Increased Expression of Proatherogenic Proteins in Immune Cell Subtypes in Tobacco Cigarette Smokers But Not in Electronic Cigarette Vapers.

It is unclear how oxidative stress triggered by smoking and vaping may alter specific immune cell subsets. In this study, we showed that tobacco cigarette smoking, but not electronic-cigarette vaping, is associated with increased expression of major proteins in the toll-like receptor 4 (TLR4) inflammasome-interleukin (IL)-6 signalling axis in monocyte subtypes and T cells. TLR4 senses oxidative stress in immune cells caspase-1 is a key protein of inflammasome activation, and IL-6R-α is the receptor for IL-6 that drives proatherogenic IL-6 signalling. These findings implicate the non-nicotine, pro-oxidant toxicants in tobacco cigarette smoke as instigators of increased expression of key proteins in the TLR4-inflammasome-IL-6 axis that contribute to atherogenesis.

A Novel Role for IL-6 Receptor Classic Signaling: Induction of RORγt+Foxp3+ Tregs with Enhanced Suppressive Capacity.

BACKGROUND: New therapies blocking the IL-6 receptor (IL-6R) have recently become available and are successfully being used to treat inflammatory diseases like arthritis. Whether IL-6 blockers may help patients with kidney inflammation currently remains unknown. METHODS: To learn more about the complex role of CD4+ T cell-intrinsic IL-6R signaling, we induced nephrotoxic nephritis, a mouse model for crescentic GN, in mice lacking T cell-specific IL-6Ra. We used adoptive transfer experiments and studies in reporter mice to analyze immune responses and Treg subpopulations. RESULTS: Lack of IL-6Ra signaling in mouse CD4+ T cells impaired the generation of proinflammatory Th17 cells, but surprisingly did not ameliorate the course of GN. In contrast, renal damage was significantly reduced by restricting IL-6Ra deficiency to T effector cells and excluding Tregs. Detailed studies of Tregs revealed unaltered IL-10 production despite IL-6Ra deficiency. However, in vivo and in vitro, IL-6Ra classic signaling induced RORγt+Foxp3+ double-positive Tregs (biTregs), which carry the trafficking receptor CCR6 and have potent immunoregulatory properties. Indeed, lack of IL-6Ra significantly reduced Treg in vitro suppressive capacity. Finally, adoptive transfer of T cells containing IL-6Ra-/- Tregs resulted in severe aggravation of GN in mice. CONCLUSIONS: Our data refine the old paradigm, that IL-6 enhances Th17 responses and suppresses Tregs. We here provide evidence that T cell-intrinsic IL-6Ra classic signaling indeed induces the generation of Th17 cells but at the same time highly immunosuppressive RORγt+ biTregs. These results advocate caution and indicate that IL-6-directed therapies for GN need to be cell-type specific.

Hepatic leptin receptor expression can partially compensate for IL-6Rα deficiency in DEN-induced hepatocellular carcinoma.

OBJECTIVE: The current obesity pandemic represents a major health burden, given that it predisposes to the development of numerous obesity-associated disorders. The obesity-derived adipokines not only impair systemic insulin action but also increase the incidence of hepatocellular carcinoma (HCC), a highly prevalent cancer with poor prognosis. Thus, worldwide incidences of HCC are expected to further increase, and defining the molecular as well as cellular mechanisms will allow for establishing new potential treatment options. The adipose tissue of obese individuals increases circulating leptin and interleukin-6 (IL-6) levels, which both share similar signaling capacities such as Signal Transducer and Activator of Transcription 3 (STAT3) and Phosphoinositide 3-kinase (PI3K)/Akt activation. While mouse models with deficient IL-6 signaling show an ameliorated but not absent Diethylnitrosamine (DEN)-induced HCC development, the morbid obesity in mice with mutant leptin signaling complicates the dissection of hepatic leptin receptor (LEPR) and IL-6 signaling in HCC development. Here we have investigated the function of compensating hepatic LEPR expression in HCC development of IL-6Rα-deficient mice. METHODS: We generated and characterized a mouse model of hepatic LEPR deficiency that was intercrossed with IL-6Rα-deficient mice. Cohorts of single and double knockout mice were subjected to the DEN-HCC model to ascertain liver cancer development and characterize metabolic alterations. RESULTS: We demonstrate that both high-fat diet (HFD)-induced obesity and IL-6Rα deficiency induce hepatic Lepr expression. Consistently, double knockout mice show a further reduction in tumor burden in DEN-induced HCC when compared to control and single LepRL-KO/IL-6Rα knock out mice, whereas metabolism remained largely unaltered between the genotypes. CONCLUSIONS: Our findings reveal a compensatory role for hepatic LEPR in HCC development of IL-6Rα-deficient mice and suggest hepatocyte-specific leptin signaling as promoter of HCC under obese conditions.

Effects of Interleukin-6 on posterior capsular opacification.

The purpose of this work was to determine the effects of interleukin-6 (IL-6) on the development of posterior capsular opacification (PCO) in vitro and in vivo. Western blot and real-time PCR were used to test the IL-6-induced epithelial-mesenchymal transition (EMT) marker α-smooth muscle actin (α-SMA), the extracellular matrix (ECM) markers fibronectin (Fn) and type I collagen (COL-1), transforming growth factor ß2 (TGF-ß2), and the activation and role of the JAK/STAT3 signaling pathway in human lens epithelial cells (HLECs). Immunocytofluorescence staining was performed to detect gp130 and IL-6Rα expression in HLECs. Rat PCO models were then established to examine the impact of STAT3 knockdown by shRNA adeno-associated virus on PCO development, and immunohistochemical staining was performed to detect the expression of Fn in the anterior and posterior capsule in vivo. We found that IL-6 promotes the expression of Fn, COL-1, TGF-ß2, p-JAK2 and p-STAT3 in HLECs but exerts little effect on α-SMA. The JAK/STAT3 inhibitor WP1066 effectively suppressed the IL-6-induced expression of Fn and COL-1 in lens epithelial cells. STAT3 knockdown effectively inhibited the development of PCO in rats and significantly reduced the expression of Fn in the anterior and posterior capsule. These data suggest that IL-6 contributes to the development of PCO by promoting TGF-ß2 activation and ECM synthesis through a JAK/STAT3 signaling-dependent mechanism. Furthermore, inhibiting JAK/STAT3 signaling effectively impairs both PCO development in rats and ECM synthesis in the lens capsule.

Obesity exacerbates colitis-associated cancer via IL-6-regulated macrophage polarisation and CCL-20/CCR-6-mediated lymphocyte recruitment.

Colorectal cancer (CRC) is one of the most lethal cancers worldwide in which the vast majority of cases exhibit little genetic risk but are associated with a sedentary lifestyle and obesity. Although the mechanisms underlying CRC and colitis-associated colorectal cancer (CAC) remain unclear, we hypothesised that obesity-induced inflammation predisposes to CAC development. Here, we show that diet-induced obesity accelerates chemically-induced CAC in mice via increased inflammation and immune cell recruitment. Obesity-induced interleukin-6 (IL-6) shifts macrophage polarisation towards tumour-promoting macrophages that produce the chemokine CC-chemokine-ligand-20 (CCL-20) in the CAC microenvironment. CCL-20 promotes CAC progression by recruiting CC-chemokine-receptor-6 (CCR-6)-expressing B cells and γδ T cells via chemotaxis. Compromised cell recruitment as well as inhibition of B and γδ T cells protects against CAC progression. Collectively, our data reveal a function for IL-6 in the CAC microenvironment via lymphocyte recruitment through the CCL-20/CCR-6 axis, thereby implicating a potential therapeutic intervention for human patients.

Functional interleukin-6 receptor-α is located in tanycytes at the base of the third ventricle.

Interleukin (IL)-6- /- mice develop mature onset obesity, whereas i.c.v. injection of IL-6 decreases obesity in rodents. Moreover, levels of IL-6 in cerebrospinal fluid (CSF) were reported to be inversely correlated with obesity in humans. Tanycytes lining the base of the third ventricle (3V) in the hypothalamus have recently been reported to be of importance for metabolism. In the present study, we investigated whether tanycytes could respond to IL-6 in the CSF. With immunohistochemistry using a well characterised antibody directed against the ligand binding receptor for IL-6, IL-6 receptor α (IL-6Rα), it was found that tanycytes, identified by the two markers, vimentin and dopamine- and cAMP-regulated phosphoprotein of 32 kDa, contained IL-6Rα. There were fewer IL-6Rα on another type of ventricle-lining cells, ependymal cells, as identified by the marker glucose transporter-1. To demonstrate that the immunoreactive IL-6Rα were responsive to IL-6, we injected IL-6 i.c.v. This treatment increased immunoreactive phosphorylated signal transducer and activator of transcription-3 (pSTAT3) in tanycytes after 5 minutes and in cells in the medial part of the arcuate nucleus after 5 and 15 minutes. Intracerebroventricular injection of leptin exerted similar effects. As expected, i.p. injection of leptin also induced pSTAT3 staining in the hypothalamus, whereas i.p. IL-6 injection had little effect on this parameter. Intracerebroventricular or i.p. injection of vehicle only had no effect on pSTAT3-immunoreactivity. In summary, there are functional IL-6Rα on tanycytes at the bottom of the 3V, in agreement with the possibility that ventricular administration of IL-6 decreases obesity in mice via an effect on this cell type.

Molecular Dynamics Simulations Reveal Key Roles of the Interleukin-6 Alpha Receptor in the Assembly of the Human Interleukin-6 Receptor Complex.

Human interleukin-6 (hIL-6) is a pleiotropic cytokine with three distinct receptor epitopes, termed sites I, II, and III, which function to assemble a signaling complex. hIL-6 signals via a glycoprotein 130 (gp130) homodimer after initially forming a heterodimer with the nonsignaling α-receptor (IL-6Rα). The molecular description of the assembly of the hIL-6 signaling complex remains elusive because available structures provide descriptions of hIL-6 in its free and fully bound receptor forms, but not for intermediate steps that are crucial in the stepwise assembly of the signaling complex. In this report, molecular dynamics simulations provide atomic details describing the functional role of the initial hIL-6/IL-6Rα complex in facilitating subsequent interactions with gp130, which have not been previously shown. IL-6Rα binding to hIL-6 rigidifies the flexible N-terminus of the hIL-6 AB-loop through interactions with the D2 domain of IL-6Rα. This rigidification combined with repositioning of residues involved in gp130 receptor recognition promotes gp130 binding at site III. Binding of gp130 receptors at sites II and III is coupled with the release of the hIL-6 N-terminal AB-loop interaction and a pivoting of IL-6Rα around the hIL-6 helix bundle to the state of the hIL-6/IL-6Rα/gp130 complex.

Blinded sample size recalculation in clinical trials incorporating historical data.

Recruiting sufficient patients within an acceptable time horizon is an issue for most clinical trials and is especially challenging in the field of rare diseases. It is therefore an attractive option to include historical data from previous (pilot) trials in the current study thus reducing the recruitment burden. In clinical trials with binary endpoint, the required sample size does not only depend on the type I error rate, the power, and the treatment group difference but additionally on the overall event rate. However, there is usually some uncertainty in the planning phase about the value of this nuisance parameter. We present methods for blinded sample size recalculation in the setting of two-arm superiority trials with historical control data where the overall rate is estimated mid-course and the sample size is recalculated accordingly. The operating characteristics of the method are investigated in terms of actual type I error rate, power, and expected sample size. Application is illustrated with a clinical trial example in patients with systemic sclerosis, a rare connective tissue disorder.

Update of sarilumb to treat rheumatoid arthritis based on randomized clinical trials: a systematic review.

INTRODUCTION: Sarilumab is a human monoclonal antibody against Interleukin 6 α (IL-6α) receptor. Compared to tocilizumab, another IL-6 α receptor antibody, sarilumab has a different structure and higher affinity. Areas covered: In a systematic literature review, we examined all sarilumab randomized clinical trials (RCTs) in rheumatoid arthritis. The 6 reviewed RCTs included patients who were inadequate MTX, DMARD and/or TNFi responders. Sarilumab 150-200 mg every 2 weeks improved RA signs, symptoms, function and decreased radiological progression up to 52 weeks. The most common adverse events were infections and neutropenia, the latter of which will require careful observation in future trials. Examination of the effect of sero-positivity, disease duration, presence of erosions, use of previous biologic and comparisons to other biologics etc are still needed to complete understanding of this drug's profile. Long term studies, too, will be needed to assess long term tolerability Expert commentary: Results support the use of sarilumab to treat RA patients with inadequate response to MTX, other DMARDs and TNFis, although further studies are needed to fully assess its toxicity and understand the specific place of sarilumab in the RA armamentarium.

Trans-presentation of IL-6 by dendritic cells is required for the priming of pathogenic TH17 cells.

The cellular sources of interleukin 6 (IL-6) that are relevant for differentiation of the TH17 subset of helper T cells remain unclear. Here we used a novel strategy for the conditional deletion of distinct IL-6-producing cell types to show that dendritic cells (DCs) positive for the signaling regulator Sirpα were essential for the generation of pathogenic TH17 cells. Using their IL-6 receptor α-chain (IL-6Rα), Sirpα+ DCs trans-presented IL-6 to T cells during the process of cognate interaction. While ambient IL-6 was sufficient to suppress the induction of expression of the transcription factor Foxp3 in T cells, trans-presentation of IL-6 by DC-bound IL-6Rα (called 'IL-6 cluster signaling' here) was needed to prevent premature induction of interferon-γ (IFN-γ) expression in T cells and to generate pathogenic TH17 cells in vivo. Our findings should guide therapeutic approaches for the treatment of TH17-cell-mediated autoimmune diseases.

Differential T Cell Cytokine Receptivity and Not Signal Quality Distinguishes IL-6 and IL-10 Signaling during Th17 Differentiation.

How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4(+) T cells differentially express IL-6 and IL-10 receptors in an activation state-dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6-mediated Th17 subset maturation. IL-10 cannot support Th17 differentiation because of insufficient cytokine receptivity rather than signal quality. Enforced expression of IL-10Rα on naive T cells permits an IL-10-generated STAT3 signal equivalent to that of IL-6 and equally capable of promoting Th17 formation. Similarly, naive T cell IL-10Rα expression also allows IL-10 to mimic the effects of IL-6 on both Th1/Th2 skewing and Tfh cell differentiation. Our results demonstrate a key role for the regulation of receptor expression rather than signal quality or duration in differentiating the functional outcomes of IL-6 and IL-10 signaling, and identify distinct signaling properties of these cytokines in T cells compared with myeloid cells.

Interleukin-6 receptor alpha blockade improves skin lesions in a murine model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterized by antinuclear autoantibodies (ANA) and immunocomplexes, commonly affecting kidneys, skin, heart, lung or even the brain. We have shown that JunB(Δep) mice develop a SLE phenotype linked to increased epidermal Interleukin (IL)-6 secretion. Blocking of IL-6 receptor alpha (IL-6Rα) is considered as therapeutic strategy for the treatment of SLE. JunB(Δep) and wild-type mice were treated for short (5 weeks) or long term (21 weeks) with the IL-6Rα-blocking antibody MR16-1. Skin and kidney of mice were investigated by histology and immunofluorescence, and in addition, kidneys were analysed by electron microscopy. Furthermore, soluble IL-6R (sIL-6R), antihistone and antinucleosome antibodies levels were measured and associated with disease parameters. Treatment with MR16-1 resulted in significant improvement of SLE-like skin lesions in JunB(Δep) mice, compared to untreated mice. The sIL-6R amount upon long-term treatment with MR16-1 was significantly higher in JunB(Δep) versus untreated JunB(Δep) (P = 0.034) or wild-type mice (P = 0.034). MR16-1 treatment over these time spans did not significantly improve kidney pathology of immunoglobulin deposits causing impaired function. Significantly higher antihistone (P = 0.028) and antinucleosome antibody levels (P = 0.028) were measured in MR16-1-treated JunB(Δep) mice after treatment compared to levels before therapy. In conclusion, blockade of IL-6Rα improves skin lesions in a murine SLE model, but does not have a beneficial effect on autoimmune-mediated kidney pathology. Inhibition of IL-6R signalling might be helpful in lupus cases with predominant skin involvement, but combinatorial treatment might be required to restrain autoantibodies.

Remodeling of plasma lipoproteins in patients with rheumatoid arthritis: Interleukin-6 receptor-alpha inhibition with tocilizumab.

PURPOSE: Rheumatoid arthritis (RA) is associated with increased cardiovascular risk, mediated in part by elevated circulating interleukin-6 levels and proinflammatory changes in plasma lipoproteins. We hypothesized that RA patients acquire inflammation-induced modifications to the protein cargo of circulating lipoproteins that may be reversed by tocilizumab, an interleukin-6 receptor-alpha inhibitor. EXPERIMENTAL DESIGN: Size-exclusion chromatography and reverse-phase protein arrays using 29 antibodies against 26 proteins were applied at baseline and after tocilizumab treatment to analyze the distributions of apolipoproteins, enzymes, lipid transfer proteins, and other associated proteins in plasma lipoprotein fractions from 20 women with RA. RESULTS: A 30% reduction in high-density lipoprotein (HDL)-associated serum amyloid A4 and complement C4 occurred with tocilizumab. Levels of C-reactive protein, associated or comigrating with HDL and low-density lipoprotein (LDL) peaks, were reduced on treatment by approximately 80% and 24%, respectively. Reductions in lipoprotein-associated phospholipase A2, lipoprotein (a), and cholesteryl ester transfer protein in the LDL fraction suggest reductions in LDL-associated proatherogenic factors. Elevations in very low-density lipoprotein (VLDL) enriched with apolipoprotein E were equally observed. CONCLUSIONS AND CLINICAL RELEVANCE: Tocilizumab treatment led to reductions in proinflammatory components and proatherogenic proteins associated with HDL. Whether changes in the proteome of VLDL, LDL, and HDL induced by anti-inflammatory tocilizumab treatment in RA patients modify cardiovascular disease risk requires further investigation.

Modular organization of Interleukin-6 and Interleukin-11 α-receptors.

Interleukin (IL)-6 and IL-11 are the only canonical members of the IL-6 family of cytokines that induce signaling through a homodimer of the common ß-receptor glycoprotein (gp)130. A pre-requisite for signal transduction is the initial binding of the cytokines to their unique α-receptors, IL-6R and IL-11R. The cell-type specific expression of the two receptors determines the target cells of IL-6 and IL-11, because gp130 is ubiquitously expressed. However, ciliary neurotrophic factor (CNTF) and IL-27p28/IL-30 have been described as additional ligands for the IL-6R, underlining a remarkable plasticity among the cytokines of the IL-6 family and their receptors. In this study, we show that neither IL-6 nor IL-11 can bind to and signal through the α-receptor of the respective other cytokine. We further create eight chimeric IL-6/IL-11 receptors, which are all biologically active. We find that the domains D1 to D3, which contain the cytokine binding module (CBM), determine which cytokine can activate the chimeric receptor, whereas the stalk region, the transmembrane region, or the intracellular region do not participate in the ligand selectivity of the receptor and are therefore interchangeable between IL-6R and IL-11R. These results suggest a modular organization of the IL-6R and IL-11R, and a similar signal transduction complex of the two cytokines.

T Follicular Helper Cell-Dependent Clearance of a Persistent Virus Infection Requires T Cell Expression of the Histone Demethylase UTX.

Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection.

Interleukin-6 (IL-6) mediated the increased contraction of distal colon in streptozotocin-induced diabetes in rats via IL-6 receptor pathway.

Colonic dysmotility occurs in diabetes and blood plasma interleukin (IL)-6 levels are significantly elevated in type 1 diabetes mellitus. The aim of this study was to investigate whether IL-6 and the IL-6 receptor pathway mediates colonic dysfunction in type 1 diabetes mellitus. Male SD rats were treated with a single intraperitoneally injected dose of streptozotocin (STZ), and those displaying sustained high blood glucose were selected as diabetes mellitus models. Longitudinal muscle strips of colon were prepared to monitor colonic contraction in vitro. Contractile responses of strips of colon were recorded following treatment with IL-6 in control animals, and following anti IL-6 antibody treatment in STZ-induced diabetes in rats. Concentration of IL-6 in plasma and colon were determined by ELISA. Expressions of IL-6 α-receptor and IL-6 ß-receptor in colon tissues were determined by immunohistochemistry or Western blot analysis. The non-diabetes rats treated with IL-6 and the untreated diabetes rats showed increased contraction of distal colon, whereas the diabetes rats treated with anti-IL-6 antibody showed decreased contraction of distal colon compared with the untreated diabetes rats. The IL-6 levels of plasma but not colon increased in diabetes rats. The expression of IL-6 α-receptor increased in diabetes rats. These results indicate that diabetes rats show an increase in the contractions of distal colon partly via the IL-6-IL-6 receptor pathway.

The expression of interleukin-6 and its receptor in various brain regions and their roles in exploratory behavior and stress responses.

We examined the involvement of interleukin-6 (IL-6) and its receptor IL-6Rα on behavior and stress responses in mice. In the open field, both wild-type (WT) and IL-6 deficient mice displayed similar levels of locomotor activity; however, IL-6 deficient mice spent more time in the central part of the arena compared to control WT mice. After behavioral testing, mice were subjected to stress and then sacrificed. The levels of IL-6 and its receptor in their brains were determined. Immunohistochemical labeling of brain sections for IL-6 showed a high level of expression in the subventricular zone of the lateral ventricles and in the border zone of the third and fourth ventricles. Interestingly, 95% of the IL-6-expressing cells had an astrocytic phenotype, and the remaining 5% were microglial cells. A low level of IL-6 expression was observed in the olfactory bulb, hypothalamus, hippocampus, cerebral cortex, cerebellum, midbrain and several brainstem structures. The vast majority of IL-6-expressing cells in these structures had a neuronal phenotype. Stress increased the number of IL-6-immunoreactive astrocytes and microglial cells. The levels of the IL-6Rα receptor were increased in the hypothalamus of stressed mice. Therefore, in this study, we describe for the first time the distribution of IL-6 in various types of brain cells and in previously unreported regions, such as the subventricular zone of the lateral ventricle. Moreover, we provide data on regional distribution and expression within specific cell phenotypes. This highly differential expression of IL-6 indicates its specific roles in the regulation of neuronal and astrocytic functions, in addition to the roles of IL-6 and its receptor IL-6Rα in stress responses.

Sarilumab Plus Methotrexate in Patients With Active Rheumatoid Arthritis and Inadequate Response to Methotrexate: Results of a Phase III Study.

OBJECTIVE: To evaluate the efficacy and safety of sarilumab in combination with methotrexate (MTX) for the treatment of rheumatoid arthritis (RA). METHODS: Adults with moderate-to-severe RA and an inadequate response to MTX were randomized (1:1:1) to receive sarilumab (doses of 150 mg or 200 mg) or placebo every 2 weeks in conjunction with weekly MTX for 52 weeks. Co-primary end points were the proportion of patients achieving American College of Rheumatology 20% (ACR20) improvement responses at week 24, change from baseline in the Health Assessment Questionnaire (HAQ) disability index (DI) at week 16, and change from baseline in the modified Sharp/van der Heijde score (SHS) of radiographic damage at week 52. RESULTS: Baseline characteristics were similar among the groups. For all 3 co-primary end points, the sarilumab 150 mg and 200 mg groups demonstrated statistically significant improvements as compared with the placebo group (ACR20 response rate at week 24, 58.0%, 66.4%, and 33.4%, respectively [P < 0.0001]; least squares mean change in HAQ DI at week 16, -0.53, -0.55, and -0.29, respectively [P < 0.0001]; and mean change in SHS at week 52, 0.90, 0.25, and 2.78, respectively [P < 0.0001]). The most common treatment-emergent adverse event was infection. In the sarilumab 150 mg, sarilumab 200 mg, and placebo groups, the incidence of serious infections was 2.6%, 4.0%, and 2.3%, respectively. Elevations in alanine aminotransferase levels >3-fold the upper limit of normal occurred in 9.5%, 8.0%, and 2.1% of patients, respectively; in 24 patients, this led to discontinuation of treatment. Elevated total cholesterol levels were observed in 36.8%, 43.0%, and 18.3% of patients, respectively. In patients receiving 150 mg and 200 mg sarilumab, neutrophil counts of 0.5 to <1.0 × 10(9) /liter were observed in 5.1% and 7.8% of patients, respectively, while neutrophil counts of <0.5 × 10(9) /liter were observed in 0.9% and 0.7% of patients, respectively; none of the patients receiving placebo experienced changes in neutrophil counts. CONCLUSION: In RA patients treated with sarilumab (150 mg or 200 mg every 2 weeks) in combination with MTX, both doses provided sustained clinical efficacy, as shown by significant improvements in symptomatic, functional, and radiographic outcomes. Sarilumab was generally well tolerated. The adverse events observed in this study were consistent with the effects of interleukin-6 signaling blockade.

An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo.

In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor α (sIL-6Rα) and IL-6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, ZIL-6_13 with an affinity (KD) for IL-6 of ∼500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, ZIL-6_13 was fused to either the N- or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira®). One AffiMab construct with ZIL-6_13 positioned at the N-terminus of the heavy chain, denoted ZIL-6_13-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the ZIL-6_13-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.