cell pellet was resuspended in 500 µL of 76 mM citric acid buffer, pH 2.3 and incubated at RT for 6 min to elute any bound phages. Cells were spun at 10,000 g for 5 min, and the supernatant was transferred to 500 µL of 1 M Tris-HCl buffer, pH 7.5.

6.2.2.2 Infection and amplification of eluted phage

The 10 µL of eluted phage was kept for the following output titration, and the remainder was transferred to 10 mL of log phase E. coli, prepared by sub-culturing the overnight culture in 2YT with Tetracycline, and incubated non-shaking for 30 min at 37 °C. The infected E. coli was centrifuged at 2000 × g for 10 min, and the supernatant was discarded. The cell pellet was resuspended in 250 µL of 2YT media and spread evenly onto one 150 mm 2YT Agar plate containing 2% Glucose (v/v) and Ampicillin (1:1000). The plate was incubated overnight at 30 °C.

6.2.2.3 Titration of eluted phage

The 10 µL eluted phage was added to 90 µL of PBS in 1.5 mL tube and serially diluted 1 in 10 up to 10-4. The tip was changed between each dilution as phage particles are sticky. Each diluted phage (1 µL) was transferred to a new tube containing 100 µL of log phage E. coli and incubated non-shaking for 30 min at 37 °C. The 20 µL phage-infected E. coli for each dilution was spread over 1/4 of the 100 mm 2YT Agar plate containing 2% Glucose (v/v) and Ampicillin (1:1000). The plate was incubated overnight at 37 °C. Next day, the quadrant that has many and countable colonies were selected to calculate the eluted titre in colony forming units (cfu), as follows: Output titre (cfu) = Number of colonies from 1 µL sample × (100 µL/20 µL) × 1000 µL/mL × dilution factor.

6.2.2.4 Rescue of phage-infected E. coli

Described previously in section 2.3.17.5.

6.2.2.5 Titration of rescued phage

Described previously in section 2.3.17.6.

6.2.2.6 Analysis of enriched phage pools by flow cytometry

Adherent positive and KD EphA2 U87 cells were detached from one confluent T75 flask. The cells were centrifuged at 400 × g for 4 min and washed with PBS thrice at RT and aliquoted equally in 6 98

Eppendorf tubes for each cell. Then cells were blocked with 500 µL of 2% MPBS (w/v) and incubated with rotation for 30 min at 4 °C. In parallel, 20 µL of MJ-SM scFv phage library and phage pools obtained from rounds 1 to 3 were blocked with 1000 µL 2% MPBS (w/v) and placed on ice for 30 min. Tubes, including 106 cells/mL, were centrifuged and resuspended with each diluted scFv phage libraries. Two tubes of cells were incubated with 2% MPBS (w/v) only. After 1 hr rotation at 4 °C, tubes containing cells were centrifuged at 400 ×g, 4 °C for 4 min. The washing step was repeated three times using ice-cold PBS. Then cells were incubated with 200 µL mouse anti-M13 monoclonal antibody (GE Healthcare Life Sciences) diluted 1/500 in 2% MPBS (w/v) at 4 °C for 1 h. The control cells were incubated with 2% MPBS (w/v) only. The washing step was repeated three times using ice-cold PBS and the cell pellets, excluding the control cell tube, were resuspended with 200 µL goat anti-mouse Dylight 594 (Abcam) diluted 1/500 in 2% MPBS for 1 h at 4 °C. The washing step was repeated three times. The cell pellets were resuspended in 1 mL ice-cold PBS and flow cytometry was applied using Sysmex Partec Cube 6.

6.2.2.7 Analysis of single phage clones by monoclonal phage ELISA

Round three of phage-infected E. coli glycerol stock was used for the analysis of individual phage clones. In this regard, 40 µL of glycerol stock was added on the edge of one 150 mm 2YT Agar plate containing 2% Glucose (v/v) and Ampicillin (1:1000) for making a 4×4 streak plate dilution. This was repeated three times to have 4 plates, and then plates were incubated at 37 °C overnight. On day two, each well of 96 well round-bottom plate comprising 150 µL 2YT containing 2% Glucose (v/v) and Ampicillin (1:1000) was inoculated with each single clone achieved from the overnight plates. The H7 to H12 wells were free of clone and acted as a negative control. The plate was wrapped in cling wrap and incubated overnight at 37 °C with shaking. On day three, 5 µL of the previous night culture was transferred into the corresponding wells of a new 96-well round bottom culture plate containing 150 µL 2YT containing 2% Glucose (v/v) and Ampicillin (1:1000). The plate then was incubated with shaking for 3 h at 220 rpm, 37 °C. After incubation, 50 µL M13KO7 helper phage with 4×108 pfu/mL diluted in 10 mL 2YT was added into each well of the second plate and incubated non-shaking for 30 min at 37 °C, followed by shaking for 30 min. The plate was centrifuged at 3200 × g for 10 min at RT. The supernatant was discarded, and the pellet was resuspended with 200 µL of 2YT kanamycin (1:1000) and ampicillin (1:1000), and incubated overnight with shaking at 220 rpm, 30 °C.

The first monoclonal phage ELISA was performed by immobilising recombinant human EphA2-ECD and AFBP as a positive and negative antigen, respectively. The antigens were diluted to 3 µg/mL in PBS, and 200 µL of each antigen was separately added to all the wells of one Maxisorp plate a day

99 before conducting ELISA. The plates were incubated overnight at RT. The remainder of the procedure was described by details in section 2.3.19.

The second monoclonal phage ELISA was conducted using fixed adherent positive and KD EphA2 U87 cells as a positive and negative control. Cells were grown in a 96-well transparent bottom sterile tissue culture plate (Corning) 3 d before ELISA experiment to reach the 90% confluency. On the day of cell-based ELISA experiment, the media of plate wells were discarded, and the wells were washed three times with ice-cold PBS and then fixed with 100 µL of 2% paraformaldehyde (PFA) diluted in PBS for 10 min at 4 °C. The cells were fixed on the plate to preserve their receptors and to reduce loss during washing steps. The PFA was discarded and gently washed with cold PBS, and finally, the cells were blocked in 2% MPBS for the rest of the ELISA. The rest of the procedure was explained in section 2.3.19, whereas all the procedure was performed manually and gently at 4 °C without any centrifugation.

6.3 Results 6.3.1 Phage pool enrichment analysis by flow cytometry using EphA2 positive and KD U87 cells

The enrichment of sub-libraries for specific phage binders after each round of selection was monitored by flow cytometry using EphA2 positive and KD U87 cells (Figure 6.1). The phage pool analysis for positive EphA2 cells indicated enrichment of phage binders with increasing selection rounds (Figure 6.1, A). This was initiated from round 2, suggesting the enrichment of specific phage binders was becoming higher round by round. The results for EphA2 KD U87 cells (Figure 6.1, B) indicated the enrichment of phage binders to KD cells for EphA2 as well, suggesting the enrichment of non-specific phage binders during three rounds of biopanning. The result was consistent with the monoclonal phage ELISA outcome. The results suggest that the usage of EphA2 KD U87 cells in cell-based biopanning as a subtraction step may not have been successful in this particular biopanning campaign.

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Figure 6.1: Flow cytometry analysis of phage pools enrichment after three rounds of biopanning using A) positive EphA2 U87 cell line, B) EphA2 knock down U87 human glioblastoma cell line. Phage pools displayed enhancement of binding to U87 cells by increasing the selection rounds (panel A). As flow cytometry results show in panel B, non-specific phage binders were amplified during three rounds of biopanning. Antibody binding was detected using DyLight 594 anti-human IgG secondary antibody. Secondary antibody fluorescence (X-axis) was measured using 561 nm laser and 630/22 nm filter. Number of events is shown on Y axis. The data were analysed using FCS Express Flow version 4 (De Novo Software™).

6.3.2 Monoclonal phage ELISA for human EphA2 scFv-phage binders

To assess the specificity of each phage clone to the EphA2, monoclonal phage ELISA was carried out using immobilised recombinant human EphA2-ECD and positive EphA2 U87 cells.

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There were no phage antibody clones observed that exhibited high absorbance (A450 ≥ 1.0) in the phage ELISA against purified human EphA2-ECD (Figure 6.2, A). Results showed that the difference of phage clone absorbance is not significant (A450 ≥ 1.0) between recombinant human EphA2-ECD and AFBP, suggesting that the Round 3 enriched clones do not to bind to recombinant human EphA2- ECD.

Figure 6.2, B shows the binding of phage clones to the U87 cell surface by whole cell ELISA. Two phage clones (D6 and D7) showed a significant absorbance indicating binding to positive U87 cells; however, no binding to the KD EphA2 U87 cells was evident. This suggests these phage binders were probably enriched against an irrelevant antigen, which is present on both EphA2 positive and negative U87 cells, warranting no further characterisation. An overlay of both monoclonal phage ELISA data was performed to observe the similarity of phage clones binding to both fixed U87 cells and recombinant human EphA2-ECD. As illustrated in Figure 6.2, C, there was no profile similarity noted.

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Figure 6.2 Monoclonal Phage ELISA to screen individual phage clones binding to EphA2. A) Binding of phage clones obtained against positive EphA2 U87 cells to human EphA2–ECD, B) Binding of phage clones obtained against positive EphA2 U87 cells to U87 cells expressing EphA2, and C) Overlaying the monoclonal phage ELISA bar charts against positive EphA2 U87 cells against the immobilised recombinant human EphA2-ECD and U87 cells.

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6.4 Discussion

Selection of antibody phage libraries on recombinant, purified protein antigens has been widely used to present the immobilised target on solid surface to the phage antibody library. However as has been previously mentioned, conformational changes can occur to the protein upon immobilisation resulting in loss of specific epitopes; furthermore immobilisation may result in antigen presentation in particular orientations that may hide or mask an epitope, preventing binding of phage antibodies upon biopanning with a phage antibody library. These impediments can be circumvented by the use of cell- based biopanning methodologies, to isolate antibodies from phage libraries that bind conformational epitopes; however, this approach has its own challenges, such as high background due to the binding of non-specific binders, the abundance of irrelevant antigens present on the cell surface and the relatively low abundance of the target antigen. .

To isolate the EphA2 mAbs, cell-based biopanning was employed using the positive and KD EphA2 U87 cells. A low cell passage number was used since passaging of cancer cell lines may change their membrane protein expression profile, and therefore, may result in change or loss of the original antigenic determinants in the cancerous tissue [304].

The suspension form of adherent U87 cells was prepared for three rounds of biopanning to reduce the isolation of high phage binders to culture-flask plastic, and to minimise the problems caused by Fetal Bovine Serum (FBS) proteins being present in the adherent culture medium [302]. This also provides a larger surface area for exposing the target receptor to phage library [300].

The EphA2 KD cell was used in the subtraction biopanning step. This step depletes the phage library of phage antibodies that bind to the host cell membrane surface antigens [305, 306]. This should theoretically remove or minimise the number binders to common, shared epitopes between the two cell lines, and allowing the selection of phage antibodies that specifically bind the target protein upon biopanning with antigen positive cells [183, 187, 191, 192, 198]. However, the depletion step using KD EphA2 U87 cells did not remove the high-affinity binders to similar epitopes in both positive and negative EphA2 cell lines, as observed in phage pool analysis using EphA2 KD cells (Figure 6.1, B). It was not expected to observe the enrichment of certain phage antibody clones against the EphA2 KD cell. This suggests that there may have been a number of binders to common antigens/epitopes on both positive and negative cells that were not removed in the two subtraction steps.

The low density of EphA2 on the U87 cells may be another reason for not being able to isolate EphA2- specific antibodies. This might be due to the low-level expression of EphA2 on U87 cells in comparison with other GBM cell lines [96, 141]. Furthermore, some EphA2 receptor antigenic determinants may be masked by the presence of abundant carbohydrates and other endogenous 106 proteins at high density on the cell surface. Additionally, the appropriate tumour cell is not always accessible for selection and the cell used in biopanning may have a different expression profile compared to that in tissue which can be affected by the extracellular matrix and cellular surroundings [262]. Since the project was focused on glioblastoma, the only available GBM cell line, U87, was used for biopanning.

We performed all the incubations of cells with phage antibodies at 4 °C to minimise internalization of phage antibodies; however, an alternative strategy might be to promote receptor-mediated endocytosis. Endocytosis of the phage antibody could be accomplished by incubation at 37 °C to allow receptor-mediated endocytosis to occur into positive EphA2 U87 cells. As a consequence, the endocytosed phage binders could be recovered through cell lysis and amplified for the next round of biopanning [307]. In this case, the negative selection would not be necessary and eliminate the difficulties to find the negative cell line for EphA2. This would allow the discovery of not only specific EphA2 phage binders to the cell surface but also functional internalizing phage binders. Transiently expressing the target receptor might also assist in increasing the density of the cell surface receptor [198, 308].

In a reported study, a two-combinatorial selection strategy was used, including recombinant protein- based and cell-based biopanning, [37] to identify Insulin-like growth factor 2 receptor (IGF2R) specific peptides. Using a two-selection approach could be a better strategy for targeting EphA2. The first round could be cell-based biopanning using the transiently expressed EphA2 and allow for internalization and the second round could be antigen biopanning by immobilization of recombinant EphA2 extracellular domain. The internalization strategy would prevent to lose the specific phage binder through stringent washing steps [309], and the immobilised recombinant ECD may allow selecting the specific phage binder to only EphA2 receptor and not to other Eph family receptors as they co-express on neuronal cells [310].

6.5 Conclusion

The selection of the appropriate cells expressing the target receptor is very important for cell-based biopanning, and it would be better to use characterised cell lines with a high expression level of a target protein in this selection strategy. Also, the cells used for flow cytometry could not be necessarily the best option to use in cell-based biopanning. Stringent washing can assist in removing non-specific binders; however, it might lead to removing the potential specific phage antibody binders. The use of a standard selection strategy for all membrane receptors for mAb isolation is not feasible since each receptor has varying complexity and unique structure. A combinatorial selection strategy could be a better strategy to increase the chance of successful antibody selection. Though the 107

EphA2 receptor is a single integral membrane protein, it is a difficult target for antibody discovery due to the high homology of this receptor with other Eph family receptors co-expressed on neural cells. Furthermore, transient expression of EphA2 receptor in a neuronal cell is challenging due to a low expression efficiency in comparison with other cell lines commonly used for increasing the density of the receptor. Using the other tissue primary cell lines, expressing EphA2 at a high level may result in a better cell selection outcome through cell-based biopanning before the validation of isolated mAbs in neural tissue.

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Chapter 7: Application of antibodies to detect Circulating Tumour Cells in brain cancer

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7.1 Introduction

Circulating Tumour Cells (CTCs) are cancer cells that have been released from the primary tumour and are carried to remote sites through circulating blood. They infiltrate from blood into a distant tissue, and after having the opportunity to adapt themselves to a new environment, they then gradually develop as a new clone to form a metastasis [311, 312]. They are understood to be the reason for most cancer-related deaths. Although metastatic related disease progression is highly uncommon in primary brain tumours, CTCs still exist in patient blood samples and as such, represent a highly promising source of diagnostic material [313]. Tissue biopsy is considered an invasive procedure and repeating this procedure is not always practical. By contrast, blood sampling for diagnostics can be repeatedly performed over time. Liquid biopsy can reveal the CTCs in patient blood and provides valuable information regarding patients’ profiles and early cancer detection, including cancer grading and progression, prognosis, metastatic risk, management of appropriate treatment and monitoring the treatment outcome [311, 314, 315]. The future of prognosis through detecting CTCs would be very promising by monitoring the low number of CTCs without need for surgical staging. The importance of CTC number for prognosis, and the changes observed in the CTC number after response to treatment have been described in different cancers [316-320]. CTC features of cancer patients have shown that the genomic profiles of CTCs are remarkably consistent with those of primary tumours, suggesting that characterising a patient’s CTCs can provide a snapshot of the molecular profile of the patient’s primary tumour [321, 322]. These biomarkers, or molecular signatures, could be utilized as diagnostic, prognostic and predictive tools to facilitate precise tumour classification, treatment management, and to enlighten about patient survival [50]. Antibodies as reagents can specifically detect the CTC biomarkers by conjugation to Raman reporters. Surface-enhanced Raman scattering (SERS) is a spectroscopic methodology that has detection sensitivity down to single molecule by amplifying Raman reporter signals, which have been attached on a single nanoparticle [323, 324]. Glioblastoma is an aggressive primary neuroepithelial tumour in adults with a median survival of 9 to 12 months, and accounts for approximately 60% of all astrocyte tumours [325]. Many biomarkers have been reported for glioblastoma. Among them, EphA2[56], EphA3[57] and epidermal growth factor receptor (EGFR) [45, 46] are overexpressed on the surface of high-grade glioma cells, and they have been identified as potential prognostic biomarkers in GBM. The modification of GBM biomarkers, such as tyrosine receptor kinases (RTKs), occurs in more than 80% of primary GBM through mutation and amplification [326]. The two signalling pathways used by RTKs in GBM, including RAS/RAF/MAPK have genetic alteration, which leads to manifestation

110 of pro-oncogenic properties [326]. Different genetic alterations of EGFR occurs in approximately 57% of GBM tumours [327]. Among these genetic alterations, EGFR oncogene amplification accounts as a dominant mutation in GBM [26, 328-330] and can be observed particularly in patients with a primary subtype of GBM and much less in secondary GBM [331]. Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is highly over-expressed in glioma and is an ideal candidate for immunotherapy of GBM [332, 333]. It has been reported that EphA2 is exclusively expressed in GBM but not in the normal brain tissue [96]. Furthermore, its high expression correlates with neovascularisation and apoptosis in astrocyte brain tumours [96, 97, 140]. Expression of EphA3 is associated with other GSC (gastric stem cell) biomarkers expression, including integrin α6 and CD 133[58, 334]. The high expression of EphA3 has been reported in mesenchymal glioblastoma and makes it an attractive therapeutic target for GBM. In this chapter, the preliminary feasibility of using these three GBM cell surface biomarkers to differentiate CTCs using Raman spectroscopy was investigated by using commercial antibodies and glioblastoma patient-derived cell lines differentially expressing these three biomarkers. Firstly, their expression was confirmed on the cell lines using flow cytometry for each biomarker on positive and negative cells to ensure the binding and specificity of each antibody. The primary GBM cells were grown as single neurospheres to be able to express the biomarkers. Each antibody was conjugated to individual Raman reporters to evaluate the expression level of each biomarker by reading the intensity of Raman reporters. Multiplexing of biomarkers was performed to study the heterogeneity of GBM on available cells.

It was originally envisaged that anti-EphA2 antibodies isolated from our immunoglobulin gene libraries using phage display technology would be used in these studies. However, as outlined in previous chapters, EphA2 presented as a difficult target for a number of reasons. Although an antibody that binds EphA2 was successfully isolated (mAb AGH001), binding studies proved that mAb AGH001 was not specific for EphA2, and therefore was not be used in the studies presented in this chapter. In its place, a commercial, mouse anti-EphA2 polyclonal antibody preparation was used.

7.2 Materials and methods 7.2.1 Antibodies

Primary antibodies: Panitumumab (anti-EGFR) is a fully human mAb specific to the EGFR, provided by NBF-UQ (the ordered expression vector was transfected in CHO cells and purified by NBF). Mouse anti•human EphA2 mAb (MAB3035, Bio-Techne), and Rabbit EPHA3 antibody-N-terminal region (OAAB04655, Ava Systems Biology) were purchased.

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Secondary antibodies: Anti-human IgG (Fc specific)-FITC (Sigma), Goat Anti-Mouse IgG H&L-Cy5 (Abcam), and FITC Goat Anti-Rabbit IgG (BD pharmingen) were purchased.

7.2.2 Cell lines

The characterised GBM patient-derived cell lines PB1 and WK1 [335, 336] were kindly provided by Prof. Bryan Day from Brain Cancer Research Unit, QIMR Berghofer Medical Research Institute. EphA2 KD WK1 cell was kindly supplied by Dr Brett Stringer at QIMR Berghofer Medical Research Institute, and had been generated using CRISPR/Cas9 with single guide RNA (sgRNA) technique. PB1 is derived from left frontal tumour 57-year male diagnosed of a high-grade glial tumour with features of glioblastoma multiform (WHO grade IV), and WK1 is derived from right parieto-occipital tumour of 77-year male glioblastoma patient (WHO grade IV) (https://www.qimrberghofer.edu.au/our-research/commercialisation/q-cell/).

7.2.3 Adherent cell culture

The primary cell lines were cultured as an adherent monolayer on 1% Matrigel (Corning)-coated flasks using Stempro NSC SFM kit (Life Technologies). The growth media consisted of KnockOut™ DMEM ⁄ F-12 Basal Medium supplemented with StemPro® NSC SFM Supplement, FGF Basic Recombinant Human (10 µg), EGF Recombinant Human (10 µg) and 20 mM/mL GlutaMAX (GibCo). Penicillin-Streptomycin (10,000 U/mL) was added to the culture medium, and the complete medium was filtered before usage. Then cells were grown at 37 °C with 7.5% CO2/95% humidified air atmosphere. Cells were passaged using 3 mL Accutase solution (Sigma) for T75 flask, followed by 5 min incubation at 37 °C. Then 1.5 mL soybean trypsin inhibitor (Sigma) was added to the flask to stop Accutase dissociation activity. Centrifugation was performed at 211 ×g, at 20 ⁰C for 4 min. The cell pellet was resuspended with warm media to a new 1% Matrigel-coated flask. A bank of primary GBM cells was prepared in low passage numbers.

7.2.4 Single neurosphere cell culture

The primary GBM cells were cultured similar to adherent cells with the exclusion of coating the flasks with Matrigel to allow cells to form neurospheres. Tumour sphere cells were enzymatically passaged using Accutase to break them to single neurosphere. Briefly, the tumoursphere cells were transferred to a 50 mL tube and incubated for 6 min to allow the tumoursphere to settle (based on the gravity). Subsequently, the medium was aspirated from the top, with the cells remaining at the bottom of the tube with a minimal volume of the medium. Accutase (1 mL) was then added, followed by 5 min incubation at RT prior to pipetting. The Accutase was diluted by adding 3 mL medium before transferring the contents of the tube into a new tube. The cells were centrifuged at 1500 rpm for 4 112 min, and the pellet was resuspended with complete medium and spread into a new non-coated Matrigel flask. Single GBM sphere-form cells were maintained for 13 d to allow the expression of biomarkers. During this period, the old medium was replaced with a complete fresh medium based on gravity without centrifugation, followed by gentle pipetting every 2-3 d for 30 times to prevent the formation of clusters of the neural cells.

7.2.5 Flow cytometry

The protocol was described in section 2.3.25 and flow cytometry was performed using BD Accuri C6.

7.2.6 Gold nanoparticles synthesis

A 100 mL conical flask was washed more than three times with dish detergent liquid until no signs of residual water spread on the inner surface of the glassware. All the glassware and apparatus needed for Gold nanoparticles (AuNPs) synthesis were washed and rinsed carefully. Glassware and magnetic stirrer bar were washed with fresh Aqua regia for at least 20 min. Aqua regia was prepared by mixing

Hydrochloric acid (HCl)/ Nitric acid (HNO3) 3:1(v/v). The neck of the flask was rinsed with Aqua regia after the solution turned bright red/orange and then covered with cling wrap. The solution was neutralised by NaOH (Chem Supply) to pH 7.4 before disposing.

Following this, 1 mL of 1% (w/v) Gold (III) chloride trihydrate (HAuCl4.3H2O, Sigma-Aldrich) was added to the flask containing 99 mL of Milli-Q water. The mixture was heated using hotplate at 310 ºC with 500 rpm spinning speed. The flask was covered by a cling wrap to prevent evaporation during boiling. 1% Trisodium citrate dehydrate (1 mL) (w/v) (C6H5Na3O7.2H2O, UNIVAR) was added to the mixture after the solution began boiling and kept for boiling another 20 min. The solution colour changed from dark blue to dark red within 30 seconds. The Au-NP solution was cooled to room temperature. The solution was used immediately for the functionalisation step, and the other solution was stored at 4 ºC. The size of the synthesised AuNPs was determined using UV-VIS.

7.2.7 Raman reporter preparation

1mM of each Raman reporter-surface marker (SERS) were prepared as follows: 1) 4-mercaptobenzoic acid (MBA) (MW 154.19, Sigma-Aldrich, cat#706329-1G) for anti- EGFR; 2) 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA) (MW 226.14, TG1, cat#5211-44-9) for anti-EphA3,

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3) 5, 5′-Dithiobis-2-nitrobenzoic acid (DTNB, Ellman′s reagent) (MW396.35, Sigma-Aldrich, cat#D8130-5G) for anti-EphA2.

7.2.8 SERS-NPs functionalisation

From each 1 mM Raman Reporter (10 µL) was added into 1 mL AuNPs suspension, followed by addition of 2 µL of 1.0 mM 3, 3′-Dithiodipropionic acid di (N-hydroxysuccinimide ester, Sigma- Aldrich). The mixture was incubated at 25 oC for 5 to 7 h with shaking at 350 rpm and centrifuged at 5400 ×g for 10 min. The supernatant was discarded and 200 µL of 0.1 mM PBS buffer was added to the pellet. A cuvette was rinsed with water and Aqua regia before drying off with nitrogen gas. AuNPs-Reporter solution (60 µL) was transferred to a clean cuvette to measure the SERS signal intensity based on the single acquisition by Peak 1.3 software version 2.37 (Snowy Range). Distinct vibrational Raman peaks for Raman reporter are as following: MBA (1080 cm-1); DTNB (1337 cm- 1); TFMBA (1380 cm-1). Anti-EGFR, anti-EphA2 and anti-EphA3 antibodies (1 µL of 10 µg/20 µL) were separately added to 200 µL AuNPs-Reporter solution MBA, DTNB and TFMBA Raman reporters, respectively. The mixture was incubated at 25 ºC for 30 min, before centrifuging at 600×g for 6 min. The supernatant was removed, and the pellet was resuspended with 200 µL of 0.1% Bovine Serum Albumin (BSA) to block non-specific binding. The solution was incubated at 4 ºC for overnight.

7.2.9 SERS-NPs labelling on single neurosphere primary glioblastoma cell lines

The three cell pellets, including WK1, EphA2 KD WK1 and PB1 were resuspended in 2 mL of 0.1% (w/v) BSA-PBS, and 500 µL of each was distributed into four tubes. The multiplex solutions were prepared by adding 30 µL of the selected Raman reporter-coated gold nanoparticles (Ab-SERS-label) to each tube of single neurosphere cells. The mixture was incubated at 37 ºC for 30 min. The mixture solution was spun at 400 ×g for 1 min, and the cell pellet was gently washed with 200 µL of 1% BSA- PBS buffer. The washing step was repeated 4 times, and the cell pellet was resuspended in 60 µL of 1% (w/v) BSA-PBS buffer.

7.2.10 SERS measurement

The entire sample was transferred into a clean cuvette for SERS measurement. The Peak 1.3 software version 2.37 (Snowy Range) was employed for obtaining 150 measurements of each sample. These continual measurements were taken to provide a different portion of cells during their Brownian motion.

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7.3 Results

7.3.1 Non-adherent sphere cell culture

Patient-derived glioblastoma cells were used to examine the binding of commercial antibodies to the cell surface biomarkers. When the cells grew in adherent condition, the flow cytometry results did not show binding of EphA2 and EphA3 antibodies. However, the binding was observed when the cells were cultured without matrigel, indicating the expression of the biomarkers. In non-adherent conditions, the tumorsphere of cells was formed by proliferation of one progenitor cell (GSCs), which led to the spherical formation of each cancer cell [337]. Since the cells were fused together, the inner cells die due to the lack of oxygen and nutrients; therefore, it was technically challenging to detect the surface biomarkers by flow cytometry. Due to this phenomenum, the cells were maintained as single GSCs by frequently pipetting to break the tumorsphere cells to single progenitor cells. In this strategy, the expression of GBM markers was possible, and their presence was confirmed by binding of the specific antibodies to the biomarkers using flow cytometry.

7.3.2 Binding specificity of commercial antibodies to the EphA2, EphA3 and EGFR on the surface of glioblastoma primary cells

Flow cytometry was performed to check the expression of biomarkers-of-interest on neurosphere- grown GBM cells, through observing the binding of commercial antibodies to the target biomarkers. A positive shift in the histogram was observed when the anti-EphA2 antibody bound to WK1 cells, indicating EphA2expression (Figure 7.1, a). In contrast, there was no positive shift observed for EphA2 KD WK1 (Figure 7.1, b). Flow cytometry binding of EphA3 antibody on WK1 displayed a positive shift in fluorescent intensity (Figure 7.1, c), resulting in two peaks on EphA3 antibody histogram. This could be due to the detection of anti-EphA3 in two populations of WK1 cells; one in neurosphere form and the other in cluster shape (Figure 7.1, c). Binding of EphA3 antibody was detected by a very small shift on PB1 cells, which are characterised as low-level EphA3 expression (Figure 7.1, d). Binding of anti-EGFR antibody was examined on three different cell lines, and results showed the positive shift in the histogram, indicating the expression of EGFR on all three cell lines (Figure 7.1, e, f and g). These results were consistent with QIMR flow cytometry results (https://www.qimrberghofer.edu.au/wp-content/uploads/Q-Cell_Cell-line-flow-cytometry.pdf ), and also data recently reported [335].

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Figure 7.1 Binding analysis of commercial antibodies to GBM markers. Analysis of anti-EphA2 binding on (a) WK1 cells and (b) EphA2 KD WK1 cells; anti-EphA3 binding of on (c) WK1 and (d) PB1 cells; anti-EGFR binding on (e) WK1, (f) EphA2 KD WK1, (g) PB1 cells.

7.3.3 The specificity test of each Ab-SERS for EphA2, EphA3 and EGFR surface markers

To examine the specificity of each Ab-SERS label, the expression level of each biomarker was tested on the corresponding positive and negative primary GBM cells. The expression level of each biomarker was achieved through Raman spectroscopy by reading the produced intensity of each SERS conjugated to the relevant antibody. The EphA2 expression level was 884.69 a.u. for WK1 (Figure 7.2, a), whereas this was 95.07 a.u. for EphA2 KD WK1 (Figure 7.2, b). The measured expression of EphA3 on WK1 was 857.11 a.u. (Figure 7.2, c), while this was 252.59 a.u. on PB1 (Figure 7.2, d). EGFR expression level was 1237.05 and 837.28 a.u. on WK1 (Figure 7.2, e) and PB1 (Figure 7.2, f), respectively. The higher intensity of SERS detection for each biomarker represented the higher marker expression. These results indicated the specificity of each Ab-SERS to detect the corresponding cell surface biomarker. Results were

117 consistent with flow cytometry analysis, representing the expression of EGFR biomarker on all cell lines, EphA3 biomarker on WK1 GBM cell and EphA2 biomarker on both WK1 and PB1 cells.

Figure 7.2 Specificity test of Ab-SERS labels on single neurosphere primary glioblastoma cells. EphA2 expression on a) WK1, b) EphA2 KD WK1; EphA3 expression on c) WK1, d) PB1; EGFR expression on e) WK1 and f) PB1 cells.

7.3.4 Heterogeneity profiling of multiple biomarkers for each glioblastoma primary single cell

The signal distribution curve of three biomarker expression levels was achieved by multiplexing the three Ab-SERS on a single cell line. The Raman signal plotted from measurements of each marker

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(frequency vs Raman intensity), presenting the intensity distribution of expressed markers through all measured events. The wider the curve for each marker, the more the heterogeneity of the sample is indicated. Multiplexing on EphA2 KD WK1 (Figure 7.3, a) indicated the lower distribution of EphA2 by displaying the narrow signal distribution, which was consistent with the flow cytometry and anti- EphA2 SERS results. EphA3 showed the lower signal distribution in PB1 multiplexing (Figure 7.3, b) compared with EphA2 and EGFR signal distributions. This was consistent with flow cytometry and anti-EphA3 SERS results. WK1 multiplexing (Figure 7.3, c) indicated the heterogeneity of this cell for expressing the all three glioblastoma cell surface biomarkers; EphA2, EphA3 and EGFR.

This technology also represents the heterogeneity of each biomarker in different GBM cells. Since each cell expresses different biomarkers on its surface, thus this expression pattern for a combination of biomarkers is exclusive to that cell.

Figure 7.3 Multiple biomarker heterogeneity profiling on a) EphA2 KD WK1, b) PB1 and c) WK1 cells

7.4 Discussion

Glioblastoma or grade IV glioma is an aggressive, malignant brain tumour arising from astrocytes. The median survival in patients suffering from glioblastoma is less than 15 months [14]. The

119 diagnosed patient with GBM undergoes surgery to remove the tumour tissue, followed by radiotherapy and TMZ administration [338]. However, GBM is incurable and patients need to undertake other treatments to prolong the survival and relieve the GBM symptoms. Identifying the GBM biomarkers are significantly crucial for early diagnosis, prognosis and treatment management, and has been extensively reported [339-341]. Phage display technology can provide the tools for the isolation of antibodies that bind specifically to the cell and organ-specific biomarkers in their native conformation [341, 342]. The generated antibodies can form the basis of therapeutic and diagnostic reagents. Specifically, they can be used to detect CTCs collected by a blood test, or in other words “liquid biopsy” to sequence the tumour before, during and after treatment without having to perform serial biopsies. Since CTC flow are in very low abundance, such as a single tumour cell in a million blood cells, it is challenging to detect these cells in the bloodstream. By conjugating GBM surface markers antibodies to Raman reporters, it makes it possible to detect CTCs in the blood stream. This strategy may reduce the overall mortality rate of the disease by earlier detection of CTCs, providing more accurate information on cancer progress, prognostic factors, real-time monitoring of therapies, resistant mechanisms, identification of therapeutic targets and finally trend of metastases in patients suffering cancer [343]. Multiplexing of EphA2, EphA3 and EGFR antibodies using corresponding Ramen reporters in different GBM cells showed that each GBM cell has its unique expression pattern. Multiplexing of Raman reporters gives the opportunity to provide multiple signals simultaneously with high sensitivity to profile the multiple markers’ heterogeneity on each glioblastoma cell. Results revealed that multiple RTKs are likely maintaining distinct cell subpopulations, suggesting that a combination of two or more pathway inhibitors might be necessary to be applied in treating GBM more efficiently. Antibodies utilised through the methodology presented can assist in characterising the sample cell without requiring a large number of cells, which restricts the application and use of flow cytometry [344]. One suggested reason for the unsuccessful therapeutic strategy of targeting a particular receptor tyrosine kinase in glioblastoma is that probably other RTKs might become activated simultaneously in the same tumour [345]. Reports have shown that concurrent phosphorylation of multiple RTKs could occur in GBM and initiate resistance in single-RTK inhibition via receptor tyrosine kinase switching in a cell [346]. Heterogeneity is a characteristic of high-grade glioma. The tumour cells express several biomarkers such as tyrosine kinase receptors including EGFR, EphA2, EphA3, PDGFRa and CD44, therefore intra-tumour heterogeneity could be analysed by employing a cocktail of biomarker antibodies at a given point in time. Thus, the treatment strategy could be guided by the expression level of 120 biomarkers. It would be possible to investigate the heterogeneity of only one biomarker expression pattern by multiplexing GBM tumour cell subpopulations and demonstrate the unique pattern of amplification for each biomarker. However, this method may not be identical to another patient. The application of CTCs for early diagnosis of GBM is extremely promising, but it also has an application in monitoring patient responses to surgery and therapy. Measuring the CTC after surgery and following treatment can significantly help to monitor the response of a patient to the drug, the management of treatment and the recurrence of cancer, which together could substantially prolong the median survival rate in GBM cancer therapy.

7.5 Conclusion

Employing antibodies with Ramen reporters can provide an opportunity for early detection of CTCs in patients suffering GBM. Additionally, it facilitates monitoring of treatment and consequent changes of patients’ CTC phenotype without having to perform highly invasive surgery; thus this brings a more personalized and customized therapy for individual patients. Detection of biomarkers utilizing antibodies and Raman spectroscopy may simplify the profiling of inter- and intra-tumour heterogeneity.

Application of these antibodies in the above-mentioned approach might reduce the rate of mortality and morbidity in patients bearing glioblastoma through providing valuable information about disease status based on predictive prognosis, to assist with better management strategies in clinical decision making.

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Chapter 8. General discussion and conclusion

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8.1 General discussion

Monoclonal antibodies (mAbs) have in recent years become the major class of biologic medicines. The rate of approval for mAbs is outstripping that of any other therapeutic entity. mAbs are able to treat a variety of diseases, including various types of cancers, inflammatory diseases including rheumatoid arthritis, psoriasis, ankylosing spondylitis and Cohn’s disease. As well, they are approved for the treatment of infectious disease (e.g. Palivizumab for the treatment of Respiratory Syncytial Virus infection). More recently the advent of immunotherapy, utilising check point inhibition by mAbs, whereby a patients’ exhausted T-cells residing within tumours are re-activated to recognise and kill tumour cells, has led to spectacular results in the treatment of metastatic melanoma. The combination of check point inhibitors Ipilimumab (anti-CTLA4) and Nivolumab (anti-PD-1) are also now showing promise in the treatment of advanced/metastatic Non-Small Cell Lung Carcinoma (NSCLC). In the area of theranostics, much research is being carried out on the use of antibody fragments such as scFvs and Fabs, labelled with fluorescent or radiolabels, for the diagnosis and therapy of cancer. A cancer type, which requires immediate attention in terms of a more focussed research effort globally, is brain cancer. At present, there are no antibody therapies that are able to effectively treat brain cancer. Glioblastoma multiforme (GBM) is a particularly aggressive tumour, and to this day life expectancy upon diagnosis is around 12-15 months.

This thesis is focussed on GBM, and the development of antibodies that might effectively treat GBM in the future. GBM is a highly malignant brain tumour, which naturally occurs in both humans and dogs. EphA2, one of the Eph tyrosine kinase family receptors, is highly expressed in GBM cells [134] and plays crucial roles in the oncogenic properties of GBM cells [114]. The current lack of effective treatments for glioblastoma means that there is an urgent need for new therapies, and targeted therapy using mAbs directed towards EphA2 is a promising avenue for new therapies. mAbs which cross- react with both human and canine EphA2 will allow comparative oncology studies in canine models which are directly translatable to human GBM studies.

There are a number of technologies available for the discovery, isolation and generation of mAbs. Phage display technology provides an ideal platform for generating novel mAbs of human origin. The technique is described in detail in Chapter 1, and was used to create Humira, at present the biggest selling drug globally, with global revenue in excess of US $12B. The National Biologics Facility at the AIBN, University of Queensland has a number of naïve human immunoglobulin gene libraries of high quality and diversity. The Mahler/Jones library was utilised in this thesis for the isolation of mAbs against brain tumour targets. These antibodies are in the scFv format, and must be reformatted into complete IgG1 antibodies for testing and characterisation.

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The aim of the work in reported in this thesis was to isolate specific antibodies with cross-reactivity to both canine and human EphA2 receptors using the phage display technique. Various strategies were employed for the presentation of the EphA2 antigen prior to biopanning with the phage antibody library. General methods and materials used frequently throughout the thesis are described in Chapter 2. The four result chapters include specific experimental protocols and research outcomes.

Chapter 3 is entitled “Isolation of canine and human monoclonal antibodies against recombinant canine and human EphA2 Extracellular Domain (ECD)”. Recombinant ECD of canine and human EphA2 were expressed in CHO cells and immobilised onto immunotubes for screening the binders from a naïve human scFv phage library. Several binders were isolated and reformatted to human whole IgG1. The most promising mAb, AGH001, had nanomolar affinity to both human and canine EphA2-ECD; however, mAb AGH001 bound to another unknown molecule on EphA2 KD U87 cells. It was therefore presumed that it binds an epitope on EphA2 which is shared with other EphA family receptors which are highly conserved or other similar receptors. For example, the fibronectin domain, which is naturally present in the structure of many membrane proteins, is a potential epitope of mAb AGH001.

Next, we decided to target the ligand-binding domain (LBD) of EphA2 specifically, as this domain is characteristic for each EphA receptor in EphA/ephrinA complex. Therefore, in Chapter 4 entitled “Isolation of canine and human monoclonal antibodies against recombinant canine and human EphA2 Ligand- Binding Domain (LBD)”, the recombinant canine and human EphA2-LBD were produced as soluble antigen to be used for isolating binders from the phage antibody library. Simultaneously, three separate soluble biopanning campaigns were performed and described in this chapter. Several binders were isolated and reformatted to full-length human IgG1. Only HuB1 and CrossA11 mAbs conserved their affinity and cross-reactivity to recombinant canine and human EphA2-LBDs after reformatting. Their binding was examined against EphA2-ECD, since LBD is a part of ECD. However, there was no binding observed for CrossA11 mAb; this could be due to the different conformation of the LBD in recombinant EphA2-LBD and recombinant EphA2-ECD. Thus, CrossA11 mAb can only bind to the conformation of the LBD which was presented to the library during biopanning. Both mAbs bound to EphA2 KD cells, and we assumed that their epitopes probably overlapped partially with other EphA LBDs since EphA2 has around 50% homology with several LBD of EphA receptors (Appendix 6).

We were interested in identifying the epitope of mAb AGH001 on EphA2 to support our conclusion that mAb AGH001 binds to a site within EphA2-ECD that is conserved in other EphA receptors. Chapter 5 entitled “Identifying the epitope of mAb AGH001 within the extracellular domain of the

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EphA2 receptor” was dedicated to identifying the binding site of mAb AGH001 on EphA2-ECD. Different recombinant domains of human and canine EphA2-ECD were constructed, including CRD, both LBD and CRD together and two Fn domains. All the constructs were successfully expressed, except CRD due to the complexity of its structure. No binding was observed for either mAb AGH001 or the positive control antibody, mAb 4B3, to any domain except the whole ECD. This suggested that the recombinant versions of each sub-domain have a different conformation than the whole ECD. There is a possibility that the mAb AGH001 epitope is located in another domain of EphA2-ECD, for instance, in the CRD or in the junction between the CRD and Fn domains. Since these domains are conserved among Eph receptors and they are co-expressed in cells expressing EphA2, they may potentially be the specific binding site for mAb AGH001. Western blot experiments showed that mAb AGH001 has a conformational epitope compared with mAb 4B3 which has a linear epitope. It was concluded that soluble antigen biopanning was not practical for isolating antibodies specific for the native conformation of EphA2, so the next attempt was to use cell-based biopanning to preserve the conformation of native EphA2 during biopanning.

Chapter 6 is entitled “Isolation of human monoclonal antibodies against native EphA2 through cell- based biopanning”. In this chapter, cell-based biopanning was used in an attempt to isolate mAbs that bind specifically to the native human EphA2 ectodomain. The EphA2 KD U87 cells were used in the subtraction step to remove cross-reactive binders to related Eph receptors. However, the number of binders to common epitopes to both positive and negative cells were very high in phage library, which did not lead to the isolation of specific EphA2 binders.

If the isolation of antibodies to EphA2 was successful, they would have been further developed for comparative oncology studies. One potential use of the antibodies would be for diagnostic purposes to detect circulating glioblastoma tumour cells in blood samples.

Chapter 7 was entitled “Applications of antibodies to detect circulating tumour cells in brain cancer”. The research reported in this chapter demonstrated the success of employing antibodies in detection of CTCs. Commercial, mouse-derived, human target-specific polyclonal antibodies were used since attempts to isolate human-mAbs by phage display were unsuccessful. Raman reporters were conjugated to commercial antibodies, including anti-EphA2, anti-EphA3 and anti-EGFR. The expression of each GBM biomarker with high sensitivity was shown in both positive and negative glioblastoma patient-derived cell lines for each biomarker. Additionally, the heterogeneity of GBM cells was displayed by the varying expression levels of these three biomarkers. This methodology gives the opportunity through providing valuable information about disease status based on predictive prognosis, to assist with better management strategies in clinical decision making.

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8.2 General conclusion

In conclusion, the research outcomes reported in this thesis highlight that the isolation of antibodies by selectively targeting EphA2 is challenging, since this receptor belongs to a large family with closely related proteins. Furthermore, this protein is a multi-domain receptor, and it is not surprising to isolate an antibody against any of the domains. Soluble antigen biopanning, of either the whole ECD or sub-domains of the ECD, was not a successful strategy for presenting antigen to a phage antibody library, since the conformation of the receptor protein through expression and purification might be altered, leading to the isolation of binders with specificity to different epitopes compared with native epitopes. However, phage antibody display experimental outcomes need to be assessed on a case by case basis, and it cannot be generalised that one biopanning strategy and campaign will be successful for different antigens. Cell-based biopanning promises much potential, and should be optimised to ensure the target receptor is highly expressed, and non-specific binding to irrelevant antigens is minimised.

The mAb AGH001 isolated in this study binds to recombinant human and canine EphA2-ECD with nanomolar affinity. Binding studies with EphA2 KD cells show that it does not bind only to EphA2, and must also bind to other related receptor. Therefore, this antibody may still be useful as a biopan- EphA receptor antibody as a research reagent; however the epitope needs to be identified to realise its full potential. A pull-down immunoprecipitation assay is an appropriate methodology to identify the mAb AGH001 specificity, and this should be further pursued in future work. Employing mAbs with SERS as we showed is capable of efficiently capture the CTCs and has demonstrated a great potential for future detection of CTCs in clinical application.

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Supplementary for Chapter 3

Appendix 1. Full-length sequences of human and canine EphA2 receptors by GeneArt. Yellow highlight represents the secretion tag and red colour shows the last residue of EphA2 ECD

Human EphA2 sequence 5'NheIsite-Kozak- MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQN IMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKE TFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFY LAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGG EEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSP EGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIV YSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLV TSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNV RRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVG VVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFT TEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEAGI MGQFSHHNIIRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGM KYLANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPE AISYRKFTSASDVWSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIY QLMMQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTV SEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQV NTVGIPI-STOP-BamHI-3'

Canine EphA2 sequence 5'NheI site-Kozak- MERPGPRACLALLWGCVLASAAAAQGKEVVLLDFAAAKGELGWLTHPYGKGWDLMQNI MDDMPIYMYSVCNVVAGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKE TFNLYYAESDVDYGTNFQKRQFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLSRKGFY LAFQDIGACVALLSVRVYYKKCPELLQGLARFPETIAGSDAPSLATVAGTCVDHAVVPPGG EEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKSEASESPCLECPVHTVLSS EGATFCDCEEGYFRAPQDLLSMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGRDDIV YSVTCEQCWPESGECGPCEASVRYSEPPHALTRTSVTVSDLEPHMNYTFAVEARNGVSELV ASRSFRTASVSINQTEPPKVKLEGRTTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNV RRTEGFSVTLDDLAPDTTYLIQVQALTQEGQGAGSKIHEFQTLSTEASGNLAVIGGVAVGIV LLLVLAGIGLFIHRRRKSLRTRQSSEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFTTEI HPSCVTRQKVIGAGEFGEVYKGTLKASGKKEVPVAIKTLKAGYTEKQRVDFLSEASIMGQF SHHNIIRLEGVVSKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGMKYL ANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISY RKFTSASDVWSYGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLM MQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVSEW LESIKMQQYTEHFLAAGYTAIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTV GIPI-STOP-BamHI-3'

143

Appendix 2. Multiple sequence alignment of EphA-ECDs using CLUSTAL O (1.2.4)

SP|P29317|EPHA2_HUMAN ------AQGKEVVLLDFAAAGGELGWLTHPYGKGW 29 SP|P21709|EPHA1_HUMAN ------KEVTLMDTSKAQGELGWLLDPPKDGW 26 SP|P29320|EPHA3_HUMAN ------ELIPQPSNEVNLLDSKTIQGELGWISYPSH-GW 32 SP|P54764|EPHA4_HUMAN ------VTGSRVYPANEVTLLDSRSVQGELGWIASPLEGGW 35 SP|P54756|EPHA5_HUMAN PASLAGCYSAPRRAPLWTCLLLCAALRTLLASPSNEVNLLDSRTVMGDLGWIAFPKN-GW 59 SP|Q9UF33|EPHA6_HUMAN ------WP------GDCSHVSNNQVVLLDTTTVLGELGWKTYPLN-GW 35 SP|Q15375|EPHA7_HUMAN ------QAAKEVLLLDSKAQQTELEWISSPPN-GW 28 SP|P29322|EPHA8_HUMAN ------ARGEVNLLDTSTIHGDWGWLTYPAH-GW 27 SP|Q5JZY3|EPHAA_HUMAN ------EEVILLDSKASQAELGWTALPSN-GW 25 :* *:* : * * **

SP|P29317|EPHA2_HUMAN DLMQNIM-NDMPIYMYSVCNVMS-GDQDNWLRTNWVYRGE-AERIFIELKFTVRDCNSFP 86 SP|P21709|EPHA1_HUMAN SEQQQIL-NGTPLYMYQDCPMQGRRDTDHWLRSNWIYRGEEASRVHVELQFTVRDCKSFP 85 SP|P29320|EPHA3_HUMAN EEISGVDEHYTPIRTYQVCNVMD-HSQNNWLRTNWVPRNS-AQKIYVELKFTLRDCNSIP 90 SP|P54764|EPHA4_HUMAN EEVSIMDEKNTPIRTYQVCNVME-PSQNNWLRTDWITREG-AQRVYIEIKFTLRDCNSLP 93 SP|P54756|EPHA5_HUMAN EEIGEVDENYAPIHTYQVCKVME-QNQNNWLLTSWISNEG-ASRIFIELKFTLRDCNSLP 117 SP|Q9UF33|EPHA6_HUMAN DAITEMDEHNRPIHTYQVCNVME-PNQNNWLRTNWISRDA-AQKIYVEMKFTLRDCNSIP 93 SP|Q15375|EPHA7_HUMAN EEISGLDENYTPIRTYQVCQVME-PNQNNWLRTNWISKGN-AQRIFVELKFTLRDCNSLP 86 SP|P29322|EPHA8_HUMAN DSINEVDESFQPIHTYQVCNVMS-PNQNNWLRTSWVPRDG-ARRVYAEIKFTLRDCNSMP 85 SP|Q5JZY3|EPHAA_HUMAN EEISGVDEHDRPIRTYQVCNVLE-PNQDNWLQTGWISRGR-GQRIFVELQFTLRDCSSIP 83 . : *: *. * : . ::** :.*: . . ::. *::**:***.*:*

SP|P29317|EPHA2_HUMAN GGAS--SCKETFNLYYAESDLDYGTNF---QKRLFTKIDTIAPDEITVSSDFEARHVKLN 141 SP|P21709|EPHA1_HUMAN GGAGPLGCKETFNLLYMESDQDVGIQL---RRPLFQKVTTVAADQSFTIRDLVSGSVKLN 142 SP|P29320|EPHA3_HUMAN LVLG--TCKETFNLYYMESDDDHGVKF---REHQFTKIDTIAADESFTQMDLGDRILKLN 145 SP|P54764|EPHA4_HUMAN GVMG--TCKETFNLYYYESDNDKERFI---RENQFVKIDTIAADESFTQVDIGDRIMKLN 148 SP|P54756|EPHA5_HUMAN GGLG--TCKETFNMYYFESDDQNGRNI---KENQYIKIDTIAADESFTELDLGDRVMKLN 172 SP|Q9UF33|EPHA6_HUMAN WVLG--TCKETFNLFYMESDESHGIKF---KPNQYTKIDTIAADESFTQMDLGDRILKLN 148 SP|Q15375|EPHA7_HUMAN GVLG--TCKETFNLYYYETDYDTGRNI---RENLYVKIDTIAADESFTQGDLGERKMKLN 141 SP|P29322|EPHA8_HUMAN GVLG--TCKETFNLYYLESDRDLGAST---QESQFLKIDTIAADESFTGADLGVRRLKLN 140 SP|Q5JZY3|EPHAA_HUMAN GAAG--TCKETFNVYYLETEADLGRGRPRLGGSRPRKIDTIAADESFTQGDLGERKMKLN 141 . ******: * *:: . *: *:* *: . *: :***

SP|P29317|EPHA2_HUMAN VEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLA 201 SP|P21709|EPHA1_HUMAN VERCSLGRLTRRGLYLAFHNPGACVALVSVRVFYQRCPETLNGLAQFPDTLPGP--AGLV 200 SP|P29320|EPHA3_HUMAN TEIREVGPVNKKGFYLAFQDVGACVALVSVRVYFKKCPFTVKNLAMFPDTVP-MDSQSLV 204 SP|P54764|EPHA4_HUMAN TEIRDVGPLSKKGFYLAFQDVGACIALVSVRVFYKKCPLTVRNLAQFPDTITGADTSSLV 208 SP|P54756|EPHA5_HUMAN TEVRDVGPLSKKGFYLAFQDVGACIALVSVRVYYKKCPSVVRHLAVFPDTITGADSSQLL 232 SP|Q9UF33|EPHA6_HUMAN TEIREVGPIERKGFYLAFQDIGACIALVSVRVFYKKCPFTVRNLAMFPDTIPRVDSSSLV 208 SP|Q15375|EPHA7_HUMAN TEVREIGPLSKKGFYLAFQDVGACIALVSVKVYYKKCWSIIENLAIFPDTVTGSEFSSLV 201 SP|P29322|EPHA8_HUMAN TEVRSVGPLSKRGFYLAFQDIGACLAILSLRIYYKKCPAMVRNLAAFSEAVTGADSSSLV 200 SP|Q5JZY3|EPHAA_HUMAN TEVREIGPLSRRGFHLAFQDVGACVALVSVRVYYKQCRATVRGLATFPATAAESAFSTLV 201 .* .:* : ::*::***:: ***:*::*:::::::* :. ** * : *

SP|P29317|EPHA2_HUMAN TVAGTCVDHAVVPPG-GEEPRMHCAVDGEWLVPIGQCLCQAGYEKV--EDACQACSPGFF 258 SP|P21709|EPHA1_HUMAN EVAGTCLPHARASPRPSGAPRMHCSPDGEWLVPVGRCHCEPGYEEGGSGEACVACPSGSY 260 SP|P29320|EPHA3_HUMAN EVRGSCVNNSKE----EDPPRMYCSTEGEWLVPIGKCSCNAGYEER--GFMCQACRPGFY 258 SP|P54764|EPHA4_HUMAN EVRGSCVNNSEE----KDVPKMYCGADGEWLVPIGNCLCNAGHEER--SGECQACKIGYY 262 SP|P54756|EPHA5_HUMAN EVSGSCVNHSVT----DEPPKMHCSAEGEWLVPIGKCMCKAGYEEK--NGTCQVCRPGFF 286 SP|Q9UF33|EPHA6_HUMAN EVRGSCVKSAEE----RDTPKLYCGADGDWLVPLGRCICSTGYEEI--EGSCHACRPGFY 262 SP|Q15375|EPHA7_HUMAN EVRGTCVSSAEEEA--ENAPRMHCSAEGEWLVPIGKCICKAGYQQK--GDTCEPCGRGFY 257 SP|P29322|EPHA8_HUMAN EVRGQCVRHSEE----RDTPKMYCSAEGEWLVPIGKCVCSAGYEER--RDACVACELGFY 254 SP|Q5JZY3|EPHAA_HUMAN EVAGTCVAHSEGEP--GSPPRMHCGADGEWLVPVGRCSCSAGFQER--GDFCEACPPGFY 257 * * *: : *:::*. :*:****:*.* *. *.:: * * * :

SP|P29317|EPHA2_HUMAN KFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMG 318 SP|P21709|EPHA1_HUMAN RMDMDTPHCLTCPQQSTAESEGATICTCESGHYRAPGEGPQVACTGPPSAPRNLSFSASG 320 SP|P29320|EPHA3_HUMAN KALDGNMKCAKCPPHSSTQEDGSMNCRCENNYFRADKDPPSMACTRPPSSPRNVISNINE 318 SP|P54764|EPHA4_HUMAN KALSTDATCAKCPPHSYSVWEGATSCTCDRGFFRADNDAASMPCTRPPSAPLNLISNVNE 322 144

SP|P54756|EPHA5_HUMAN KASPHIQSCGKCPPHSYTHEEASTSCVCEKDYFRRESDPPTMACTRPPSAPRNAISNVNE 346 SP|Q9UF33|EPHA6_HUMAN KAFAGNTKCSKCPPHSLTYMEATSVCQCEKGYFRAEKDPPSMACTRPPSAPRNVVFNINE 322 SP|Q15375|EPHA7_HUMAN KSSSQDLQCSRCPTHSFSDKEGSSRCECEDGYYRAPSDPPYVACTRPPSAPQNLIFNINQ 317 SP|P29322|EPHA8_HUMAN KSAPGDQLCARCPPHSHSAAPAAQACHCDLSYYRAALDPPSSACTRPPSAPVNLISSVNG 314 SP|Q5JZY3|EPHAA_HUMAN KVSPRRPLCSPCPEHSRALENASTFCVCQDSYARSPTDPPSASCTRPPSAPRDLQYSLSR 317 : * ** :: .: * *: .. * : ** ***:*

SP|P29317|EPHA2_HUMAN AK--VELRWTPPQDSGGREDIVYSVTCEQCWP---ESGECGPCEASVRYSEPPHGLTRTS 373 SP|P21709|EPHA1_HUMAN TQ--LSLRWEPPADTGGRQDVRYSVRCSQCQGTAQDGGPCQPCGVGVHFSPGARGLTTPA 378 SP|P29320|EPHA3_HUMAN TS--VILDWSWPLDTGGRKDVTFNIICKKCGW---NIKQCEPCSPNVRFLPRQFGLTNTT 373 SP|P54764|EPHA4_HUMAN TS--VNLEWSSPQNTGGRQDISYNVVCKKCGA--GDPSKCRPCGSGVHYTPQQNGLKTTK 378 SP|P54756|EPHA5_HUMAN TS--VFLEWIPPADTGGRKDVSYYIACKKCNS---HAGVCEECGGHVRYLPRQSGLKNTS 401 SP|Q9UF33|EPHA6_HUMAN TA--LILEWSPPSDTGGRKDLTYSVICKKCGL---DTSQCEDCGGGLRFIPRHTGLINNS 377 SP|Q15375|EPHA7_HUMAN TT--VSLEWSPPADNGGRNDVTYRILCKRCSW---EQGECVPCGSNIGYMPQQTGLEDNY 372 SP|P29322|EPHA8_HUMAN TS--VTLEWAPPLDPGGRSDITYNAVCRRCPW---ALSRCEACGSGTRFVPQQTSLVQAS 369 SP|Q5JZY3|EPHAA_HUMAN SPLVLRLRWLPPADSGGRSDVTYSLLCLRCGRE-GPAGACEPCGPRVAFLPRQAGLRERA 376 : : * * * : ***.*: : * :* * * : .*

SP|P29317|EPHA2_HUMAN VTVSDLEPHMNYTFTVEARNGVSGLVTS--RSFRTASVSINQTEPPKV---RLEGRSTTS 428 SP|P21709|EPHA1_HUMAN VHVNGLEPYANYTFNVEAQNGVSGLGSS-GHASTSVSISMGHAESLSGLSLRLVKKEPRQ 437 SP|P29320|EPHA3_HUMAN VTVTDLLAHTNYTFEIDAVNGVSELSSP-PRQFAAVSITTNQAAPSPVLTIKKDRTSRNS 432 SP|P54764|EPHA4_HUMAN VSITDLLAHTNYTFEIWAVNGVSKYNPN-PDQSVSVTVTTNQAAPSSIALVQAKEVTRYS 437 SP|P54756|EPHA5_HUMAN VMMVDLLAHTNYTFEIEAVNGVSDLSPG-ARQYVSVNVTTNQAAPSPVTNVKKGKIAKNS 460 SP|Q9UF33|EPHA6_HUMAN VIVLDFVSHVNYTFEIEAMNGVSELSFS-PKPFTAITVTTDQDAPSLIGVVRKDWASQNS 436 SP|Q15375|EPHA7_HUMAN VTVMDLLAHANYTFEVEAVNGVSDLSRS-QRLFAAVSITTGQAAPSQVSGVMKERVLQRS 431 SP|P29322|EPHA8_HUMAN LLVANLLAHMNYSFWIEAVNGVSDLSPE-PRRAAVVNITTNQAAPSQVVVIRQERAGQTS 428 SP|Q5JZY3|EPHAA_HUMAN ATLLHLRPGARYTVRVAALNGVSGPAAAAGTTYAQVTVSTGPGAPWEEDEIRRDRVEPQS 436 : : .*:. : * **** .:: . .

SP|P29317|EPHA2_HUMAN LSVSWSIPPP---QQSRVWKYEVTYRKKG-DSNSYNVRRTEGFSVTLDDLAPDTTYLVQV 484 SP|P21709|EPHA1_HUMAN LELTWAGSRPR--SPGANLTYELHVLNQD--EERYQMV--LEPRVLLTELQPDTTYIVRV 491 SP|P29320|EPHA3_HUMAN ISLSWQEPEH---PNGIILDYEVKYYEKQEQETSYTILRARGTNVTISSLKPDTIYVFQI 489 SP|P54764|EPHA4_HUMAN VALAWLEPDR---PNGVILEYEVKYYEKDQNERSYRIVRTAARNTDIKGLNPLTSYVFHV 494 SP|P54756|EPHA5_HUMAN ISLSWQEPDR---PNGIILEYEIKYFEK-DQETSYTIIKSKETTITAEGLKPASVYVFQI 516 SP|Q9UF33|EPHA6_HUMAN IALSWQAPAF---SNGAILDYEIKYYEKEHEQLTYSSTRSKAPSVIITGLKPATKYVFHI 493 SP|Q15375|EPHA7_HUMAN VELSWQEPEH---PNGVITEYEIKYYEKDQRERTYSTVKTKSTSASINNLKPGTVYVFQI 488 SP|P29322|EPHA8_HUMAN VSLLWQEPEQ---PNGIILEYEIKYYEKDKEMQSYSTLKAVTTRATVSGLKPGTRYVFQV 485 SP|Q5JZY3|EPHAA_HUMAN VSLSWREPIPAGAPGANDTEYEIRYYEKGQSEQTYSMVKTGAPTVTVTNLKPATRYVFQI 496 : : * . **: :: * * * : *:.::

SP|P29317|EPHA2_HUMAN QALTQEG---QGAGSKVHEFQTLSPEGSGNLAV------514 SP|P21709|EPHA1_HUMAN RMLTPLG---PGPFSPDHEFRTSPPVSRGLTGGE------522 SP|P29320|EPHA3_HUMAN RARTAAG---YGTNSRKFEFETSPDSFSISGESS-----Q--- 521 SP|P54764|EPHA4_HUMAN RARTAAG---YGDFSEPLEVTTNTVPSRIIGDGA-----NST- 528 SP|P54756|EPHA5_HUMAN RARTAAG---YGVFSRRFEFETTPVFAASSDQSQ-----IP-- 549 SP|Q9UF33|EPHA6_HUMAN RVRTATG---YSGYSQKFEFETGDETSDMAAEQG-----QILV 528 SP|Q15375|EPHA7_HUMAN RAFTAAG---YGNYSPRLDVATLEEATGKMFEATAVSSEQNPV 528 SP|P29322|EPHA8_HUMAN RARTSAG---CGRFSQAMEVETGKPRPRYDTRT------515 SP|Q5JZY3|EPHAA_HUMAN RAASPGPSWEAQSFNPSIEVQTLGEAASGSRDQS------PA 532 : : . :. *

145

Appendix 3. The percentage of homology between EphA2-ECD and other EphA-ECDs using Clustal Omega (EMBL-EBI, https://www.ebi.ac.uk/Tools/msa/clustalo/).

Percent Identity EphA1-ECD 47.43 EphA3-ECD 46.76 EphA4-ECD 45.01 EphA2-ECD EphA5-ECD 45.78 EphA6-ECD 44.51 EphA7-ECD 46.09 EphA8-ECD 44.99 EphA10-ECD 44.20

146

Appendix 4. Screen capture of the MS-MS results for human and canine EphA2-ECD. Peptides in green represent confident peptide matches, red = poor match, grey = unmatched and yellow = moderate confidence.

Canine EphA2-ECD

Human EphA2-ECD

147

Supplementary for chapter 4

Appendix 5: Multiple sequence alignment of EphA-LBDs using CLUSTAL O (1.2.4)

sp|P21709|EphA1-LBD27-209 EVTLMDTSKAQGELGWLLDPPKDGWSEQQQIL-NGTPLYMYQDCPMQGRRDTDHWLRSNW 59 sp|P29317|EphA2-LBD28-206 EVVLLDFAAAGGELGWLTHPYGKGWDLMQNIM-NDMPIYMYSVCNVMS-GDQDNWLRTNW 58 sp|O09127|EphA8-LBD30-208 EVNLLDTSTIHGDWGWLTYPA-HGWDSINEVDESFRPIHTYQVCNVMS-PNQNNWLRTNW 58 sp|P29320|EphA3-LBD29-207 EVNLLDSKTIQGELGWISYPS-HGWEEISGVDEHYTPIRTYQVCNVMD-HSQNNWLRTNW 58 sp|Q9UF33|3EphA6-LBD4-212 QVVLLDTTTVLGELGWKTYPL-NGWDAITEMDEHNRPIHTYQVCNVME-PNQNNWLRTNW 58 sp|Q5JZY3|EphA10-LBD35-216 EVILLDSKASQAELGWTALPS-NGWEEISGVDEHDRPIRTYQVCNVLE-PNQDNWLQTGW 58 sp|P54764|EphA4-LBD30-209 EVTLLDSRSVQGELGWIASPLEGGWEEVSIMDEKNTPIRTYQVCNVME-PSQNNWLRTDW 59 sp|P54756|EphA5-LBD60-238 EVNLLDSRTVMGDLGWIAFPK-NGWEEIGEVDENYAPIHTYQVCKVME-QNQNNWLLTSW 58 sp|Q15375|EphA7-LBD32-210 EVLLLDSKAQQTELEWISSPP-NGWEEISGLDENYTPIRTYQVCQVME-PNQNNWLRTNW 58 sp|P21709|EphA1-LBD27-209 IYRGEEASRVHVELQFTVRDCKSFPGGAGPLGCKETFNLLYMESDQDVGIQL---RRPLF 116 sp|P29317|EphA2-LBD28-206 VYRGE-AERIFIELKFTVRDCNSFPGGAS--SCKETFNLYYAESDLDYGTNF---QKRLF 112 sp|O09127|EphA8-LBD30-208 VPRDG-ARRVYAEIKFTLRDCNSIPGVLG--TCKETFNLHYLESDRDLGAST---QESQF 112 sp|P29320|EphA3-LBD29-207 VPRNS-AQKIYVELKFTLRDCNSIPLVLG--TCKETFNLYYMESDDDHGVKF---REHQF 112 sp|Q9UF33|3EphA6-LBD4-212 ISRDA-AQKIYVEMKFTLRDCNSIPWVLG--TCKETFNLFYMESDESHGIKF---KPNQY 112 sp|Q5JZY3|EphA10-LBD35-216 ISRGR-GQRIFVELQFTLRDCSSIPGAAG--TCKETFNVYYLETEADLGRGRPRLGGSRP 115 sp|P54764|EphA4-LBD30-209 ITREG-AQRVYIEIKFTLRDCNSLPGVMG--TCKETFNLYYYESDNDKERFI---RENQF 113 sp|P54756|EphA5-LBD60-238 ISNEG-ASRIFIELKFTLRDCNSLPGGLG--TCKETFNMYYFESDDQNGRNI---KENQY 112 sp|Q15375|EphA7-LBD32-210 ISKGN-AQRIFVELKFTLRDCNSLPGVLG--TCKETFNLYYYETDYDTGRNI---RENLY 112

sp|P21709|EphA1-LBD27-209 QKVTTVAADQSFTIRDLVSGSVKLNVERCSLGRLTRRGLYLAFHNPGACVALVSVRVFYQ 176 sp|P29317|EphA2-LBD28-206 TKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYK 172 sp|O09127|EphA8-LBD30-208 LKIDTIAADESFTGADLGVRRLKLNTEVRGVGPLSKRGFYLAFQDIGACLAILSLRIYYK 172 sp|P29320|EphA3-LBD29-207 TKIDTIAADESFTQMDLGDRILKLNTEIREVGPVNKKGFYLAFQDVGACVALVSVRVYFK 172 sp|Q9UF33|3EphA6-LBD4-212 TKIDTIAADESFTQMDLGDRILKLNTEIREVGPIERKGFYLAFQDIGACIALVSVRVFYK 172 sp|Q5JZY3|EphA10-LBD35-216 RKIDTIAADESFTQGDLGERKMKLNTEVREIGPLSRRGFHLAFQDVGACVALVSVRVYYK 175 sp|P54764|EphA4-LBD30-209 VKIDTIAADESFTQVDIGDRIMKLNTEIRDVGPLSKKGFYLAFQDVGACIALVSVRVFYK 173 sp|P54756|EphA5-LBD60-238 IKIDTIAADESFTELDLGDRVMKLNTEVRDVGPLSKKGFYLAFQDVGACIALVSVRVYYK 172 sp|Q15375|EphA7-LBD32-210 VKIDTIAADESFTQGDLGERKMKLNTEVREIGPLSKKGFYLAFQDVGACIALVSVKVYYK 172

sp|P21709|EphA1-LBD27-209 RCPETLN 183 sp|P29317|EphA2-LBD28-206 KCPELLQ 179 sp|O09127|EphA8-LBD30-208 KCPAMVR 179 sp|P29320|EphA3-LBD29-207 KCPFTVK 179 sp|Q9UF33|3EphA6-LBD4-212 KCPFTVR 179 sp|Q5JZY3|EphA10-LBD35-216 QCRATVR 182 sp|P54764|EphA4-LBD30-209 KCPLTVR 180 sp|P54756|EphA5-LBD60-238 KCPSVVR 179 sp|Q15375|EphA7-LBD32-210 KCWSIIE 179

148

Appendix 6. The percentage of homology between EphA2-LBD and other EphA-ligand binding domains.

Percent Identity

EphA1- LBD 55.87

EphA3- LBD 57.87

EphA4- LBD 55.31

EphA2-LBD EphA5- LBD 56.18

EphA6- LBD 56.74

EphA7- LBD 56.18

EphA8- LBD 57.30

EphA10- LBD 53.37

Appendix 7. Screen capture of the MS-MS results for human and canine EphA2-LBD. Peptides in green represent confident peptide matches, red = poor match, grey = unmatched and yellow = moderate confidence.

Human EphA2-LBD

QGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDN WLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRL FTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYK KCPHHHHHH 149

Canine EphA2-LBD

QGKEVVLLDFAAAKGELGWLTHPYGKGWDLMQNIMDDMPIYMYSVCNVVAGDQDN WLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDVDYGTNFQKRQ FTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLSRKGFYLAFQDIGACVALLSVRVYY KKCPHHHHHH

Supplementary for chapter 5

Appendix 8. Alignment of cysteine-rich domain among EphA family receptors using Clustal Omega (EMBL-EBI, https://www.ebi.ac.uk/Tools/msa/clustalo/).

sp|P21709|EphA1-Cys-191-329 CVALVSVRVFYQRCPETLNGLAQFPDTLPGP--AGLVEVAGTCLPHARASPRPSGAPRMH 58

sp|P29317|EphA2-Cys-188-325 CVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVP-PGGEEPRMH 59

sp|Q15375|EphA7-Cys-192-328 CIALVSVKVYYKKCWSIIENLAIFPDTVTGSEFSSLVEVRGTCVSSAEE--EAENAPRMH 58

sp|P29322|EphA8-Cys-191-325 CLAILSLRIYYKKCPAMVRNLAAFSEAVTGADSSSLVEVRGQCVRHSEE----RDTPKMY 56

sp|P54756|EphA5-Cys-220-354 CIALVSVRVYYKKCPSVVRHLAVFPDTITGADSSQLLEVSGSCVNHSVT----DEPPKMH 56

sp|P29320|EphA3-Cys-189-322 CVALVSVRVYFKKCPFTVKNLAMFPDTVP-MDSQSLVEVRGSCVNNSKE----EDPPRMY 55

sp|P54764|EphA4-Cys-191-325 CIALVSVRVFYKKCPLTVRNLAQFPDTITGADTSSLVEVRGSCVNNSEE----KDVPKMY 56

sp|P21709|EphA1-Cys-191-329 CSPDGEWLVPVGRCHCEPGYEEGGSGEACVACPSGSYRMDMDTPHCLTCPQQSTAESEGA 118 sp|P29317|EphA2-Cys-188-325 CAVDGEWLVPIGQCLCQAGYEKV--EDACQACSPGFFKFEASESPCLECPEHTLPSPEGA 117 sp|Q15375|EphA7-Cys-192-328 CSAEGEWLVPIGKCICKAGYQQK--GDTCEPCGRGFYKSSSQDLQCSRCPTHSFSDKEGS 116

sp|P29322|EphA8-Cys-191-325 CSAEGEWLVPIGKCVCSAGYEER—RDACVACELGFYKSAPGDQLCARCPPHSHSAAPAA 150

114 sp|P54756|EphA5-Cys-220-354 CSAEGEWLVPIGKCMCKAGYEEK—NGTCQVCRPGFFKASPHIQSCGKCPPHSYTHEEAS 114 sp|P29320|EphA3-Cys-189-322 CSTEGEWLVPIGKCSCNAGYEER--GFMCQACRPGFYKALDGNMKCAKCPPHSSTQEDGS 113 sp|P54764|EphA4-Cys-191-325 CGADGEWLVPIGNCLCNAGHEER--SGECQACKIGYYKALSTDATCAKCPPHSYSVWEGA 114 sp|P21709|EphA1-Cys-191-329 TICTCESGHYRAPGEGPQVAC 139

sp|P29317|EphA2-Cys-188-325 TSCECEEGFFRAPQDPASMPC 138

sp|Q15375|EphA7-Cys-192-328 SRCECEDGYYRAPSDPPYVAC 137

sp|P29322|EphA8-Cys-191-325 QACHCDLSYYRAALDPPSSAC 135

sp|P54756|EphA5-Cys-220-354 TSCVCEKDYFRRESDPPTMAC 135

sp|P29320|EphA3-Cys-189-322 MNCRCENNYFRADKDPPSMAC 134

sp|P54764|EphA4-Cys-191-325 TSCTCDRGFFRADNDAASMPC 135

Appendix 9. The percentage of homology between EphA2-CRD and other EphA-Cysteine-rich domains

Percent Identity

EphA1-CRD 49.26

EphA3-CRD 50.00

EphA4-CRD 53.33

EphA2- EphA5-CRD 52.59

CRD EphA7-CRD 48.91

EphA8-CRD 45.93

151