Alkhalifah, Mohammed Ali (2007) Recombinant antibodies against Clostridium difficile toxin A. PhD thesis. http://theses.gla.ac.uk/2156/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Recombinant antibodies against Clostridium difficile Toxin A University Of Glasgow Faculty Biomedical Life Sciences of and Division Infection Immunity of and June 2007 Mohammed Ali AlKhalifah Supervisor: Dr. R. Aitken A thesis for the degree submitted of Doctor of ý, j6hilosqphy Author's declaration This thesis is the the original work of author except where otherwise stated Mohammed A. AlKhalihfah Acknowledgments I best Dr. Robert thanks to express my gratitude and most sincere my supervisor Aitken for his continued encouragement, supervision and profound enthusiasm, discussion, this thesis. stimulating patience and reading of, and commenting on I help Dr. Gill Douce greatly appreciate and acknowledge the of as my assessor for her helpful advice. I to thank Mehdi Golchin Subash Verma for helpful discussions, wish and friendliness humour. and I thak must sincerely my parents, my wife and my children whose support and have helpted thoughout encouragement me my studies. ,7 1 thank friend Ali Abdul Jabbar Al Homood for his also my cusin and efforts and before during this continuing support and work. My thanks to infection immunity for their help also go all and staff members and during the collaboration work Finally, I thank the Ministry Health, Saudi Arabia for financial of my support and to this allowing me carry out research work. Table Contents of Chapter I General Introduction I General Introduction 3 1.1 Pathogenicity 8 1.2 Toxin structure 1.3 Toxin mode of action 9 1.3.1 Receptor binding and receptor 11 1.3.2 Toxin A to the entry cell 12 1.3.3 Toxin function A- 15 difficile Toxin 1.4 Humoral inumme Clostridium and cellular response against 16 1.5 Methods for control 16 1.5.1 Antibiotics 18 1.5.2 Immunotherapy 22 1.6 Toxinotypes importance and strains of 26 1.7 Antibodies CDR and conformation 27 1.8 Engineered antibodies 28 1.8.1 Humanization of antibodies -- 1.8.1.1 Chimeric antibodies _28 29 1.8.1.2 CDR- grafted antibodies 29 1.8.1.3 Transgenic Antibodies 30 1.9 Recombinant Antibodies display and phage 33 1.9.1 Creation libraries of antibody 33 1.9.1.1 Immune libraries -1 33 1.9.1.2 Non-immune libraries 34 1.9.1.3 Synthetic libraries 35 1.9.2 Formats for in display antibodies phage 36 1.10 Phage display 41 1.10.1 Selection Phage of antibody 42 1.10.2 Screening of phage antibodies 43 1.10.3 Antibody Expression 1.10.4 Affinity maturation _44 45 1.11 Aims 46 Chapter 2 46 MaterWs and methods 47 Materials and methods 47 2.1 Bacterial strain 47 2.2 Confirmation insert Toxin A C-terminal of encoding region 48 2.3 Growth bacteria the Toxin A of and expression of carboxy-terminal region _ 48 2.4 Purification the by of protein affinity chromatography 49 2.5 Protein assay 49 2.6 SDS-PAGE analysis 50 2.7 Electroblotting 2.8 Recognition in ELISA 50 51 2.9 Binding Toxin A to of native and recombinant cellular receptors 52 2.10 Haemagglutination Toxin A activity of recombinant 52 2.11 Sensitivity F9 Vero lines to Toxin A of and cell native . IV 53 2.12 Kinetics Toxin A F9 Vero of action of native on and cell Toxin A 2.13 Inhibition Toxin A and recombinant of native with polyclonal anti-Toxin 53 A in vitro 54 2.14 Inhibition Toxin A Fab fragments of native with polyclonal 54 2.14.1 Digestion A of polyclonal anti-Toxin antibody with papain 54 2.14.2 SDS-PAGE gel electrophoresis 54 2.14.3 Electroblotting 2.14.4 Detection Toxin A by Fab fragment in ELISA of native _55 55 2.14.5 Inhibition Toxin A Fab fragments in of native with polyclonal vitro 57 2.15 Tomlinson libraries scFv 61 2.15.1 KM13 helper phage, 63 2.15.2 Objectives 63 2.15.3 Tomlinson Library 1 63 2.15.3.1 Propagation KM13 helper of phage 64 2.15.3.2 Titration KM 13 helper of phage stock 65 2.15.3.3 Growing library the 65 2.15.3.4 Titration libraries of phage 66 2.15.3.5 Growing TG1 HB2151 and cells 66 2.15.4 Experiments before selection 66 2.15.4.1 Growing positive controls 66 2.15.4.2 Production of soluble positive control 2.15.4.3 Detection by Western blot 67 of anti-BSA and anti-ubiquitin scFvs 67 2.16 Antibody selection 2.16.1 Panning library I C- terminal domain of Toxin A 67 against purified recombinant 67 2.16.1.1 First round of selection 68 Infecting TG I cell with eluted phage antibodies 69 Rescue of selected phage antibodies 69 Titration of rescued phage 70 2.16.1.2 Second round of selection 70 2.16.1.3 Third round of selection 70 2.16.2 Screening by ELISA phage 1 70 2.16.2.1 Screening by ELISA (polyclonal ELISA) phage particles 71 2.16.2.2 Screening by ELISA (monoclonal ELISA) phage particles 71 Monoclonal Phage ELISA U__ 2.17 Production fragments 72 of soluble antibody 2.18 Genetic 74 characterization of selected scFvs 74 2.18.1 Plasmid digestion 2.18.2 Sequence 74 of selected clones 2.19 SDS-PAGE Western blot 75 and analysis of selected clones 2.20 High level 75 expression and purification of soluble scFv 2.20.1 Purification by 76 the of scFvs affinity chromatography - 2.21 Biological immunochemical 77 activity and characterization of purified scFv _ 2.21.1 Recognition Toxin A by 77 of native selected scFv 2.21.2 Haernagglutination inhibition 77 assay 2.21.2.1 Preparation Rabbit 78 of erythrocytes 2.21.2.2 Haernagglutination inhibition 78 2.21.3 Rabbit haemolysis 78 erythrocyte assay 2.21.4 Indirect immunofluorescent 79 fixed test antibody with cells 2.21.5 Indirect immunofluorescent 80 in test antibody with cells suspension _ 2.21.6 SDS-polyacrylamide 81 Western blotting gel electrophoresis and 2.21.7 Expression levels 82 of selected scFvs 2.22 Epitope 82 analysis 2.22.1 Epitope 83 saturation curve 2.22.2 Epitope 84 different saturation experiment using two scFvs 2.22.3 Blocking 86 by of monoclonal phage antibody epitopes scFv of the same specificity _ 2.22.3.1 Preparation 86 of phage antibodies 2.22.3.2 Optimization 87 blocking by ELISA of epitope 2.22.3.3 88 Blocking by of phage antibody scFv V 88 2.23 Assay for toxin neutralizing activity 88 2.23.1 Flow binding toxin cytometric analysis of 89 2.23.2 Inhibition Toxin A in of cytotoxicity vitro 89 2.23.2.1 Preparation F9 line of cell 89 2.23.2.2 Addition Toxin A- of antibody mixtures 90 2.24 Epitope mapping 90 2.24.1 Materials and methods 2.24.1.1 Bacteria and plasmids 90 2.24.1.2 Preparation of pET767 91 2.24.2 Polymerase chain reaction , 91 2.24.2.1 PCR primers 94 2.24.2.2 PCR reactions 95 2.24.3 Elution DNA from and purification of agarose gels 95 2.24.4 Restriction digestion ligation enzyme and 96 2.24.5 DNA sequencing 98 2.24.6 Cloning 98 2.24.6.1 Cloning into 2.1 pCR vector -TOPO 98 2.24.6.2 Cloning into pCG806 98 2.24.6.3 Ligation 99 2.24.6.4 Transformation 99 2.24.6.5 Preparation DNA duplex of synthetic 99 2.24.6.6 Phosphorylation of annealed oligonucleotides 100 2.25 Protein expression 100 2.25.1 SDS-PAGE and electrobloting 102 Chapter 3 102 Results 103 3 Results 3.1 Confirmation Toxin A C-terminal binding domain 103 of sequences encoding - 3.2 Purification the by 105 of protein affinity chromatography 3.3 SDS-electrophoresis 105 ý 3.4 Electroblotting 107 analysis 3.5 Recognition in ELISA 107 3.6 Binding Toxin A to 110 of native and recombinant cellular receptors 3.7 Haemagglutination Toxin A 110 activity of recombinant 3.8 Sensitivity F9 Vero lines Toxin A 113 to of and cell native 3.9 Kinetics Toxin A F9 Vero lines 113 of action of native on and cell 3.10 Inhibition 114 the Toxin A of action of native 3.11 Inhibition Toxin A Fab fragments 119 of native with polyclonal 3.11.1 Digestion 119 A of polyclonal anti-Toxin antibody with papain 3.11.1.1 SDS-PAGE gel electrophoresis 3.11.2 Electroblotting 122 3.11.3 Detection Toxin A by Fab fragment in ELISA 122 of native 3.11.4 Inhibition Toxin A Fab fragments in 125 of native with polyclonal vitro 3.12 Tomlinson library 125 scFv 3.12.1 Growing library 125 the 3.12.2 Detection 127 by Western blot of anti-BSA and anti-ubiquitin scFvs 3.12.3 Phage display against the C-terminal domain Toxin A 127 of 3.12.4 Screening 130 by ELISA polyclonal phage 3.12.5 Screening 130 by ELISA monoclonal phage 3.12.6 Screening 133 by ELISA monoclonal soluble scFv antibodies 3.13 Genetic 135 characterization of selected scFvs . 3.14 Sequence 135 of selected clones 136 3.15 Detection Toxin A by of native selected scFv 143 3.16 Haernagglutination inhibition 143 3.17 Haemolysis assay 145 3.18 Indirect immunofluorescent fixed test antibody with cells 3.19 Indirect immunofluorescent 147 test antibody with suspended cells 3.20 Characterization icFvs 149 of selected 3.20.1 SDS-polyacrylamide Westem blotting 149 gel electrophoresis and 3.21 Expression levels 149 of selected scFvs 3.22 Purification the by 152 of scFvs affinity chromatography 3.23 Epitope 153 saturation curve 3.24 Epitope two different 153 saturation experiment using scFvs 3.25 Competitive ELISA 157 with monoclonal phage antibodies and scFv 3.25.1 Preliminary 157 experiments 3.25.2 Competitive ELISA 160 experiments 3.26 Flow Toxin A-receptor interaction 162 cytometric analysis of 3.27 Inhibition Toxin A by 164 of cytotoxicity scFvs 3.28 Epitope 167 mapping 3.28.1 Cloning three Toxin A into 2.1 167 of regions of pCR vector -TOPO 3.28.1.1 Restriction 167 analysis 3.28.2 Cloning Toxin A fragments into 171 a maltose-binding protein vector 3.28.3 Cloning DNA linker into MBP 171 of pCG806 vector 3.29 Characterisation MBP fusions 177 of Chapter 4 1025 4 Discussion 185 . 4.1 Purification C-terminal domain Toxin A and characterization of recombinant of 185 4.2 Screening the Tomlinson library 192 of 4.3 Biological A 195 activity of anti-Toxin scFvs. 4.4 Epitope A 200 mapping of anti-Toxin scFv 4.5 Conclusion 202 5 Appendices 204 5.1 Luria-Bertani (LB) broth 204 or agar 5.2 Preparation 204 of antibiotic solutions 5.3 SOC 204 medium 5.4 Citric buffer 205 acid phosphate 6 Bibliography 206 List tables of 24 Table 1.1: RFLP for typing toxin characteristics used of genes Table 1.2: C. difficile 25 toxin strains with variant genes 1- Table 2.1: Inhibition Toxin A Fab fragments in of native with polyclonal vitro design: 56 experimental Table 2.2: Design 85 of epitope saturation experiment Table 2.3: Bacterial for 92 strains used epitope mapping Table 2.4: List for of primers used epitope mapping and characterization of scFv 93 genes Table 2.5: List 97 their target of restriction enzymes and sequences Table 3.1: Recoveries during 129 of phage selection Table 3.2 List the the in : of primer pairs and predicted size of products used cloning four Toxin A 168 of regions of Table 3.3: List the detected fragments 183 of scFvs and List figures of 14 Figure 1.1: Summary Toxin A the of proposed mode of action of 32 Figure 1.2: The formats various of engineered antibodies Figure 2.1: Structure 59 of phagemid pIT2 60 Figure 2.2: DNA sequence of pIT2 vector Figure 2.3 Principle (Goletz 2002) 62 : of proteolytic selection et al., Figure 3.2: Complete Toxin A from nucleic acid sequence encoding recombinant 104 pET767 Ll-! - Figure 3.3: SDS-PAGE Toxin A by analysis of recombinant affinity chromatography 106 Figure 3.4: Western blotting Toxin A by analysis of recombinant purified affinity 108 chromatography Figure 3.5: Detection Toxin A in ELISA. 109 of recombinant Figure 3.6: Immunofluorescence detect binding to the assay of recombinant and Toxin A F9 ill to mative receptors on cells Figure 3.7: Haemagglutination Toxin A 112 activity of recombinant Figure 3.8: Sensitivity F9 lines to Toxin A 115 of cell native Figure 3.9: Sensitivity F9 lines to Toxin A 116 of cell native Figure 3.10: Sensitivity Vero lines to Toxin A 117 of cell native Figure 3.11: Effective time the Toxin A F9 of exposure on action of native on and 'Yero 118 cells. Figure 3.12: Inhibition Toxin A by A 120 of polyclonal anti-Toxin and rTA Figure 3.13: Non SDS-PAGE digested A reducing analysis of papain anti-Toxin antiserum. 121 Ll- Figure 3.14: Western blotting digested Toxin A analysis of papain anti antiserum -123 Figure 3.15: Reaction intact IgG Fab fragments ELISA of Toxin A in and with native r 124 Figure 3.16: Inhibition Toxin A by digested A of papain polyclonal anti-Toxin antiserum. 126 , Figure 3.17: Polyclonal ELISA phage Tomlinson library I during of against panning recomninantToxin A 131 ,