Suzanne Herren

TLR4/MD-2 Monoclonal Antibody Therapy Affords Protection in Experimental Models of Septic Shock

Overactivation of the immune system upon acute bacterial infection leads to septic shock. Specific bacterial products potently stimulate immune cells via toll-like receptors (TLRs). Gram-negative bacteria induce a predominantly TLR4-driven signal through LPS release. To neutralize LPS signaling in experimental models of sepsis, we generated mAbs toward the TLR4/myeloid differentiation protein-2 (MD-2) complex. The binding properties of an array of selected rat mAbs differed in respect to their specificity for TLR4/MD-2 complex. The specificity of one such mAb, 5E3, to murine TLR4 was confirmed by its recognition of an epitope within the second quarter of the ectodomain. 5E3 inhibited LPS-dependent cell activation in vitro and prevented proinflammatory cytokine production in vivo following LPS challenge in a dose-dependent manner. Furthermore, 5E3 protected mice from lethal shock-like syndrome when applied using both preventative and therapeutic protocols. Most notably, in the colon ascendens stent peritonitis model of polymicrobial abdominal sepsis, administration of a single dose of 5E3 (50 μg) protected mice against mortality. These results demonstrate that neutralizing TLR4/MD-2 is highly efficacious in protecting against bacterial infection-induced toxemia and offers TLR4/MD-2 mAb treatment as a potential therapy for numerous clinical indications.

The distribution of IL-13 receptor α1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4

To study the expression of IL‐13 receptor α1 (IL‐13Rα1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL‐13Rα1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38− B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD−CD38− B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up‐regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL‐13Rα1 expression levels on monocytes. While IL‐13Rα1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL‐13Rα1 might have functions unrelated to the capacity to form a type II IL‐4 / IL‐13R with IL‐4Rα.

IL-18-binding protein expression by endothelial cells and macrophages is up-regulated during active Crohn's disease.

The pathogenesis of Crohn’s disease (CD) remains under intense investigation. Increasing evidence suggests a role for mature IL-18 in the induction of proinflammatory cytokines and Th1 polarization in CD lesions. The aim of this study was to investigate the contribution of the IL-18-neutralizing (a and c) and non-neutralizing (b and d) isoforms of IL-18-binding protein (IL-18BP) during active CD. Intestinal endothelial cells and macrophages were the major source of IL-18BP within the submucosa, and this IL-18BP production was also found to be relevant to other types of endothelial cells (HUVEC) and macrophages (peripheral monocytes). IL-18BP messenger transcript and protein were significantly increased in surgically resected specimens from active CD compared with control patients, correlating with an up-regulation of IL-18. Analysis of the expression of the four IL-18BP isoforms as well as being free or bound to IL-18 was reported and revealed that unbound IL-18BP isoforms a and c and inactive isoform d were present in specimens from active CD and control patients while isoform b was not detected. IL-18/IL-18BP complex was also detected. Interestingly, although most was complexed, free mature IL-18 could still be detected in active CD specimens even in the presence of the IL-18BP isoform a/c. These results demonstrate that the appropriate neutralizing isoforms are present in the intestinal tissue of patients with active CD and highlights the complexity of IL-18/IL-18BP biology.

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.

Pivotal Involvement of Fcγ Receptor IIA in the Neutralization of Lipopolysaccharide Signaling via a Potent Novel Anti-TLR4 Monoclonal Antibody 15C1

The mammalian Toll-like receptor (TLR) family has evolved to sense pathogens in the environment and protect the host against infection. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria and induces a signaling cascade that, when exaggerated, has been associated with severe sepsis. We have generated a TLR4-specific monoclonal antibody, 15C1, which neutralizes LPS-induced TLR4 activation in a dose-dependent manner. 15C1 potently blocks the effects of LPS on a panel of primary cells and cell lines in vitro. The binding of 15C1 was mapped to an epitope in the second portion of the extracellular region of TLR4, which has been shown previously to be functionally important in the recognition of LPS. Furthermore, we demonstrate a novel mechanism of inhibition, as the effects of 15C1 are partially Fc-dependent, involving the regulatory Fcγ receptor IIA (CD32A). In addition to introducing 15C1 as a potent clinical candidate for use in the treatment of LPS-mediated indications, our work demonstrates a newly discovered pathway whose manipulation is pivotal in achieving optimal neutralizing benefit.

Critical role of CD23 in allergen-induced bronchoconstriction in a murine model of allergic asthma

CD23‐deficient and anti‐CD23 monoclonal antibody‐treated mice were used to investigate the role of the low‐affinity receptor for IgE (CD23) in allergic airway inflammation and airway hyperresponsiveness (AHR). While there were no significant differences in ovalbumin (OVA)‐specific IgE titers and tissue eosinophilia, evaluation of lung function demonstrated that CD23− / − mice showed an increased AHR to methacholine (MCh) when compared to wild‐type mice but were completely resistant to the OVA challenge. Anti‐CD23 Fab fragment treatment of wild‐type mice did not affect the MCh‐induced AHR but significantly reduced the OVA‐induced airway constriction. These results imply a novel role for CD23 in lung inflammation and suggest that anti‐CD23 Fab fragment treatment may be of therapeutic use in allergic asthma.

A novel monoclonal antibody, C41, reveals IL-13Rα1 expression by murine germinal center B cells and follicular dendritic cells

Responsiveness to IL‐13 involves at least two chains, IL‐4Rα and IL‐13Rα1. Although mouse B cells express IL‐4Rα, little is known about their expression of IL‐13Rα chains. To investigate this topic further, we have generated a monoclonal antibody (C41) specific for murine IL‐13Rα1. Using C41, IL‐13Rα1 expression was detected on germinal center (GC) B cells by flow cytometry and immunohistochemistry. In addition, IL‐13Rα1 was observed on follicular dendritic cells, but not interdigitating dendritic cells in the T cell areas. Furthermore, resting B cells also expressed IL‐13Rα1, and in the presence of IL‐13 produced increased amounts of IgM in response to in vitro CD40 stimulation. However, C41 was unable to neutralize this bioactivity. The distribution of IL‐13Rα1 on murine B cells and during GC reactions suggests a role for IL‐13 during B cell differentiation.

Selective Antibody Intervention of Toll-like Receptor 4 Activation through Fc γ Receptor Tethering

Background: Dysregulated leukocyte activation via Toll-like receptor 4 (TLR4) is central to numerous inflammatory disorders. Results: A novel mechanism of action involving Fc γ receptor tethering allows anti-TLR4 blocking antibodies to achieve increased potency on inflammatory leukocytes. Conclusion: This novel mechanism of action allows selective targeting of TLR4 activation during inflammation. Significance: The data provide a novel mechanism to dampen TLR4-mediated inflammatory disorders. Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). 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 inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.

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

Background: The IL-6 signaling complex consists of a hexameric structure essential for IL-6 cis- and trans-signaling. Results: mAb 25F10 targets site IIb of IL-6R and disrupts hexamer assembly to selectively block trans-signaling. Conclusion: Cis- and trans-signaling in mice utilize distinct mechanisms to mediate assembly of the IL-6R complex. Significance: Therapeutic targeting of site IIb of IL-6R provides a unique mode of action for IL-6 inhibition. 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.

A specific anti-citrullinated protein antibody profile identifies a group of rheumatoid arthritis patients with a toll-like receptor 4-mediated disease

BackgroundIncreased 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.MethodsThe 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.ResultsRASF 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.ConclusionTLR4 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.