The alternative pathway (AP) of complement activation is implicated in several renal pathologies. These include membranoproliferative glomerulonephritis (MPGN) type II and atypical haemolytic uraemic syndrome (aHUS). Factor H (FH), comprised of 20 short consensus repeats (SCRs), is the primary regulator of the AP at the glomerular basement membrane (GBM) as evidenced by the fact that impaired factor H function is linked to both diseases. The current treatment options for MPGN type II and aHUS are limited and patients ultimately develop renal failure. There is a clear need for kidney targeted therapeutics to control AP dysregulation. I sought to generate novel recombinant FH fusion proteins to replace dysfunctional FH and to deliver the complement regulatory domain of factor H (SCRs 1-5) to the GBM. A protein containing the regulatory SCRs (FH1-5) and one containing the regulatory and cell-surface binding SCRs (FH1-5/18-20) was generated and expressed in a eukaryotic expression system. C3b binding and full complement regulatory function of FH1-5 and FH1- 5/18-20 was confirmed by in vitro assays. To target the GBM a B cell hybridoma (mAb3) was obtained which secretes an antibody that binds to the NC1 domains of collagen type IV alpha-3 chain, expressed predominately in the GBM. The mAb3 variable regions (Fv) were successfully cloned after elimination of an abundantly expressed endogenous light chain transcript. A mAb3 single chain FV (scFv) was cloned downstream of FH1-5 creating a FH1-5/mAb3 scFv fusion protein. As an alternative a fusion protein incorporating the scFv of the 1G6 monoclonal antibody (FH1-5/1G6 scFv) was generated. Low expression levels of the FH1-5/scFv proteins limited the functional analysis. Binding of both proteins to C3b and binding of FH1-5/1G6 scFv but not FH1-5/mAb3 scFv to bovine and murine NC1 domains was demonstrated using cell culture supernatants. These reagents remain in the early stage of development but have potential to be attractive alternatives for the treatment of aHUS and MPGN type II. In addition to MPGN type II, FH1-5/scFv may find application in the treatment or prevention of anti-GBM nephritis and renal ischaemia-reperfusion injury.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:594505 |
| Date | January 2013 |
| Creators | Hunze, Eva-Maria |
| Publisher | University of Newcastle upon Tyne |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10443/2031 |
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