I03-Macromolecular Crystallography
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Abstract: Psoriasis (PSO) and psoriatic arthritis (PsA) are Th17-driven inflammatory diseases where interleukin (IL)-17 cytokines like IL-17A and IL-17F promote disease pathogenesis. There are six IL-17 isoforms (IL-17A–F) and five IL-17 receptors (IL-17RA–E; Liu et al., 2020 ). IL-17 cytokines primarily function as homodimers, although IL-17A and IL-17F can heterodimerize ( Brembilla et al., 2018 ). IL-17A and IL-17F genes share the same chromosomal location (6p12), their proteins share ∼55% sequence homology, and both cytokines signal through the IL-17RA/RC heterodimeric complex ( Brembilla et al., 2018 , Liu et al., 2020 ). Inhibition of the IL-17 pathway using IL-17A targeted (secukinumab and ixekizumab) or IL-17RA targeted (brodalumab) biologics is effective in PSO and PsA. In contrast to these therapies, bimekizumab (BKZ) is a novel humanized IgG1 antibody that selectively inhibits IL-17F in addition to IL-17A with binding affinities of 23 pM and 3.2 pM, respectively ( Adams et al., 2020 ). The clinical efficacy of BKZ warrants increased utility in patients with PSO, thereby necessitating a deeper molecular understanding of its mechanism of action ( Reich et al., 2021b , Waters et al., 2021 , Wilson et al., 2022 ).
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Apr 2024
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I02-Macromolecular Crystallography
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Stevan
Shaw
,
Tim
Bourne
,
Chris
Meier
,
Bruce
Carrington
,
Rich
Gelinas
,
Alistair
Henry
,
Andrew
Popplewell
,
Ralph
Adams
,
Terry
Baker
,
Steve
Rapecki
,
Diane
Marshall
,
Adrian
Moore
,
Helen
Neale
,
Alastair
Lawson
Open Access
Abstract: Interleukin-6 (IL-6) is a critical regulator of the immune system and has been widely implicated in autoimmune disease. Here, we describe the discovery and characterization of olokizumab, a humanized antibody to IL-6. Data from structural biology, cell biology and primate pharmacology demonstrate the therapeutic potential of targeting IL-6 at “Site 3”, blocking the interaction with the signaling co-receptor gp130.
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Apr 2014
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Ralph
Adams
,
Laura
Griffin
,
Joanne E.
Compson
,
Mark
Jairaj
,
Terry
Baker
,
Tom
Ceska
,
Shauna
West
,
Oliver
Zaccheo
,
Emma
Davé
,
Alastair Dg.
Lawson
,
David P.
Humphreys
,
Sam
Heywood
Open Access
Abstract: We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1–7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 – pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.
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Jun 2016
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I04-Macromolecular Crystallography
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Ayla A.
Wahid
,
Rhys W.
Dunphy
,
Alex
Macpherson
,
Beth G.
Gibson
,
Liudmila
Kulik
,
Kevin
Whale
,
Catherine
Back
,
Thomas M.
Hallam
,
Bayan
Alkhawaja
,
Rebecca L.
Martin
,
Ingrid
Meschede
,
Maisem
Laabei
,
Alastair D. G.
Lawson
,
V. Michael
Holers
,
Andrew G.
Watts
,
Susan J.
Crennell
,
Claire L.
Harris
,
Kevin J.
Marchbank
,
Jean M. H.
Van Den Elsen
Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.
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Aug 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Daniel J
Shaw
,
Lorna C.
Waters
,
Sarah L
Strong
,
Monika-Sarah E. D.
Schulze
,
Gregory M
Greetham
,
Michael
Towrie
,
Anthony W.
Parker
,
Christine E.
Prosser
,
Alistair J.
Henry
,
Alistair D. G.
Lawson
,
Mark D.
Carr
,
Richard J.
Taylor
,
Neil T.
Hunt
,
Frederick W
Muskett
Diamond Proposal Number(s):
[29404]
Open Access
Abstract: Knowledge of protein dynamics is fundamental to the understanding of biological processes, with NMR and 2D-IR spectroscopy being two of the principal methods for studying protein dynamics. Here, we combine these two methods to gain a new understanding of the complex mechanism of a cytokine:receptor interaction. The dynamic nature of many cytokines is now being recognised as a key property in the signalling mechanism. Interleukin-17’s (IL-17) are proinflammatory cytokines which, if unregulated, are associated with serious autoimmune diseases such as psoriasis, and although there are several therapeutics on the market for these conditions, small molecule therapeutics remain elusive. Previous studies, exploiting crystallographic methods alone, have been unable to explain the dramatic differences in affinity observed between IL-17 dimers and their receptors, suggesting there are factors that cannot be fully explained by the analysis of static structures alone. Here, we show that the IL-17 family of cytokines have varying degrees of flexibility which directly correlates to their receptor affinities. Small molecule inhibitors of the cytokine:receptor interaction are usually thought to function by either causing steric clashes or structural changes. However, our results, supported by other biophysical methods, provide evidence for an alternate mechanism of inhibition, in which the small molecule rigidifies the protein, causing a reduction in receptor affinity. The results presented here indicate an induced fit model of cytokine:receptor binding, with the more flexible cytokines having a higher affinity. Our approach could be applied to other systems where the inhibition of a protein-protein interaction has proved intractable, for example due to the flat, featureless nature of the interface. Targeting allosteric sites which modulate protein dynamics, opens up new avenues for novel therapeutic development.
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Jun 2023
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Ralph
Adams
,
Callum
Joyce
,
Mikhail
Kuravskiy
,
Katriona
Harrison
,
Zainab
Ahdash
,
Matthew
Balmforth
,
Kelda
Chia
,
Cinzia
Marceddu
,
Matthew
Coates
,
James
Snowden
,
Emmanuel
Goursaud
,
Karelle
Ménochet
,
Jean
Van Den Elsen
,
Richard J.
Payne
,
Alastair D. G.
Lawson
,
Anthony
Scott-Tucker
,
Alex
Macpherson
Diamond Proposal Number(s):
[29404, 5956]
Open Access
Abstract: Background: Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering.
Methods: Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion.
Results: By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity.
Conclusions: This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.
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May 2023
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I03-Macromolecular Crystallography
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Alex
Macpherson
,
Maisem
Laabei
,
Zainab
Ahdash
,
Melissa A
Graewert
,
James R
Birtley
,
Monika-Sarah
Schulze
,
Susan
Crennell
,
Sarah A
Robinson
,
Ben
Holmes
,
Vladas
Oleinikovas
,
Per H.
Nilsson
,
James
Snowden
,
Victoria
Ellis
,
Tom Eirik
Mollnes
,
Charlotte M.
Deane
,
Dmitri
Svergun
,
Alastair D. G.
Lawson
,
Jean M. H.
Van Den Elsen
Diamond Proposal Number(s):
[20029]
Open Access
Abstract: Bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce high affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.
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Feb 2021
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I03-Macromolecular Crystallography
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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
Shi
,
Jean
Van Den Elsen
,
Alastair D. G.
Lawson
Open Access
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.
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Sep 2021
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