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We present a comprehensive analysis of all ring systems (both heterocyclic and nonheterocyclic) in clinical trial compounds and FDA-approved drugs. We show 67% of small molecules in clinical trials comprise only ring systems found in marketed drugs, which mirrors previously published findings for newly approved drugs. We also show there are approximately 450 000 unique ring systems derived from 2.24 billion molecules currently available in synthesized chemical space, and molecules in clinical trials utilize only 0.1% of this available pool. Moreover, there are fewer ring systems in drugs compared with those in clinical trials, but this is balanced by the drug ring systems being reused more often. Furthermore, systematic changes of up to two atoms on existing drug and clinical trial ring systems give a set of 3902 future clinical trial ring systems, which are predicted to cover approximately 50% of the novel ring systems entering clinical trials.

Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors., Small molecules stabilising a distorted TNF trimer can inhibit TNF signaling, but the underlying mechanism is unclear. Here, the authors characterize the inhibitor-bound TNF-receptor complex structurally and biochemically, showing that the inhibitors alter TNF-receptor binding stoichiometry and cluster formation.

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 chemical synthesis, with a co-crystal structure of a chemically synthesised knob domain in complex with antigen showing structural equivalence to the biological product. For drug discovery, following immunisation of cattle, knob domain peptides can be synthesised directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate non-biological modifications. We demonstrate that, through rational design with non-natural amino acids, 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 cyclisation, exploiting the unusual proximity of the N- and C-termini to synthesise 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 characterisation of their molecular structures, in vitro pharmacology and pharmacokinetics.

Over the last 10 years considerable progress has been made in the application of small molecules to modulating protein-protein interactions (PPIs), and the navigation from “undruggable” to a host of candidate molecules in clinical trials has been well-charted in recent, comprehensive reviews. Structure-based design has played an important role in this scientific journey, with three dimensional structures guiding medicinal chemistry efforts. However, the importance of two additional dimensions: movement and time is only now being realised, as increasing computing power, closely aligned with wet lab validation, is applied to the challenge. Protein dynamics are fundamental to biology and disease, and application to PPI drug discovery has massively widened the scope for new chemical entities to influence function from allosteric, and previously unreported, sites. In this forward-looking perspective we highlight exciting, new opportunities for small molecules to modulate disease biology, by adjusting the frequency profile of natural conformational sampling, through the stabilisation of clinically desired conformers of target proteins.

Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having identical variable domains that are different for each set of IgG1 and IgG4P constructs. Relative aggregation propensities were determined at pH 7.4, representing physiological conditions, and pH 5.0, representing commonly used storage conditions. Our work indicates that the net charge state of variable domains relative to the net charge state of the constant domains is predominantly responsible for the different native state aggregation behavior of IgG1 and IgG4P mAbs. This observation suggests that the global net charge of a multi domain protein is not a reliable predictor of aggregation propensity. Furthermore, we demonstrate a design strategy in the frameworks of variable domains to reduce the native state aggregation propensity of mAbs identified as being aggregation-prone. Importantly, substitution of specifically identified residues with alternative, human germline residues, to optimize Fv charge, resulted in decreased aggregation potential at pH 5.0 and 7.4, thus increasing developability.

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer–small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF–TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein–protein interactions., TNF can be inhibited by small molecules that stabilize the TNF trimer in an asymmetric conformation. Here, the authors develop a monoclonal antibody that selectively binds this inactive form of TNF, enabling both target engagement assessment and structural characterization of TNF binding to TNF receptor 1.

eLife 10, e63586 (1-27) (2021). doi:10.7554/eLife.63586, 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 �� 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., Published by eLife Sciences Publications, Cambridge

The proinflammatory cytokines IL-17A and IL-17F have been identified as key drivers of a range of human inflammatory diseases, such as psoriasis, which has led to several therapeutic antibodies targeted at IL-17A. The two cytokines have been shown to tightly associate as functional homo and hetero dimers, which induce signalling via the formation of a cell surface signalling complex with a single copy of both IL-17RA and IL-17RC. Striking differences in affinity have been observed for IL-17RA binding to IL-17AA, IL-17AF and IL-17FF, however, the functional significance and molecular basis for this has remained unclear. We have obtained comprehensive backbone NMR assignments for full length IL-17AA (79%), IL-17AF (93%) and IL-17FF (89%), which show that the dimers adopt almost identical backbone topologies in solution to those observed in reported crystal structures. Analysis of the line widths and intensities of assigned backbone amide NMR signals has revealed striking differences in the conformational plasticity and dynamics of IL-17AA compared to both IL-17AF and IL-17FF. Our NMR data indicate that a number of regions of IL-17AA are interconverting between at least two distinct conformations on a relatively slow timescale. Such conformational heterogeneity has previously been shown to play an important role in the formation of many high affinity protein-protein complexes. The locations of the affected IL-17AA residues essentially coincides with the regions of both IL-17A and IL-17F previously shown to undergo significant structural changes on binding to IL-17RA. Substantially less conformational exchange was revealed by the NMR data for IL-17FF and IL-17AF. We propose that the markedly different conformational dynamic properties of the distinct functional IL-17 dimers plays a key role in determining their affinities for IL-17RA, with the more dynamic and plastic nature of IL-17AA contributing to the significantly tighter affinity observed for binding to IL-17RA. In contrast, the dynamic properties are expected to have little influence on the affinity of IL-17 dimers for IL-17RC, which has recently been shown to induce only small structural changes in IL-17FF upon binding.

To overcome limited germline combinatorial diversity, bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce affinity-maturated 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.

Interleukin (IL)-17A is a key driver of inflammation and the principal target of anti-IL-17 therapeutic monoclonal antibodies. IL-17A, and its structurally similar family member IL-17F, have been shown to be functionally dysregulated in certain human immune-mediated inflammatory diseases such as psoriasis, psoriatic arthritis, and axial spondyloarthritis. Given the overlapping biology of these two cytokines, we postulated that dual neutralization of IL-17A and IL-17F may provide a greater depth of clinical response in IL-17-mediated diseases than IL-17A inhibition alone. We identified 496.g1, a humanized antibody with strong affinity for IL-17A but poor affinity for IL-17F. Affinity maturation of 496.g1 to 496.g3 greatly enhanced the affinity of the Fab fragment for IL-17F while retaining strong binding to IL-17A. As an IgG1, the affinity for IL-17A and IL-17F was 3.2 pM and 23 pM, respectively. Comparison of 496.g3 IgG1 with the commercially available anti-IL-17A monoclonal antibodies ixekizumab and secukinumab, by surface plasmon resonance and in a human in vitro IL-17A functional assay, showed that 496.g3 and ixekizumab display equivalent affinity for IL-17A, and that both antibodies are markedly more potent than secukinumab. In contrast to ixekizumab and secukinumab, 496.g3 exhibited the unique feature of also being able to neutralize the biological activity of IL-17F. Therefore, antibody 496.g3 was selected for clinical development for its ability to neutralize the biologic function of both IL-17A and IL-17F and was renamed bimekizumab (formerly UCB4940). Early clinical data in patients with psoriasis, in those with psoriatic arthritis, and from the Phase 2 studies in psoriasis, psoriatic arthritis, and ankylosing spondylitis, are encouraging and support the targeted approach of dual neutralization of IL-17A and IL-17F. Taken together, these findings provide the rationale for the continued clinical evaluation of bimekizumab in patients with immune-mediated inflammatory diseases.

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 present the first X-ray crystal structures of disulphide-linked human C3d dimers and show they undergo structurally-stabilising N-terminal domain swapping when in complex with the Staphylococcus aureus immunomodulator Sbi. Through binding studies and flow cytometric analyses we uncover the physiologically-relevant roles of these dimers in crosslinking complement receptor 2 and modulating B cell activation to potentially promote anergy. This potential induction of cellular tolerance by C3d dimers could contribute to Sbi-mediated S. aureus immune evasion as well as limit autoreactive immune responses under physiological conditions. Thus, insights gained from our findings could inform the design of novel therapies for autoimmune disorders and enhance our understanding surrounding the importance of complement in the fluid phase.

Complement component C5 is the target of the monoclonal antibody Eculizumab, and the focus of a sustained drug discovery effort to prevent complement-induced inflammation in a range of autoimmune diseases. The immune evasion protein OmCI binds to, and potently inactivates, C5; this tight-binding interaction can be exploited to affinity-purify C5 protein from serum, offering a vastly simplified protocol compared to existing methods. However, breaking the high-affinity interaction requires conditions which risk denaturing or activating C5. We performed structure-guided in silico mutagenesis to identify prospective OmCI residues that contribute significantly to the binding affinity. We tested our predictions in vitro, using site directed mutagenesis, and characterised mutants using a range of biophysical techniques as well as functional assays. Our biophysical analyses suggest the C5-OmCI interaction is complex with potential for multiple binding modes.We present single mutations that lower the affinity of OmCI for C5 and combinations of mutations that significantly decrease, or entirely abrogate, formation of the complex. The affinity attenuated forms of OmCI are suitable for affinity purification and allow elution under mild conditions that are non-denaturing or activating to C5. We present the rational design, biophysical characterisation and experimental validation of affinity reduced forms of OmCI as tool reagents to enable the affinity purification of C5.

Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions.

Tackling PPIs, particularly by stabilizing clinically favored conformations of target proteins, with orally available, bona fide small molecules remains a significant but immensely worthwhile challenge for the pharmaceutical industry. Success may be more likely through the application of nature's learnings to build intrinsic rigidity into the design of clinical candidates.

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.

Protein drug targets vary from highly structured to completely disordered; either way dynamics governs function. Hence, understanding the dynamical aspects of how protein targets function can enable improved interventions with drug molecules. Computational approaches offer highly detailed structural models of protein dynamics which are becoming more predictive as model quality and sampling power improve. However, the most advanced and popular models still have errors owing to imperfect parameter sets and often cannot access longer timescales of many crucial biological processes. Experimental approaches offer more certainty but can struggle to detect and measure lightly populated conformations of target proteins and subtle allostery. An emerging solution is to integrate available experimental data into advanced molecular simulations. In the future, molecular simulation in combination with experimental data may be able to offer detailed models of important drug targets such that improved functional mechanisms or selectivity can be accessed.

Tackling PPIs, particularly by stabilizing clinically favored conformations of target proteins, with orally available, bona fide small molecules remains a significant but immensely worthwhile challenge for the pharmaceutical industry. Success may be more likely through the application of nature's learnings to build intrinsic rigidity into the design of clinical candidates.

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.

Double electron-electron resonance in conjunction with site-directed spin labeling has been used to probe natural conformational sampling of the human tumor necrosis factor α trimer. We suggest a previously unreported, predeoligomerization conformation of the trimer that has been shown to be sampled at low frequency. A model of this trimeric state has been constructed based on crystal structures using the double-electron-electron-resonance distances. The model shows one of the protomers to be rotated and tilted outward at the tip end, leading to a breaking of the trimerous symmetry and distortion at a receptor-binding interface. The new structure offers opportunities to modulate the biological activity of tumor necrosis factor α through stabilization of the distorted trimer with small molecules.

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.

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.

Camel antibodies have been widely investigated, but work has focused upon the unique heavy chain antibodies found across camelid species. These are homodimers, devoid of light chains and the first constant heavy chain domain. Camelid species also display conventional hetero-tetrameric antibodies with identical pairs of heavy and light chains; in Camelus dromedarius these constitute 25% of circulating antibodies. Few investigations have been made on this subset of antibodies and complete conventional camel IgG sequences have not been reported.Here we study the sequence diversity of functional variable and constant regions observed in 57 conventional heavy, 18 kappa and 35 lambda light chains of C. dromedarius and Camelus bactrianus. We detail sequences of the full kappa and lambda light chain, variable and CH1 region for IgG1a and IgG1b and the CH2 and CH3 region for IgG1a. The majority (60%) of IgG1 variable region sequences aligned with the human IgHV3 family (clan III) and had leader sequences beginning with MELG whereas the remaining sequences aligned with the IgHV4 (clan II) and had leader sequences beginning with MRLL. Distinct differences in CDR length were observed between the two; where CDR1 was typically 5 and 7 residues and CDR2 at 17 and 16 residues, respectively. CDR3 length of IgHV4 (range 11 to 20) was closer to that typical of VHH antibodies than that of IgHV3 (range 3 to 18 residues). Designed oligonucleotide primers have enabled identification of paired heavy and light chains of conventional camel antibodies from individual B cell clones.

Specific, high affinity protein-protein interactions lie at the heart of many essential biological processes, including the recognition of an apparently limitless range of foreign proteins by natural antibodies, which has been exploited to develop therapeutic antibodies. To mediate biological processes, high affinity protein complexes need to form on appropriate, relatively rapid timescales, which presents a challenge for the productive engagement of complexes with large and complex contact surfaces (∼600–1800 Å2). We have obtained comprehensive backbone NMR assignments for two distinct, high affinity antibody fragments (single chain variable and antigen-binding (Fab) fragments), which recognize the structurally diverse cytokines interleukin-1β (IL-1β, β-sheet) and interleukin-6 (IL-6, α-helical). NMR studies have revealed that the hearts of the antigen binding sites in both free anti-IL-1β Fab and anti-IL-6 single chain variable exist in multiple conformations, which interconvert on a timescale comparable with the rates of antibody-antigen complex formation. In addition, we have identified a conserved antigen binding-induced change in the orientation of the two variable domains. The observed conformational heterogeneity and slow dynamics at protein antigen binding sites appears to be a conserved feature of many high affinity protein-protein interfaces structurally characterized by NMR, suggesting an essential role in protein complex formation. We propose that this behavior may reflect a soft capture, protein-protein docking mechanism, facilitating formation of high affinity protein complexes on a timescale consistent with biological processes.

We have analyzed the rings, ring systems, and frameworks in drugs listed in the FDA Orange Book to understand the frequency, timelines, molecular property space, and the application of these rings in different therapeutic areas and target classes. This analysis shows that there are only 351 ring systems and 1197 frameworks in drugs that came onto the market before 2013. Furthermore, on average six new ring systems enter drug space each year and approximately 28% of new drugs contain a new ring system. Moreover, it is very unusual for a drug to contain more than one new ring system and the majority of the most frequently used ring systems (83%) were first used in drugs developed prior to 1983. These observations give insight into the chemical novelty of drugs and potentially efficient ways to assess compound libraries and develop compounds from hit identification to lead optimization and beyond.

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.

Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe.

A number of secreted cytokines, such as interleukin-6 (IL-6), are attractive targets for the treatment of inflammatory diseases. We have determined the solution structure of mouse IL-6 to assess the functional significance of apparent differences in the receptor interaction sites (IL-6Rα and gp130) suggested by the fairly low degree of sequence similarity with human IL-6. Structure-based sequence alignment of mouse IL-6 and human IL-6 revealed surprising differences in the conservation of the two distinct gp130 binding sites (IIa and IIIa), which suggests a primacy for site III-mediated interactions in driving initial assembly of the IL-6/IL-6Rα/gp130 ternary complex. This is further supported by a series of direct binding experiments, which clearly demonstrate a high affinity IL-6/IL-6Rα-gp130 interaction via site III but only weak binding via site II. Collectively, our findings suggest a pathway for the evolution of the hexameric, IL-6/IL-6Rα/gp130 signaling complex and strategies for therapeutic targeting. We propose that the signaling complex originally involved specific interactions between IL-6 and IL-6Rα (site I) and between the D1 domain of gp130 and IL-6/IL-6Rα (site III), with the later inclusion of interactions between the D2 and D3 domains of gp130 and IL-6/IL-6Rα (site II) through serendipity. It seems likely that IL-6 signaling benefited from the evolution of a multipurpose, nonspecific protein interaction surface on gp130, now known as the cytokine binding homology region (site II contact surface), which fortuitously contributes to stabilization of the IL-6/IL-6Rα/gp130 signaling complex.

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 VH/VL interface deviation, which indicates that the modelling precision may be improved by taking into account simultaneous CDR loops conformation and VH/VL 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.

The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 CH1 and IgG4 CH1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L–H) bond, did not affect thermal stability. Introducing the IgG1 type L–H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L–H interchain DSB with the IgG4 type L–H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 CH1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format.

Although antibody-based therapeutics have become firmly established as medicines for serious diseases, the value of antibodies as tools in the early stages of small-molecule drug discovery is only beginning to be realized. In particular, antibodies may provide information to reduce risk in small-molecule drug discovery by enabling the validation of targets and by providing insights into the design of small-molecule screening assays. Moreover, antibodies can act as guides in the quest for small molecules that have the ability to modulate protein-protein interactions, which have traditionally only been considered to be tractable targets for biological drugs. The development of small molecules that have similar therapeutic effects to current biologics has the potential to benefit a broader range of patients at earlier stages of disease.

SUMMARY Aims: Lacosamide (LCM; SPM 927, Vimpat®) is an antiepileptic drug (AED) used as adjunctive treatment for adults with partial-onset seizures. LCM has a different mode of action from traditional sodium channel blocking AEDs in that it selectively enhances slow inactivation of sodium channels without affecting fast inactivation. Initial investigations suggested that LCM might have an additional mode of action by binding to the collapsin response mediator protein 2 (CRMP-2), which is further investigated here. Methods: LCM binding to native and cloned human CRMP-2 was determined using radioligand binding experiments and surface plasmon resonance measurements. Results: No specific binding of [3H]LCM (free concentration 100–1450 nM) to isolated or membrane bound human CRMP-2 expressed in mammalian cell systems and bacteria was observed. Surface plasmon resonance analysis also showed that LCM, over a concentration range of 0.39–100 μM, does not specifically bind to human CRMP-2. Conclusion: The diverse drug binding methods employed here are well suited to detect specific binding of LCM to CRMP-2 in the micromolar range, yet the results obtained were all negative. Results of this study suggest that LCM does not specifically bind to CRMP-2.

Background: Intestinal inflammation associated with inflammatory bowel disease (IBD) is typically characterized by an inflammatory cell infiltrate and pro-inflammatory cytokine production. Of particular interest, the frequency of colony stimulating factor-1 (CSF-1)-expressing cells is increased in active lesions. In this study, we have investigated the role of CSF-1 in mucosal inflammation, using a murine model of colitis induced by dextran sulfate sodium (DSS). Methods: A neutralizing anti-CSF-1 antibody was administered to Balb/c mice that received DSS in their drinking water. Signs of colitis, such as clinical disease score, cellular infiltrate, and cytokine production, were assessed. Results: Administration of a neutralizing anti-CSF-1 antibody significantly inhibited DSS-induced colitis. Clinical symptoms, such as weight loss and the appearance of diarrhea or fecal blood, were reduced by CSF-1 blockade; histologic scores were also improved. The cellular infiltrate of macrophages and T cells was inhibited and a trend toward reduced production of pro-inflammatory cytokines was noted. Conclusions: This is the first study to demonstrate that CSF-1 plays an important role in mediating intestinal mucosal inflammation and therefore may prove to be an attractive therapeutic target for intestinal diseases such as inflammatory bowel disease. (Inflamm Bowel Dis 2006)

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.

We demonstrate the importance of optimizing the balance of light chain (LC) and heavy chain (HC) expression to achieve high level production of Fab' fragments in the Escherichia coli periplasm. The LC:HC balance has been controlled by varying the codon usage of the signal peptide (SP) and 5' mature domain coding regions. Different SP coding regions have been identified from a codon wobble-based library using alkaline phosphatase (AP) as a reporter gene. A plasmid system that enables random combination of these variant SP coding regions is used to construct optimized Fab' expression plasmids. These small plasmid libraries facilitated selection of optimal Fab' expression plasmids and resulted in increases of periplasmic yield, up to 580 mgL(-1) from E. coli fermentations and will enable rapid variable region subcloning and selection of future Fab(') expression plasmids.

We have analyzed the rings, ring systems, and frameworks in drugs listed in the FDA Orange Book to understand the frequency, timelines, molecular property space, and the application of these rings in different therapeutic areas and target classes. This analysis shows that there are only 351 ring systems and 1197 frameworks in drugs that came onto the market before 2013. Furthermore, on average six new ring systems enter drug space each year and approximately 28% of new drugs contain a new ring system. Moreover, it is very unusual for a drug to contain more than one new ring system and the majority of the most frequently used ring systems (83%) were first used in drugs developed prior to 1983. These observations give insight into the chemical novelty of drugs and potentially efficient ways to assess compound libraries and develop compounds from hit identification to lead optimization and beyond.

Single chain Fv chimeric receptors, or T-bodies, are described with intracellular sequences comprising the costimulatory signaling domain of CD28 in series with the ζ-chain from the TCR complex. Using an engineered human single chain Fv derived from P67, an mAb with specificity for human CD33, and a spacer comprising an Ab hinge region with either Fcγ or part of the CD28 extracellular region, fusion molecules were constructed to test the ability of single chain designs to mediate both primary signaling and costimulation from one extracellular binding event. Constructs with the CD28 signaling domain proximal and the ζ-chain distal to the membrane were found to express more efficiently in Jurkat than constructs with the opposite orientation and were capable of mediating up to 20 times more IL-2 production on stimulation with solid phase Ag when compared with transfectants expressing chimeric receptors with ζ-chain intracellular signaling domains only. IL-2 production was specific to Ag challenge and was completely inhibited by incubation with free Ab of the same specificity as the extracellular binding site of the construct, but not by an isotype-matched control Ab. The CD28 intracellular domain of these fusion proteins was shown to be capable of binding the p85 subunit of phosphatidylinositol 3′-kinase. These constructs represent the first of a new generation of single gene multidomain chimeric receptors capable of mediating both primary and costimulatory signaling specifically from a single extracellular recognition event.

Heavy chain antibodies differ in structure to conventional antibodies lacking both the light chain and the first heavy chain constant domain (CH1). Characteristics of the antigen-binding variable heavy domain of the heavy chain antibody (VHH) including the smaller size, high solubility and stability make them an attractive alternative to more traditional antibody fragments for detailed NMR-based structural analysis. Here we report essentially complete backbone and side chain (15)N, (13)C and (1)H assignments for a free VHH. Analysis of the backbone chemical shift data obtained indicates that the VHH is comprised predominantly of β-sheets corresponding to nearly 60% of the protein backbone.

The identification of the optimal T-cell effector subtype is a crucial issue for adoptive cell therapy with chimeric receptor-modified T cells. The ideal T cell population must be able to home toward tumor site, exert prolonged antitumoral activity, and display minimal toxicity against normal tissues. Therefore, we characterized the in vitro antitumoral properties of three effector T-cell populations: Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs), cytokine-induced killer (CIK) cells, and γ9δ2 T (GDT) cells, after transduction with a chimeric receptor specific for the CD33 antigen, broadly expressed on acute myeloid leukemia cells. EBV-CTLs, CIK, and GDT cells were generated and transduced with high efficiency with a retroviral vector coding for an anti-CD33-ζ chimeric receptor without alterations of their native phenotype. Anti-CD33-ζ chimeric receptor-redirected T cells displayed analogous in vitro chemotactic activity toward CXCL12. In addition, anti-CD33-ζ chimeric receptor-expressing EBV-CTLs, CIK, and GDT cells showed potent and similar cytotoxicity against several CD33 leukemic targets both in short-term 4-hours- Chromium-release assays (mean killing vs primary leukemic cells at effector:target ratio of 5:1; 50%, 61%, and 50% for EBV-CTLs, CIK, and GDT cells, respectively) and in long-term assays, where they were cocultured with leukemic cells for 6 days on stromal mesenchymal cells (mean survival of primary leukemic cells at effector:target ratio of 1:100; 18%, 16%, and 29% for EBV-CTLs, CIK, and GDT cells, respectively). Moreover, all effector cells acquired consistent capability to proliferate in vitro after contact with CD33cells and to release high and comparable levels of immunostimulatory cytokines, while secreting similar low amount of immunoregulatory cytokines as the unmanipulated counterpart. Our results indicate that expression of an anti-CD33-ζ chimeric receptor potently and similarly increase the antileukemic functions of different effector T-cell subtypes, underlying the impossibility to identify a more potent T-cell population through in vitro analysis, and consistently with recent observations that have emerged from clinical trials with chimeric receptor-modified T cells, suggesting the need to perform such type of studies in the human setting. © 2011 by Lippincott Williams & Wilkins.

Intestinal inflammation associated with inflammatory bowel disease (IBD) is typically characterized by an inflammatory cell infiltrate and pro-inflammatory cytokine production. Of particular interest, the frequency of colony stimulating factor-1 (CSF-l)-expressing cells is increased in active lesions. In this study, we have investigated the role of CSF-1 in mucosal inflammation, using a murine model of colitis induced by dextran sulfate sodium (DSS).A neutralizing anti-CSF-1 antibody was administered to Balb/c mice that received DSS in their drinking water. Signs of colitis, such as clinical disease score, cellular infiltrate, and cytokine production, were assessed.Administration of a neutralizing anti-CSF-1 antibody significantly inhibited DSS-induced colitis. Clinical symptoms, such as weight loss and the appearance of diarrhea or fecal blood, were reduced by CSF-1 blockade; histologic scores were also improved. The cellular infiltrate of macrophages and T cells was inhibited and a trend toward reduced production of pro-inflammatory cytokines was noted.This is the first study to demonstrate that CSF-1 plays an important role in mediating intestinal mucosal inflammation and therefore may prove to be an attractive therapeutic target for intestinal diseases such as inflammatory bowel disease.