Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman’s Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

BackgroundDisulfide-rich peptides (DRPs) are found throughout nature. They are suitable scaffolds for drug development due to their small cores, whose disulfide bonds impart extraordinary chemical and biological stability. A challenge in developing a DRP therapeutic is to engineer binding to a specific target. This challenge can be overcome by (i) sampling the large sequence space of a given scaffold through a phage display library and by (ii) panning multiple libraries encoding structurally distinct scaffolds. Here, we implement a protocol for defining these diverse scaffolds, based on clustering structurally defined DRPs according to their conformational similarity.ResultsWe developed and applied a hierarchical clustering protocol based on DRP structural similarity, followed by two post-processing steps, to classify 806 unique DRP structures into 81 clusters. The 20 most populated clusters comprised 85% of all DRPs. Representative scaffolds were selected from each of these clusters; the representatives were structurally distinct from one another, but similar to other DRPs in their respective clusters. To demonstrate the utility of the clusters, phage libraries were constructed for three of the representative scaffolds and panned against interleukin-23. One library produced a peptide that bound to this target with an IC50 of 3.3 μM.ConclusionsMost DRP clusters contained members that were diverse in sequence, host organism, and interacting proteins, indicating that cluster members were functionally diverse despite having similar structure. Only 20 peptide scaffolds accounted for most of the natural DRP structural diversity, providing suitable starting points for seeding phage display experiments. Through selection of the scaffold surface to vary in phage display, libraries can be designed that present sequence diversity in architecturally distinct, biologically relevant combinations of secondary structures. We supported this hypothesis with a proof-of-concept experiment in which three phage libraries were constructed and panned against the IL-23 target, resulting in a single-digit μM hit and suggesting that a collection of libraries based on the full set of 20 scaffolds increases the potential to identify efficiently peptide binders to a protein target in a drug discovery program.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1350-9) contains supplementary material, which is available to authorized users.

Methods: To model the chronic nature of IBD we chose to study the TNFDARE mouse model of Crohn's-like ileitis. This model stably expresses TNF over the lifespan of the animal resulting in the development of progressively worsening specific inflammation in the terminal ileum faithfully recapitulating the histological characteristics and CD4 T cell profile of CD. Netrin-1 treatment studies were performed in these mice followed by histologic analysis of the ileum, flow cytometric profiling of cell types in the ileum and RT-PCR and protein analysis was performed on whole ileum tissue. CD4 T cells were isolated from TNFDARE mice and utilized in in vivo and in vitro migration assays. Results: Netrin-1 treatment almost completely reversed histological disease and dramatically decreased the number of effector CD4 T cells in the ileum of TNFDARE mice without significantly affecting other cell populations studied. Following netrin-1 treatment, RT-PCR analysis demonstrated a robust reduction in the expression of Th1 chemokine receptors, CXCR3 and CCR5, in TNFDARE ileum. ELISA of whole ileal tissue showed a significant decrease in the Th1 cytokine IFNϒ in TNFDARE ileum following netrin-1 treatment. These observations suggest that netrin-1 attenuates the CD4 Th1 response. CD4 T cell proliferation, release of IFNϒ or differentiation to Th1 phenotype were unaffected by netrin-1 treatment. However, netrin-1 prevented TNFDARE CD4 T cell migration towards specific Th1 chemokines in vitro. Homing assays performed in vivo demonstrated netrin-1 blockade of TNFDARE CD4 T cell trafficking to the inflamed ileum, pointing to a direct effect of netrin-1 on CD4 T cell migration. Conclusions: Based on these findings, we conclude that netrin-1 can suppress chronic intestinal inflammation as observed in CD. Our initial observations suggest that netrin-1 inhibits CD4 Th1 T cell migration to the inflamed intestine which indicates that netrin-1 signaling in CD4 T cells may be an exciting pathway that warrants further investigation. P-125 YI Dysregulated Estrogen Receptor Expression in Mucosal T Cells Leads to Female Sex Bias in an Experimental Model of Chronic IleitisBackground: Crohn's disease (CD) patients exhibit an increased frequency of Foxp3expressing regulatory T cells (CD4 + CD25 + Foxp3 + , Tregs) in the intestinal mucosa, which show suppressive function ex vivo despite their failure to regulate chronic activation of effector T cells in situ. This suggests that factors present in the inflamed intestinal mucosa can disrupt normal Treg function in vivo. CD is disproportionate in female patients, suggesting that estrogen (E2) may contribute to worsened clinical disease. Despite this linkage, E2 has paradoxically been shown to be immunoprotective via induction of Foxp3 expression in conventional T cells (CD4 + CD25 2 , Tconv). The goal of this study was to determine the mechanistic contribution of E2 signaling to T cells in female ileitis subjects and to test the hypothesis that enhanced E2 signaling to estrogen receptor beta (ERb) subverts...

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Polycythemia Vera (PV) is a rare blood disease where mutations in JAK2 kinase confer constitutive JAK2 activity leading to abnormally elevated erythropoiesis that is independent of erythropoietin. PV patients present with iron deficiency at diagnosis due to increased iron utilization for erythropoiesis (Ginzburg YZ, Leukemia 2018) which worsens after repeated therapeutic phlebotomy (TP) performed to maintain hematocrit below 45%. The resulting suppression of hepcidin, the body's main negative regulator of iron metabolism, fuels expanded erythropoiesis resulting in a continued need for TP and thereby exacerbating patients' iron deficiency. Rusfertide targets iron exporter membrane protein ferroportin to trigger its degradation, preventing iron export from cells responsible for dietary iron absorption and cells that store and recycle iron. The resulting pharmacodynamic effect of lowered serum iron has disease-modifying effects in PV (Ginzburg YZ, Leukemia 2018). Rusfertide essentially eliminated the need for therapeutic phlebotomy in all PV patients (Kremyanskaya M, Blood 2020 136 Suppl 1: 33). Rusfertide also reversed iron deficiency, as indicated by increased serum ferritin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) in these patients. We present results from studies in a mouse PV model with JAK2-V617F mutation as in human PV (Mullaly A, Cancer Cell 2010; JAX stock #031658). We show that rusfertide analog Peptide A is efficacious in lowering hematocrit (HCT) while modulating other hematological parameters. Further, we show redistribution of iron away from erythropoiesis and renormalization of iron homeostasis as evidenced by ferrokinetic parameters. PV mice were treated over 6-weeks (thrice per week) with Vehicle or Peptide A at 2.5 or 7.5 mg/kg. At the end of 6 weeks, hematology parameters HCT, hemoglobin, RBC counts, were elevated in the PV-Vehicle group as compared to wild type (WT-Vehicle) mice (Table 1). Hematology parameters in PV-2.5 mg/kg group were lowered to WT-Vehicle values. In PV-7.5 mg/kg group, these parameters were lower than WT-Vehicle values, indicating that excessive iron restriction (EIR) leads to the expected anemic conditions. MCH and mean corpuscular hemoglobin concentration (MCHC) in PV-Vehicle group and PV-2.5 mg/kg treated were comparable to WT-Vehicle, indicating a lack of EIR. For the PV-7.5 mg/kg treated group, MCH and MCHC were significantly lower than WT-Vehicle, suggesting EIR at a high dose impacts hemoglobin concentration of RBC. To investigate the impact of iron restriction with Peptide A on erythroblast precursor cells in bone marrow, we conducted flow-cytometry analysis by gating on CD71 and TER-119 expression, and measuring intracellular iron using Ferro Far Red (FFR) dye. The CD71 + early precursor cell population did not change with Peptide A treatment however, the CD71 -/TER-119 + late precursor cell population was significantly lowered (~4-fold and 7.5-fold, in 2.5 and 7.5 mg/kg Peptide A treated PV groups respectively). Iron levels of CD71 + cells were dose-dependently and statistically significantly reduced in the Peptide A treated groups as compared to PV-Vehicle group. Iron levels of CD71 - cells were marginally lowered only in the PV-7.5 mg/kg group. We investigated the nature of iron redistribution induced by Peptide A, by using flow assay to assess iron concentration in splenic macrophages (F4/80 +/CD11b +). Iron was ~2-fold higher in the PV-7.5 mg/kg group as compared to PV-vehicle, and marginally higher in PV-2.5 mg/kg group. Total tissue iron concentration in the spleen was elevated in a dose-related manner in Peptide A treated groups compared to PV-Vehicle group, and in commensuration serum ferritin was increased. Serum iron was ~2-fold lower in PV-Vehicle group as compared to WT-Vehicle indicating iron depletion due to increased iron utilization for erythropoiesis. Serum iron measured after clearance of Peptide A from circulation (48 hr post-dose), was marginally increased for both Peptide A treated groups compared to PV-Vehicle. These data demonstrate that treatment with rusfertide and analogs, restricts iron from erythropoiesis by sequestering it in macrophage storage compartments. These effects along with normalization of iron homeostasis contribute to usefulness of rusfertide dose titration treatment in maintaining HCT <45% and improving symptoms related to iron deficiency in human PV. Figure 1 Figure 1. Disclosures Taranath: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhao: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Vengalam: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tang: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dion: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Su: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tovera: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhandari: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cheng: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mattheakis: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company.