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

Abstract

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.

The allosteric modulation of complement C5 by knob domain peptides

“…Using this approach, we obtained four knob domain peptides: K8, K57, K92, and K149, which we have shown to display tight binding to human C5. Previously we reported equilibrium dissociation constants of 17.8 nM for K8, 1.4 nM for K57, <0.6 nM for K92, and 15.5 nM for K149 ( Macpherson et al, 2020 ).…”

“…By the end of 2019, over 60 peptide drugs have received regulatory approval, with an estimated 400 more in active development globally ( Lau and Dunn, 2018 ; Lee et al, 2019 ). As a potential route to discover therapeutic peptides, we previously reported a method for deriving peptides from the ultra-long heavy chain complementarity determining region 3 (ul-CDRH3), which are unique to a subset of bovine antibodies ( Macpherson et al, 2020 ). We have shown that knob domains, a cysteine-rich mini-domain common to all ul-CDRH3, can bind antigen autonomously when removed from the antibody scaffold ( Macpherson et al, 2020 ).…”

“…As a potential route to discover therapeutic peptides, we previously reported a method for deriving peptides from the ultra-long heavy chain complementarity determining region 3 (ul-CDRH3), which are unique to a subset of bovine antibodies ( Macpherson et al, 2020 ). We have shown that knob domains, a cysteine-rich mini-domain common to all ul-CDRH3, can bind antigen autonomously when removed from the antibody scaffold ( Macpherson et al, 2020 ). This allows peptide affinity maturation to be performed in vivo, harnessing the cow’s immune system to produce peptides with complex stabilising networks of disulphide bonds.…”

“…For the discovery of knob domain peptides, immunisation of cattle is followed by cell sorting of B-cells using fluorescently labelled antigen. A library of antigen-specific CDRH3 sequences is created by performing a reverse transcription polymerase chain reaction (RT PCR) on the B-cell lysate, followed by a PCR using primers specific to the conserved framework regions which flank CDRH3 ( Macpherson et al, 2020 ). Upon sequencing, ul-CDRH3s are immediately evident and the knob domains can be expressed recombinantly as cleavable fusion proteins ( Macpherson et al, 2020 ).…”

“…This method for the discovery of knob domain peptides was established using complement component C5, and we reported peptides which bound C5 with affinities in the pM–low nM range ( Macpherson et al, 2020 ). Herein, we use these novel peptides to probe the structural and functional aspects of C5 activation.…”