An antibody-assisted structural and functional study of Complement C5
: (Alternative Format Thesis)

  • Alex Macpherson

Student thesis: Doctoral ThesisPhD


Complement C5 is an integral component of the terminal pathway of the complement cascade. Cleavage of C5, by either of the C5 convertases, generates the release of C5a, the most potent anaphylatoxin of the complement cascade, and formation of C5b, the keystone of the lytic terminal complement complex.
Preventing activation of C5 is the primary goal of immune evasion molecules from a range of pathogens, as well as a number of licensed and clinical stage drugs, which seek to prevent complement induced inflammation and autolysis in diseases such as paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome.
This study uses antibodies as to probe the structural biology and molecular pharmacology of C5. The new antibody formats developed for this purpose are from a structurally unique antibody subset, found exclusively in bovines. In cows, around 10 % of IgM and IgG antibodies have ultralong CDRH3, where a knob domain, a series of mini loops stabilised by 2-5 disulphide bonds, is presented on a β-ribbon stalk, over 40 Å from the adjacent complementarity determining regions. Viewed in isolation, the knob domains resemble the cysteine-rich peptides or knottins, which occur in various forms throughout host immunity (the defensins) and host defence (as venoms and toxins).
We first present the rational design of a modified form of the immune evasion molecule OmCI, to permit the single-step affinity purification of C5 protein from serum. Protein purified in this manner was then used for bovine antibody discovery. We show, for the first time, that knob domains can function autonomously to bind antigen independently of the bovine antibody scaffold. This creates a new, low molecular weight antibody fragment of just 3-6 kDa, around one third the size of a camelid VHH, and so small as to be considered a peptide. These novel molecules were used to probe C5 and, through co-crystal structures and solution biophysics methods, we explore the molecular basis for the allosteric modulation of C5. We observed intricate modulation of C5 biology, with one knob domain selectively antagonising C5 activation by the alternative pathway, indicating that the two C5 convertases are not functionally equivalent and that a targetable mechanistic difference exists.
Finally, we show that, uniquely for antibody fragments, knob domains are amenable to solid phase chemical synthesis. We present methods for antibody-derived peptide medicinal chemistry, with protocols for solid phase synthesis and modification of knob domains, together with characterisation of their molecular structures, in vitro pharmacology and in vivo pharmacokinetics.
Date of Award26 May 2021
Original languageEnglish
Awarding Institution
  • University of Bath
SponsorsUCB Pharma Ltd
SupervisorJean Van Den Elsen (Supervisor), Susan Crennell (Supervisor) & Alastair Lawson (Supervisor)

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