Search

ObjectivesAnti-citrullinated protein antibodies (ACPAs) form immune complexes with citrullinated proteins binding toll-like receptor (TLR) 4, which has been proposed as a mediator of rheumatoid arthritis (RA). NI-0101 is a first-in-class humanised monoclonal antibody blocking TLR4, as confirmed by inhibition of in vivo lipopolysaccharide-induced cytokine release in healthy volunteers. This study was design to confirm preclinical investigations supporting a biomarker-driven approach for treatment of patients with RA who present positive for these immune complexes.MethodsPlacebo-controlled, double-blind, randomised (2:1) trial of the tolerability and efficacy of NI-0101 (5 mg/kg, every 2 weeks for 12 weeks) versus placebo in ACPA-positive RA patients with inadequate response to methotrexate. Efficacy measures included Disease Activity Score (28-joint count) with C reactive protein (DAS28-CRP), European League Against Rheumatism (EULAR) good and moderate responses, and American College of Rheumatology (ACR) 20, ACR50 and ACR70 responses. Subgroup analyses defined on biomarkers were conducted. Pharmacokinetics, pharmacodynamics and safety were reported.Results90 patients were randomised (NI-0101 (61) and placebo (29)); 86 completed the study. No significant between-group difference was observed for any of the efficacy endpoints. Subgroup analyses using baseline parameters as covariants did not reveal any population responding to NI-0101. Treatment-emergent adverse events occurred in 51.7% of patients who received placebo versus 52.5% for NI-0101.ConclusionsWe demonstrate for the first time that in RA, a human immune-mediated inflammatory disease, blocking the TLR4 pathway alone does not improve disease parameters. Successful targeting of innate immune pathways in RA may require broader and/or earlier inhibitory approaches.

Toll-like receptor-4 (TLR4) pathways are major contributors to pathological inflammatory responses induced by tissue damage. NI-0101 is the first monoclonal antibody (mAb) blocking TLR4 signaling. This activity is independent of the ligand type and concentration, therefore, potentially blocking any TLR4 ligands. A phase I single ascending dose study was conducted in 73 healthy volunteers to evaluate NI-0101 tolerability, preliminary safety, pharmacokinetics (PKs), and pharmacodynamics (PDs), in absence and in presence of a systemic challenge with lipopolysaccharide (LPS), a TLR4 ligand. NI-0101 was well tolerated without safety concern. The PK profile was characterized by a half-life of ∼10 days at high concentrations and by a rapid elimination at low concentrations due to expected target-mediated drug disposition. NI-0101 prevented cytokine release following ex vivo and in vivo LPS administration and prevented the C-reactive protein (CRP) increase and the occurrence of flu-like symptoms expected following the in vivo administration of LPS.

Background: Interferon beta (IFNb) reduces relapse frequency and disability progression in patients with multiple sclerosis (MS). Objectives: Early identification of prognostic biomarkers of IFNb-treated patients will allow more effective management of MS. Methods: The IMPROVE study evaluated subcutaneous IFNb versus placebo in 180 patients with relapsing–remitting MS. Magnetic resonance imaging scans, clinical assessments, and blood samples were obtained at baseline and every 4 weeks from every participant. Thirty-nine biomarkers (32 transcripts; seven proteins) were studied in 155 patients from IMPROVE. Therapeutic response was defined by absence of new combined unique lesions, relapses, and sustained increase in Expanded Disability Status Scale over 1 year. A machine learning approach was used to examine the association between biomarker expression and treatment response. Results: While baseline levels of individual genes were relatively poor predictors, combinations of three genes were able to identify subjects with sub-optimal therapeutic responses. The triplet CASP2/IRF4/IRF6, previously identified in an independent dataset, was tested among other combinations. This triplet showed acceptable predictive accuracy (0.68) and specificity (0.88), but had relatively low sensitivity (0.22) resulting in an area under the curve (AUC) of 0.63. Other combinations of biomarkers resulted in AUC of up to 0.80 (e.g. CASP2/IL10/IL12Rb1). Conclusions: Baseline expression, or induction ratios, of specific gene combinations correlate with future therapeutic response to IFNb, and have the potential to be prognostically useful.

Platelet interaction with type III collagen is mediated by several platelet receptors that recognize specific sequences in collagen. We previously described an octapeptide KP*GEP*GPK within the alpha(1)III-CB4 fragment that binds to platelets and specifically inhibits platelet aggregation induced by type III collagen. In this study, we demonstrated that the octapeptide prevented platelet contact and spreading on type III collagen and subendothelium under static and flow conditions. Platelets adhered to the immobilized octapeptide, and anti-bodies directed against other platelet collagen receptors (glycoprotein (GP) Ia/IIa, GP IV, p65, p47) did not impair this adhesion. The platelet octapeptide receptor was identified by ligand blotting as a protein doublet with molecular masses of 68 and 72 kDa and does not correspond to any other already known platelet collagen receptors (GP Ia, GP IV GP VI, and p65). Our results indicate that a specific type III collagen receptor, expressed on the platelet surface, is involved in the first stages of platelet type III collagen interaction.

The adhesion of a human microvascular endothelial cell line to its own matrix was studied in comparison with adhesion of the same cells to fibronectin or thrombospondin-1. These endothelial cells adhered preferentially to their matrix whereas an equal cell number was attached to fibronectin or thrombospondin-1. The adhesion of cells to thrombospondin-1 was mediated by the N-terminal heparin binding domain of thrombospondin-1 as shown by the use of a recombinant fragment, N18. Cells adhering to their matrix displayed a morphology and a cytoskeleton organization very similar to that observed in vivo with an apical immunostaining for actin stress fibers and a fine basal labeling for vinculin. Cells on fibronectin were extensively spread and rapidly assembled stress fibers and focal contacts. Cells adherent to thrombospondin-1 presented large lamellae rich in actin but devoid of vinculin and only few actin fibers were observed. Depending on the substratum used, adhering endothelial cells displayed also different tyrosine phosphorylation patterns on electrophoresis. Our observations indicate that endothelial cells adhering to their matrix present an activation state intermediate between that induced by a "hyperadhesive" protein like fibronectin and that generated by a moderate, indeed anti-adhesive, protein like thrombospondin-1.

Background Increased expression of toll-like receptor 4 (TLR4) and its endogenous ligands, is characteristic of rheumatoid arthritis (RA) synovitis. In this study, we evaluated how these TLR4 ligands may drive pathogenic processes and whether the fine profiling of anti-citrullinated protein antibodies (ACPA) based on their target specificity might provide a simple means to predict therapeutic benefit when neutralizing TLR4 in this disease. Methods The capacity of RA synovial fluids (RASF) to stimulate cytokine production in monocytes from patients with RA was analyzed by ELISA. The presence of TLR4 activators in RASF was determined by measuring the levels of ACPA, ACPA subtypes with reactivity to specific citrullinated peptides and other TLR4 ligands. Neutralization of TLR4 signaling was investigated using NI-0101, a therapeutic antibody that targets TLR4. Results RASF exhibited a heterogeneous capacity to induce production of proinflammatory cytokines by monocytes isolated from patients with RA. Such cytokine responses were significantly modified by TLR4 blockade achieved using NI-0101. The analysis of the content of RASF and matched sera demonstrated that ACPA fine specificities in patient samples predict cellular response to anti-TLR4 exposure in vitro. Conclusion TLR4 represents a possible therapeutic target in RA. Our study demonstrates that TLR4 inhibition in an ex vivo model of RA pathogenesis can significantly modulate cytokine release and does so in specific subgroups of RA patient-derived samples. It also suggests that ACPA fine profiling has the potential to identify RA patients with a predominantly TLR4-driven pathotype that could be used to predict preferential response to TLR4 antagonism. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1128-5) contains supplementary material, which is available to authorized users.

Background Innate immunity is implicated in RA pathogenesis and is likely initiated via TLR pathways. NI-0101 is the first humanized monoclonal antibody (mAb) that blocks TLR4 signaling independently of ligand type (i.e., exogenous/endogenous) and concentration. Objectives To determine preliminary tolerability, safety, pharmacokinetic (PK)/pharmacodynamic (PD) profiles after single administrations of different NI-0101 doses to healthy volunteers (HV), as well as to investigate NI-0101 ability to block TLR4-mediated inflammatory cytokine production induced by endogenous TLR4 ligands. Methods A PK/PD guided single ascending dose Phase 1 study was conducted in 73 HV, in the presence of in vivo and ex vivo challenges with the exogenous TLR4 ligand, lipopolysaccharide (LPS). In parallel, monocytes obtained from RA patients were stimulated with endogenous TLR4 ligands or synovial fluids (SF) of RA patients in presence and absence of NI-0101. The correlation of TLR4 blockade with level of endogenous TLR4 ligands in SF was assessed. Finally, the anti-mouse TLR4 surrogate mAb, 5E3, was tested in murine models of RA (IL-1Rn -/- mice and collagen induced arthritis). Results NI-0101 was administered up to a single dose of 15 mg/kg in the Phase 1 study in HV and showed no safety concerns. The predictable PK profile was biphasic, similar to other therapeutic IgG targeting cell surface receptors. NI-0101 administration inhibited ex vivo and in vivo LPS-induced cytokine release (complete inhibition from a dose of 1 mg/kg), as well as prevented CRP increase and the occurrence of flu-like symptoms following LPS administration to HV. NI-0101 PK/PD profiles allowed, through modeling and simulation of multiple administration of NI-0101, to identify an appropriate dose range to be tested in Phase 2. NI-0101 interaction with TLR4 and Fcγ Receptor, in vitro, efficiently blocked TLR4 activation by citrullinated protein immune complexes (cP-IC) present in SF of RA patient sub-populations. NI-0101 blocked the activation of monocytes stimulated with SF from RA patients. This inhibition correlated with the presence of endogenous TLR4 ligands in the synovial fluids (e.g., anti citrullinated protein antibodies, citrullinated fibrinogen immune complex). Therapeutic administration of 5E3 ameliorated disease progression in both mouse models of arthritis. Conclusions NI-0101 blocks TLR4 activation induced by in vivo LPS challenges in HV and by cP-IC in RA patient cells. Taken together, these data strongly support the potential of TLR4 as a valid therapeutic target in RA, and provide an opportunity to evaluate specific endogenous TLR4 ligands as biomarkers of treatment response in Phase 2. Disclosure of Interest E. Monnet Employee of: Novimmune, L. Shang Employee of: Novimmune, G. Lapeyre Employee of: Novimmune, K. deGraaf Employee of: Novimmune, E. Hatterer Employee of: Novimmune, V. Buatois Employee of: Novimmune, G. Elson Employee of: Novimmune, W. Ferlin Employee of: Novimmune, C. Gabay Consultant for: Novimmune, J. Sokolove Consultant for: Novimmune, S. Jones Consultant for: Novimmune, E. Choy Consultant for: Novimmune, I. McInnes Consultant for: Novimmune, M. Kosco-Vilbois Employee of: Novimmune, C. de Min Employee of: Novimmune

Aims Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking TLR4 activation induced by multiple ligands on inflammatory leukocytes represents an elegant strategy when targeting the underlying causes of human diseases. A ligand-independent anti-TLR4 neutralizing antibody, which utilizes a novel mechanism of action to enhance its inhibitory effects on inflammatory leukocytes, is evaluated as a strategy for TLR4 neutralization in rheumatoid arthritis (RA). Methods and results An anti-human TLR4 mAb, NI-0101, binds to human TLR4 in a region involved in receptor dimerization. Using exogenous, endogenous or chemical ligands, NI-0101 was shown to block TLR4 signaling regardless of activator. The mechanism of action of the antibody was further explored using confocal microscopy and fluorescence resonance energy transfer (FRET) analysis. The results revealed that NI-0101 harnesses the clustering of TLR4 and Fc gamma receptors (Fc γ R) in lipid rafts to achieve increased potency on inflammatory cells. The advantage of the novel mechanism of action was tested with a newly reported TLR4 ligand in RA, immune complexes containing citrullinated fibrinogen (cFb-IC), and synovial fluid samples from RA patients. The results demonstrated that NI-0101 is well suited to block TLR4-dependent pro-inflammatory cytokine production (e.g., IL-6, TNF). Conclusions Our data demonstrate that NI-0101 is superior in blocking multiple ligand-induced TLR4 activation on Fc γ R + inflammatory cells. The results, thus, suggest that this novel mechanism of action is well-suited to block TLR4 activation in RA patients where immune complexes are thought to drive disease.

SummaryTIIICBP is a new platelet receptor involved in platelet-type III collagen and platelet-subendothelium interactions. This receptor is composed of a doublet of 72-68 kDa proteins. In this study, the major protein (68 kDa) was purified and used to produce monoclonal antibodies. One of these antibodies, 7F4, binds to platelets as confirmed by flow cytometry. 7F4 inhibited platelet contact, spreading and aggregation induced by type III collagen. Under flow conditions, 7F4 prevented platelet interactions with type III collagen, endothelial cell matrix and the KOGEOGPK type III collagen octapeptide: the specific sequence recognized by TIIICBP. On the other hand, 7F4 had no effect on platelet-type I collagen interactions. TIIICBP was also detected on lymphocytes, granulocytes and monocytes. TIIICBP was expressed on endothelial cells and fibroblasts but not on smooth-muscle cells. These results show that TIIICBP is expressed on several cell types and participates in cell adhesion to the subendothelium.

The role of glycoprotein Ia/IIa was studied during platelet contact and aggregation induced by type I and type III collagen. The anti-glycoprotein Ia/IIa (6F1) antibody inhibited type I collagen-induced aggregation but did not inhibit the first contact between platelets and collagen. In contrast, it was without effect either on type III collagen-induced contact or platelet interaction with the subendothelium in a static assay. Platelet aggregation induced by type III collagen was only slightly slowed down by 6F1 but pp72 spleen tyrosine kinase phosphorylation was not modified even at concentrations of 6F1 that completely blocked platelet activation induced by type I collagen. Our results indicate that glycoprotein Ia/IIa is not a primary binding site for type I or type III collagen on the platelet membrane. This receptor is more specifically involved in type I collagen-induced platelet spreading and aggregation.