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.
Highlights
The smallest autonomous, naturally occurring, functional antibody domains reported have been the variable regions of camelid heavy-chain antibodies (VHH) [1] and the variable regions of the immunoglobulin new antigen receptor (VNAR), derived from sharks [2], resulting in heavy-chain variable region fragments, of some 12–15 kDa [3, 4]
Sequencing of polyclonal libraries of antigen-enriched heavy-chain complementarity-determining region 3 (CDRH3) can be used as a rapid approach to discover knob domains
We present a method for obtaining isolated knob domains from bovine antibodies, using standard fluorescence-activated cell sorting (FACS), PCR, and mammalian transient expression
Summary
The smallest autonomous, naturally occurring, functional antibody domains reported have been the variable regions of camelid heavy-chain antibodies (VHH) [1] and the variable regions of the immunoglobulin new antigen receptor (VNAR), derived from sharks [2], resulting in heavy-chain variable region fragments, of some 12–15 kDa [3, 4]. In 1997, the first report of bovine antibodies featuring an ultralong heavy-chain complementarity-determining region 3 (CDRH3) was published [5], with subsequent crystal structures revealing a conserved anti-parallel β-ribbon ‘stalk’ presenting a disulphide-stabilised ‘knob’ domain of 3–6 KDa [6]. The knob domain, shown, comprises several small loops stapled by 2 to 5 disulphide bonds and is held some 25–45 Å clear of the surface of the other complementarity-determining region (CDR) loops [6,7,8]. Mutation studies with whole IgG have shown that binding can occur predominantly through the knob domain with limited input from neighbouring CDRs [6], but the surrounding architecture of the antibody scaffold is still thought to play a critical.
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