Interleukin-1ß suppression dampens inflammatory leucocyte production and uptake in atherosclerosis.

AIMS: Targeting vascular inflammation represents a novel therapeutic approach to reduce complications of atherosclerosis. Neutralizing the pro-inflammatory cytokine interleukin-1ß (IL-1ß) using canakinumab, a monoclonal antibody, reduces the incidence of cardiovascular events in patients after myocardial infarction (MI). The biological basis for these beneficial effects remains incompletely understood. We sought to explore the mechanisms of IL-1ß-targeted therapies. METHODS AND RESULTS: In mice with early atherosclerosis (ApoE-/- mice on a high-cholesterol diet for 6 weeks), we found that 3 weeks of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3)-inflammasome inhibition or anti-IL-1ß treatment (using either MCC950, an NLRP3-inflammasome inhibitor which blocks production and release of active IL-1ß, or a murine analogue of canakinumab) dampened accumulation of leucocytes in atherosclerotic aortas, which consequently resulted in slower progression of atherosclerosis. Causally, we found that endothelial cells from atherosclerotic aortas lowered expression of leucocyte chemoattractants and adhesion molecules upon NLRP3-inflammasome inhibition, indicating that NLRP3-inflammasome- and IL-1ß-targeted therapies reduced blood leucocyte recruitment to atherosclerotic aortas. In accord, adoptive transfer experiments revealed that anti-IL-1ß treatment mitigated blood myeloid cell uptake to atherosclerotic aortas. We further report that anti-IL-1ß treatment and NLRP3-inflammasome inhibition reduced inflammatory leucocyte supply by decreasing proliferation of bone marrow haematopoietic stem and progenitor cells, demonstrating that suppression of IL-1ß and the NLRP3-inflammasome lowered production of disease-propagating leucocytes. Using bone marrow reconstitution experiments, we observed that haematopoietic cell-specific NLRP3-inflammasome activity contributed to both enhanced recruitment and increased supply of blood inflammatory leucocytes. Further experiments that queried whether anti-IL-1ß treatment reduced vascular inflammation also in post-MI accelerated atherosclerosis documented the operation of convergent mechanisms (reduced supply and uptake of inflammatory leucocytes). In line with our pre-clinical findings, post-MI patients on canakinumab treatment showed reduced blood monocyte numbers. CONCLUSIONS: Our murine and human data reveal that anti-IL-1ß treatment and NLRP3-inflammasome inhibition dampened vascular inflammation and progression of atherosclerosis through reduced blood inflammatory leucocyte (i) supply and (ii) uptake into atherosclerotic aortas providing additional mechanistic insights into links between haematopoiesis and atherogenesis, and into the beneficial effects of NLRP3-inflammasome- and IL-1ß-targeted therapies.

A dysregulated interleukin-18-interferon-γ-CXCL9 axis impacts treatment response to canakinumab in systemic juvenile idiopathic arthritis.

OBJECTIVES: The monoclonal IL-1ß antibody canakinumab is approved for the treatment of systemic juvenile idiopathic arthritis (SJIA). Its efficacy has been proven in several trials, but not all patients show a complete and sustained response to therapy. We aimed to analyse the association of baseline serum biomarkers with treatment outcome in patients with SJIA treated with canakinumab. METHODS: Serum samples from 54 patients with active SJIA without recent macrophage activation syndrome (MAS) treated with canakinumab in an open-label response characterization study were subjected to a multiplexed bead array assay. Interesting targets from these analyses were validated by ELISA. Clinical treatment outcomes included modified paediatric ACR (pACR) 30 and 90 responses, clinically inactive disease (CID) within 15 days of treatment and sustained complete response, defined as pACR100 or CID within 15 days of treatment plus no future flare or MAS. RESULTS: In canakinumab-naïve patients most biomarkers were elevated when compared with healthy controls at baseline and some rapidly decreased by day 15 [IL-1 receptor antagonist (IL-1RA), IL-6, IL-18 and S100A12]. Responders had higher IL-18 and IFN-γ levels and lower chemokine (C-X-C motif) ligand 9 (CXCL9) levels at baseline, emphasized by the IL-18: CXCL9 and IFN-γ: CXCL9 ratios. These ratios had significant accuracy in predicting treatment responses. CONCLUSION: Differential regulation of the IL-18-IFN-γ-CXCL9 axis is observed in patients with SJIA. Higher IL-18: CXCL9 and IFN-γ: CXCL9 ratios at baseline are associated with a better clinical response to canakinumab treatment in SJIA. Future studies are needed to validate these findings and determine their generalizability to patients with recent MAS.

The long and winding road in pharmaceutical development of canakinumab from rare genetic autoinflammatory syndromes to myocardial infarction and cancer.

Interleukin-1beta (IL-1ß) is an ancient and evolutionary conserved cytokine, which orchestrates innate immune responses triggered by infections in vertebrates. While temporally limited induction of IL-1ß protects the organism against traumatic or infectious insults, its chronic production in unabated inflammation causes or enhances clinical manifestations of disease in almost all organ systems. Therefore, pharmacological targeting of IL-1ß in a variety of clinical inflammatory conditions may provide symptomatic relief or profound disease modification. The discovery of proteolytic processing of the inactive pro-IL-1ß to mature, active and secreted IL-1ß by the inflammasome/caspase 1 complex entailed a number of drug discovery programs aiming towards low molecular weight inhibitors across the Pharma industry. Approved and marketed IL-1 pathway drugs today, however, are protein-based injectable drugs ("biologics") targeting either IL-1ß, or the IL-1 receptor. Canakinumab is a human monoclonal antibody that binds human IL-1ß with high affinity and neutralizes its biological activity. This review describes the unique preclinical and clinical development journey of canakinumab starting from a rare genetic autoinflammatory disease and a systemic juvenile form of arthritis to further rare monogenetic periodic fever syndromes, and leading to non-orphan diseases, such as gout, myocardial infarction, and lung cancer.

Combination Therapy for Treating Advanced Drug-Resistant Acute Lymphoblastic Leukemia.

Drug-resistant acute lymphoblastic leukemia (ALL) patients do not respond to standard chemotherapy, and an urgent need exists to develop new treatment strategies. Our study exploited the presence of B-cell activating factor receptor (BAFF-R) on the surface of drug-resistant B-ALL cells as a therapeutic target. We used anti-BAFF-R (VAY736), optimized for natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), to kill drug-resistant ALL cells. VAY736 antibody and NK cell treatments significantly decreased ALL disease burden and provided survival benefit in vivo However, if the disease was advanced, the ADCC efficacy of NK cells was inhibited by microenvironmental transforming growth factor-beta (TGFß). Inhibiting TGFß signaling in NK cells using the TGFß receptor 1 (R1) inhibitor (EW-7197) significantly enhanced VAY736-induced NK cell-mediated ALL killing. Our results highlight the potential of using a combination of VAY736 antibody with EW-7197 to treat advance-stage, drug-resistant B-ALL patients.

Treatment of primary Sjögren's syndrome with ianalumab (VAY736) targeting B cells by BAFF receptor blockade coupled with enhanced, antibody-dependent cellular cytotoxicity.

OBJECTIVES: To evaluate the efficacy and safety of ianalumab (VAY736), a B cell-depleting, B cell activating factor receptor-blocking, monoclonal antibody, in patients with active primary Sjögren's syndrome (pSS) in a double-blind, placebo-controlled, phase II, single-centre study. METHODS: Patients with pSS, EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) ≥6, were randomised to ianalumab single infusion at either 3 mg/kg (n=6), 10 mg/kg (n=12) or placebo (n=9). Outcomes were measured blinded at baseline and weeks 6, 12, 24, and unblinded at end of study (EoS) when B cell numbers had recovered. Clinical outcomes included ESSDAI, EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI), salivary flow rate, ocular staining score, physician global assessment and patient assessments of fatigue and general quality of life. Laboratory-based measures included circulating leucocyte subsets and markers of B cell activity. RESULTS: A similar trend showing positive therapeutic effect by ianalumab was observed across the primary clinical outcome (ESSDAI) and all secondary clinical outcomes (ESSPRI, Multidimensional Fatigue Inventory, Short Form-36, global assessments by physician and patient) versus the placebo-treated group. Rapid and profound B cell depletion of long-lasting duration occurred after a single infusion of ianalumab at either dose. Serum Ig light chains decreased, with return to baseline levels at EoS. Changes in some clinical outcomes persisted through to EoS in the higher dose group. Adverse effects were largely limited to mild to moderate infusion reactions within 24 hours of ianalumab administration. CONCLUSIONS: Overall results in this single-dose study suggest potent and sustained B cell depletion by ianalumab could provide therapeutic benefits in patients with pSS without major side effects.

Stage-dependent differential effects of interleukin-1 isoforms on experimental atherosclerosis.

AIMS: Targeting interleukin-1 (IL-1) represents a novel therapeutic approach to atherosclerosis. CANTOS demonstrated the benefits of IL-1ß neutralization in patients post-myocardial infarction with residual inflammatory risk. Yet, some mouse data have shown a prominent role of IL-1α rather than IL-1ß in atherosclerosis, or even a deleterious effect of IL-1 on outward arterial remodelling in atherosclerosis-susceptible mice. To shed light on these disparate results, this study investigated the effect of neutralizing IL-1α or/and IL-1ß isoforms starting either early in atherogenesis or later in ApoE-/- mice with established atheroma. METHODS AND RESULTS: The neutralization of IL-1α or of both IL-1 isoforms impaired outward remodelling during early atherogenesis as assessed by micro-computed tomographic and histologic assessment. In contrast, the neutralization of IL-1ß did not impair outward remodelling either during early atherogenesis or in mice with established lesions. Interleukin-1ß inhibition promoted a slant of blood monocytes towards a less inflammatory state during atherogenesis, reduced the size of established atheromata, and increased plasma levels of IL-10 without limiting outward remodelling of brachiocephalic arteries. CONCLUSION: This study established a pivotal role for IL-1α in the remodelling of arteries during early experimental atherogenesis, whereas IL-1ß drives inflammation during atherogenesis and the evolution of advanced atheroma in mice.

Interleukin-1ß has atheroprotective effects in advanced atherosclerotic lesions of mice.

Despite decades of research, our understanding of the processes controlling late-stage atherosclerotic plaque stability remains poor. A prevailing hypothesis is that reducing inflammation may improve advanced plaque stability, as recently tested in the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial, in which post-myocardial infarction subjects were treated with an IL-1ß antibody. Here, we performed intervention studies in which smooth muscle cell (SMC) lineage-tracing Apoe-/- mice with advanced atherosclerosis were treated with anti-IL-1ß or IgG control antibodies. Surprisingly, we found that IL-1ß antibody treatment between 18 and 26 weeks of Western diet feeding induced a marked reduction in SMC and collagen content, but increased macrophage numbers in the fibrous cap. Moreover, although IL-1ß antibody treatment had no effect on lesion size, it completely inhibited beneficial outward remodeling. We also found that SMC-specific knockout of Il1r1 (encoding IL-1 receptor type 1) resulted in smaller lesions nearly devoid of SMCs and lacking a fibrous cap, whereas macrophage-selective loss of IL-1R1 had no effect on lesion size or composition. Taken together, these results show that IL-1ß has multiple beneficial effects in late-stage murine atherosclerosis, including promotion of outward remodeling and formation and maintenance of an SMC- and collagen-rich fibrous cap.

Neutralization of Interleukin-1ß following Diffuse Traumatic Brain Injury in the Mouse Attenuates the Loss of Mature Oligodendrocytes.

Traumatic brain injury (TBI) commonly results in injury to the components of the white matter tracts, causing post-injury cognitive deficits. The myelin-producing oligodendrocytes (OLs) are vulnerable to TBI, although may potentially be replaced by proliferating oligodendrocyte progenitor cells (OPCs). The cytokine interleukin-1ß (IL-1ß) is a key mediator of the complex inflammatory response, and when neutralized in experimental TBI, behavioral outcome was improved. To evaluate the role of IL-1ß on oligodendrocyte cell death and OPC proliferation, 116 adult male mice subjected to sham injury or the central fluid percussion injury (cFPI) model of traumatic axonal injury, were analyzed at two, seven, and 14 days post-injury. At 30 min post-injury, mice were randomly administered an IL-1ß neutralizing or a control antibody. OPC proliferation (5-ethynyl 2'- deoxyuridine (EdU)/Olig2 co-labeling) and mature oligodendrocyte cell loss was evaluated in injured white matter tracts. Microglia/macrophages immunohistochemistry and ramification using Sholl analysis were also evaluated. Neutralizing IL-1ß resulted in attenuated cell death, indicated by cleaved caspase-3 expression, and attenuated loss of mature OLs from two to seven days post-injury in brain-injured animals. IL-1ß neutralization also attenuated the early, two day post-injury increase of microglia/macrophage immunoreactivity and altered their ramification. The proliferation of OPCs in brain-injured animals was not altered, however. Our data suggest that IL-1ß is involved in the TBI-induced loss of OLs and early microglia/macrophage activation, although not the OPC proliferation. Attenuated oligodendrocyte cell loss may contribute to the improved behavioral outcome observed by IL-1ß neutralization in this mouse model of diffuse TBI.

Early changes in gene expression and inflammatory proteins in systemic juvenile idiopathic arthritis patients on canakinumab therapy.

BACKGROUND: Canakinumab is a human anti-interleukin-1ß (IL-1ß) monoclonal antibody neutralizing IL-1ß-mediated pathways. We sought to characterize the molecular response to canakinumab and evaluate potential markers of response using samples from two pivotal trials in systemic juvenile idiopathic arthritis (SJIA). METHODS: Gene expression was measured in patients with febrile SJIA and in matched healthy controls by Affymetrix DNA microarrays. Transcriptional response was assessed by gene expression changes from baseline to day 3 using adapted JIA American College of Rheumatology (aACR) response criteria (50 aACR JIA). Changes in pro-inflammatory cytokines IL-6 and IL-18 were assessed up to day 197. RESULTS: Microarray analysis identified 984 probe sets differentially expressed (≥2-fold difference; P < 0.05) in patients versus controls. Over 50% of patients with ≥50 aACR JIA were recognizable by baseline expression values. Analysis of gene expression profiles from patients achieving ≥50 aACR JIA response at day 15 identified 102 probe sets differentially expressed upon treatment (≥2-fold difference; P < 0.05) on day 3 versus baseline, including IL-1ß, IL-1 receptors (IL1-R1 and IL1-R2), IL-1 receptor accessory protein (IL1-RAP), and IL-6. The strongest clinical response was observed in patients with higher baseline expression of dysregulated genes and a strong transcriptional response on day 3. IL-6 declined by day 3 (≥8-fold decline; P < 0.0001) and remained suppressed. IL-18 declined on day 57 (≥1.5-fold decline, P ≤ 0.002). CONCLUSIONS: Treatment with canakinumab in SJIA patients resulted in downregulation of innate immune response genes and reductions in IL-6 and clinical symptoms. Additional research is needed to investigate potential differences in the disease mechanisms in patients with heterogeneous gene transcription profiles. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00886769 (trial 1). Registered on 22 April 2009; NCT00889863 (trial 2). Registered on 21 April 2009.

An Activating Janus Kinase-3 Mutation Is Associated with Cytotoxic T Lymphocyte Antigen-4-Dependent Immune Dysregulation Syndrome.

Heterozygous mutations in the cytotoxic T lymphocyte antigen-4 (CTLA-4) are associated with lymphadenopathy, autoimmunity, immune dysregulation, and hypogammaglobulinemia in about 70% of the carriers. So far, the incomplete penetrance of CTLA-4 haploinsufficiency has been attributed to unknown genetic modifiers, epigenetic changes, or environmental effects. We sought to identify potential genetic modifiers in a family with differential clinical penetrance of CTLA-4 haploinsufficiency. Here, we report on a rare heterozygous gain-of-function mutation in Janus kinase-3 (JAK3) (p.R840C), which is associated with the clinical manifestation of CTLA-4 haploinsufficiency in a patient carrying a novel loss-of-function mutation in CTLA-4 (p.Y139C). While the asymptomatic parents carry either the CTLA-4 mutation or the JAK3 variant, their son has inherited both heterozygous mutations and suffers from hypogammaglobulinemia combined with autoimmunity and lymphoid hyperplasia. Although the patient's lymph node and spleen contained many hyperplastic germinal centers with follicular helper T (TFH) cells and immunoglobulin (Ig) G-positive B cells, plasma cell, and memory B cell development was impaired. CXCR5+PD-1+TIGIT+ TFH cells contributed to a large part of circulating T cells, but they produced only very low amounts of interleukin (IL)-4, IL-10, and IL-21 required for the development of memory B cells and plasma cells. We, therefore, suggest that the combination of the loss-of-function mutation in CTLA-4 with the gain-of-function mutation in JAK3 directs the differentiation of CD4 T cells into dysfunctional TFH cells supporting the development of lymphadenopathy, hypogammaglobulinemia, and immunodeficiency. Thus, the combination of rare genetic heterozygous variants that remain clinically unnoticed individually may lead to T cell hyperactivity, impaired memory B cell, and plasma cell development resulting finally in combined immunodeficiency.

Therapeutic Administration of a Monoclonal Anti-Il-1ß Antibody Protects Against Experimental Melioidosis.

BACKGROUND: Melioidosis, caused by the gram-negative bacterium Burkholderia pseudomallei, is a common cause of community-acquired sepsis in Southeast Asia and Northern Australia. The NLRP3 inflammasome and its downstream product interleukin-1 beta (IL-1ß) have been proposed to play crucial roles in melioidosis. In this study, we characterized the role of IL-1ß more closely and we assessed its therapeutic potential. METHODS: mRNA expression of inflammasome components was determined in isolated leukocytes of 32 healthy controls and 34 patients with sepsis caused by B pseudomallei.Wild-type (WT), NLRP3-deficient (Nlrp3), and Asc mice were infected with B pseudomallei. In additional experiments, infected WT mice were treated with an anti-IL-1ß antibody. After 24, 48, and 72 hours (h) mice were sacrificed and organs were harvested. Furthermore, survival studies were performed. RESULTS: Patients with melioidosis exhibited lower mRNA levels of caspase-1, NLRP3, and ASC. Bacterial dissemination and organ damage were increased in B pseudomallei-infected Nlrp3 and Asc mice, together with a reduced pulmonary cell influx. Anti-IL-1ß treatment of B pseudomallei challenged mice resulted in strongly reduced bacterial counts, organ damage, and pulmonary granulocyte influx together with reduced mortality. Postponement of anti-IL-1ß treatment for 24 h postinfection still protected mice during melioidosis. CONCLUSION: Expression of caspase-1, NLRP3, and ASC is altered in melioidosis patients. In mice, both NLRP3 and ASC contribute to the host defense against melioidosis. Anti-IL-1ß treatment protects mice against B pseudomallei infection and might be a novel treatment strategy in melioidosis.

Diffuse traumatic axonal injury in mice induces complex behavioural alterations that are normalized by neutralization of interleukin-1ß.

Widespread traumatic axonal injury (TAI) results in brain network dysfunction, which commonly leads to persisting cognitive and behavioural impairments following traumatic brain injury (TBI). TBI induces a complex neuroinflammatory response, frequently located at sites of axonal pathology. The role of the pro-inflammatory cytokine interleukin (IL)-1ß has not been established in TAI. An IL-1ß-neutralizing or a control antibody was administered intraperitoneally at 30 min following central fluid percussion injury (cFPI), a mouse model of widespread TAI. Mice subjected to moderate cFPI (n = 41) were compared with sham-injured controls (n = 20) and untreated, naive mice (n = 9). The anti-IL-1ß antibody reached the target brain regions in adequate therapeutic concentrations (up to ~30 µg/brain tissue) at 24 h post-injury in both cFPI (n = 5) and sham-injured (n = 3) mice, with lower concentrations at 72 h post-injury (up to ~18 µg/g brain tissue in three cFPI mice). Functional outcome was analysed with the multivariate concentric square field (MCSF) test at 2 and 9 days post-injury, and the Morris water maze (MWM) at 14-21 days post-injury. Following TAI, the IL-1ß-neutralizing antibody resulted in an improved behavioural outcome, including normalized behavioural profiles in the MCSF test. The performance in the MWM probe (memory) trial was improved, although not in the learning trials. The IL-1ß-neutralizing treatment did not influence cerebral ventricle size or the number of microglia/macrophages. These findings support the hypothesis that IL-1ß is an important contributor to the processes causing complex cognitive and behavioural disturbances following TAI.

Preclinical characterization and clinical development of ILARIS(®) (canakinumab) for the treatment of autoinflammatory diseases.

Interleukin-1beta (IL-1ß) is a pro-inflammatory cytokine which is part of the first line innate response in vertebrates and is induced in injury, infection, and immunity. While temporally limited induction of IL-1ß is believed to protect the organisms against traumatic or infectious insults, its aberrant expression in chronic inflammation is detrimental. Therefore, pharmacological neutralization of IL-1ß in chronic inflammatory diseases is a meaningful strategy to treat inflammation and to alleviate respective clinical symptoms in man. Canakinumab is a high-affinity human monoclonal antibody designed to target human IL-1ß in inflammatory diseases. Indeed, canakinumab has shown excellent efficacy in rare genetic autoinflammatory diseases or pathological conditions associated with aberrant production of IL-1ß. This review focuses on the molecular and clinical mode of action and pharmaceutical development of canakinumab in (auto)inflammatory diseases.

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.

Reducing Vascular Calcification by Anti-IL-1ß Monoclonal Antibody in a Mouse Model of Familial Hypercholesterolemia.

BACKGROUND: Given the link between cholesterol and activation of inflammation via interleukin 1ß (IL-1ß), we tested the effects of IL-1ß inhibition on atherosclerotic calcification in mice. Patients with familial hypercholesterolemia develop extensive aortic calcification and calcific aortic stenosis. Although statins delay this process, low-density lipoprotein (LDL) cholesterol lowering alone is not enough to avert it. Data suggest that vascular inflammation initiated by hypercholesterolemia is followed by unchecked mineralization at sites of atherosclerotic plaques. The LDL-receptor (LDLR)-deficient (Ldlr(-/-)) and LDLR-attenuated Pcsk9(Tg) mice are available animal models for pharmacological testing. METHODS: A mouse monoclonal antibody (mAb) against IL-1ß or placebo was administered subcutaneously in Ldlr(-/-) and Pcsk9(Tg) models fed a Western diet. Drug level, anthropometric, lipid, and glucose profiles were determined. Expressions of proprotein convertase subtilisin/kexin type 9 (PCSK9), serum amyloid A1, and cytokine were measured by enzyme-linked immunosorbent assay. Aortic calcification was determined by microcomputerized tomography (micro-CT) and X-ray densitometry, and aortic flow velocity was assessed by ultrasound. RESULTS: Circulating levels of IL-1ß in Ldlr(-/-) mice were significantly greater (2-fold) than observed in Pcsk9(Tg) mice. Placebo- and mAb-treated mice did not differ in their growth, lipid, glucose profiles, and other cytokines. Calcifications were significantly diminished in mAb-treatment Ldlr(-/-) mice (a reduction of ∼ 75% by X-ray and ∼ 90% by micro-CT) and reduced insignificantly in mAb-treatment Pcsk9(Tg) mice, whereas aortic flow velocity was unchanged in both models. CONCLUSIONS: Herein, we demonstrate that aortic calcifications can be inhibited by an IL-1ß mAb in LDLR-deficient mice. These results have a translational component to prevent vascular calcification in human and represent new evidence to rationalize targeting inflammation in cardiovascular disease.

The molecular mode of action and species specificity of canakinumab, a human monoclonal antibody neutralizing IL-1ß.

Interleukin-1ß (IL-1ß) plays a key role in autoinflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA) or cryopyrin-associated periodic syndrome (CAPS). Canakinumab, a human monoclonal anti-IL-1ß antibody, was recently approved for human use under the brand name Ilaris®. Canakinumab does not cross-react with IL-1ß from mouse, rat, rabbit, or macaques. The crystal structure of the canakinumab Fab bound to human IL-1ß was determined in an attempt to rationalize the species specificity. The X-ray analysis reveals a complex surface epitope with an intricate network of well-ordered water molecules at the antibody-antigen interface. The canakinumab paratope is largely pre-organized, as demonstrated by the structure determination of the free Fab. Glu 64 of human IL-1ß is a pivotal epitope residue explaining the exquisite species specificity of canakinumab. We identified marmoset as the only non-human primate species that carries Glu 64 in its IL-1ß and demonstrates full cross-reactivity of canakinumab, thereby enabling toxicological studies in this species. As demonstrated by the X-ray structure of the complex with IL-1ß, canakinumab binds IL-1ß on the opposite side with respect to the IL-1RAcP binding site, and in an approximately orthogonal orientation with respect to IL-1RI. However, the antibody and IL-1RI binding sites slightly overlap and the VH region of canakinumab would sterically interfere with the D1 domain of IL-1RI, as shown by a structural overlay with the IL-1ß:IL-1RI complex. Therefore, direct competition with IL-1RI for IL-1ß binding is the molecular mechanism of neutralization by canakinumab, which is also confirmed by competition assays with recombinant IL-1RI and IL-1RII.

Biomarkers in systemic juvenile idiopathic arthritis: a comparison with biomarkers in cryopyrin-associated periodic syndromes.

PURPOSE OF REVIEW: This review summarizes biomarkers in systemic juvenile idiopathic arthritis (sJIA). Broadly, the markers are classified under protein, cellular, gene expression and genetic markers. We also compare the biomarkers in sJIA to biomarkers in cryopyrin-associated periodic syndrome (CAPS). RECENT FINDINGS: Recent publications showing the similarity of clinical response of sJIA and CAPS to anti-interleukin 1 therapies prompted a comparison at the biomarker level. SUMMARY: sJIA traditionally is classified under the umbrella of juvenile idiopathic arthritis. At the clinical phenotypic level, sJIA has several features that are more similar to those seen in CAPS. In this review, we summarize biomarkers in sJIA and CAPS and draw upon the various similarities and differences between the two families of diseases. The main differences between sJIA and CAPS biomarkers are genetic markers, with CAPS being a family of monogenic diseases with mutations in NLRP3. There have been a small number of publications describing cellular biomarkers in sJIA with no such studies described for CAPS. Many of the protein marker's characteristics of sJIA are also seen to characterize CAPS. The gene expression data in both sJIA and CAPS show a strong upregulation of innate immunity pathways. In addition, we describe a strong similarity between sJIA and CAPS at the gene expression level in which several genes that form a part of the erythropoiesis signature are upregulated in both sJIA and CAPS.

Effector-mediated eradication of precursor B acute lymphoblastic leukemia with a novel Fc-engineered monoclonal antibody targeting the BAFF-R.

B-cell activating factor receptor (BAFF-R) is expressed on precursor B acute lymphoblastic leukemia (pre-B ALL) cells, but not on their pre-B normal counterparts. Thus, selective killing of ALL cells is possible by targeting this receptor. Here, we have further examined therapeutic targeting of pre-B ALL based on the presence of the BAFF-R. Mouse pre-B ALL cells lacking BAFF-R function had comparable viability and proliferation to wild-type cells, but were more sensitive to drug treatment in vitro. Viability of human pre-B ALL cells was further reduced when antibodies to the BAFF-R were combined with other drugs, even in the presence of stromal protection. This indicates that inhibition of BAFF-R function reduces fitness of stressed pre-B ALL cells. We tested a novel humanized anti-BAFF-R monoclonal antibody optimalized for FcRγIII-mediated, antibody-dependent cell killing by effector cells. Antibody binding to human ALL cells was inhibitable, in a dose-dependent manner, by recombinant human BAFF. There was no evidence for internalization of the antibodies. The antibodies significantly stimulated natural killer cell-mediated killing of different human patient-derived ALL cells. Moreover, incubation of such ALL cells with these antibodies stimulated phagocytosis by macrophages. When this was tested in an immunodeficient transplant model, mice that were treated with the antibody had a significantly decreased leukemia burden in bone marrow and spleen. In view of the restricted expression of the BAFF-R on normal cells and the multiple anti-pre-B ALL activities stimulated by this antibody, a further examination of its use for treatment of pre-B ALL is warranted.

Pharmacokinetics, biocompatibility and bioavailability of a controlled release monoclonal antibody formulation.

The sustained and localized delivery of monoclonal antibodies has become highly relevant, because of the increasing number of investigated local delivery applications in recent years. As the local delivery of antibodies is associated with high technological hurdles, very few successful approaches have been reported in the literature so far. Alginate-based delivery systems were previously described as promising sustained release formulations for monoclonal antibodies (mAbs). In order to further investigate their applicability, a single-dose animal study was conducted to compare the biocompatibility, the pharmacokinetics and the bioavailability of a human monoclonal antibody liquid formulation with two alginate-based sustained delivery systems after subcutaneous administration in rats. 28 days after injection, the depot systems were still found in the subcutis of the animals. A calcium cross-linked alginate formulation, which was injected as a hydrogel, was present as multiple compartments separated by subcutaneous tissue. An in situ forming alginate formulation was recovered as a single compact and cohesive structure. It can be assumed that the multiple compartments of the hydrogel formulation led to almost identical pharmacokinetic profiles for all tested animals, whereas the compact nature of the in situ forming system resulted in large interindividual variations in pharmacokinetics. As compared to the liquid formulation the hydrogel formulations led to lower mAb serum levels, and the in situ forming system to a shift in the time to reach the maximum mAb serum concentration (Tmax) from 2 to 4 days. Importantly, it was shown that after 28 days only marginal amounts of residual mAb were present in the alginate matrix and in the tissue at the injection site indicating nearly complete release. In line with this finding, systemic drug bioavailability was not affected by using the controlled release systems. This study successfully demonstrates the suitability and underlines the potential of polyanionic systems for local and controlled mAb delivery.

Interleukin-1 mediates neuroinflammatory changes associated with diet-induced atherosclerosis.

BACKGROUND: Systemic inflammation contributes to brain pathology in cerebrovascular disease through mechanisms that are poorly understood. METHODS AND RESULTS: Here we show that atherosclerosis, a major systemic inflammatory disease, is associated with severe cerebrovascular inflammation in mice and that this effect is mediated by the proinflammatory cytokine interleukin-1 (IL-1). Apolipoprotein E-deficient mice fed Paigen or Western diets develop vascular inflammation, microglial activation, and leukocyte recruitment in the brain, which are absent in apolipoprotein E-deficient mice crossed with IL-1 type 1 receptor-deficient mice. Systemic neutralization of IL-1ß with an anti-IL-1ß antibody reversed aortic plaque formation (by 34% after a Paigen and 45% after a Western diet) and reduced inflammatory cytokine expression in peripheral organs. Central, lipid accumulation-associated leukocyte infiltration into the choroid plexus was reversed by IL-1ß antibody administration. Animals fed a Western diet showed 57% lower vascular inflammation in the brain than that of mice fed a Paigen diet, and this was reduced further by 24% after IL-1ß antibody administration. CONCLUSIONS: These results indicate that IL-1 is a key driver of systemically mediated cerebrovascular inflammation and that interventions against IL-1ß could be therapeutically useful in atherosclerosis, dementia, or stroke. (J Am Heart Assoc. 2012;1:e002006 doi: 10.1161/JAHA.112.002006.).