Modular design of bi- and multi-specific knob domain fusions.

Introduction: The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.

Chimeric antigens displaying GPR65 extracellular loops on a soluble scaffold enabled the discovery of antibodies, which recognized native receptor.

GPR65 is a proton-sensing G-protein coupled receptor associated with multiple immune-mediated inflammatory diseases, whose function is relatively poorly understood. With few reagents commercially available to probe the biology of receptor, generation of an anti-GPR65 monoclonal antibody was desired. Using soluble chimeric scaffolds, such as ApoE3, displaying the extracellular loops of GPR65, together with established phage display technology, native GPR65 loop-specific antibodies were identified.

Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery.

Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα.

Phage Display of Bovine Ultralong CDRH3.

Phage display is an in vitro technique used in the discovery of monoclonal antibodies that has been used successfully in the discovery of both camelid VHH and shark variable new antigen receptor domains (VNAR). Bovines also contain a unique "ultralong CDRH3" with a conserved structural motif, comprising a knob domain and β-stalk. When removed from the antibody scaffold, either the entire ultralong CDRH3 or the knob domain alone, is typically capable of binding an antigen, to produce antibody fragments that are smaller than both VHH and VNAR.

Serum albumin binding knob domains engineered within a V framework III bispecific antibody format and as chimeric peptides.

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.

A computational method for predicting the aggregation propensity of IgG1 and IgG4(P) mAbs in common storage buffers.

The propensity for some monoclonal antibodies (mAbs) to aggregate at physiological and manufacturing pH values can prevent their use as therapeutic molecules or delay time to market. Consequently, developability assessments are essential to select optimum candidates, or inform on mitigation strategies to avoid potential late-stage failures. These studies are typically performed in a range of buffer solutions because factors such as pH can dramatically alter the aggregation propensity of the test mAbs (up to 100-fold in extreme cases).

Rings in Clinical Trials and Drugs: Present and Future.

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.

The proximity of the N- and C- termini of bovine knob domains enable engineering of target specificity into polypeptide chains.

Cysteine-rich knob domains can be isolated from the ultralong heavy-chain complementarity-determining region (CDR) 3, which are unique to a subset of bovine antibodies, to create antibody fragments of ~4 kDa. Advantageously, the N- and C- termini of these small binding domains are in close proximity, and we propose that this may offer a practical route to engineer extrinsic binding specificity into proteins. To test this, we transplanted knob domains into various loops of rat serum albumin, targeting sites that were distal to the interface with the neonatal Fc receptor.

Insights Into the Structure-Function Relationships of Dimeric C3d Fragments.

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.

The Chemical Synthesis of Knob Domain Antibody Fragments.

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.

Modulating Target Protein Biology Through the Re-mapping of Conformational Distributions Using Small Molecules.

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.

Conformational dynamics in interleukin 17A and 17F functional complexes is a key determinant of receptor A affinity and specificity.

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.

The allosteric modulation of complement C5 by knob domain peptides.

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.

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF.

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.

Structural insights into the disruption of TNF-TNFR1 signalling by small molecules stabilising a distorted TNF.

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.

Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies.

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds.

Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability.

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.

Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer.

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.

The rational design of affinity-attenuated OmCI for the purification of complement C5.

Complement component C5 is the target of the mAb eculizumab and is 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 with existing methods. However, breaking the high-affinity interaction requires conditions that risk denaturing or activating C5.

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

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.

Computational Exploration of Conformational Transitions in Protein Drug Targets.

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.