Molecular Characterization and Inhibition of Antibodies Elicited Against Galactosyltransferase ...

185 Pages·2016·2.64 MB·English

by  John M. Stewart

Loma Linda University
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Loma Linda University Electronic Theses, Dissertations & Projects
6-2015
Molecular Characterization and Inhibition of
Antibodies Elicited Against Galactosyltransferase
Knockout Pig Xenografts
John M. Stewart
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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
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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

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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.

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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
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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

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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

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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
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