SELECTION AND ENGINEERING OF ANTI-PANIE VHH SINGLE DOMAIN ANTIBODIES AND THEIR FUSION TO CELLULOSE BINDING MODULES A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph By TED INGEMAR JAKOB FJALLMAN In partial fulfilment of requirements for the degree of Doctor of Philosophy May, 2008 © Ted Ingemar Jakob Fjallman, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-42557-2 Our file Notre reference ISBN: 978-0-494-42557-2 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. 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Canada ABSTRACT SELECTION AND ENGINEERING OF ANTI-PANIE VHH SINGLE DOMAIN ANTIBODIES AND THEIR FUSION TO CELLULOSE BINDING MODULES Ted Ingemar Jakob Fjallman Advisor: University of Guelph, 2008 Professor J. Christopher Hall Plant pathogens from the genera Pythium, Phytophthora and Fusarium are responsible for 80% of root disease epidemics in hydroponic greenhouses worldwide. Many of these pathogens secrete virulence factors belonging to the Nepl-Like-Protein (NLP) family of necrosis-inducing proteins. This thesis describes the development of a platform, consisting of antibody-cellulose binding modules bound to cellulose, for combating epidemics of plants through the capture of PaNie (NLPPya) secreted by Pythium aphanidermatum. Four recombinant single domain antibodies (VHHS) with affinities to PaNie ranging from 800 nM to 20.2 uM were developed by selection from a naive ribosome display library and a hyperimmune phage display library. Three of these antibodies bind to a synthesized peptide containing the highly conserved NPP1 domain of the NLP family. All four antibodies have varied complementarity determining regions and framework regions representing three of the four VHH subfamilies. The genes of the three highest affinity VHHS were fused to a cellulose binding module (CBM) with an intervening thrombin cleavage site. All VHH-CBM fusions were functional and the highest affinity binder P10-CBM was analysed in more detail. P10-CBM could be purified directly from culture using cellulose beads and cleaved with thrombin to yield fully functional P10 VHH. P10-CBM was bound to paper to create a bioactive filter paper for the capture of PaNie. The data obtained suggest that the affinity of paper-bound P10- CBM is the same as for soluble P10-CBM. P10-CBM is also able to bind PaNie while attached to the cellulose surfaces of tobacco roots as shown by confocal laser scanning microscopy. Improvements of the affinity of the VHH-CBM-based platforms and the utility of the antibodies for the elucidation of NLP-associated disease are discussed. Features of PaNie and the VnH-PaNie interaction suggest that NLPs may be pore forming toxins. In the future, VHH-CBMS could be produced in situ by non-pathogenic biocontrol agents and may be used to remove pathogenic toxins from hydroponic systems used in greenhouses and space-based advanced life support systems. ACKNOWLEDGEMENTS "Science never ends, but our lives do. So, learn to pass on the torch" This thesis is dedicated to all the people who never hesitated to pass part of their life's torch to me and brighten my path. Thank you for the deep thoughts, the laughs and above all the patience and honesty. My development as a researcher would not have been so insightful were it not for Chris' trust and patience. I hope you realize what a gift you have given me, by letting me work things out my own way. Your impact on society through your teaching and guidance works on a deep level in the lives of everyone who has worked with you. You have taught me how to be an effective human being, not just a knowledgeable scholar. My gratitude also extends to my advisors, examiners and teachers throughout these years. Special thanks to Profs Michael D. McLean and John C. Sutton for giving expert advice for my laboratory and greenhouse work and for reading my thesis thoroughly. Mike, you always forced me to be humble about my science and I thank you for that. Drs Roger MacKenzie and Mehdi Arbabi-Gharoudi; you made it possible for me to push myself to the limit and focus like I had never focused before. Of course none of this would have been possible without the funding from Flowers Canada, CresTech, NSERC NRC, CRC Chair and Syngenta. What got me through the rough parts was no doubt the sisterly and brotherly companionship of the 2002-2008 Hall lab crew. You all give me great hope for the i future. Nina, you can slap me with bologni any day... seriously your humour kept me sane at times; Shokouh, your kindred spirit awakens my thirst for knowledge and my joy of sharing; Yongqing, your care and objectivity blend so harmoniously, I hope one day I will pick up some of that wisdom; Tsafrir, patience incarnate -just seeing you in the hallway had a soothing effect on me; Greg, your integrity is a role model for me; Adam, your honesty is so refreshing. The last three years of my PhD were frustrating and even painful at times, but Cindy, you made me see that painful events only turn into suffering if you let them. In fact, you have given me so much renewed energy to tackle problems, by helping me redefine what a problem is. You renewed my interest in philosophy and at the same time made me a more pragmatic scientist - a combination I have struggled to attain all my life. Last but not least, thank you to my family for a free upbringing that gave me the confidence and skills to be a freethinker. It is the fact that whenever we had discussions and got the encyclopedia out to settle disputes, which made me value objectivity as the one tool that can bridge the different perceptions in this world. Thanks to all of you, I am now resolved that I can positively contribute to the future of humanity. I have grown so much, but I have not forgotten the creative child in me; thus, I want to say "Tack" (thanks in Swedish) with the following illustration. It is intended to show that a little part of everyone contributed to this thesis. Congratulations to you! ii CO <y -T (D on '^ • «* O w Q - CD .£ ^ CD -~ S3 • rs 3 GO o - O EG rt -^ g ^ co :cd CO ^ f <-C>J a Ec o y & ^ I 2 g ° Z ^ c 5 ^ ffi CD ^ .r ST M CD » -PS -Si S3 O ? '^ 5t > C * r — 5b CO O > iii Table of Contents ACKNOWLEDGEMENTS i TABLE OF CONTENTS iv LIST OF TABLES vm LIST OF FIGURES ix LIST OF ABBREVIATIONS xin 1. GENERAL INTRODUCTION AND RATIONALE 1 2. LITERATURE REVIEW AND RESEARCH OBJECTIVES 5 2.1. Antibody technology 5 2.1.1. Conventional immunoglobulins 5 2.1.2. Recombinant antibodies 7 2.1.3. Recombinant antibody applications 11 2.1.4. Selection of recombinant antibodies from antibody libraries 14 2.1.5. Affinity maturation 19 2.1.6. Antibody expression systems 21 2.2. Cellulose-protein scaffolds 31 2.2.1. Structure of cellulose in plants 31 2.2.2. Enzymatic digestion of plant cellulose 33 2.2.3. Cellulose binding modules 35 2.3. Epidemiology of Pythium aphanidermatum 39 2.3.1. Physiology of Pythium aphanidermatum 39 2.3.2. Associated disease 41 2.3.3. Occurrence and pathogenicity 42 iv 2.3.4. Mechanism of action 45 2.3.5. Current detection and control strategies 50 2.4. Research objectives 54 3. SCREENING OF A NAIVE LLAMA RIBOSOME DISPLAY LIBRARY AGAINST AN NLP PEPTIDE 55 3.1. Introduction 55 3.2. Materials and methods 56 3.2.1. Peptide design & preparation 56 3.2.2. Selection of antibodies by ribosome display 57 3.2.3. Cloning and expression of VHH sequences 62 3.2.4. Cloning and expression of PaNie 63 3.2.5. Anti-peptide ELISA 65 3.2.6. Anti-PaNie inhibition ELISA 65 3.3. Results 66 3.3.1. NLPsg2 peptide characteristics 66 3.3.2. Ribosome display selection and sequence analysis 74 3.3.3.