The Chemical Synthesis of Knob Domain Antibody Fragments

Alex Macpherson*, James R Birtley, Robert J Broadbridge, Kevin Brady, Monika-Sarah E D Schulze, Yalan Tang, Callum Joyce, Kenneth Saunders, Gregory Bogle, John Horton, Sebastian Kelm, Richard D Taylor, Richard J Franklin, Matthew D Selby, Maisem Laabei, Toska Wonfor, Adam Hold, Phil Stanley, Douangsone Vadysirisack, Jiye ShiJean van den Elsen, Alastair D G Lawson

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

10 Citations (Scopus)


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 co-crystal 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.

Original languageEnglish
Pages (from-to)1757-1769
Number of pages13
JournalACS Chemical Biology
Issue number9
Early online date18 Aug 2021
Publication statusPublished - 17 Sept 2021

Bibliographical note

Publisher Copyright:
© 2021 The Authors. Published by American Chemical Society.


  • Amino Acid Sequence
  • Animals
  • Cattle
  • Complementarity Determining Regions/chemistry
  • Immunoglobulin Fragments/blood
  • Male
  • Models, Molecular
  • Peptides, Cyclic/blood
  • Protein Binding
  • Protein Domains
  • Protein Folding
  • Rats, Sprague-Dawley
  • Solid-Phase Synthesis Techniques
  • Tandem Mass Spectrometry
  • Thermodynamics


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