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Beyond Antibodies: Development of a Novel Molecular Scaffold Based on Human Chaperonin 10

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Beyond Antibodies: Development of a Novel Molecular Scaffold Based on Human Chaperonin 10 Beyond Antibodies: Development of a Novel Molecular Scaffold Based on Human Chaperonin 10 Abdulkarim Mohammed Alsultan B.Pharm, M.Biotech A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Australian Institute for Bioengineering and Nanotechnology (AIBN) Abstract This study reports the development of a new molecular scaffold based on human Chaperonin 10 (hCpn10), for the development of protein-based new molecular entities (NMEs) of diagnostic and therapeutic potential. The aims were to establish the fundamental basis of a molecular design for the scaffold, to enable the display of non-native peptides with binding activity to selected targets, while maintaining structural integrity and native heptameric conformation. Recently there has been much global interest in developing NMEs of protein and peptide origin, as therapeutic agents and diagnostic reagents. A class of NMEs are based on molecular scaffolds, whereby peptides with binding activity to a given target are displayed on a protein backbone or scaffold. The molecular scaffold provides a rigid folding unit which spatially brings together several exposed peptide loops, forming an extended interface that ensures tight binding of the target. Monoclonal antibodies (mAbs) are in essence molecular scaffolds, whereby complementarity determining regions, otherwise known as CDR peptide loops, extend from a framework and contact antigen. mAbs have been widely utilised in the life sciences and biopharmaceutical industries for the development of diagnostic probes and biologic medicines, respectively. However the current patent landscape surrounding mAbs is complex and commercialisation of newly developed antibodies can be hampered by licensing agreements and royalty stacking. Molecular scaffolds are an alternative to antibodies, and some of the scaffolds in development include lipocalins, fibronectin domain, DARPins consensus repeat domain and avimers, to name a few. hCpn10 is a homo-oligomer composed of seven subunits. Each hCpn10 monomer is composed of a β-barrel core structure that is flanked by two flexible loops, known as the β-hairpin and mobile loop. It is proposed the mobile could be substituted with peptides and so increase the apparent affinity to a given target compared to that of naked peptides, due to avidity conferred by the heptameric structure. This study focussed on assessing the properties of hCpn10 and determining whether this molecule could be utilised as a scaffold for the development of NMEs that have binding activity to selected targets. i In the first example of the utility of hCpn10 as a molecular scaffold, the E-76 peptide, which binds to Factor VIIa (FVIIa) of the extrinsic coagulation cascade pathway, was substituted into the mobile loop region of hCpn10 creating a NME designated CE76 with anticoagulant activity. Anticoagulants prevent blood from clotting and have clinical applications associated with coronary heart disease and stroke. Common anticoagulants include heparin, warfarin, and dicoumarol. Although these compounds have proved beneficial they are not without complications and side effects. An NME has been created through display of the E-76 peptide on the hCpn10 scaffold, creating the highly avid CE76, which binds FVIIa with sub- nanomolar affinity. Prior to creation of CE76 NME, molecular dynamic (MD) simulation studies were conducted in order to optimise linkers at the N- and C-junctures of the mobile loop, so as to optimise mobile loop orientation and conserve native-like heptamer formation. Simulation studies revealed that inserting proline residues at both ends of the mobile loop region stabilized the new CE76 variants (named CE76P1) and facilitated heptamer assembly. Characterisation by size exclusion chromatography confirmed CE76P1 formed a stable heptamer, and bound specifically to the exosite of FVIIa, and subsequently inhibited the activation of Factor X (IC50 1.5 nM). CE76P1 prologed the extrinsic pathway 4-fold higher than E-76 alone, while not affecting the intrinsic coagulation cascade as determined by prothrombin time (PT) and thromboelastography (TEG) assays. CE76P1 has the potential to be a new biologic drug with potent anticoagulant properties. The utility of hCpn10 as a molecular scaffold was further assessed by incorporating P7 peptide into the mobile loop, creating a NME designated CP7. P7 peptide binds to CD44, which is overexpressed in cancers of the breast, colon, prostate and brain. Binding studies revealed that CP7 bound specifically to recombinant CD44 (KD 1.32 nM), while no binding was detected to surface protein CD24. Immunofluorescence imaging and flow cytometry studies showed increased binding activity of CP7 to breast adenocarcinoma MDA-MB-486 cells compared to P7 peptide alone. Further expression studies using peptides that bind VEGFR and FGFR, were incorporated into hCpn10 scaffold also resulted in correct assembly of heptamer. ii Based on these studies associated with the incorporation of selected peptides into the hCpn10 scaffold, it is evident that hCpn10 can be utilised as a molecular scaffold for the developing of NMEs with therapeutic and diagnostic potential. The demonstration that human Cpn10 is a useful molecular scaffold for the creation of NMEs in a “beyond antibody” approach offers the opportunity to create new intellectual property in the area of biologic medicines and bioanalytical reagents/diagnostics. iii 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, 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 research higher degree 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. iv Publications during candidature No publications Publications included in this thesis No publications included v Contributions by others to the thesis Dr. Herbert Treutlein, of Computist Bio-Nanotech, RMIT University, contributed to the research by conducting MD simulations presented in Chapter 3. Dr. Sharon Du of the Translational Research Institute (TRI), School of Medicine, University of Queensland, contributed in designing clotting assays presented in Chapter 4. Ms. Dinora Roche Recinos contributed to CR2VEGF-P2 expression in Chapter 6. Statement of parts of the thesis submitted to qualify for the award of another degree None vi Acknowledgements “The one who is not thankful for the few blessings will not be thankful for the many, and the one who is not thankful to the people will not be thankful to Allah” Prophet Mohammed (Peace be upon him and his progeny) First and foremost, I am grateful and thankful to Almighty Allah “GOD” for His limitless mercy, as well as to the Messenger of Allah Prophet Mohammed and his progeny peace be upon them all for surrounding me with infinite blessing. I express my deepest thanks to my guardians, supporters and advisors Associate Professor Stephen Mahler (principal supervisor) and Dr. David Chin (co-supervisor) for their encouragements and continued guidance during my study. It has been a privilege to work with you, to be under your supervisions and to learn more from you both. To all my colleagues past and current members of Gray Group and National Biologics Facility (NBF) at the Australian Institute for Bioengineering and Nanotechnology (AIBN), especially: Professor Peter Gray (Group leader), Martina Jones, Christopher Howard, Sumukh Kumble, Matthew Smede, Vanessa Sandford, Liam Nolan, Christopher de Bakker, Karin Taylor, Kar Man Leung, Jeff Hou, Kebaneilwe Lebani, Marianne Gillard, Camila Orellana, Dinora Roche Recinos, Emily Chan, Gency Gunasingh, Jay Heise-Seabrook, Jiang Eric Zhu, Kym Hoger, Lyndon Raftery, Michael Song, Michael Yeh, Mohammed AlFaleh, Neetika Arora, Steve Goodall, Tim Ruder, Trent Munro, Veronica Martinez, Xiaoli Chen, Zainab Almansour, Nadya Shale, Edwin Huang, Aidan Bell, and David Crowley, “a small thing in your eyes is so big in main”, so thank you all for your help and your advices that you generously gave at various stages in my journey. Enormous thanks to Sumukh Kumble for assisting in proofreading most of my thesis, and special thanks to Andrea Schaller for helping in proofreading the modelling in Chapter 3, and thank you both for
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