The Contribution of IgG Fc Glycans on Antibody-Mediated Immune Suppression

100 Pages·2016·3.42 MB·English

The Contribution of IgG Fc Glycans on
Antibody-Mediated Immune Suppression (AMIS) to
Murine Erythrocytes
by
Danielle Marjoram
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Laboratory Medicine and Pathobiology
University of Toronto
© Copyright by Danielle Marjoram 2016
The Contribution of IgG Fc Glycans on Antibody-Mediated
Immune Suppression (AMIS) to Murine Erythrocytes
Danielle Marjoram
Master of Science
Laboratory Medicine and Pathobiology
University of Toronto
2016
Abstract
The ability of anti-D to prevent hemolytic disease of the fetus and newborn (HDFN) is an
example of antibody-mediated immune suppression (AMIS). A recombinant monoclonal
antibody to replace anti-D is desired, however, none of the developed therapies have been as
effective and some antibodies have unexpectedly led to enhanced immune responses. The
requirement for IgG glycosylation for AMIS was analyzed for five antibodies reactive with
erythrocytes in a murine model. The antibodies recognized the HOD (hen egg lysozyme [HEL]
linked to ovalbumin and the human Duffy transmembrane protein) antigen. It was observed that
glycosylation in the Fc region of polyclonal AMIS-inducing antibodies was not essential for
complete suppression. In contrast, monoclonal AMIS-inducing antibodies were partially
sensitive to removal of the glycan structure. These results may help explain the superiority of
polyclonal antibodies to monoclonal therapeutics for AMIS induction and could influence the
development of effective therapies to replace anti-D.
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Acknowledgments
First, I would like to thank my supervisor Dr. Alan Lazarus for his guidance and support during
my time as a graduate student. Dr. Lazarus provided the perfect balance of constructive criticism
and praise, which motivated me to always strive to be the best scientist possible. When I faced
challenges with the project, Dr. Lazarus was approachable and would provide insights and
solutions to the issues at hand. Dr. Lazarus is an excellent supervisor because he displays strong
leadership skills and encourages his lab members to achieve success. I have gained valuable
skills from my time in the Lazarus lab.
I would like to thank Dr. Lidice Bernardo for being an excellent mentor and contributing to my
growth as a scientist during my program. She was always welcoming and patient with the
numerous questions I presented to her. Lidice taught me valuable techniques and skills for the
laboratory, but she also provided insights into science as a career. Without her help on the AMIS
project, I would have had a much more difficult time completing my research. I am truly grateful
for all of her assistance during my time in the lab.
I would also like to thank Mr. Andrew Crow for providing useful knowledge about various
techniques and always being available to help with any issues in the lab. Thank you to Ms. Joan
Legarda for helping me settle into the lab, organizing meetings, and helping with orders. I would
like to thank Dr. Ben Yu for teaching me how to perform deglycosylation reactions and helping
to analyze the UPLC data. I would like to thank Dr. Yoelys Cruz-Leal for her assistance with the
clearance experiments. Thank you to Dr. Alaa Amash, Melissa Menard, and Dr. Lin Wang for
being welcoming and providing a positive work environment.
I would like to thank my committee members Dr. John Semple and Dr. Gerald Prud’homme for
taking the time to assist me with the completion of my program. Their questions and insights
during the committee meetings were highly beneficial for my progress throughout my graduate
education.
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Table of Contents
Acknowledgments .......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Tables ................................................................................................................................. ix
List of Figures ..................................................................................................................................x
Abbreviations ................................................................................................................................ xii
Chapter 1 ..........................................................................................................................................1
Hemolytic Disease of the Fetus and Newborn (HDFN) .............................................................1
1.1 Clinical manifestations of HDFN ........................................................................................2
1.1.1 Maternal alloimmunization ......................................................................................2
1.1.2 Detection ..................................................................................................................3
1.1.3 Potential complications associated with HDFN ......................................................3
1.2 HDFN prophylaxis ...............................................................................................................4
1.2.1 Anti-D ......................................................................................................................4
Chapter 2 ..........................................................................................................................................6
Anti-D Prophylaxis .....................................................................................................................6
2.1 Clinical applications of anti-D .............................................................................................7
2.1.1 Anti-D for HDFN prophylaxis .................................................................................7
2.1.2 Anti-D for ITP treatment .........................................................................................8
2.2 Monoclonal anti-D development .........................................................................................8
2.2.1 Limitations of monoclonal antibody therapies ........................................................9
2.2.2 Rozrolimupab .........................................................................................................10
Chapter 3 ........................................................................................................................................11
Antibody-Mediated Immune Suppression (AMIS) ...................................................................11
3.1 Proposed mechanisms of AMIS .........................................................................................11
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3.1.1 FcγRIIB-mediated B cell inhibition .......................................................................11
3.1.2 Epitope masking .....................................................................................................11
3.1.3 Red cell clearance ..................................................................................................12
3.1.4 Alternative mechanisms .........................................................................................13
3.2 Experimental models of AMIS ..........................................................................................14
3.2.1 SRBC model of RBC immunization ......................................................................14
3.2.2 HOD mouse model of RBC immunization ............................................................15
3.2.3 Vaccines .................................................................................................................16
3.2.4 Anti-platelet antibodies ..........................................................................................16
Chapter 4 ........................................................................................................................................18
IgG N-linked Glycosylation ......................................................................................................18
4.1 Function .............................................................................................................................19
4.1.1 Sialic acid residues .................................................................................................19
4.1.2 Fucose residues ......................................................................................................20
Chapter 5 ........................................................................................................................................21
RATIONALE AND HYPOTHESIS .........................................................................................21
5.1 Brief Background ...............................................................................................................21
5.2 Rationale ............................................................................................................................21
5.3 Hypotheses .........................................................................................................................22
Chapter 6 ........................................................................................................................................23
MATERIALS AND METHODS ..............................................................................................23
6.1 Mice ...................................................................................................................................23
6.1.1 HOD-RBC collection .............................................................................................23
6.1.2 Sera collection ........................................................................................................23
6.1.3 Tail vein injection ..................................................................................................23
6.2 Antibodies ..........................................................................................................................24
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6.3 Deglycosylation .................................................................................................................24
6.3.1 Glycan removal ......................................................................................................24
6.3.2 Validation of deglycosylation reaction ..................................................................25
6.3.3 Binding of deglycosylated antibodies to HOD-RBCs ...........................................25
6.4 AMIS induction .................................................................................................................26
6.4.1 Assessing antibody binding to HOD-RBCs ...........................................................26
6.5 Serologic detection of HEL-specific antibodies ................................................................26
6.6 Glycan analysis ..................................................................................................................27
6.6.1 Lectin-based ELISA ...............................................................................................27
6.6.2 Ultra performance liquid chromatography (UPLC) ...............................................27
6.7 Lectin-based fractionation .................................................................................................28
6.7.1 Separation of antibodies containing sialic acid ......................................................28
6.7.2 Analysis of fractions by lectin-based ELISA .........................................................29
6.7.3 Binding of sialic acid-depleted polyclonal anti-HEL to HOD-RBCs ....................29
6.7.4 In vivo AMIS induction with sialic acid-depleted polyclonal anti-HEL
antibody ..................................................................................................................30
6.8 In vivo red cell clearance ....................................................................................................30
6.8.1 Optimization of HOD-RBCs labeling with PKH26 ...............................................30
6.8.2 In vivo HOD-RBC clearance .................................................................................31
6.9 Statistical Analysis .............................................................................................................31
Chapter 7 ........................................................................................................................................32
RESULTS .................................................................................................................................32
7.1 Induction of AMIS with red cell antibodies ......................................................................32
7.1.1 The immune response to transfused HOD-RBCs in mice .....................................33
7.2 Induction of AMIS with polyclonal HEL-specific antibodies ...........................................35
7.2.1 Confirmation of N-linked glycan removal .............................................................35
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7.2.2 Binding of anti-HOD antibodies to HOD-RBCs after deglycosylation .................35
7.2.3 Lectin-based ELISA to detect IgG glycan structures ............................................35
7.2.4 UPLC analysis of polyclonal anti-HEL .................................................................37
7.2.5 AMIS induction with deglycosylated polyclonal red cell antibodies ....................38
7.2.6 AMIS induction with sialic acid-depleted polyclonal HEL-specific antibodies ...40
7.3 Induction of AMIS with 4B7 HEL-specific antibody ........................................................42
7.3.1 Confirmation of N-linked glycan removal for 4B7 ...............................................42
7.3.2 4B7 binding to HOD-RBCs after deglycosylation ................................................42
7.3.3 Lectin-based ELISA to detect 4B7 glycan structures ............................................42
7.3.4 UPLC analysis of 4B7 monoclonal anti-HEL antibody ........................................44
7.3.5 AMIS induction with deglycosylated 4B7 anti-HEL antibody ..............................45
7.4 Induction of AMIS with 6D7 HEL-specific antibody .......................................................47
7.4.1 Confirmation of N-linked glycan removal for 6D7 ...............................................47
7.4.2 6D7 binding to HOD-RBCs after deglycosylation ................................................47
7.4.3 Lectin-based ELISA to detect 6D7 glycan structures ............................................47
7.4.4 UPLC analysis of 6D7 monoclonal anti-HEL antibody ........................................49
7.4.5 AMIS induction with deglycosylated 6D7 anti-HEL antibody .............................50
7.5 Induction of AMIS with MIMA 29 Duffy-specific antibody ............................................52
7.5.1 Confirmation of N-linked glycan removal from MIMA 29 ...................................52
7.5.2 Binding of MIMA 29 to HOD-RBCs after deglycosylation ..................................52
7.5.3 Lectin-based ELISA to detect MIMA 29 glycan structures ..................................52
7.5.4 UPLC analysis of MIMA 29 monoclonal anti-Duffy antibody .............................54
7.5.5 AMIS induction with deglycosylated MIMA 29 anti-Duffy antibody ..................55
7.6 Induction of AMIS with CBC-512 monoclonal RBC antibody .........................................57
7.6.1 Confirmation of N-linked glycan removal from CBC-512 ....................................57
7.6.2 Binding of CBC-512 to HOD-RBCs after deglycosylation ...................................57
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7.6.3 UPLC analysis of CBC-512 monoclonal anti-Duffy antibody ..............................57
7.6.4 AMIS induction with deglycosylated CBC-512 anti-Duffy antibody ...................59
7.7 In vivo red cell clearance ....................................................................................................61
7.7.1 RBC clearance with deglycosylated anti-Duffy antibody; MIMA 29 ...................61
7.7.2 RBC clearance with deglycosylated anti-Duffy antibody; CBC-512 ....................63
Chapter 8 ........................................................................................................................................66
DISCUSSION ...........................................................................................................................66
8.1 The Fc glycan is not likely required for AMIS induction by polyclonal RBC
antibodies ...........................................................................................................................66
8.2 Fc glycan removal impacts early stages of AMIS for HEL- specific monoclonal
antibodies ...........................................................................................................................68
8.3 Deglycosylation has differential effects for the ability of two Duffy-specific
antibodies to induce AMIS ................................................................................................69
8.4 AMIS can occur independently of red cell clearance ........................................................70
Chapter 9 ........................................................................................................................................72
SUMMARY ..............................................................................................................................72
Chapter 10 ......................................................................................................................................73
FUTURE DIRECTIONS ..........................................................................................................73
REFERENCES ..............................................................................................................................74
Appendix ........................................................................................................................................84
SUPPLEMENTAL FIGURES ..................................................................................................84
viii
List of Tables
Table 1. Summary of proposed mechanisms of AMIS induction 17
Table 2. Previously established AMIS induction by anti-RBC antibodies 32
Table 3. Summary of deglycosylated anti-HOD antibodies ability to mediate AMIS 65

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List of Figures
Figure 1. The molecular structure of the IgG conserved N-linked glycan. 16
Figure 2. The erythrocyte-specific HOD antigen present in HOD transgenic mice. 18
Figure 3. The PNGase F enzyme cleavage site for IgG N-linked glycans. 25
Figure 4. Sialic acid lectin-based fractionation schematic. 29
Figure 5. The immune response to transfused HOD-RBCs in C57BL/6 mice. 34
Figure 6. Analysis of wildtype and deglycosylated polyclonal anti-HEL antibodies. 36
Figure 7. Glycan analysis of wildtype and deglycosylated polyclonal anti-HEL antibodies.
37
Figure 8. AMIS induction with wildtype and deglycosylated polyclonal anti-HEL antibody.
39
Figure 9. AMIS induction with sialic acid-depleted polyclonal anti-HEL. 41
Figure 10. Analysis of wildtype and deglycosylated monoclonal HEL-specific 4B7 (IgG1).
43
Figure 11. Glycan analysis of wildtype and deglycosylated 4B7 monoclonal anti-HEL
antibody. 44
Figure 12. AMIS induction with wildtype and deglycosylated 4B7 monoclonal anti-HEL
antibody. 46
Figure 13. Analysis of wildtype and deglycosylated monoclonal HEL-specific 6D7 (IgG1).
48
Figure 14. Glycan analysis of wildtype and deglycosylated 6D7 monoclonal anti-HEL
antibody. 49
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