The allosteric modulation of complement C5 by knob domain peptides

Abstract

Bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce high affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.

The chemical synthesis of knob domain antibody fragments

“…Of the four knob domains which we reported to tightly bind C5, three were functionally modifying and two were demonstrably allosteric (as defined by partial antagonism at asymptotic concentrations) 7 . Notably, one knob domain, K92, demonstrated that selective allosteric inhibition of the alternative pathway can be achieved through C5 7 .…”

“…Of the four knob domains which we reported to tightly bind C5, three were functionally modifying and two were demonstrably allosteric (as defined by partial antagonism at asymptotic concentrations) 7 . Notably, one knob domain, K92, demonstrated that selective allosteric inhibition of the alternative pathway can be achieved through C5 7 . Co-crystal structures of the C5-knob domain complexes highlighted the molecular interactions underpinning binding and showed that the knob domain peptides adopted 3-strand β-sheet topologies and were constrained by varying numbers of disulphide bonds 7 .…”

“…We have previously shown that knob domains can function autonomously to bind antigen independently of the bovine antibody scaffold 6, 7 . This creates antibody fragments of just 3-6 kDa, so small as to be considered peptides.…”

“…Notably, one knob domain, K92, demonstrated that selective allosteric inhibition of the alternative pathway can be achieved through C5 7 . Co-crystal structures of the C5-knob domain complexes highlighted the molecular interactions underpinning binding and showed that the knob domain peptides adopted 3-strand β-sheet topologies and were constrained by varying numbers of disulphide bonds 7 . Broadly similar folds are endorsed by the cysteine-rich defensin 26 and various venom peptides 27 which are ubiquitous as tools of innate immunity and defence, across all clades, and which are also the focus of drug discovery efforts 28, 29 .…”

“…Here, we produce previously reported C5 modifying knob domain peptides 6, 7 by chemical synthesis and explore their biological activity, structure and in vivo pharmacokinetics. This study presents antibody-derived medicinal chemistry as a simple approach to modify knob domains to aid the discovery of drug candidates.…”