Tuning the Reactivity of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Activators for Optimal in Vivo Efficacy.

Dimethyl fumarate 1 is approved for the treatment of multiple sclerosis but is also associated with off-target activation of the niacin receptor. By using a tetrazolone or triazolone bioisostere approach to the fumarate and vinyl sulfone series of Nrf2 activators, we have optimized the electrophilicity of the double bond to tune the on-target Nrf2 activation with PK properties to achieve efficacy in animal models of multiple sclerosis. The study linked highly potent, highly electrophilic molecules to low plasma stability and, subsequently, limited efficacy. By contrast, a sulfonylvinyltriazolone 17 retains on-target potency but shows much weaker electrophilic potential. As a consequence, in vivo high exposures of 17 are obtained, resulting in efficacy in the EAE model similar to that observed for DMF. 17 (R079) is Ames negative, is not cytotoxic to cells, and shows little inhibition of either the niacin receptor or a panel of off-target receptors.

Pharmacologic inhibition of PKCα and PKCθ prevents GVHD while preserving GVL activity in mice.

Allogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell-mediated graft-vs-leukemia (GVL) effects but often leads to severe graft-vs-host disease (GVHD). Given that protein kinase Cθ (PKCθ), in cooperation with PKCα, is essential for T-cell signaling and function, we have evaluated PKCθ and PKCα as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKCα(-/-)/θ(-/-) donor T cells to induce GVHD was further reduced compared with PKCθ(-/-) T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production, and migration to GVHD target organs. Treatment with a specific inhibitor for both PKCθ and PKCα impaired donor T-cell proliferation, migration, and chemokine/cytokine production and significantly decreased GVHD in myeloablative preclinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKCθ and PKCα spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKCα and θ contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKCα and PKCθ signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.

XOMA 052, a potent, high-affinity monoclonal antibody for the treatment of IL-1ß-mediated diseases.

Interleukin-1ß (IL-1ß) is a potent mediator of inflammatory responses and plays a role in the differentiation of a number of lymphoid cells. In several inflammatory and autoimmune diseases, serum levels of IL-1ß are elevated and correlate with disease development and severity. The central role of the IL-1 pathway in several diseases has been validated by inhibitors currently in clinical development or approved by the FDA. However, the need to effectively modulate IL-1ß-mediated local inflammation with the systemic delivery of an efficacious, safe and convenient drug still exists. To meet these challenges, we developed XOMA 052 (gevokizumab), a potent anti-IL-1ß neutralizing antibody that was designed in silico and humanized using Human Engineering™ technology. XOMA 052 has a 300 femtomolar binding affinity for human IL-1ß and an in vitro potency in the low picomolar range. XOMA 052 binds to a unique IL-1ß epitope where residues critical for binding have been identified. We have previously reported that XOMA 052 is efficacious in vivo in a diet-induced obesity mouse model thought to be driven by low levels of chronic inflammation. We report here that XOMA 052 also reduces acute inflammation in vivo, neutralizing the effect of exogenously administered human IL-1ß and blocking peritonitis in a mouse model of acute gout. Based on its high potency, novel mechanism of action, long half-life, and high affinity, XOMA 052 provides a new strategy for the treatment of a number of inflammatory, autoimmune and metabolic diseases in which the role of IL-1ß is central to pathogenesis.

Kinetic approach to pathway attenuation using XOMA 052, a regulatory therapeutic antibody that modulates interleukin-1beta activity.

Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1beta (IL-1beta) antibody XOMA 052 is a potent inhibitor of IL-1beta activity that reduces the affinity of IL-1beta for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1beta bound by XOMA 052 is 20-100-fold lower than that of IL-1beta in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1beta while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1beta activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody.target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.

XOMA 052, an anti-IL-1{beta} monoclonal antibody, improves glucose control and {beta}-cell function in the diet-induced obesity mouse model.

Recent evidence suggests that IL-1beta-mediated glucotoxicity plays a critical role in type 2 diabetes mellitus. Although previous work has shown that inhibiting IL-1beta can lead to improvements in glucose control and beta-cell function, we hypothesized that more efficient targeting of IL-1beta with a novel monoclonal antibody, XOMA 052, would reveal an effect on additional parameters affecting metabolic disease. In the diet-induced obesity model, XOMA 052 was administered to mice fed either normal or high-fat diet (HFD) for up to 19 wk. XOMA 052 was administered as a prophylactic treatment or as a therapy. Mice were analyzed for glucose tolerance, insulin tolerance, insulin secretion, and lipid profile. In addition, the pancreata were analyzed for beta-cell apoptosis, proliferation, and beta-cell mass. Mice on HFD exhibited elevated glucose and glycated hemoglobin levels, impaired glucose tolerance and insulin secretion, and elevated lipid profile, which were prevented by XOMA 052. XOMA 052 also reduced beta-cell apoptosis and increased beta-cell proliferation. XOMA 052 maintained the HFD-induced compensatory increase in beta-cell mass, while also preventing the loss in beta-cell mass seen with extended HFD feeding. Analysis of fasting insulin and glucose levels suggests that XOMA 052 prevented HFD-induced insulin resistance. These studies provide new evidence that targeting IL-1beta in vivo could improve insulin sensitivity and lead to beta-cell sparing. This is in addition to previously reported benefits on glycemic control. Taken together, the data presented suggest that XOMA 052 could be effective for treating many aspects of type 2 diabetes mellitus.

IL-25 regulates Th17 function in autoimmune inflammation.

Interleukin (IL)-25 is a member of the IL-17 family of cytokines. However, unlike the other members of this family, IL-25 promotes T helper (Th) 2 responses. We now show that IL-25 also regulates the development of autoimmune inflammation mediated by IL-17-producing T cells. We have generated IL-25-deficient (il25-/-) mice and found that they are highly susceptible to experimental autoimmune encephalomyelitis (EAE). The accelerated disease in the il25-/- mice is associated with an increase of IL-23 in the periphery and a subsequent increase in the number of inflammatory IL-17-, IFNgamma-, and TNF-producing T cells that invade the central nervous system. Neutralization of IL-17 but not IFNgamma in il25-/- mice prevented EAE, suggesting that IL-17 is a major disease-promoting factor. IL-25 treatment at several time points during a relapse-remitting model or chronic model of EAE completely suppressed disease. IL-25 treatment induced elevated production of IL-13, which is required for suppression of Th17 responses by direct inhibition of IL-23, IL-1beta, and IL-6 expression in activated dendritic cells. Thus, IL-25 and IL-17, being members of the same cytokine family, play opposing roles in the pathogenesis of organ-specific autoimmunity.

Interleukin 25 regulates type 2 cytokine-dependent immunity and limits chronic inflammation in the gastrointestinal tract.

The cytokine interleukin (IL) 25 has been implicated in the initiation of type 2 immunity by driving the expression of type 2 cytokines such as IL-5 and IL-13, although its role in the regulation of immunity and infection-induced inflammation is unknown. Here, we identify a dual function for IL-25: first, in promoting type 2 cytokine-dependent immunity to gastrointestinal helminth infection and, second, in limiting proinflammatory cytokine production and chronic intestinal inflammation. Treatment of genetically susceptible mice with exogenous IL-25 promoted type 2 cytokine responses and immunity to Trichuris. IL-25 was constitutively expressed by CD4+ and CD8+ T cells in the gut of mouse strains that are resistant to Trichuris, and IL-25-deficient mice on a genetically resistant background failed to develop a type 2 immune response or eradicate infection. Furthermore, chronically infected IL-25(-/-) mice developed severe infection-induced intestinal inflammation associated with heightened expression of interferon-gamma and IL-17, identifying a role for IL-25 in limiting pathologic inflammation at mucosal sites. Therefore, IL-25 is not only a critical mediator of type 2 immunity, but is also required for the regulation of inflammation in the gastrointestinal tract.

IL-23 provides a limited mechanism of resistance to acute toxoplasmosis in the absence of IL-12.

IL-23 and IL-12 are heterodimeric cytokines which share the p40 subunit, but which have unique second subunits, IL-23p19 and IL-12p35. Since p40 is required for the development of the Th1 type response necessary for resistance to Toxoplasma gondii, studies were performed to assess the role of IL-23 in resistance to this pathogen. Increased levels of IL-23 were detected in mice infected with T. gondii and in vitro stimulation of dendritic cells with this pathogen resulted in increased levels of mRNA for this cytokine. To address the role of IL-23 in resistance to T. gondii, mice lacking the p40 subunit (common to IL-12 and IL-23) and mice that lack IL-12 p35 (specific for IL-12) were infected and their responses were compared. These studies revealed that p40(-/-) mice rapidly succumbed to toxoplasmosis, while p35(-/-) mice displayed enhanced resistance though they eventually succumbed to this infection. In addition, the administration of IL-23 to p40(-/-) mice infected with T. gondii resulted in a decreased parasite burden and enhanced resistance. However, the enhanced resistance of p35(-/-) mice or p40(-/-) mice treated with IL-23 was not associated with increased production of IFN-gamma. When IL-23p19(-/-) mice were infected with T. gondii these mice developed normal T cell responses and controlled parasite replication to the same extent as wild-type mice. Together, these studies indicate that IL-12, not IL-23, plays a dominant role in resistance to toxoplasmosis but, in the absence of IL-12, IL-23 can provide a limited mechanism of resistance to this infection.

c-Rel regulation of the cell cycle in primary mouse B lymphocytes.

Surface-expressed BCR mediates the proliferation and expansion of antigen-specific B lymphocytes during a humoral immune response. Although several studies extensively characterize BCR proliferative signaling, the mechanisms linking these pathways to the cell cycle remain elusive. Using knockout mice, we show that c-Rel, a proto-oncogenic member of the NF-kappaB transcription factor family, is essential to BCR-mediated proliferation and cell cycle progression. Splenic B cells obtained from gene-targeted c-Rel knockout mice display a defective proliferation response to antigen receptor cross-linking, resulting in G(1) arrest. At the molecular level, we see that BCR stimulation of resting c-Rel(-/-) B cells fails to induce proper cyclin D3 and cyclin E expression, thereby negatively impacting G(1) phase cyclin-dependent kinase (CDK) activity. c-Rel-deficient B cells also exhibit incomplete phosphorylation of the Retinoblastoma protein (pRb) and poor expression of E2Fs, thus impeding the G(1) to S phase transition. Down-regulation of the pRb-related p130 protein during the G(0) to G(1) transition and removal of the CDK inhibitor p27(KIP1) in late G(1) parallel that of wild-type cells, suggesting that Rel-deficient B cells can exit the G(0) resting state and enter G(1) phase normally. Finally, we demonstrate that restoration of proliferation can be achieved partially upon reintroduction of cyclin E using a protein transduction method to reconstitute primary B cells. Collectively, these studies emphasize the importance of c-Rel in lymphocyte proliferation and oncogenesis, and highlight a requirement for c-Rel in establishing an effective humoral immune response.