TOWARDS THE DEVELOPMENT OF NOVEL BISPECIFIC ANTIBODIES TO INHIBIT KEY CELL SURFACE RECEPTORS INTEGRAL FOR THE GROWTH AND MIGRATION OF TUMOUR CELLS Andrew Lai Bachelor of Science, UNSW 2008 Master of Biotechnology, QUT 2010 Bachelor of Applied Science (Hons), QUT 2012 Submitted for the degree of Doctor of Philosophy Institute of Health and Biomedical Innovation Faculty of Health Queensland University of Technology 2016 Keywords Breast cancer, extracellular matrix, insulin-like growth factor, metastasis, migration, therapeutics, phage display, single chain variable fragments, vitronectin Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells i Abstract Metastatic breast cancer, or breast cancer which has spread from the primary tumour to distal secondary sites, remains a major killer of women today. Researchers have observed that the relationship between tumour cells and its surrounding environment plays an important role in cancer progression. One such interaction is between the Insulin-like growth factor (IGF) system and the integrin system, which has been demonstrated to be involved in cancer cell metabolic activity and migration. Therefore, the aim of this project was to translate this knowledge into the generation of bispecific antibody fragments (BsAb) targeting both systems, in order to disrupt their roles in cancer growth and metastasis. To screen for IGF-1R and αv integrin binding ScFv, a phage display enrichment procedure using the Tomlinson ScFv libraries was conducted. After the panning cycles, 192 clones were screened for binding using ELISA, of which 16 were selected for sequencing. Analysis of the results revealed 1 IGF-R and 3 αv integrin unique binding ScFv, which were all subsequently expressed in a bacterial expression system. The ScFv were purified to a high degree using Ni-NTA and protein-L agarose affinity chromatography and were further assessed using size exclusion chromatography to detect the presence of any ScFv aggregates. Functional characterisation studies using MCF-7 breast cancer cells indicated that the IGF-1R targeting ScFv was able to inhibit both IGF-I and VN dependent metabolic activity and migration. Cell-based assays were also used to characterise the anti-αv integrin ScFv, however, no inhibition of cell attachment or migration was observed. Investigation into possible reasons for the inability to inhibit cell processes revealed the original αv integrin sample used for panning may not have been fit for purpose. Thus, while the anti-IGF-1R ScFv shows potential, the lack of a functional anti-αv integrin ScFv made the generation of a bi-specific antibody fragment unachievable within this project. In light of these results, further characterisation should be conducted to assess the potential therapeutic treatment of breast cancer using the generated ScFv. ii Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells Table of Contents Keywords .................................................................................................................................. i Abstract .................................................................................................................................... ii Table of Contents .................................................................................................................... iii List of Figures ........................................................................................................................ vii List of Tables .......................................................................................................................... ix List of Abbreviations ................................................................................................................x Statement of Original Authorship ...........................................................................................xv Acknowledgements ............................................................................................................... xvi Literature Review ............................................................................. 1 1.1 Breast cancer ...................................................................................................................2 1.2 Targeted cancer therapies ...............................................................................................3 1.3 Insulin-like growth factor system ...................................................................................6 1.4 Integrins ..........................................................................................................................8 1.5 IGF-1R and αv integrin signalling pathway interaction .................................................9 1.6 IGF-1R and VN binding integrin targeting strategies ..................................................10 1.7 Antibody structure and fragmentation ..........................................................................14 1.8 Bispecific antibodies .....................................................................................................15 1.9 Antibody generation .....................................................................................................18 1.10 Phage display ................................................................................................................19 1.10.1 KM13 phage physiology and structure ...............................................................21 1.10.2 Phage display selection ......................................................................................22 1.11 Conclusion and knowledge gaps ..................................................................................23 1.12 Project outline ...............................................................................................................24 1.12.1 Hypothesis ..........................................................................................................24 1.12.2 Aims ...................................................................................................................24 Materials and Methods .................................................................. 26 2.1 Introduction ..................................................................................................................27 2.2 Materials .......................................................................................................................27 2.3 Cloning .........................................................................................................................27 2.3.1 General PCR .......................................................................................................27 2.3.2 Mini-prep plasmid isolation ...............................................................................28 2.3.3 Sanger sequencing of plasmids ..........................................................................29 2.4 Mammalian cell culture ................................................................................................30 2.4.1 Propagation of MCF-7 cell lines ........................................................................30 2.4.2 Cell counting ......................................................................................................30 2.4.3 Cryopreservation of cell lines .............................................................................30 2.4.4 Mycoplasma Testing ..........................................................................................31 2.5 Protein isolation from adherent cell cultures ................................................................32 Towards the development of novel bispecific antibodies to inhibit key cell surface receptors integral for the growth and migration of tumour cells iii 2.5.1 Denaturing conditions ........................................................................................ 32 2.5.2 Non-denaturing conditions ................................................................................ 32 2.6 Protein Quantification .................................................................................................. 33 2.7 Protein detection .......................................................................................................... 33 2.7.1 SDS-PAGE and Chemiluminescence Western .................................................. 33 2.7.2 Quantitative Westerns ........................................................................................ 34 2.7.3 Silver staining .................................................................................................... 34 Phage Display selection and screening.......................................... 36 3.1 Introduction .................................................................................................................. 37 3.2 Experimental procedures .............................................................................................. 40 3.2.1 Materials ............................................................................................................ 40 3.2.2 Phage display libraries ....................................................................................... 40 3.2.3 E. coli strain, media, and culture conditions ...................................................... 40 3.2.4 KM13 helper phage amplification ....................................................................