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Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining 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., Competing Interests: AM, ZA, JB, MS, BH, VO, JS, VE, AL employee of UCB and may hold shares and/or stock options, ML, MG, SC, SR, PN, DS, Jv No competing interests declared, TM T.E.M is a Board member of Ra Pharmaceuticals, Inc, CD Reviewing editor, eLife, (© 2021, Macpherson et al.)

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)-Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD-IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests. Alex Macpherson, Fabien Lecomte, Richard D. Taylor, Tim Norman, Marta Westwood, Ben Cossins, James White, Robert Griffin, Christine Prosser, Sebastian Kelm, Alistair Henry, Richard Taylor, and Alastair D. Lawson are all current or former employees and/or shareholders in UCB Pharma. Nicolas Basse is an employee and/or shareholder in Sanofi. Hervé Deboves is an employee and/or shareholder in Evotec. John Porter is an employee and/or shareholder in Midatech Pharma. Katharine Cain is an employee and/or shareholder in Vertex. Amy H. Sullivan and David Fox III are/were employees and/or shareholders in Beryllium Discovery. This work was funded by UCB Pharma.

We have enumerated all linear combinations of ring systems from FDA approved drugs, up to three rings in length and up to four bonds linkers to give an in silico database of approximately 14 million molecules. This virtual library was compared with molecular databases of published and commercially available compounds to assess the prevalence of drug ring combinations in modern medicinal chemistry and to identify areas of under-represented, but clinically validated, chemical space. From the 10 trillion molecular comparisons, we found that less than 1% of the possible combinations of drug ring systems appear in commercially available libraries. This key observation highlights significant opportunities to design new fragment-like and lead-like libraries aimed at improving success rates and reducing risk in small molecule drug discovery, as, based on our previous analysis ( Taylor J. Med. Chem. 2014 , 57 , 5845 - 5849 ), approximately 70% of all new drugs are made up of only ring systems that have been used in existing drugs.

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the "junctional epitope" nature of VHH6, a camelid single domain antibody recognizing the IL-6-gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions., Competing Interests: With the exception of U.W. and J.K., all authors are or have been UCB employees at the time this project was running. A.D.G.L., T.S.B., S.L., C.R.V., M.W., D.M.M., R.A. and S.R. hold shares and/or share options in UCB.

Therapeutic and diagnostic applications of monoclonal antibodies often require careful selection of binders that recognize specific epitopes on the target molecule to exert a desired modulation of biological function. Here we present a proof-of-concept application for the rational design of an epitope-specific antibody binding with the target protein Keap1, by grafting pre-defined structural interaction patterns from the native binding partner protein, Nrf2, onto geometrically matched positions of a set of antibody scaffolds. The designed antibodies bind to Keap1 and block the Keap1-Nrf2 interaction in an epitope-specific way. One resulting antibody is further optimised to achieve low-nanomolar binding affinity by in silico redesign of the CDRH3 sequences. An X-ray co-crystal structure of one resulting design reveals that the actual binding orientation and interface with Keap1 is very close to the design model, despite an unexpected CDRH3 tilt and V H /V L interface deviation, which indicates that the modelling precision may be improved by taking into account simultaneous CDR loops conformation and V H /V L orientation optimisation upon antibody sequence change. Our study confirms that, given a pre-existing crystal structure of the target protein-protein interaction, hotspots grafting with CDR loop swapping is an attractive route to the rational design of an antibody targeting a pre-selected epitope., Competing Interests: The authors declare competing financial interests: All authors are employees of UCB.

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.

An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

Earlier studies proved the success of using chemically functionalised multi-walled carbon nanotubes (f-MWNTs) as nanocarriers to the brain. Little insight into the kinetics of brain distribution of f-MWNTs in vivo has been reported. This study employed a wide range of qualitative and quantitative techniques with the aim of shedding the light on f-MWNT's brain distribution following intravenous injection. γ-Scintigraphy quantified the uptake of studied radiolabelled f-MWNT in the whole brain parenchyma and capillaries while 3D-single photon emission computed tomography/computed tomography imaging and autoradiography illustrated spatial distribution within various brain regions. Raman and multiphoton luminescence together with transmission electron microscopy confirmed the presence of intact f-MWNT in mouse brain, in a label-free manner. The results evidenced the presence of f-MWNT in mice brain parenchyma, in addition to brain endothelium. Such information on the rate and extent of regional and cellular brain distribution is needed before further implementation into neurological therapeutics can be made., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

The protein-protein interaction (PPI) target class is particularly challenging, but offers potential for "first in class" therapies. Most known PPI small molecules are orthosteric inhibitors but many PPI sites may be fundamentally intractable to this approach. One potential alternative is to consider more attractive, remote small molecule pockets; however, on the whole, allostery is poorly understood and difficult to discover and develop. Here we review the literature in order to understand the basis for allostery, especially as it can apply to PPIs. We suggest that the upfront generation of sophisticated and experimentally validated dynamic models of target proteins can aid in target choice and strategy for allosteric intervention to produce the required functional effect.

The Wnt signaling pathway is of central importance in embryogenesis, development and adult tissue homeostasis, and dysregulation of this pathway is associated with cancer and other diseases. Despite the developmental and potential therapeutic significance of this pathway, many aspects of Wnt signaling, including the control of the master transcriptional co-activator β-catenin, remain poorly understood. In order to explore this aspect, a diverse immune llama VHH phagemid library was constructed and panned against β-catenin. VHH antibody fragments from the library were expressed intracellularly, and a number of antibodies were shown to possess function-modifying intracellular activity in a luciferase-based Wnt signaling HEK293 reporter bioassay. Further characterization of one such VHH (named LL3) confirmed that it bound endogenous β-catenin, and that it inhibited the Wnt signaling pathway downstream of the destruction complex, while production of a control Ala-substituted complementarity-determining region (CDR)3 mutant demonstrated that the inhibition of β-catenin activity by the parent intracellular antibody was dependent on the specific CDR sequence of the antibody.

As the considerable technical challenges involved with generating crystal structures of G (guanine nucleotide- binding) protein-coupled receptors (GPCRs) are starting to be successfully addressed, opportunities to apply fragment-based drug discovery (FBDD) to this class of target are becoming a reality. GPCRs represent a large and important family of drug targets with considerable clinical and commercial interest. While their general seven transmembrane helix bundle structures are amenable to therapeutic intervention with small molecules, to date successful drugs have primarily been discovered using traditional competitive or function-based screening. With advances in biophysical screening techniques such as Surface Plasmon Resonance (SPR) and Target-Immobilised NMR Screening (TINS), being matched to developments in molecular dynamics simulations, virtual screening and stabilisation of biologically relevant conformations of GPCRs, structure-based approaches using fragment starting points are beginning to be applied to the discovery of new generations of small molecules.