Kevin M. Klucher

IL-28, IL-29 and their class II cytokine receptor IL-28R

Cytokines play a critical role in modulating the innate and adaptive immune systems. Here, we have identified from the human genomic sequence a family of three cytokines, designated interleukin 28A (IL-28A), IL-28B and IL-29, that are distantly related to type I interferons (IFNs) and the IL-10 family. We found that like type I IFNs, IL-28 and IL-29 were induced by viral infection and showed antiviral activity. However, IL-28 and IL-29 interacted with a heterodimeric class II cytokine receptor that consisted of IL-10 receptor β (IL-10Rβ) and an orphan class II receptor chain, designated IL-28Rα. This newly described cytokine family may serve as an alternative to type I IFNs in providing immunity to viral infection.

An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity.

Type III IFNs (IFN-λ/IL-28/29) are cytokines with type I IFN-like antiviral activities, which remain poorly characterized. We herein show that most cell types expressed both types I and III IFNs after TLR stimulation or virus infection, whereas the ability of cells to respond to IFN-λ was restricted to a narrow subset of cells, including plasmacytoid dendritic cells and epithelial cells. To examine the role of type III IFN in antiviral defense, we generated IL-28Rα-deficient mice. These mice were indistinguishable from wild-type mice with respect to clearance of a panel of different viruses, whereas mice lacking the type I IFN receptor (IFNAR−/−) were significantly impaired. However, the strong antiviral activity evoked by treatment of mice with TLR3 or TLR9 agonists was significantly reduced in both IL-28RA−/− and IFNAR−/− mice. The type I IFN receptor system has been shown to mediate positive feedback on IFN-αβ expression, and we found that the type I IFN receptor system also mediates positive feedback on IFN-λ expression, whereas IL-28Rα signaling does not provide feedback on either type I or type III IFN expression in vivo. Finally, using bone-marrow chimeric mice we showed that TLR-activated antiviral defense requires expression of IL-28Rα only on nonhemopoietic cells. In this compartment, epithelial cells responded to IFN-λ and directly restricted virus replication. Our data suggest type III IFN to target a specific subset of cells and to contribute to the antiviral response evoked by TLRs.

Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes†

Interleukin‐28A (IL‐28A), IL‐28B and IL‐29 are a family of class II cytokines that stimulate antiviral responses through a heterodimeric receptor that is distinct from the type I interferon (IFN) receptor. To better understand how this newly described family of cytokines regulates the antiviral state, we compared various cellular responses elicited by IL‐29 and IFN‐α. Here we show that these cytokines stimulate similar patterns of signal transducer and activator of transcription 1 (STAT‐1), ‐2, ‐3, and ‐5 phosphorylation and nearly identical patterns of gene expression when analyzed in two distinct cell types by microarray analysis. Interestingly, the IL‐29 receptor is preferentially expressed on primary hepatocytes within normal liver and pegylated forms of IL‐29 and IFN‐α induced equivalent 2′5′ oligoadenylate synthetase (OAS) and MX1 gene expression in this cell type. Pegylated IL‐29 also produced a significant reduction in human hepatitis B and hepatitis C viral load in vitro and reduced the cytopathic effect caused by the fully replicating flavivirus, West Nile virus. In conclusion, IL‐29 and IFN‐α stimulate identical antiviral responses despite their utilization of different receptors. This fact, combined with significant receptor expression in hepatitis virus‐infected livers, suggests that IL‐29 may have therapeutic value against chronic viral hepatitis in human patients. (HEPATOLOGY 2006;44:896–906.)

Interferon-lambda contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses.

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-α, IFN-β and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-λ uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR10/0) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-λ might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-λ readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR10/0 mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-λ failed to induce Mx1 in the liver of IFNAR10/0 mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-λ receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-α/β and IFN-λ were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR10/0 mice. From these results we conclude that IFN-λ contributes to inborn resistance against viral pathogens infecting the lung but not the liver.

A Multicenter Phase I Trial of PX-866, an Oral Irreversible Phosphatidylinositol 3-Kinase Inhibitor, in Patients with Advanced Solid Tumors

Purpose: The objectives of the study were to evaluate the maximum tolerated dose (MTD), safety, pharmacodynamics, pharmacokinetics, and antitumor activity of PX-866 in patients with incurable cancers. Experimental Design: This was a phase I, open-label, dose-escalation study. Drug was administered orally once per day either on an intermittent (arm 1; days 1–5 and 8–12 of a 28-day cycle) or continuous (arm 2; days 1–28 of a 28-day cycle) schedule. Additional patients were treated at the arm 2 MTD in a food effects substudy. Results: Eighty-four patients were treated in the arm 1 (n = 51), arm 2 (n = 20), and food effects (n = 13) cohorts. The most frequent study drug–related adverse events were gastrointestinal disorders (69.0%), with diarrhea being the most common (48.8%). The MTD was 12 and 8 mg for arm 1 and 2, respectively. The dose-limiting toxicities (DLT) consisted of grade III diarrhea (n = 3) and grade III elevated aspartate aminotransferase (AST; n = 1). The pharmacokinetics profile was dose proportional, with no evidence of drug accumulation. PX-866–associated inhibition of platelet pAKTSER473 was observed at the arm 2 MTD. The best response per Response Evaluation Criteria in Solid Tumors (RECIST) was stable disease in 22% of evaluable patients in arm 1, 53% in arm 2, and 11% in the food effects cohort. Eight patients were on study for 4 or more months. Conclusions: This first-in-human study shows that PX-866, an irreversible small-molecule inhibitor of phosphatidylinositol 3-kinase (PI3K), was well tolerated and was associated with prolonged stable disease, particularly when using a continuous dosing schedule. Clin Cancer Res; 18(15); 4173–82. ©2012 AACR.

IL‐28A (IFN‐λ2) modulates lung DC function to promote Th1 immune skewing and suppress allergic airway disease

IL‐28 (IFN‐λ) cytokines exhibit potent antiviral and antitumor function but their full spectrum of activities remains largely unknown. Recently, IL‐28 cytokine family members were found to be profoundly down‐regulated in allergic asthma. We now reveal a novel role of IL‐28 cytokines in inducing type 1 immunity and protection from allergic airway disease. Treatment of wild‐type mice with recombinant or adenovirally expressed IL‐28A ameliorated allergic airway disease, suppressed Th2 and Th17 responses and induced IFN‐γ. Moreover, abrogation of endogenous IL‐28 cytokine function in IL‐28Rα−/− mice exacerbated allergic airway inflammation by augmenting Th2 and Th17 responses, and IgE levels. Central to IL‐28A immunoregulatory activity was its capacity to modulate lung CD11c+ dendritic cell (DC) function to down‐regulate OX40L, up‐regulate IL‐12p70 and promote Th1 differentiation. Consistently, IL‐28A‐mediated protection was absent in IFN‐γ−/− mice or after IL‐12 neutralization and could be adoptively transferred by IL‐28A‐treated CD11c+ cells. These data demonstrate a critical role of IL‐28 cytokines in controlling T cell responses in vivo through the modulation of lung CD11c+ DC function in experimental allergic asthma.

Interferon Lambda as a Potential New Therapeutic for Hepatitis C

Interferon lambdas (IFN‐λ) are Type III interferons with biological activity, including induction of antiviral genes, similar to Type I IFNs, but signal through a distinct receptor complex. The expression pattern for the IFN‐λ receptor is more cell specific than the widely distributed IFN‐α receptor, suggesting in vivo, IFN‐λ may have fewer side effects than IFN‐α, such as less hematologic toxicities. A PEGylated form of IFN‐λ (PEG‐rIL‐29) was well tolerated in animals and did not result in hematologic toxicity. Clinical data from initial studies of PEG‐rIL‐29 has demonstrated antiviral effects in patients with hepatitis C without producing hematologic toxicity. These preclinical and early clinical data support PEG‐rIL‐29 as a potential new therapeutic agent for treatment of patients with hepatitis C.

Pharmacokinetics and pharmacodynamics of pegylated interferon lambda-1 in cynomolgus monkeys.

Type III lambda interferons (IFNs) have activity similar to type I IFNs, but a more restricted receptor distribution. A pegylated human IFN lambda-1 (pegIFNλ) is under development for chronic hepatitis C. Induction of receptor signaling (STAT1 phosphorylation) and expression of interferon-stimulated genes (ISGs) by pegIFNλ were assessed in, respectively, cynomolgus monkey leukocyte subsets and hepatocytes stimulated in vitro. ISG induction by pegIFNλ or IFNα was also assessed in peripheral leukocytes and liver biopsies after single and repeat (x3) dosing of pegIFNλ (0.03, 0.3, 3.0 mg/kg) or unpegylated IFNα-2b (10(7) IU/kg). Single-dose pharmacokinetics of pegIFNλ were evaluated. Strong ISG induction occurred in cultured hepatocytes and liver biopsies with both pegIFNλ and IFNα. However, STAT1 phosphorylation, MHC class 1 upregulation, and ISG induction in leukocytes only occurred with IFNα. Serum neopterin was unaffected by pegIFNλ; however, β-2-microglobulin was elevated at all doses. The terminal half-life of pegIFNλ was 23 h with a 59 mL/kg volume of distribution, consistent with other pegylated IFNs. Serum exposure was dose-proportional across the dosing range. These data demonstrate the suitability of cynomolgus monkeys for the preclinical evaluation of pegIFNλ. Additionally, the absence of pegIFNλ pharmacologic activity in leukocytes is consistent with its low receptor expression in blood.

Abstract 762: ONT-10, a liposomal vaccine targeting hypoglycosylated MUC1, induces a potent cellular and humoral response and suppresses the growth of MUC1 expressing tumors

MUC1 is highly expressed in multiple tumor histologies and is hypoglycosylated relative to normal tissue, with prematurely terminated carbohydrate modifications including αN-acetylgalactosamine (Tn). The aberrant glycosylation state of MUC1 makes this antigen immunologically distinct in tumor relative to normal tissues and an attractive vaccine target. ONT-10 is a liposomal formulated vaccine that incorporates a synthetic glycopeptide antigen that includes two complete 20 amino acid tandem repeats from human MUC1, including six glycosylated sites modified by Ser- or Thr-O- Tn, and is incorporated into the liposomal drug product via two C-terminal lipid serines. The ONT-10 drug product is formulated with the novel, fully synthetic lipid A adjuvant, penta erythritol lipid A (PET Lipid A). PET Lipid A displays a similar spatial distribution of lipid chains as natural Lipid A, but is composed of pure 6-acyl moieties, resulting in enhanced potency as an activator of Toll-like Receptor 2 and 4 (TLR2, TLR4). In preclinical studies, immunization of mice with ONT-10 induces a prominent T cell and antibody response to MUC1 and results in a substantial growth inhibition of MUC1 expressing tumors. T cell recall assays show a Th1 cytokine pattern with strong IFNγ production in spleen derived CD4 and CD8 cells in response to Tn-MUC1. At doses as low as 5μg/mouse ONT-10 results in a high titer anti-MUC1 antibody response composed principally of IgG2b and IgG3 that show preferential binding to Tn-MUC1 and selectively target tumor cells that express human MUC1. In a preventative B16-MUC1 tumor model, vaccination with 5μg ONT-10 potently suppresses tumor growth, resulting in 99% mean tumor growth inhibition relative to controls with 9/12 animals tumor free four weeks after tumor cell implantation. Similarly, in a MC38-MUC1 tumor model 5μg ONT-10 results in an 90% reduction in tumor growth relative to controls. Based on these preclinical data, we propose that the combination of the glycopeptide antigen structure and improved Lipid A adjuvant in ONT-10 offer advantages relative to previous MUC1 targeted vaccines by potently activating both humoral and cellular immunity against hypoglycosylated MUC1 and that these features may provide additional therapeutic benefit to patients with MUC1 expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 762. doi:10.1158/1538-7445.AM2011-762

Phase 1 dose escalation of ONT-10, a therapeutic MUC1 vaccine, in patients with advanced cancer

Meeting abstracts Mucin 1 (MUC1), a glycoprotein normally expressed at low levels on the apical borders of secretory epithelial cells, is overexpressed and aberrantly glycosylated in many cancers. ONT 10 is a therapeutic peptide vaccine incorporating a synthetic glycolipopeptide MUC1 antigen,