posted on 2021-08-18, 16:33authored byAlex 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 Shi, Jean van den Elsen, Alastair D. G. Lawson
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.