The chemical synthesis of knob domain antibody fragments

DOI: 10.1021/acschembio.1c00472

Authors: Alex Macpherson (UCB Pharma) , James R. Birtley (UCB Pharma) , Robert J. Broadbridge (Peptide Protein Research) , Kevin Brady (UCB Pharma) , Monika-Sarah E. D. Schulze (UCB Pharma) , Yalan Tang (UCB-Ra Pharma) , Callum Joyce (UCB Pharma) , Kenneth Saunders (UCB Pharma) , Gregory Bogle (Covance Ltd) , John Horton (Peptide Protein Research) , Sebastian Kelm (UCB Pharma) , Richard D. Taylor (UCB Pharma) , Richard J. Franklin (UCB Pharma) , Matthew D. Selby (UCB Pharma) , Maisem Laabei (University of Bath) , Toska Wonfor (University of Bath) , Adam Hold (UCB Pharma) , Phil Stanley (UCB Pharma) , Douangsone Vadysirisack (UCB-Ra Pharma) , Jiye Shi (UCB Pharma) , Jean Van Den Elsen (University of Bath) , Alastair D. G. Lawson (UCB Pharma)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Acs Chemical Biology , VOL 16 , PAGES 1757 - 1769

State: Published (Approved)
Published: September 2021

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 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.

Journal Keywords: Disulfides; Peptides and proteins; Monomers; Enzyme-linked immunosorbent assays; Assays

Subject Areas: Biology and Bio-materials, Chemistry, Medicine


Instruments: I03-Macromolecular Crystallography

Added On: 06/04/2022 14:57

Documents:
acschembio.1c00472.pdf

Discipline Tags:

Health & Wellbeing Biochemistry Chemistry Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)