An anti-TL1A antibody for the treatment of asthma and inflammatory bowel disease.

TL1A is an attractive therapeutic target for the treatment of mucosal inflammation associated with inflammatory bowel disease (IBD) and asthma. Blockade of the TL1A pathway has been shown to reduce inflammatory responses while leaving baseline immunity intact, and to be beneficial in animal models of colitis and asthma. Given the therapeutic potential of blocking this pathway in IBD and asthma, we developed C03V, a human antibody that binds with high affinity to soluble and membrane-bound TL1A. In an assay measuring apoptosis induced by exogenous TL1A, C03V was 43-fold more potent than the next most potent anti-TL1A antibody analyzed. C03V also potently inhibited endogenous TL1A activity in a primary cell-based assay. This potency was linked to the C03V-binding epitope on TL1A, encompassing the residue R32. This residue is critical for the binding of TL1A to its signaling receptor DR3 but not to its decoy receptor DcR3, and explains why C03V inhibited TL1A-DR3 binding to a much greater extent than TL1A-DcR3 binding. This characteristic may be advantageous to preserve some of the homeostatic functions of DcR3, such as TL1A antagonism. In colitis models, C03V significantly ameliorated microscopic, macroscopic and clinical aspects of disease pathology, and in an asthma model it significantly reduced airways inflammation. Notable in both types of disease model was the reduction in fibrosis observed after C03V treatment. C03V has the potential to address unmet medical needs in asthma and IBD.

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model.

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor ß-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor ß-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1ß, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.

A novel role for Glucocorticoid-Induced TNF Receptor Ligand (Gitrl) in early embryonic zebrafish development.

Tumour necrosis factor ligand and receptor superfamily (TNFSF and TNFRSF) members have diverse and well-studied functions in the immune system. Additional, non-immunological roles, such as in the morphogenesis of bone, tooth, hair and skin have also been described for some members. GITRL and its receptor GITR are well-described as co-regulators of the mammalian immune response. Here, we describe the identification and cloning of their zebrafish homologues and demonstrate a novel role for the ligand, but not the receptor, in early vertebrate development. The assignment of zebrafish Gitrl and Gitr was supported by homology and phylogenetic analysis. The ligand exhibited an oscillating pattern of mRNA expression during the first 36 hours post fertilization, during which time gitr mRNA was not detected, and morpholino oligonucleotide-mediated knock-down of gitrl, but not of gitr, resulted in disruption of early embryogenesis, most clearly revealed during gastrulation, which corresponded to the earliest peak in gitrl mRNA expression (5.25-10 hpf). We found Stat3 signalling to be altered in the gitrl-morphants, suggesting that one possible role for Gitrl during embryogenesis may be modulation of Jak/Stat signalling.

A novel class of anti-IL-12p40 antibodies: potent neutralization via inhibition of IL-12-IL-12Rß2 and IL-23-IL-23R.

While current therapeutic antibodies bind to IL-12 and IL-23 and inhibit their binding to IL-12Rß1, we describe a novel antibody, termed 6F6, that binds to IL-12 and IL-23 and inhibits the interaction of IL-12 and IL-23 with their cognate signalling receptors IL-12Rß2 and IL23R. This antibody does not affect the natural inhibition of the IL-12/23 pathway by the antagonists monomeric IL-12p40 and IL-12p80, which suggests that a dual antagonist system is possible. We have mapped the epitope of 6F6 to domain 3 of the p40 chain common to IL-12 and IL-23 and demonstrate that an antibody bound to this epitope is sufficient to inhibit engagement of the signalling receptors. Antibodies with this unique mechanism of inhibition are potent inhibitors of IL-12 induced IFN-γ production and IL-23 induced IL-17 production in vitro, and in an in vivo model of psoriasis, treatment with a humanized variant of this antibody, h6F6, reduced the inflammatory response, resulting in decreased epidermal hyperplasia. We believe that this new class of IL-12/23 neutralising antibodies has the potential to provide improved potency and efficacy as anti-inflammatory agents, particularly in diseases characterized by an overproduction of IL-12.

Allelic variation of Ets1 does not contribute to NK and NKT cell deficiencies in type 1 diabetes susceptible NOD mice.

The NOD mouse is a well characterized model of type 1 diabetes that shares several of the characteristics of Ets1-deficient targeted mutant mice, viz: defects in TCR allelic exclusion, susceptibility to a lupus like disease characterized by IgM and IgG autoantibodies and immune complex-mediated glomerulonephritis, and deficiencies of NK and NKT cells. Here, we sought evidence for allelic variation of Ets1 in mice contributing to the NK and NKT cell phenotypes of the NOD strain. ETS1 expression in NK and NKT cells was reduced in NOD mice, compared to C57BL/6 mice. Although NKT cells numbers were significantly correlated with ETS1 expression in both strains, NKT cell numbers were not linked to the Ets1 gene in a first backcross from NOD to C57BL/6 mice. These results indicate that allelic variation of Ets1 did not contribute to variation in NKT cell numbers in these mice. It remains possible that a third factor not linked to the Ets1 locus controls both ETS1 expression and subsequently NK and NKT cell phenotypes.

Key role of the GITR/GITRLigand pathway in the development of murine autoimmune diabetes: a potential therapeutic target.

BACKGROUND: The cross-talk between pathogenic T lymphocytes and regulatory T cells (Tregs) plays a major role in the progression of autoimmune diseases. Our objective is to identify molecules and/or pathways involved in this interaction and representing potential targets for innovative therapies. Glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand are key players in the T effector/Treg interaction. GITR is expressed at low levels on resting T cells and is significantly up-regulated upon activation. Constitutive high expression of GITR is detected only on Tregs. GITR interacts with its ligand mainly expressed on antigen presenting cells and endothelial cells. It has been suggested that GITR triggering activates effector T lymphocytes while inhibiting Tregs thus contributing to the amplification of immune responses. In this study, we examined the role of GITR/GITRLigand interaction in the progression of autoimmune diabetes. METHODS AND FINDINGS: Treatment of 10-day-old non-obese diabetic (NOD) mice, which spontaneously develop diabetes, with an agonistic GITR-specific antibody induced a significant acceleration of disease onset (80% at 12 weeks of age). This activity was not due to a decline in the numbers or functional capacity of CD4(+)CD25(+)Foxp3(+) Tregs but rather to a major activation of 'diabetogenic' T cells. This conclusion was supported by results showing that anti-GITR antibody exacerbates diabetes also in CD28(-/-) NOD mice, which lack Tregs. In addition, treatment of NOD mice, infused with the diabetogenic CD4(+)BDC2.5 T cell clone, with GITR-specific antibody substantially increased their migration, proliferation and activation within the pancreatic islets and draining lymph nodes. As a mirror image, blockade of the GITR/GITRLigand pathway using a neutralizing GITRLigand-specific antibody significantly protected from diabetes even at late stages of disease progression. Experiments using the BDC2.5 T cell transfer model suggested that the GITRLigand antibody acted by limiting the homing and proliferation of pathogenic T cells in pancreatic lymph nodes. CONCLUSION: GITR triggering plays an important costimulatory role on diabetogenic T cells contributing to the development of autoimmune responses. Therefore, blockade of the GITR/GITRLigand pathway appears as a novel promising clinically oriented strategy as GITRLigand-specific antibody applied at an advanced stage of disease progression can prevent overt diabetes.

Genetic control of NKT cell numbers maps to major diabetes and lupus loci.

Natural killer T cells are an immunoregulatory population of lymphocytes that plays a critical role in controlling the adaptive immune system and contributes to the regulation of autoimmune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the nonobese diabetic (NOD) mouse strain, a well-validated model of type 1 diabetes and systemic lupus erythematosus. In this study, we report the results of a genetic linkage analysis of the genes controlling NKT cell numbers in a first backcross (BC1) from C57BL/6 to NOD.Nkrp1(b) mice. The numbers of thymic NKT cells of 320 BC1 mice were determined by fluorescence-activated cell analysis using anti-TCR Ab and CD1/alpha-galactosylceramide tetramer. Tail DNA of 138 female BC1 mice was analyzed for PCR product length polymorphisms at 181 simple sequence repeats, providing greater than 90% coverage of the autosomal genome with an average marker separation of 8 cM. Two loci exhibiting significant linkage to NKT cell numbers were identified; the most significant (Nkt1) was on distal chromosome 1, in the same region as the NOD mouse lupus susceptibility gene Babs2/Bana3. The second most significant locus (Nkt2) mapped to the same region as Idd13, a NOD-derived diabetes susceptibility gene on chromosome 2.