Human recombinant Fc constructs: production and anti-osteoclastogenic effects

Craig, Pauline Claire (2015) Human recombinant Fc constructs: production and anti-osteoclastogenic effects. PhD thesis, University of Glasgow.

Abstract

Staphylococcal protein A (SpA) produced by Staphylococcus aureus, binds Immunoglobulin G (IgG), forming SpA-IgG immune complexes (SIC), at a ratio of 2 SpA molecules to 4 IgG molecules. In vitro, SIC have been found to inhibit human osteoclastogenesis, and reduce proinflammatory cytokine secretion by human macrophages. The targeting of both osteoclasts and macrophages together may be important in diseases such as rheumatoid arthritis (RA), where inflammation and bone erosion both play roles in disease progression; resulting in considerable pain and disability. SpA is currently in clinical trials, however, as SpA forms heterogeneous IgG complexes, SpA therapy is not optimal. The aim of this project was to produce human recombinant Fc (Fragment crystallisable) constructs using different IgG subtype(s), thus optimising the immunomodulatory properties exhibited by SIC. This could lead to the development of new therapeutics, and will also give insight into Fc-mediated inhibitory mechanisms.

In this thesis, 13 human Fc constructs (5 tetramers, 5 dimers and 3 monomers) were produced in total. The methods used to generate the human Fc constructs enabled their successful production for use in in vitro cultures. However, when investigating the structure of the IgG3 tetramer (TG3), IgG3 dimer (DG3) and IgG3 monomer (MG3), it was found that TG3 and DG3 do not appear to be polymerising in the manner that was predicted.

The IgG1 tetramer (TG1) and TG3 tetramer, alongside SIC, were found to have the ability to significantly inhibit osteoclast differentiation, and the same molarities of dimers and monomers subsequently displayed reduced levels of inhibition. In studies performed using TG3, DG3, MG3 and SIC: TG3 and SIC were both shown to significantly inhibit RANK transcript levels, although they did not significantly affect the levels of RANK protein expression on the cell surface. However, the level of RANKL-mediated phosphorylated p38 (pp38) was reduced across all donors. Interestingly, TG3 and DG3 significantly reduced cell surface expression of CD115 (c-Fms; colony stimulating factor 1 receptor (CSF1R); macrophage colony-stimulating factor receptor (M-CSFR)). SIC and TG3 were able to significantly reduce transcript levels of DCSTAMP, OCSTAMP, cathepsin K and MMP-9 at certain time points, and TG3 was also shown to significantly reduce NFATc1 transcript levels. TG3, DG3, MG3 and higher amounts of SIC all displayed binding to the surface of cells, although this was only significant for DG3 and SIC.

The mechanisms of action of the human Fc constructs and SIC have not been fully elucidated, and further work is required. However, the data in this thesis suggests that these novel human Fc constructs and SIC can exert their Fc-mediated inhibitory effects by affecting pathways essential for osteoclast differentiation and function.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Recombinant, Human, IgG, Fc, Constructs, SpA, SIC, Osteoclasts, Macrophages
• Subjects: Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Immunology & Infection
• Supervisor's Name: Goodyear, Dr. Carl
• Date of Award: 2015
• Embargo Date: 15 June 2018
• Depositing User: Mrs Pauline C Craig
• Unique ID: glathesis:2015-6208
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 16 Mar 2015 10:22
• Last Modified: 15 Mar 2018 13:18
• URI: https://theses.gla.ac.uk/id/eprint/6208