The proximity of the N- and C- termini of bovine knob domains enable engineering of target specificity into polypeptide chains

Abstract

Cysteine-rich knob domains can be isolated from the ultralong heavy-chain complementarity-determining region (CDR) 3, which are unique to a subset of bovine antibodies, to create antibody fragments of ~4 kDa. Advantageously, the N- and C- termini of these small binding domains are in close proximity, and we propose that this may offer a practical route to engineer extrinsic binding specificity into proteins. To test this, we transplanted knob domains into various loops of rat serum albumin, targeting sites that were distal to the interface with the neonatal Fc receptor. Using knob domains raised against the clinically validated drug target complement component C5, we produced potent inhibitors, which exhibit an extended plasma half-life in vivo via attenuated renal clearance and neonatal Fc receptor-mediated avoidance of lysosomal catabolism. The same approach was also used to modify a Camelid V HH , targeting a framework loop situated at the opposing end of the domain to the CDRs, to produce a small, single-chain bispecific antibody and a dual inhibitor of Complement C3 and C5. This study presents new protein inhibitors of the complement cascade and demonstrates a broadly applicable method to engineer target specificity within polypeptide chains, using bovine knob domains.

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire

“…The ultralong knob domain itself has been isolated from the full IgG framework and shown to be able to bind to antigens of interest with similar affinity to the whole IgG [59,60]. Other studies have engineered knobs into protein loops to produce novel multivalent molecules [61][62][63][64][65]. In addition to binding, a recent study demonstrated that recombinant knob domains alone are able to potently neutralize SARS-CoV-2 [34] .…”

HIV envelope trimers and gp120 as immunogens to induce broadly neutralizing antibodies in cows

“…They have been engineered for use as multivalent molecules, knobbody proteins, and functional knob-only proteins [40][41][42][43][44][45][46]. They have also been engineered to bind and potently neutralize SARS-CoV-2 as recombinant knob domains, highlighting their potential as anti-viral therapeutics [47].…”

Modular design of bi- and multi-specific knob domain fusions

“…This may enable construction of head-to-tail fusions free from the binding site steric occlusion. Moreover, the proximity of N- and C-termini makes knob domains well suited for grafting onto loops of various proteins, including conventional antibody fragments ( 44 ). Five different formats of bispecific knob domain fusions have been described to date: knob domains transplanted into a framework II loop of a VHH ( 44 ), framework III loop of a Fab fragment ( 45 ), and either AB or EF loops of a full-length IgG CH3 domain ( 46 ), as well as an IgG-like bispecific format described in ( 47 ).…”

“…Moreover, the proximity of N- and C-termini makes knob domains well suited for grafting onto loops of various proteins, including conventional antibody fragments ( 44 ). Five different formats of bispecific knob domain fusions have been described to date: knob domains transplanted into a framework II loop of a VHH ( 44 ), framework III loop of a Fab fragment ( 45 ), and either AB or EF loops of a full-length IgG CH3 domain ( 46 ), as well as an IgG-like bispecific format described in ( 47 ). However, all these formats combine a knob domain with a larger antibody fragment and therefore do not leverage small size of the former in full.…”

“…Recombinant knob domain peptides express poorly both on their own and as entire CDR H3 comprising the “knob” and the “stalk” ( 37 ). Expression yields can be rescued by tethering to a larger protein, such as human Fc or thioredoxin ( 37 , 43 , 48 ), or by inserting into a loop as described above ( 37 , 44 – 46 ). Upon separation from fusion partners, the knob domain peptides retain their antigen-binding activity ( 37 , 43 ).…”