Abstract
Purpose :
The CFH-related proteins (CFHR) comprise a group of five plasma proteins structurally and functionally related to the main negative complement regulator, factor H (CFH). Mutations within the CFH/CFHR gene cluster are associated with various human diseases such as age-related macular degeneration (AMD), the leading cause of irreversible vision loss in the elderly. Deletion of the CFHR3/1 genes goes along with a decreased risk for AMD although the precise functions of the CFHR proteins still remain unclear. Antibody-dependent blocking of CFHR3 to elucidate the influence of complement activity in AMD has not been studied so far. We aim to elucidate the role of CFHR3 in complement regulation in degenerative eye diseases with the help of previously generated high-affinity monoclonal antibodies (mAbs) against CFHR3.
Methods :
MAbs were tested for specificity and avidity against human CFHR3 in indirect enzyme-linked immunosorbent assay (ELISA) and Western blot. MAbs were used for immunoprecipitation from human serum and following mass spectrometry analysis. Complement modulation was analyzed in functional in vitro hemolysis assays and complement ELISA. Sandwich ELISA for detection of CFHR3 complexes from serum of AMD patients compared to healthy controls was established. Interactions of CFH and CFHR3 with the oxidative stress marker CEP (ω-(2-carboxyethyl)pyrrole) and the complement component 3b (C3b) were tested.
Results :
Generated mAbs were highly specific for human CFHR3. MAb 269-5 showed the highest avidity and immunoprecipitated CFHR3 in complex with alternative and terminal complement components from human serum. Anti-CFHR3 mAbs inhibited the complement pathways in in vitro analyses. Binding of CFH and CFHR3 to the AMD risk factor CEP and to C3b could be shown. Both proteins competed for binding to these molecules, and mAb 269-5 interfered with this competition by inhibiting CFHR3 interaction. MAbs 269-5 and 552-3 detected CFHR3 complexes from human serum of AMD patients and healthy controls.
Conclusions :
The mAb-based inhibition of CFHR3 implies an attenuation of complement reactivity. We showed, consistent with genetic analyses, that CFHR3 is involved in complement dysregulation in AMD patients. A competition of CFH and CFHR3 to oxidative stress epitopes and C3b impairs the local complement system homeostasis in the eye. We identified CFHR3 as a promising target for immune modulation in AMD patients.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.