Inducing protein aggregation by extensional flow.

Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced protein remodeling and/or aggregation is observed both in Nature and during the large-scale industrial manufacture of proteins. Despite its ubiquity, the relationships between the type and magnitude of hydrodynamic flow, a protein's structure and stability, and the resultant aggregation propensity are unclear. Here, we assess the effects of a defined and quantified flow field dominated by extensional flow on the aggregation of BSA, ß2-microglobulin (ß2m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs). We show that the device induces protein aggregation after exposure to an extensional flow field for 0.36-1.8 ms, at concentrations as low as 0.5 mg mL-1 In addition, we reveal that the extent of aggregation depends on the applied strain rate and the concentration, structural scaffold, and sequence of the protein. Finally we demonstrate the in situ labeling of a buried cysteine residue in BSA during extensional stress. Together, these data indicate that an extensional flow readily unfolds thermodynamically and kinetically stable proteins, exposing previously sequestered sequences whose aggregation propensity determines the probability and extent of aggregation.

Rituximab causes a polarization of B cells that augments its therapeutic function in NK-cell-mediated antibody-dependent cellular cytotoxicity.

Rituximab, which binds CD20 on B cells, is one of the best-characterized antibodies used in the treatment of B-cell malignancies and autoimmune diseases. Rituximab triggers natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but little is known about the spatial and temporal dynamics of cell-cell interactions during ADCC or what makes rituximab potent at triggering ADCC. Here, using laser scanning confocal microscopy, we found that rituximab caused CD20 to cap at the B-cell surface independent of antibody crosslinking or intercellular contact. Unexpectedly, other proteins, including intercellular adhesion molecule 1 and moesin, were selectively recruited to the cap of CD20 and the microtubule organizing center became polarized toward the cap. Importantly, the frequency at which NK cells would kill target cells via ADCC increased by 60% when target cells were polarized compared with when they were unpolarized. Polarized B cells were lysed more frequently still when initial contact with NK cells occurred at the place where CD20 was capped. This demonstrates that the site of contact between immune cells and target cells influences immune responses. Together, these data establish that rituximab causes a polarization of B cells and this augments its therapeutic function in triggering NK-cell-mediated ADCC.

Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

Current approaches to fine mapping of antigen-antibody interactions.

A number of different methods are commonly used to map the fine details of the interaction between an antigen and an antibody. Undoubtedly the method that is now most commonly used to give details at the level of individual amino acids and atoms is X-ray crystallography. The feasibility of undertaking crystallographic studies has increased over recent years through the introduction of automation, miniaturization and high throughput processes. However, this still requires a high level of sophistication and expense and cannot be used when the antigen is not amenable to crystallization. Nuclear magnetic resonance spectroscopy offers a similar level of detail to crystallography but the technical hurdles are even higher such that it is rarely used in this context. Mutagenesis of either antigen or antibody offers the potential to give information at the amino acid level but suffers from the uncertainty of not knowing whether an effect is direct or indirect due to an effect on the folding of a protein. Other methods such as hydrogen deuterium exchange coupled to mass spectrometry and the use of short peptides coupled with ELISA-based approaches tend to give mapping information over a peptide region rather than at the level of individual amino acids. It is quite common to use more than one method because of the limitations and even with a crystal structure it can be useful to use mutagenesis to tease apart the contribution of individual amino acids to binding affinity.

Tozorakimab (MEDI3506): an anti-IL-33 antibody that inhibits IL-33 signalling via ST2 and RAGE/EGFR to reduce inflammation and epithelial dysfunction.

Interleukin (IL)-33 is a broad-acting alarmin cytokine that can drive inflammatory responses following tissue damage or infection and is a promising target for treatment of inflammatory disease. Here, we describe the identification of tozorakimab (MEDI3506), a potent, human anti-IL-33 monoclonal antibody, which can inhibit reduced IL-33 (IL-33red) and oxidized IL-33 (IL-33ox) activities through distinct serum-stimulated 2 (ST2) and receptor for advanced glycation end products/epidermal growth factor receptor (RAGE/EGFR complex) signalling pathways. We hypothesized that a therapeutic antibody would require an affinity higher than that of ST2 for IL-33, with an association rate greater than 107 M-1 s-1, to effectively neutralize IL-33 following rapid release from damaged tissue. An innovative antibody generation campaign identified tozorakimab, an antibody with a femtomolar affinity for IL-33red and a fast association rate (8.5 × 107 M-1 s-1), which was comparable to soluble ST2. Tozorakimab potently inhibited ST2-dependent inflammatory responses driven by IL-33 in primary human cells and in a murine model of lung epithelial injury. Additionally, tozorakimab prevented the oxidation of IL-33 and its activity via the RAGE/EGFR signalling pathway, thus increasing in vitro epithelial cell migration and repair. Tozorakimab is a novel therapeutic agent with a dual mechanism of action that blocks IL-33red and IL-33ox signalling, offering potential to reduce inflammation and epithelial dysfunction in human disease.

Short-term variations in the oxidizing power of the atmosphere.

The hydroxyl radical is the predominant atmospheric oxidant, responsible for removing a wide range of trace gases, including greenhouse gases, from the atmosphere. Determination of trends and variability in hydroxyl radical concentrations is critical to understanding whether the 'cleansing' properties of the atmosphere are changing. The variability in hydroxyl radical concentrations on annual to monthly timescales, however, is difficult to quantify. Here we show records of carbon monoxide containing radiocarbon (14CO), which is oxidized by hydroxyl radicals, from clean-air sites at Baring Head, New Zealand, and Scott Base, Antarctica, spanning 13 years. Using a model study, we correct for known variations in production of 14CO (refs 6, 7), allowing us to exploit this species as a diagnostic for short term changes in hydroxyl radical concentrations. We find no significant long-term trend in hydroxyl radical concentrations but provide evidence for recurring short-term variations of around ten per cent persisting for a few months. We also find decreases in hydroxyl radical concentrations of up to 20 per cent, apparently triggered by the eruption of Mt Pinatubo in 1991 and by the occurrence of extensive fires in Indonesia in 1997.

An in vivo platform to select and evolve aggregation-resistant proteins.

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve 'manufacturable' biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.

Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. PAI-1 has been shown to play a role in a number of diverse pathologies including cardiovascular diseases, obesity and cancer and is therefore an attractive therapeutic target. However the multiple patho-physiological roles of PAI-1, and understanding the relative contributions of these in any one disease setting, make the development of therapeutically relevant molecules challenging. Here we describe the identification and characterisation of fully human antibody MEDI-579, which binds with high affinity and specificity to the active form of human PAI-1. MEDI-579 specifically inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). We propose that MEDI-579 acts by directly competing with proteases for RCL binding and as such is able to modulate the interaction of PAI-1 with tPA and uPA in a way not previously described for a human PAI-1 inhibitor.

Investigating the Structural Compaction of Biomolecules Upon Transition to the Gas-Phase Using ESI-TWIMS-MS.

Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein's size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds. Graphical Abstract ᅟ.

A comparison of the ability of the human IgG1 allotypes G1m3 and G1m1,17 to stimulate T-cell responses from allotype matched and mismatched donors.

The immunogenicity of clinically administered antibodies has clinical implications for the patients receiving them, ranging from mild consequences, such as increased clearance of the drug from the circulation, to life-threatening effects. The emergence of methods to engineer variable regions resulting in the generation of humanised and fully human antibodies as therapeutics has reduced the potential for adverse immunogenicity. However, due to differences in sequence referred to as allotypic variation, antibody constant regions are not homogeneous within the human population, even within sub-classes of the same immunoglobulin isotype. For therapeutically administered antibodies, the potential exists for an immune response from the patient to the antibody if the allotype of patient and antibody do not match. Allotypic distribution in the human population varies within and across ethnic groups making the choice of allotype for a therapeutic antibody difficult. This study investigated the potential of human IgG1 allotypes to stimulate responses in human CD4(+) T cells from donors matched for homologous and heterologous IgG1 allotypes. Allotypic variants of the therapeutic monoclonal antibody trastuzumab were administered to genetically defined allotypic matched and mismatched donor T cells. No significant responses were observed in the mismatched T cells. To investigate the lack of T-cell responses in relation to mismatched allotypes, HLA-DR agretopes were identified via MHC associated peptide proteomics (MAPPs). As expected, many HLA-DR restricted peptides were presented. However, there were no peptides presented from the sequence regions containing the allotypic variations. Taken together, the results from the T-cell assay and MAPPs assay indicate that the allotypic differences in human IgG1 do not represent a significant risk for induction of immunogenicity.

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

The INNs and outs of antibody nonproprietary names.

An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies.

Rheumatology: a force for change in monoclonal antibodies.

Monoclonal antibodies have undergone a radical evolutionary journey over the last 25 years, with advances in molecular biology, process development and drug formulation underpinning their rise to becoming one of the most efficacious and economically important class of drug today. The success of antibodies for the treatment of rheumatological conditions, such as rheumatoid arthritis in particular, has driven the biopharmaceutical industry to optimize all aspects of therapeutic antibody discovery and development, in order to provide further benefits for patients over and above marketed treatments, via increased clinical efficacy and patient convenience. Recent advances in many of the key aspects of antibody design that are driving this evolution, such as affinity optimization, effector function modulation, pharmacokinetic engineering, as well as developments in subcutaneous drug delivery, are reviewed here.

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1.

Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding VH CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long VH CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long VH CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.

Oxidation of the alarmin IL-33 regulates ST2-dependent inflammation.

In response to infections and irritants, the respiratory epithelium releases the alarmin interleukin (IL)-33 to elicit a rapid immune response. However, little is known about the regulation of IL-33 following its release. Here we report that the biological activity of IL-33 at its receptor ST2 is rapidly terminated in the extracellular environment by the formation of two disulphide bridges, resulting in an extensive conformational change that disrupts the ST2 binding site. Both reduced (active) and disulphide bonded (inactive) forms of IL-33 can be detected in lung lavage samples from mice challenged with Alternaria extract and in sputum from patients with moderate-severe asthma. We propose that this mechanism for the rapid inactivation of secreted IL-33 constitutes a 'molecular clock' that limits the range and duration of ST2-dependent immunological responses to airway stimuli. Other IL-1 family members are also susceptible to cysteine oxidation changes that could regulate their activity and systemic exposure through a similar mechanism.

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity.

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.In our analyses, we observed distinct differences in the pattern of beneficial mutations in antibodies derived from phage and ribosome display selections, and discovered the lead antibody Jedi067 had a ~3700-fold improvement in KD over the parent KENB061. We constructed a homology model of the Fv region of Jedi067 to map the specific positions where mutations occurred in the CDR3 loops. For VL CDR3, positions 94 to 97 carry greater diversity in the ribosome display variants compared with the phage display. The positions 95a, 95b and 96 of VLCDR3 form part of the interface with VH in this model. The model shows that positions 96, 98, 100e, 100f, 100 g, 100h, 100i and 101 of the VHCDR3 include residues at the VH and VL interface. Importantly, Leu96 and Tyr98 are conserved at the interface positions in both phage and ribosome display indicating their importance in maintaining the VH-VL interface. For antibodies derived from ribosome display, there is significant diversity at residues 100a to 100f of the VH CDR3 compared with phage display. A unique deletion of isoleucine at position 102 of the lead candidate, Jedi067, also occurs in the VHCDR3.As anticipated, recombining the mutations via ribosome display led to a greater structural diversity, particularly in the heavy chain CDR3, which in turn led to antibodies with improved potencies. For this particular analysis, we also found that VH-VL interface positions provided a source of structural diversity for those derived from the ribosome display selections. This greater diversity is a likely consequence of the presence of a larger pool of recombinants in the ribosome display system, or the evolutionary capacity of ribosome display, but may also reflect differential selection of antibodies in the two systems.

Engineering approaches to develop the next generation of antibodies to respiratory targets.

Chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) represent a significant health burden worldwide and are a major unmet medical need. Asthma affects over 300 million people and leads to 250,000 deaths per year, with an increasing prevalence particularly in developing countries. Although a large proportion of asthmatics are maintained on beta agonists and corticosteroids, there still remains a group of patients where these medicines fail to modulate symptoms and who may therefore benefit from monoclonal antibody based drugs that are aimed at controlling the disease. COPD is a cigarette smoke-driven chronic inflammatory airway disease with an increasing global prevalence. Given that current therapies fail to prevent disease progression or mortality, this patient population is also a focus for the development of monoclonal antibody therapies. At present anti-IgE (omalizumab, Xolair®) is the only monoclonal antibody based drug approved in the respiratory space for the treatment of asthma. However, an increasing number of antibodies targeting key mediators/pathways of disease are in clinical development for both asthma and COPD, including targeting the Th2 pathway for asthma (anti-IL-4/5/13) and the pro-inflammatory cytokine IL-1 for COPD. This review will examine the antibody engineering approaches used to develop the next generation of antibodies, with a focus on respiratory disease.

Monovalent IgG4 molecules: immunoglobulin Fc mutations that result in a monomeric structure.

Antibodies have become the fastest growing class of biological therapeutics, in part due to their exquisite specificity and ability to modulate protein-protein interactions with a high biological potency. The relatively large size and bivalency of antibodies, however, limits their use as therapeutics in certain circumstances. Antibody fragments, such as single-chain variable fragments and antigen binding-fragments, have emerged as viable alternatives, but without further modifications these monovalent formats have reduced terminal serum half-lives because of their small size and lack of an Fc domain, which is required for FcRn-mediated recycling. Using rational engineering of the IgG4 Fc domain to disrupt key interactions at the CH3-CH3 interface, we identified a number of point mutations that abolish Fc dimerization and created half-antibodies, a novel monovalent antibody format that retains a monomeric Fc domain. Introduction of these mutations into an IgG1 framework also led to the creation of half-antibodies. These half-antibodies were shown to be soluble, thermodynamically stable and monomeric, characteristics that are favorable for use as therapeutic proteins. Despite significantly reduced FcRn binding in vitro, which suggests that avidity gains in a dimeric Fc are critical to optimal FcRn binding, this format demonstrated an increased terminal serum half-life compared with that expected for most alternative antibody fragments.

Development of a homogeneous high-throughput screening assay for biological inhibitors of human rhinovirus infection.

Infection with human rhinovirus (HRV) is thought to result in acute respiratory exacerbations of chronic obstructive pulmonary disorder (COPD). Consequently, prevention of HRV infection may provide therapeutic benefit to these patients. As all major group HRV serotypes infect cells via an interaction between viral coat proteins and intercellular adhesion molecule-1 (ICAM-1), it is likely that inhibitors of this interaction would prevent or reduce infections. Our objective was to use phage display technology in conjunction with naive human antibody libraries to identify anti-ICAM-1 antibodies capable of functional blockade of HRV infection. Key to success was the development of a robust, functionally relevant high-throughput screen (HTS) compatible with the specific challenges of antibody screening. In this article, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF) assay based on the inhibition of soluble ICAM-1 binding to live HRV16. We describe the implementation of the method in an antibody screening campaign and demonstrate the biological relevance of the assay by confirming the activity of resultant antibodies in a cell-based in vitro HRV infection assay.