Molecular Characterization and Inhibition of Antibodies Elicited Against Galactosyltransferase ... PDF
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Loma Linda University TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works Loma Linda University Electronic Theses, Dissertations & Projects 6-2015 Molecular Characterization and Inhibition of Antibodies Elicited Against Galactosyltransferase Knockout Pig Xenografts John M. Stewart Follow this and additional works at:http://scholarsrepository.llu.edu/etd Part of theMedical Pharmacology Commons Recommended Citation Stewart, John M., "Molecular Characterization and Inhibition of Antibodies Elicited Against Galactosyltransferase Knockout Pig Xenografts" (2015).Loma Linda University Electronic Theses, Dissertations & Projects. 268. http://scholarsrepository.llu.edu/etd/268 This Dissertation is brought to you for free and open access by TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works. It has been accepted for inclusion in Loma Linda University Electronic Theses, Dissertations & Projects by an authorized administrator of TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works. For more information, please contact [email protected]. LOMA LINDA UNIVERSITY School of Medicine in conjunction with the Faculty of Graduate Studies ______________________ Molecular Characterization and Inhibition of Antibodies Elicited Against Galactosyltransferase Knockout Pig Xenografts by John M. Stewart ____________________ A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Pharmacology ___________________ June 2015 i © 2015 John M. Stewart All Rights Reserved ii Each person whose signature appears below certifies that this dissertation in his/her opinion is adequate, in scope and quality, as a dissertation for the degree Doctor of Philosophy. , Chairperson Mary Kearns-Jonker, Associate Professor of Pathology and Human Anatomy John N. Buchholz, Professor of Pharmacology Peter J. Cowan, Professor and Scientist Director, Immunology Research Center, St. Vincent’s Hospital, Melbourne Kerby C. Oberg, Professor of Pathology and Human Anatomy Kimberly J. Payne, Associate Professor of Pathology and Human Anatomy William J. Pearce, Professor of Physiology iii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my mentor, Dr. Mary Kearns- Jonker, for her direction, empathy, and patience. I am thankful to be given the opportunity to contribute to the scientific progress and the advancement of mankind. You have been guiding me through the trenches every step of the way. For this I will always be grateful. I would also like to thank my committee members for their help and guidance. They have been wonderful professors, collaborators, and even friends. Their mentorship has meant the world to me. I will always be thankful for, and fondly reminisce about, the comradery of my fellow lab members. The shared commissary and compatriotism of Nancy Appleby and Tania Fuentes. The friendship of Peter Gifford, Christine Molinaro, and the rest of the Alumni Hall staff. You have all been like family for the last four years. To my family and friends, in spite of my persistent tedium and self-involvement your love and support through this struggle has given me the strength to be tenacious. Thanks to my dad and Kelley for giving me the freedom and support to pursue my dreams. Thanks to my loving wife who has been with me every step of the way, supporting me with unceasing love, continual empathy, and perpetual manuscript, poster, and abstract revisions. This work has been published in the journal Xenotransplantation and funded, in part by NIH grant 7R01AI052079-06 (MKJ) and the Primate Center base operating grant #OD011107. iv CONTENT Approval Page .................................................................................................................... iii Acknowledgements ............................................................................................................ iv Table of Contents .................................................................................................................v List of Figures .................................................................................................................... ix List of Tables ..................................................................................................................... xi List of Abbreviations ........................................................................................................ xii Abstract .............................................................................................................................. iv Chapter 1. Introduction ..............................................................................................................1 Introduction to xenotransplantation ...................................................................1 Knocking out the galactosyltransferase gene .....................................................2 Anti-non-Gal antibodies.....................................................................................3 Non-α-1,3-Gal antigens .....................................................................................4 Antibody-mediated xenograft rejection .............................................................8 Islet xenotransplantation ............................................................................16 Renal xenotransplantation ..........................................................................16 Cardiac xenotransplantation .......................................................................17 Preamble ..........................................................................................................22 Chapter 2 – rationale and hypothesis .........................................................22 Chapter 3 – rationale and hypothesis .........................................................24 Chapter 4 – rationale and hypothesis .........................................................25 References……………………………………………………………………26 2. Xenoantibody response to porcine islet cell transplantation using GTKO, CD55, CD59, and fucosyltransferase multiple transgenic donors .........................36 Abstract ............................................................................................................38 Introduction ......................................................................................................40 Materials and Methods .....................................................................................41 v Preparation of cDNA libraries and analysis of immunoglobulin gene usage ..................................................................................................41 Flow cytometry ..........................................................................................46 Homology modeling ..................................................................................47 Results ..............................................................................................................47 Immunoglobulin heavy and light chain gene usage in untreated baboons ......................................................................................................47 Restricted xenoantibody response to porcine islet xenotransplantation ....................................................................................51 J segment usage……………………………………...…….......................59 Comparison of heavy chain structure in GTKO xenotransplantation ........60 Discussion ........................................................................................................64 Acknowledgements ..........................................................................................66 References ........................................................................................................67 3. Rhesus monkeys and baboons develop clotting factor VIII inhibitors in response to porcine endothelial cells or islets ........................................................70 Abstract ............................................................................................................72 Introduction ......................................................................................................74 Materials and Methods .....................................................................................75 Construction of an anti-non-Gal single chain xenoantibody .....................75 Expression and purification of single-chain antibody ...............................77 Animals ......................................................................................................77 Induction of anti-non-Gal xenoantibodies after immunization with porcine GTKO endothelial cells ................................................................78 Flow cytometry ..........................................................................................78 Detection of FVIII inhibitors .....................................................................79 Computational modeling ............................................................................79 Homology modeling ............................................................................80 In silico epitope prediction...................................................................80 Competition ELISA ...................................................................................81 In silico polyalanine scanning ....................................................................81 Statistics .....................................................................................................82 Results ..............................................................................................................82 The recombinant xenoantibody, H66K12, inhibits FVIII activity in vitro…… ....................................................................................................82 Serum derived xenoantibody inhibits FVIII activity in vitro .....................86 vi Computer modeling predicts induced xenoantibody binds the FVIII C1 domain ........................................................................................87 Competition ELISA confirms induced xenoantibody binds the FVIII C1 domain ........................................................................................97 In silico binding site mutation analysis…………………. .........................97 Discussion ………………………………………………………….. ...........104 Acknowledgements…………………………………………………. ...........108 References…………………………………………………………… ..........109 4. Clinically relevant small molecules inhibit anti-non-Gal IgM xenoantibody elicited in multiple pig-to-primate models of enotransplantation ........................113 Abstract ..........................................................................................................115 Introduction ....................................................................................................117 Materials and Methods ...................................................................................118 Animals ....................................................................................................118 Models of xenotransplantation .................................................................119 Immunization with GTKO pig aortic endothelial cells ......................119 Islet xenotransplantation ....................................................................119 Kidney xenotransplantation ...............................................................120 Small molecule library .............................................................................120 In vitro small molecule screening ............................................................120 In silico small molecule screening ...........................................................121 Natural antibody ELISA ..........................................................................122 Tetanus toxoid IgG ELISA ......................................................................123 Statistical analysis ....................................................................................123 Results ............................................................................................................124 Screening for clinically relevant small molecule drugs capable of inhibiting anti-non-Gal xenoantibody ......................................................124 In silico small molecule screening suggests azole antifungals bind directly to xenoantibody ..........................................................................127 Natural IgM antibodies are minimally affected by clonidine and moderately affected by gabexate mesilate and voriconazole ...................133 Clinically relevant small molecule drugs can provide added benefit in vitro in combination with anti-idiotypic antibody in vivo ...................136 Anti-idiotypic antibody and clonidine have marginal impact on natural antibodies .....................................................................................139 vii Clonidine inhibits anti-non-Gal IgM xenoantibody elicited in rhesus monkeys after transplantation with fetal pig islet-like cell clusters………………….…… ................................................................142 Clonidine selectively inhibits anti-non-Gal IgM xenoantibody elicited in baboons after GTKO pig kidney xenotransplantation ............145 Discussion ......................................................................................................148 Acknowledgements…………………………………………………. ...........152 References…………………………………………………………… ..........153 5. Conclusions………………………………………………………..… ................158 Discussion…………………………………………………………… ..........158 Future Directions……………………………………………………. ..........162 References………………………………………………………………………. ...........166 viii FIGURES Figures Page Chapter 1 1. Vascular antibody deposition causes activation of endothelial cells .....................11 2. Activated xenograft endothelial cells trigger the clotting cascade ........................14 Chapter 2 3. Flow cytometic confirmation of the GTKO/hCD55/hCD59 phenotype ................43 4. IGHV3 and IGKV gene usage in untreated baboons .............................................49 5. Restriction of IGHV genes after GTKO.Tg pig islet xenotransplantation ............52 6. Restriction of IGKV genes after GTKO.Tg pig islet xenotransplantation ............55 7. Xenoantibody response to GTKO.Tg pig islet xenotransplantation ......................57 8. Heavy chain J segment usage after GKTO.Tg pig islet xenotransplantation ........60 9. Computer modeled structural alignment of IGHV3-21 and IGHV3-66 ................62 10. Recombinant xenoantibody (H66K12) inhibits clotting factor VIII activity .........84 11. Immunization with pig endothelial cells elicits FVIII inhibitors in monkeys .......87 12. Ramachandran plot of H66K12 computer generated model ..................................91 13. Accurately predicting the interaction between FVIII C2 and Bo2C11 ..................93 14. H66K12 is predicted to interact with the FVIII C1 domain ..................................95 15. Confirmation that H66K12 binds the FVIII C1 domain ......................................100 16. Amino acid homology between pig and human FVIII ........................................102 17. Four compounds inhibit xenoantibody elicited by pig endothelial cells .............125 18. Alignment of three antibody models encoded by IGHV3-66 and IGKV1-12 .....128 19. Three of the identified drugs share a region of limited structural similarity .......131 ix
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