The preclinical pharmacology of the high affinity anti-IL-6R Nanobody? ALX-0061 supports its clinical development in rheumatoid arthritis

IntroductionThe pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology.MethodsALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control.ResultsALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration.ConclusionsALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0651-0) contains supplementary material, which is available to authorized users.     

Development of a new tank-to-wheels methodology for energy use and green house gas emissions analysis based on vehicle fleet modeling

Background, aim and scope  

Tank-to-Wheels (TtW) makes the largest contribution to the total Well-to-Wheels (WtW) energy consumption and greenhouse gas (GHG) emissions from fossil-derived transportation fuels. The most commonly adopted TtW methodologies to obtain vehicle energy consumption, energy efficiency, and GHG emissions used to date all have significant limitations. A new TtW methodology, which combines micro-scale virtual vehicle simulation with macro-scale fleet modeling, is proposed in this paper. The models capabilities are demonstrated using a case study based on data from the passenger car sector in Great Britain.     

Chemical and biological characterisation of a sensor surface for bioprocess monitoring

This paper describes the step-wise fabrication and characterisation of a multi-layer dual polarization interferometry (DPI) based biosensor utilising Protein G (ProG) as the bio-recognition layer for the detection of a fragment antibody (Fab'). The biosensor is capable of monitoring the concentration of Fab' product within the extracellular medium of a fed-batch fermentation after leakage from Escherichia coli (E.coli). The activity, stability and functionality of each sensor layer were analysed in situ using DPI, whilst the chemical identity and homogeneity of the chemical layers were assessed ex situ using X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). Two different biotin linkers were found to produce hugely differing surfaces after the capture of NeutrAvidin? (NA) and biotinylated Protein G (b-ProG). The hydrophilic (PEG)(4)-biotin linker resulted in a surface where the b-ProG layer was deposited and organised above the NA layer producing an active and stable surface, whilst the hydrophobic LC-biotin linker generated a surface where the b-ProG layer was buried within the NA layer leading to variable surfaces and poor binding of the Fab' target. The biosensor has a detection limit of 1.7 μg/ml with a dynamic range covering two orders of magnitude. The sensor can detect the onset of Fab' leakage as early as 2h following product induction, with high signal-to-noise ratios and little interference from extracellular components. Leakage of Fab' followed a biphasic profile, switching to a more rapid rate 20 h after induction, indicating accelerated product loss and the need for cultivation harvest.     

Mouse LSECtin as a model for a human Ebola virus receptor

The biochemical properties of mouse LSECtin, a glycan-binding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcβ1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcβ1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor.     

Identification of chemically diverse, novel inhibitors of 17β-hydroxysteroid dehydrogenase type 3 and 5 by pharmacophore-based virtual screening

17β-Hydroxysteroid dehydrogenase type 3 and 5 (17β-HSD3 and 17β-HSD5) catalyze testosterone biosynthesis and thereby constitute therapeutic targets for androgen-related diseases or endocrine-disrupting chemicals. As a fast and efficient tool to identify potential ligands for 17βHSD3/5, ligand- and structure-based pharmacophore models for both enzymes were developed. The models were evaluated first by in silico screening of commercial compound databases and further experimentally validated by enzymatic efficacy tests of selected virtual hits. Among the 35 tested compounds, 11 novel inhibitors with distinct chemical scaffolds, e.g. sulfonamides and triazoles, and with different selectivity properties were discovered. Thereby, we provide several potential starting points for further 17β-HSD3 and 17β-HSD5 inhibitor development. Article from the Special issue on Targeted Inhibitors.     

Rap1 mediates galanin receptor 2-induced proliferation and survival in squamous cell carcinoma

Previously we showed that galanin, a neuropeptide, is secreted by human squamous cell carcinoma of the head and neck (SCCHN) in which it exhibits an autocrine mitogenic effect. We also showed that rap1, a ras-like signaling protein, is a critical mediator of SCCHN progression. Given the emerging importance of the galanin cascade in regulating proliferation and survival, we investigated the effect of GAL on SCCHN progression via induction of galanin receptor 2 (GALR2)-mediated rap1 activation. Studies were performed in multiple SCCHN cell lines by inducing endogenous GALR2, by stably overexpressing GALR2 and by downregulating endogenous GALR2 with siGALR2. Cell proliferation and survival, mediated by the ERK and AKT signaling cascades, respectively, were evaluated by functional and immunoblot analysis. The role of rap1 in GALR2-mediated proliferation and survival was evaluated by modulating expression. Finally, the effect of GALR2 on tumor growth was determined. GALR2 stimulated proliferation and survival via ERK and AKT activation, respectively. Knockdown or inactivation of rap1 inhibited GALR2-induced, AKT and ERK-mediated survival and proliferation. Overexpression of GALR2 promoted tumor growth in vivo. GALR2 promotes proliferation and survival in vitro, and promotes tumor growth in vivo, consistent with an oncogenic role for GALR2 in SCCHN.     

An integrated approach to assessing nitroso-redox balance in systemic inflammation

Most studies examining the metabolic fate of NO during systemic inflammation have focused on measuring the quantitatively predominating, stable anions nitrite and nitrate within the circulation. However, these are not necessarily the NO-related products that govern NO metabolism and signaling in tissues. We assessed all major NO derivatives temporally in blood and vital organs during inflammation and explored their relationship to insult severity and redox status. Male rats receiving intraperitoneal endotoxin or vehicle were sacrificed for organ and blood sampling between 0 and 24 h. Endotoxin induced transient and organ-specific changes in a variety of NO metabolites. Nitrite and nitrate increased, peaking at 8 and 12 h, respectively. S- and N-nitrosation and heme-nitrosylation products also peaked at 8 h; these posttranslational protein modifications were associated with decreased myocardial function (echocardiography). Evidence of oxidative stress and systemic inflammation was also obtained. The rise in most NO derivatives was proportional to insult severity. All metabolite levels normalized within 24 h, despite evidence of persisting myocardial dysfunction and clinical unwellness. Our findings point to a complex interplay between NO production, antioxidant defense, and redox status. Although the precise (patho)physiologic roles of specific NO derivatives and their diagnostic/prognostic utility await further investigation, nitroso species in erythrocytes are the most sensitive markers of NO in systemic inflammation, detectable before clinical symptoms manifest.     

Activation of promoter activity of the catalytic subunit of γ-glutamylcysteine ligase (GCL) in brain endothelial cells by insulin requires antioxidant response element 4 and altered glycemic status: implication for GCL expression and GSH synthesis

Our recent finding that insulin increased the expression of the glutamate-cysteine ligase catalytic subunit (GCLc) with coincident increases in GCL activity and cellular glutathione (GSH) in human brain microvascular endothelial cells (IHECs) suggests a role for insulin in vascular GSH maintenance. Here, using IHECs stably transfected with promoter-luciferase reporter vectors, we found that insulin increased GCLc promoter activity, which required a prerequisite increase or decrease in medium glucose. An intact antioxidant response element-4 was essential for promoter activation, which was attenuated by inhibitors of PI3-kinase/Akt/mTOR signaling. Interestingly, only under low-glucose conditions did promoter activation correlate with increased GCLc expression and GSH synthesis. Low tert-butylhydroperoxide (tBH) concentrations similarly mediated promoter activation, but the maximal activation dose was decreased 10-fold by insulin. Insulin-tBH coadministration abrogated the low or high glucose requirement for promoter activation, suggesting possible ROS involvement. ROS production was elevated at low glucose without or with insulin; however, GSH increases were not inhibited by tempol, suggesting that ROS did not achieve the threshold for driving GCLc promoter activation and de novo GSH synthesis. The minor effect of pyruvate also ruled out a major role for hypoglycemia (± insulin)-induced metabolic stress on GSH induction under these conditions.