Complement autoantibodies in atypical haemolytic uraemic syndrome and IgA nephropathy

PhD ThesisAtypical haemolytic uraemic syndrome (aHUS) is renal disease associated with mutations and/or polymorphisms in genes encoding complement proteins, including complement factor H (CFH), factor I (CFI) and membrane cofactor protein (CD46). Recently, deficiency of CFH-related (CFHR) proteins 1 and 3 (via loss of the CFHR3/CFHR1 gene block) was linked to the generation of autoantibodies to CFH. Around 10% of aHUS patients develop CFH autoantibodies, adding aHUS to a growing list of kidney diseases with a defined autoimmune component. IgA nephropathy (IgAN) is another such renal disease, where autoantibodies target an aberrantly glycosylated IgA1. To investigate the role of CFH and CFHR copy number variation in the control of complement activation in aHUS and IgAN, I have first generated a full panel of recombinant CFHR proteins in mammalian cell culture. These were then used to generate unique monoclonal antibodies (mAbs) and ELISA protocols to screen for autoantibodies. Using carefully optimised immunisation protocols, I used my recombinant CFHR proteins to produce several highly-specific CFHR mAbs. One of which targets CFHR1 (R1/1037) and three target CFHR4 (R4/244, R4/277 and R4/123). The generation of these antibodies have allowed putative ELISA screens to be developed to measure the concentration of CFHR4 in healthy individuals and aHUS patients. My full panel of CFHR proteins also enabled screening of both aHUS and IgAN patients for the presence of autoantibodies to CFH and the CFHR proteins. Screening of aHUS plasma did not indicate the presence of any novel CFHR autoantibodies. However, IgA autoantibodies against CFHR5 (~9%) and CFH (~32%) were detected in IgAN patients. Interestingly, 64% of IgAN patients show reactivity with bovine CFH. During this PhD, I have generated a panel of unique reagents for the study of CFHR proteins in health and disease. These have allowed me to demonstrate for the first time, the presence of CFH and CFHR5 autoantibodies in a preliminary cohort of IgAN patients

Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies.

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques