Abstract
Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining 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.
Highlights
We have previously shown that antigen-specific disulphide knob domain peptides, derived from bovine antibodies have great potential for therapeutic utility
We obtained four knob domain peptides: K8, K57, K92 and K149, which we have shown to display tight binding to human C5
We present a new family of peptides, bovine antibody-derived knob domains, and show that, upon binding of the therapeutic target complement component C5 with high affinity, they display a range of inhibitory mechanisms
Summary
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. We show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Taking a hybrid biophysical approach, we present C5-knob domain cocrystal structures and, by solution methods, observed allosteric effects propagating >50. 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. As a potential route to discover therapeutic peptides, we previously reported a method for deriving peptides from the ultralong heavy chain complementarity determining region 3
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