Targeting amyotrophic lateral sclerosis by neutralizing seeding-competent TDP-43 in CSF.

In amyotrophic lateral sclerosis, a disease driven by abnormal transactive response DNA-binding protein of 43 kDa aggregation, CSF may contain pathological species of transactive response DNA-binding protein of 43 kDa contributing to the propagation of pathology and neuronal toxicity. These species, released in part by degenerating neurons, would act as a template for the aggregation of physiological protein contributing to the spread of pathology in the brain and spinal cord. In this study, a robust seed amplification assay was established to assess the presence of seeding-competent transactive response DNA-binding protein of 43 kDa species in CSF of apparently sporadic amyotrophic lateral sclerosis patients. These samples resulted in a significant acceleration of substrate aggregation differentiating the kinetics from healthy controls. In parallel, a second assay was developed to determine the level of target engagement that would be necessary to neutralize such species in human CSF by a therapeutic monoclonal antibody targeting transactive response DNA-binding protein of 43 kDa. For this, evaluation of the pharmacokinetic/pharmacodynamic effect for the monoclonal antibody, ACI-5891.9, in vivo and in vitro confirmed that a CSF concentration of ≍1100 ng/mL would be sufficient for sustained target saturation. Using this concentration in the seed amplification assay, ACI-5891.9 was able to neutralize the transactive response DNA-binding protein of 43 kDa pathogenic seeds derived from amyotrophic lateral sclerosis patient CSF. This translational work adds to the evidence of transmission of transactive response DNA-binding protein of 43 kDa pathology via CSF that could contribute to the non-contiguous pattern of clinical manifestations observed in amyotrophic lateral sclerosis and demonstrates the ability of a therapeutic monoclonal antibody to neutralize the toxic, extracellular seeding-competent transactive response DNA-binding protein of 43 kDa species in the CSF of apparently sporadic amyotrophic lateral sclerosis patients.

Improved antibody pharmacokinetics by disruption of contiguous positive surface potential and charge reduction using alternate human framework.

Optimal pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) are essential to achieve the desired pharmacological benefits in patients. To accomplish this, we followed an approach comprising structure-based mAb charge engineering in conjunction with the use of relevant preclinical models to screen and select humanized candidates with PK suitable for clinical development. Murine mAb targeting TDP-43, ACI-5891, was humanized on a framework (VH1-3/VK2-30) selected based on the highest sequence homology. Since the initial humanized mAb (ACI-5891.1) presented a fast clearance in non-human primates (NHPs), reiteration of humanization on a less basic human framework (VH1-69-2/VK2-28) while retaining high sequence homology was performed. The resulting humanized variant, ACI-5891.9, presented a six-fold reduction in clearance in NHPs resulting in a significant increase in half-life. The observed reduced clearance of ACI-5891.9 was attributed not only to the overall reduction in isoelectric point (pI) by 2 units, but importantly to a more even surface potential. These data confirm the importance and contribution of surface charges to mAb disposition in vivo. Consistent low clearance of ACI-5891.9 in Tg32 mice, a human FcRn transgenic mouse model, further confirmed its utility for early assessment and prediction of human PK. These data demonstrate that mAb surface charge is an important parameter for consideration during the selection and screening of humanized candidates in addition to maintaining the other key physiochemical and target binding characteristics.

High ionic strength dissociation assay (HISDA) for high drug tolerant immunogenicity testing.

Aim: Antidrug antibody (ADA) assessment may be challenged in studies that involve the administration of high doses of biotherapeutics and/or with long half-lives. In such cases, ADA assays with optimized drug tolerance are desired. Material & Methods: We evaluated the use of MgCl2 to develop high ionic strength dissociation assays in two investigational examples (bridging enzyme-linked immunosorbent ADA assays) to attain high drug tolerance while maintaining best possible structural integrity of ADAs. Results: Both ADA-bridging assays treated with MgCl2 showed improved drug tolerance and higher signal-to-blank values compared with overnight incubation or acid treatment. Conclusion: The use of MgCl2 treatment in ADA-bridging assays provides a sensitive, drug tolerant and easy-to-use alternative in cases where acid dissociation is not possible or unwanted.

NI-0801, an anti-chemokine (C-X-C motif) ligand 10 antibody, in patients with primary biliary cholangitis and an incomplete response to ursodeoxycholic acid.

NI-0801 is a fully human monoclonal antibody against chemokine (C-X-C motif) ligand 10 (CXCL10), which is involved in the recruitment of inflammatory T cells into the liver. The safety and efficacy of NI-0801 was assessed in patients with primary biliary cholangitis. In this open-label phase 2a study, patients with primary biliary cholangitis with an inadequate response to ursodeoxycholic acid received six consecutive intravenous administrations of NI-0801 (10 mg/kg) every 2 weeks. Patients were followed up for 3 months after the last infusion. Liver function tests, safety assessments, as well as pharmacokinetic and pharmacodynamic parameters were evaluated at different time points throughout the dosing period and the safety follow-up period. Twenty-nine patients were enrolled in the study and were treated with NI-0801. The most frequently reported adverse events included headaches (52%), pruritus (34%), fatigue (24%), and diarrhea (21%). No study drug-related serious adverse events were reported. NI-0801 administration did not lead to a significant reduction in any of the liver function tests assessed at the end of the treatment period (i.e., 2 weeks after final NI-0801 administration) compared to baseline. Conclusion: Despite clear pharmacologic responses in the blood, no therapeutic benefit of multiple administrations of NI-0801 could be demonstrated. The high production rate of CXCL10 makes it difficult to achieve drug levels that lead to sustained neutralization of the chemokine, thus limiting its targetability. (Hepatology Communications 2018;2:492-503).

Elevated circulating levels of interferon-γ and interferon-γ-induced chemokines characterise patients with macrophage activation syndrome complicating systemic juvenile idiopathic arthritis.

OBJECTIVES: Interferon-γ (IFNγ) is the pivotal mediator in murine models of primary haemophagocytic lymphohistiocytosis (pHLH). Given the similarities between primary and secondary HLH (sec-HLH), including macrophage activation syndrome (MAS), we investigate the involvement of the IFNγ pathway in MAS by evaluating levels of IFNγ and of the induced chemokines, and their relation with laboratory parameters of MAS in systemic juvenile idiopathic arthritis (sJIA) patients with MAS and in a murine MAS model. METHODS: The Luminex multiplexing assay was used to assess serum levels of interleukin (IL)-1ß, IL-6, IFNγ and of the IFNγ-induced chemokines CXCL9, CXCL10 and CXCL11 in patients with sec-HLH (n=11) and in patients with sJIA (n=54), of whom 20 had active MAS at sampling. Expression of IFNγ-induced chemokines was assessed in IL-6 transgenic mice in which MAS is induced by TLR4 stimulation with lipopolysaccharide. RESULTS: Levels of IFNγ and of IFNγ-induced chemokines were markedly elevated during active MAS and sec-HLH and were significantly higher in patients with MAS compared with active sJIA without MAS. Levels in patients with active sJIA without MAS were comparable to those of patients with clinically inactive sJIA. During MAS, ferritin and alanine transferase levels and neutrophil and platelet counts were significantly correlated with serum levels of IFNγ and CXCL9. In murine MAS, serum levels of ferritin were significantly correlated with mRNA levels of Cxcl9 in liver and spleen. CONCLUSIONS: The high levels of IFNγ and of IFNγ-induced chemokines and their correlation with the severity of laboratory abnormalities of MAS suggest a pivotal role of IFNγ in MAS.

Use of a mouse model to identify a blood biomarker for IFNγ activity in pediatric secondary hemophagocytic lymphohistiocytosis.

Life-threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon γ (IFNγ) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection-driven model of sHLH in mice, we observed that the tissue-derived levels of IFNγ are actually 500- to 2000-fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFNγ gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFNγ activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFNγ-biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFNγ activity in patients with sHLH.

Novel Insights into Interleukin 6 (IL-6) Cis- and Trans-signaling Pathways by Differentially Manipulating the Assembly of the IL-6 Signaling Complex.

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.

CC chemokine CCL5 plays a central role impacting infarct size and post-infarction heart failure in mice.

AIMS: The chemokine CCL5 plays a critical role as neutrophil and macrophage activator do in atherosclerosis and myocardial infarction. Thus, we investigated whether the treatment with a neutralizing monoclonal antibody (mAb) to mouse CCL5 would provide therapeutic benefit when provoking a coronary-associated ischaemic event. METHODS AND RESULTS: C57Bl/6 mice were submitted to left coronary artery permanent ligature. Then, various parameters were monitored for up to 21 days. At5 min and 3 days after coronary occlusion, mice received one intravenous injection of the rat anti-mouse CCL5 mAb or isotype IgG control. Infarct size was assessed histologically and by measuring serum cardiac troponin I levels. Kinetics of CCL5 tissue expression, leucocyte infiltration, matrix metalloproteinase (MMP) levels, and collagen deposition were histologically assessed. Serum chemokine levels were measured by enzyme-linked immunosorbent assay. Cardiac function and dimensions were assessed by magnetic resonance imaging (MRI). Chronic ischaemia increased both circulating and intracardiac levels of CCL5. At 24 h, treatment with the anti-CCL5 mAb resulted in a smaller infarct size and reduced circulating levels of chemokines. This effect was associated with reduction of neutrophil and macrophage infiltration within the infarcted myocardium. After 3 days of chronic ischaemia, anti-CCL5 mAb treatment reduced cardiac MMP-9. At 7 days, collagen content was significantly lower. At 21 days, neutralizing CCL5 improved mouse survival, cardiac myocyte size, and cardiac function. CONCLUSION: Treatment with anti-CCL5 mAb significantly reduced both infarct size and post-infarction heart failure in a mouse model of chronic cardiac ischaemia. Cardioprotective effects were associated with the reduction of leucocyte recruitment within infarcted hearts.

Inhibition of classic signaling is a novel function of soluble glycoprotein 130 (sgp130), which is controlled by the ratio of interleukin 6 and soluble interleukin 6 receptor.

IL-6 trans-signaling via the soluble IL-6 receptor (sIL-6R) plays a critical role in chronic inflammation and cancer. Soluble gp130 (sgp130) specifically inhibits IL-6 trans-signaling but was described to not interfere with classic signaling via the membrane-bound IL-6R. Physiological and most pathophysiological conditions are characterized by a molar excess of serum sIL-6R over IL-6 characterized by free IL-6 and IL-6 found in IL-6·sIL-6R complexes allowing both classic and trans-signaling. Surprisingly, under these conditions, sgp130 was able to trap all free IL-6 molecules in IL-6·sIL-6R·sgp130 complexes, resulting in inhibition of classic signaling. Because a significant fraction of IL-6 molecules did not form complexes with sIL-6R, our results demonstrate that compared with the anti-IL-6R antibody tocilizumab or the anti-trans-signaling monoclonal antibody 25F10, much lower concentrations of the dimeric sgp130Fc were sufficient to block trans-signaling. In vivo, sgp130Fc blocked IL-6 signaling in the colon but not in liver and lung, indicating that the colon is a prominent target of IL-6 trans-signaling. Our results point to a so far unanticipated role of sgp130 in the blockade of classic signaling and indicate that in vivo only low therapeutic concentrations of sgp130Fc guarantee blockade of IL-6 trans-signaling without affecting IL-6 classic signaling.

Although IL-6 trans-signaling is sufficient to drive local immune responses, classical IL-6 signaling is obligate for the induction of T cell-mediated autoimmunity.

IL-6-mediated T cell-driven immune responses are associated with signaling occurring through the membrane-bound cognate receptor α-chain (mIL-6Rα). Once formed, IL-6-mIL-6Rα complexes induce the homodimerization and subsequent phosphorylation of the ubiquitously expressed signal-transducing protein, gp130. This signaling event is defined as classical IL-6 signaling. However, many inflammatory processes assigned to IL-6 may be mediated via binding a naturally occurring soluble IL-6Rα, which forms an agonistic complex (IL-6/soluble IL-6Rα) capable of evoking responses on a wide range of cell types that lack mIL-6Rα (IL-6 trans-signaling). To dissect the differential contribution of the two IL-6 signaling pathways in cell-mediated inflammatory processes, we pharmaceutically targeted each using two murine models of human arthritis. Whereas intra-articular neutralization of trans-signaling attenuated local inflammatory responses, the classical pathway was found to be obligate and sufficient to induce pathogenic T cells and humoral responses, leading to systemic disease. Our data illustrate that mechanisms occurring in the secondary lymphoid organs underlying arthropathies are mediated via the classical pathway of IL-6 signaling, whereas trans-signaling contributes only at the local site, that is, in the affected tissues.

Rapid, simple and high yield production of recombinant proteins in mammalian cells using a versatile episomal system.

Many research projects in life sciences require purified biologically active recombinant protein. In addition, different formats of a given protein may be needed at different steps of experimental studies. Thus, the number of protein variants to be expressed and purified in short periods of time can expand very quickly. We have therefore developed a rapid and flexible expression system based on described episomal vector replication to generate semi-stable cell pools that secrete recombinant proteins. We cultured these pools in serum-containing medium to avoid time-consuming adaptation of cells to serum-free conditions, maintain cell viability and reuse the cultures for multiple rounds of protein production. As such, an efficient single step affinity process to purify recombinant proteins from serum-containing medium was optimized. Furthermore, a series of multi-cistronic vectors were designed to enable simultaneous expression of proteins and their biotinylation in vivo as well as fast selection of protein-expressing cell pools. Combining these improved procedures and innovative steps, exemplified with seven cytokines and cytokine receptors, we were able to produce biologically active recombinant endotoxin free protein at the milligram scale in 4-6weeks from molecular cloning to protein purification.

The IL-27 p28 subunit binds cytokine-like factor 1 to form a cytokine regulating NK and T cell activities requiring IL-6R for signaling.

IL-27 is formed by the association of a cytokine subunit, p28, with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3(-/-) and WSX-1(-/-) mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27, p28/CLF is produced by dendritic cells and is biologically active on human NK cells, increasing IL-12- and IL-2-induced IFN-gamma production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Ralpha in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells, p28/CLF induces IL-6Ralpha-dependent STAT1 and STAT3 phosphorylation. Furthermore, p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors.

Continued administration of ciliary neurotrophic factor protects mice from inflammatory pathology in experimental autoimmune encephalomyelitis.

Multiple sclerosis is an inflammatory disease of the central nervous system that leads to loss of myelin and oligodendrocytes and damage to axons. We show that daily administration (days 8 to 24) of murine ciliary neurotrophic factor (CNTF), a neurotrophic factor that has been described as a survival and differentiation factor for neurons and oligodendrocytes, significantly ameliorates the clinical course of a mouse model of multiple sclerosis. In the acute phase of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide 35-55, treatment with CNTF did not change the peripheral immune response but did reduce the number of perivascular infiltrates and T cells and the level of diffuse microglial activation in spinal cord. Blood brain barrier permeability was significantly reduced in CNTF-treated animals. Beneficial effects of CNTF did not persist after it was withdrawn. After cessation of CNTF treatment, inflammation and symptoms returned to control levels. However, slight but significantly higher numbers of oligodendrocytes, NG2-positive cells, axons, and neurons were observed in mice that had been treated with high concentrations of CNTF. Our results show that CNTF inhibits inflammation in the spinal cord, resulting in amelioration of the clinical course of experimental autoimmune encephalomyelitis during time of treatment.

Inactivation of cardiotrophin-like cytokine, a second ligand for ciliary neurotrophic factor receptor, leads to cold-induced sweating syndrome in a patient.

Ciliary neurotrophic factor (CNTF) receptor controls a pathway supporting the differentiation and survival of a wide range of neural cell types during development and in adulthood. Cardiotrophin-like cytokine (CLC)-cytokine-like factor 1 (CLF) composite cytokine is a second ligand for the CNTF alpha-component receptor (CNTFRalpha). This composite cytokine is built on the structural model of IL-12, with a complex formed by a four-helix bundle type I cytokine, CLC (also referred to as CLCF1), bound to a soluble receptor subunit, CLF (also known as CRLF1). We have reported mutations in the chaperone soluble receptor CLF, causing cold-induced sweating syndrome (CISS). In this study, we studied the CLC-mutated alleles in a patient suffering from a similar disease. This patient was compound heterozygous for two different CLC mutations. The first allele was inactivated by a stop codon at position 107 (Y107X). In the second allele, a R197L mutation in the CLC-predicted binding site to the CNTFRalpha was detected. Functional analysis of the mutated protein revealed an incapacity for R197L CLC to bind to CNTFRalpha and activate the subsequent signaling events. Structural and docking interaction studies showed that the R197L substitution destabilized the contact site between CLC and CNTFRalpha.

Detection of epsilon class switching and IgE synthesis in human B cells.

We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.

Cardiotrophin-like cytokine labelling using Bir A biotin ligase: a sensitive tool to study receptor expression by immune and non-immune cells.

The recently identified IL-6 family member cardiotrophin-like cytokine (also named novel neurotrophin-1 or B cell stimulating factor-3) forms a secreted complex with cytokine-like factor-1 which binds and activates the tripartite ciliary neurotrophic factor receptor. The striking differences between the phenotype of mice in which either the ciliary neurotrophic factor or its receptor are inactivated suggest that the cardiotrophin-like cytokine/cytokine-like factor-1 complex could be the developmentally important ciliary neurotrophic factor receptor ligand. Cardiotrophin-like cytokine is also produced in the immune system and has been reported to activate B cells in vivo and in vitro. B cells do not express the ciliary neurotrophic factor receptor suggesting the existence of an alternative receptor. We produced the cardiotrophin-like cytokine/cytokine-like factor-1 complex tagged with a Bir A biotin ligase AviTag peptide substrate. This cytokine could be efficiently biotinylated in vitro with Bir A. It was subsequently validated as a sensitive tool for ciliary neurotrophic factor receptor detection by flow cytometry and for magnetic-activated cell sorting. It was also shown to allow the detection of a specific receptor by activated B cells. Whereas binding to cells expressing the ciliary neurotrophic factor receptor could be prevented by competition with ciliary neurotrophic factor, binding to B cells was not. The biotinylated cardiotrophin-like cytokine/cytokine-like factor-1 complex therefore represents a new reagent to study ciliary neurotrophic factor and cardiotrophin-like cytokine receptor expression and for the identification of the putative cardiotrophin-like cytokine B cell receptor. It further validates the use of biotin ligase catalysed biotinylation for the detection of cytokine receptors.

Expression of biologically active mouse ciliary neutrophic factor (CNTF) and soluble CNTFRalpha in Escherichia coli and characterization of their functional specificities.

Ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine initially identified in chick embryo. It has been evaluated for the treatment of neurodegenerative diseases. CNTF also acts on non-neuronal cells such as oligodendrocytes, astrocytes, adipocytes and skeletal muscles cells. CNTF has regulatory effects on body weight and is currently in clinical trial for the treatment of diabetes and obesity. CNTF mediates its function by activating a tripartite receptor comprising the CNTF receptor alpha chain (CNTFRalpha), the leukemia inhibitory factor receptor beta chain (LIFRbeta) and gp130. Human, rat and chicken CNTF have been expressed as recombinant proteins, and most preclinical studies in murine models have been performed using rat recombinant protein. Rat and human CNTF differ in their fine specificities: in addition to CNTFR, rat CNTF has been shown to activate the LIFR (a heterodimer of LIFRbeta and gp130), whereas human CNTF can bind and activate a tripartite receptor comprising the IL-6 receptor alpha chain (IL-6Ralpha) and LIFR. To generate tools designed for mouse models of human diseases; we cloned and expressed in E. coli both mouse CNTF and the CNTFRalpha chain. Recombinant mouse CNTF was active and showed a high level of specificity for mouse CNTFR. It shares the arginine residue with rat CNTF which prevents binding to IL-6Ralpha. It did not activate the LIFR at all concentrations tested. Recombinant mouse CNTF is therefore specific for CNTFR and as such represents a useful tool with which to study CNTF in mouse models. It appears well suited for the comparative evaluation of CNTF and the two additional recently discovered CNTFR ligands, cardiotrophin-like cytokine\cytokine-like factor-1 and neuropoietin.