Investigating the Role of Immunoglobulin M in the Humoral Immune Response to Factor VIII Restricted; Files Only

Abstract

Hemophilia A is an X-linked genetic disorder characterized by a deficiency in factor VIII (FVIII), an essential protein in the blood-clotting cascade. The most significant treatment complication for patients with hemophilia A is the humoral immune response to FVIII, which primarily consists of neutralizing polyclonal anti-FVIII antibodies (i.e., inhibitors) that prevent successful replacement therapy. The mechanism of FVIII inhibitor formation and the role different immunoglobulin isotypes play in developing such an immunogenic response is not well understood. Previous laboratory data identified the presence of anti-C1 FVIII domain non- inhibitory and porcine cross-reactive immunoglobulin M (IgM) after immunization of a hemophilia A murine model with recombinant human FVIII. The persistence of these first responding IgM in the secondary humoral response to FVIII as well as the monoclonal binding to the C1 domain led to further investigation into their role in inhibitor formation. We set out to develop and characterize murine-derived anti-human FVIII IgM to evaluate their role in initiating and propagating the humoral response to FVIII. Hemophilia A mice lacking exon 16 in the F8 gene were injected retro-orbitally with recombinant B domain deleted FVIII for a total of 5 weeks. Monoclonal anti-FVIII IgM antibodies were created using hybridoma technology fusing harvested splenocytes of the immunized hemophilia A mice with an immortal myeloma cell line. Enzyme-linked immunosorbent assays (ELISA) utilizing human recombinant FVIII protein constructs were performed after purification of anti-FVIII IgMs to assess FVIII-IgM binding interactions and FVIII domain-binding specificity. IgMs were determined to be porcine- cross reactive and non-inhibitory indicating similarities to the previously characterized IgM. However, IgM demonstrated weak binding affinity to FVIII which prevented definitive determination of domain-binding specificity. Weak binding interactions were hypothesized to be the result of alternative μ’ variants of the IgM heavy chain leading to aggregation and decreased binding affinity. Further investigation of anti-FVIII IgMs is necessary to provide mechanistic insight into inhibitor development and pathogenicity. 

Table of Contents

Table of Contents

INTRODUCTION ..............................................................................................................1

Background ...........................................................................................................................................1

FVIII Procoagulant Function .................................................................................................................. 1

Treatment of Hemophilia A.................................................................................................................... 3

FVIII Inhibitor Formation.......................................................................................................................4

Characteristics of IgM Antibodies ...........................................................................................................6

Summary of the Importance of FVIII IgM Investigation...........................................................................10

MATERIALS and METHODS ...............................................................................................11

Materials ............................................................................................................................................11

Hemophilia A Murine Model .................................................................................................................12

Production of IgM Secreting Hybridomas...............................................................................................12

Anti-FVIII IgM Expression and Purification ..........................................................................................14

Nijmegen Bethesda Assay ....................................................................................................................15

Anti-FVIII IgM Binding ELISA..............................................................................................................16

Identification of the FVIII Binding Domain...........................................................................................16

IgM Excipient Stabilization Assay ........................................................................................................17

RESULTS ...........................................................................................................................18

The Low-Binding Affinity of IgM Necessitates Alternative Purification Approaches ................................19

Anti-FVIII IgMs Demonstrate Weak-Binding Interactions with FVIII and are Porcine Cross Reactive........20

IgM Binding is Not Improved in Buffers with Excipients .......................................................................21

DISCUSSION......................................................................................................................22

TABLES.............................................................................................................. ...............26

Table 1. Quantities of Purified IgM Using Affinity Chromatography Purification Technique....................26

FIGURES............................................................................................................................27

Figure 1. Schematic of Hybridoma Production Process. .........................................................................27

Figure 2. Frequency of Anti-FVIII IgM-Secreting Hybridomas. ...............................................................28

Figure 3. SDS-PAGE of IgM MAb R155 by Different Purification Approaches. ..........................................29

Figure 4. Purity of Purified IgM by SDS-PAGE .......................................................................................30

Figure 5. IgM Isotype Confirmation ELISA............................................................................................31

Figure 7. IgM Binding to FVIII. ............................................................................................................33

Figure 8. FVIII Domain-Specific Binding by Purified IgMs......................................................................34

Figure 9. Domain-Specific Binding by Purified IgMs Using Single Human Domain A2, C1, and C2 Proteins.35

Figure 10. IgM Stabilization Binding Assay. ..........................................................................................36

REFERENCES ..................................................................................................................37

SUPPLEMENTAL FIGURES................................................................................................45 

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Health Sciences, Immunology Biology, Microbiology Biology, Physiology
Keyword	IgM hemophilia A Humoral Immune Response Factor VIII Inhibitors
Committee Chair / Thesis Advisor	Batsuli, Glaivy, Emory University
Committee Members	Freeman, Amanda, Emory University Shepherd, Iain, Emory University

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