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.

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.

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.

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.

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.

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.

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.

Whole-molecule antibody engineering: generation of a high-affinity anti-IL-6 antibody with extended pharmacokinetics.

The differentiation of therapeutic monoclonal antibodies in an increasingly competitive landscape requires optimization of clinical efficacy combined with increased patient convenience. We describe here the generation of MEDI5117, a human anti-interleukin (IL)-6 antibody generated by variable domain engineering, to achieve subpicomolar affinity for IL-6, combined with Fc (fragment crystallizable) engineering to enhance pharmacokinetic half-life. MEDI5117 was shown to be highly potent in disease-relevant cellular assays. The pharmacokinetics of MEDI5117 were evaluated and compared to those of its progenitor, CAT6001, in a single-dose study in cynomolgus monkeys. The antibodies were administered, either subcutaneously or intravenously, as a single dose of 5 mg/kg. The half-life of MEDI5117 was extended by approximately 3-fold, and clearance was reduced by approximately 4-fold when compared to CAT6001. MEDI5117 therefore represents a potential 'next-generation' antibody; future studies are planned to determine the potential for affinity-driven efficacy and/or less frequent administration.