The Chemical Synthesis of Knob Domain Antibody Fragments

Abstract

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3−6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a cocrystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.

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

“…The three RSA fusions displayed t 1/2 in the range of 32–54 hours, indicating that a substantial extension in t 1/2 had been achieved for the knob domains. While comparative data for the K92 knob domain are unavailable, based on previously published PK data with a chemically synthesized K57 knob domain (K57 chem FE), 7 plasma clearance (CLp) and t 1/2 were increased for RSA-K57 Helix 4, by 22-fold and 120-fold, respectively, relative to the K57 peptide (K57 chem FE t 1/2 = 1.6 hours/CLp = .8 mL/min/kg).…”

“…Unusually for antibody fragments, knob domains are readily amenable to chemical synthesis and by this route we have exploited the proximity of the termini to produce head-to-tail cyclized knob domains, which may confer further resistance to exopeptidases in vivo. 7 In this study, we propose that the proximity of the termini also affords opportunities for protein engineering by targeting protein loops as insertion sites. Despite a knob domain comprising at least 30 amino acids, due to its folded nature, the apparent disruption to the loop might be equivalent to a much smaller linear peptide, providing a route to insert, small, high affinity binding domains into proteins, without fusing to the terminus.…”

“…As low-molecular weight therapeutic agents, knob domains display a short plasma half-life (t 1/2 ) when administered systemically. We measured a t 1/2 of 17 minutes for the unmodified K57 knob domain following administration of a 10 mg/kg intravenous (IV) dose to rats, 7 which appears symptomatic of renal clearance. 8 For therapeutic applications, it is critical that compound endures at the site of action, consequently various approaches to extend the t 1/2 of low molecular weight proteins and peptides have been explored.…”

Milking the Cow: Cattle-Derived Chimeric Ultralong CDR-H3 Antibodies and Their Engineered CDR-H3-Only Knobbody Counterparts Targeting Epidermal Growth Factor Receptor Elicit Potent NK Cell-Mediated Cytotoxicity

“…Apparently, this would suggest that the antibody scaffold including the stalk region might have a stabilizing effect for the knob region ( 12 , 14 ). Macpherson et al, however, were able to chemically synthesize solitary knob architectures by solid-phase peptide synthesis, proving that the antibody scaffold including the stalk region is not a prerequisite for the knob structure in order to function properly ( 65 ). This clearly paves the way for a multitude of engineering options.…”

Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

“…Even though both ECU and RA101295 are C5 cleavage inhibitors, our measurements reported here suggest that the residual C5a in the presence of RA101295 may be sufficient for neutrophil activation. RA101295, similar to its clinical analog zilucoplan, blocks MAC formation through a mechanism of action that disrupts C5b6 complex ( 18 , 53 ). This finding is consistent with results reporting that RA101295 completely suppressed sC5b9 levels under conditions where residual C5a was detectable using a whole blood assay.…”