Design and characterization of a protein superagonist of IL‐15 fused with IL‐15Rα and a high‐affinity T cell receptor

To avoid high systemic doses, strategies involving antigen‐specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer‐associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross‐presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high‐affinity single‐chain T cell receptor Vα‐Vβ (scTv), to deliver cytokines to intracellular tumor‐associated antigens presented as pepMHC. As typical wild‐type T cell receptors (TCRs) exhibit low affinity (K_d = 1–100 μM or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2‐K^b (SIY/K^b) with nanomolar affinity (K_d = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL‐2), interleukin 15 (IL‐15), or IL‐15/IL‐15Rα (IL‐15 linked to IL‐15Rα sushi domain, called “superfusion”). The fusions were purified with good yields and bound specifically to SIY/K^b with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine‐dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Role of IL‐18 in atopic asthma is determined by balance of IL‐18/IL‐18BP/IL‐18R

It is recognized that IL‐18 is related to development of asthma, but role of IL‐18 in asthma remains controversial and confusing. This is largely due to lack of information on expression of IL‐18 binding protein (BP) and IL‐18 receptor (R) in asthma. In this study, we found that plasma levels of IL‐18 and IL‐18BP were elevated in asthma. The ratio between plasma concentrations of IL‐18 and IL‐18BP was 1:12.8 in asthma patients. We demonstrated that 13‐fold more monocytes, 17.5‐fold more neutrophils and 4.1‐fold more B cells express IL‐18BP than IL‐18 in asthmatic blood, suggesting that there is excessive amount of IL‐18BP to abolish actions of IL‐18 in asthma. We also discovered that more IL‐18R+ monocytes, neutrophils and B cells are located in asthmatic blood. Once injected, IL‐18 eliminated IL‐18R+ monocytes in blood, but up‐regulated expression of IL‐18R in lung macrophages of OVA‐sensitized mice. Our data clearly indicate that the role of IL‐18 in asthma is very likely to be determined by balance of IL‐18/IL‐18BP/IL‐18R expression in inflammatory cells. Therefore, IL‐18R blocking or IL‐18BP activity enhancing therapies may be useful for treatment of asthma.     

In vitro and in vivo characterization of an interleukin‐15 antagonist peptide by metabolic stability, 99mTc‐labeling,and biological activity assays

Interleukin (IL)–15 is an inflammatory cytokine that constitutes a validated therapeutic target in some immunopathologies, including rheumatoid arthritis (RA). Previously, we identified an IL‐15 antagonist peptide named [K6T]P8, with potential therapeutic application in RA. In the current work, the metabolic stability of this peptide in synovial fluids from RA patients was studied. Moreover, [K6T]P8 peptide was labeled with ^99mTc to investigate its stability in human plasma and its biodistribution pattern in healthy rats. The biological activity of [K6T]P8 peptide and its dimer was evaluated in CTLL‐2 cells, using 3 different additives to improve the solubility of these peptides. The half‐life of [K6T]P8 in human synovial fluid was 5.88 ± 1.73 minutes, and the major chemical modifications included peptide dimerization, cysteinylation, and methionine oxidation. Radiolabeling of [K6T]P8 with ^99mTc showed a yield of approximately 99.8%. The ^99mTc‐labeled peptide was stable in a 30‐fold molar excess of cysteine and in human plasma, displaying a low affinity to plasma proteins. Preliminary biodistribution studies in healthy Wistar rats suggested a slow elimination of the peptide through the renal and hepatic pathways. Although citric acid, sucrose, and Tween 80 enhanced the solubility of [K6T]P8 peptide and its dimer, only the sucrose did not interfere with the in vitro proliferation assay used to assess their biological activity. The results here presented, reinforce nonclinical characterization of the [K6T]P8 peptide, a potential agent for the treatment of RA and other diseases associated with IL‐15 overexpression.     

Molecular dissection of the interactions of an antitumor interleukin‐2‐derived mutein on a phage display‐based platform

A mutein with stronger antitumor activity and lower toxicity than wild‐type human interleukin‐2 (IL‐2) has been recently described. The rationale behind its design was to reinforce the immunostimulatory potential through the introduction of four mutations that would selectively disrupt the interaction with the IL‐2 receptor alpha chain (thought to be critical for both IL‐2‐driven expansion of T regulatory cells and IL‐2‐mediated toxic effects). Despite the successful results of the mutein in several tumor models, characterization of its interactions was still to be performed. The current work, based on phage display of IL‐2‐derived variants, showed the individual contribution of each mutation to the impairment of alpha chain binding. A more sensitive assay, based on the ability of phage‐displayed IL‐2 variants to induce proliferation of the IL‐2‐dependent CTLL‐2 cell line, revealed differences between the mutated variants. The results validated the mutein design, highlighting the importance of the combined effects of the four mutations. The developed phage display‐based platform is robust and sensitive, allows a fast comparative evaluation of multiple variants, and could be broadly used to engineer IL‐2 and related cytokines, accelerating the development of cytokine‐derived therapeutics. Copyright © 2015 John Wiley & Sons, Ltd.     

IL‐1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence‐associated secretory phenotype

Interleukin‐1 alpha (IL‐1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL‐1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL‐1α in these contexts is unknown. We show IL‐1α is activated by caspase‐5 or caspase‐11 cleavage at a conserved site. Caspase‐5 drives cleaved IL‐1α release after human macrophage inflammasome activation, while IL‐1α secretion from murine macrophages only requires caspase‐11, with IL‐1β release needing caspase‐11 and caspase‐1. Importantly, senescent human cells require caspase‐5 for the IL‐1α‐dependent senescence‐associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase‐11 for the SASP‐driven immune surveillance of senescent cells in vivo. Together, we identify IL‐1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL‐1α is activated during senescence. Thus, targeting caspase‐5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.     

Critical role of inflammatory mast cell in fibrosis: Potential therapeutic effect of IL‐37

Background

Fibrosis involves the activation of inflammatory cells, leading to a decrease in physiological function of the affected organ or tissue.

Aims

To update and synthesize relevant information concerning fibrosis into a new hypothesis to explain the pathogenesis of fibrosis and propose potential novel therapeutic approaches.

Materials and Methods

Literature was reviewed and relevant information is discussed in the context of the pathogenesis of fibrosis.

Results

A number of cytokines and their mRNA are involved in the circulatory system and in organs of patients with fibrotic tissues. The profibrotic cytokines are generated by several activated immune cells, including fibroblasts and mast cells (MCs), which are important for tissue inflammatory responses to different types of injury. MC‐derived TNF, IL‐1, and IL‐33 contribute crucially to the initiation of a cascade of the host defence mechanism(s), leading to the fibrosis process. Inhibition of TNF and inflammatory cytokines may slow the progression of fibrosis and improve the pathological status of the affected subject. IL‐37 is generated by various types of immune cells and is an IL‐1 family member protein. IL‐37 is not a receptor antagonist; it binds IL‐18 receptor alpha (IL‐18Rα) and delivers the inhibitory signal by using TIR8. It has been shown that IL‐37 can be protective in inflammation and injury, and inhibits both innate and adaptive immunity.

Discussion

IL‐37 may be useful for suppression of inflammatory diseases induced by inhibiting MyD88‐dependent TLR signalling. In addition, IL‐37 downregulates NF‐κB induced by TLR2 or TLR4 through a mechanism dependent on IL‐18Rα.

Conclusion

This review summarizes current knowledge on the role of MC in inflammation and tissue/organ fibrosis, with a focus on the therapeutic potential of IL‐37‐targeting cytokines.

     

A structure-activity study on the bradykinin B1 antagonist desArg10-HOE 140: The alanine scan

It has recently been shown that the biological activity of the second generation kinin B1 receptor selective antagonist, desArg10 HOE 140, can be improved by specific amino acid substitutions. Starting from this observation, we undertook a systematic structure-activity relationship study of this antagonist, based on the alanine-scan technique, in order to obtain useful information for the rational design of more analogues. Our data indicate that the sequence of desArg10 HOE 140 does not tolerate the replacement by Ala of most of its residues, with the exception of Ser in position 7 and, to a lesser extent, D-Arg in position 1 and Hyp in position 4. The most critical residues appear to be Pro in position 3 and the C-terminal dipeptide DTic-Oic; Ala replacement at these positions resultes in a total loss of activity. Moreover, replacement by Ala of Gly in position 5 reverts the activity of desArg10 HOE 140 to that of an agonist.     

X‐ray crystal structure localizes the mechanism of inhibition of an IL‐36R antagonist monoclonal antibody to interaction with Ig1 and Ig2 extra cellular domains

Cellular signaling via binding of the cytokines IL‐36α, β, and γ along with binding of the accessory protein IL‐36RAcP, to their cognate receptor IL‐36R is believed to play a major role in epithelial and immune cell‐mediated inflammation responses. Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses. We report here the molecular structure of a complex between an extracellular portion of human IL‐36R and a Fab derived from a high affinity anti‐IL‐36R neutralizing monoclonal antibody at 2.3 Å resolution. This structure, the first of IL‐36R, reveals similarities with other structurally characterized IL‐1R family members and elucidates the molecular determinants leading to the high affinity binding of the monoclonal antibody. The structure of the complex reveals that the epitope recognized by the Fab is remote from both the putative ligand and accessory protein binding interfaces on IL‐36R, suggesting that the functional activity of the antibody is noncompetitive for these binding events.     

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The Apc^Min/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, Apc^Min/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc^Min/+ WT and Apc^Min/+ TLR4^?/? mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of Apc^Min/+ TLR4^?/? mice compared with Apc^Min/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.     

Role of interleukin‐15 in cardiovascular diseases

Interleukin (IL)‐15 is a recently identified cytokine, which belongs to the interleukin‐2(IL‐2) family, and plays an important role in innate and adaptive immunoreaction. Given the fact that the structure of IL‐15 is partially similar to IL‐2, they share some common biological effects, including immunoregulation. IL‐2 was proven to protect cardiac function in mouse myocardial infarction models. Cardiovascular diseases (CVDs) dominate the cause of mortality worldwide. Besides atherosclerosis, inflammation is also widely involved in the pathogenesis of many CVDs including hypertension, heart failure (HF) and aneurysm. IL‐15, as a pro‐inflammatory cytokine, is up‐regulated in some cardiovascular diseases, such as myocardial infarction and atherosclerosis. The current understanding of IL‐15, including its signal pathway and cellular function, was described. Furthermore, IL‐15 has a protective effect in myocardial infarction and myocarditis by decreasing cardiomyocyte death and improving heart function. The inhibited effect of IL‐15 in ductus arteriosus (DA) should be focused on. IL‐15 promoted atherogenesis. IL‐15 may be a good target in treatment of cardiovascular diabetology. Finally, future research direction of IL‐15 deserves attention. Since IL‐15 plays several roles in CVDs, understanding the role of the IL‐15/IL‐15R system may provide a scientific basis for the development of new approaches that use IL‐15 for the treatment of CVDs.     

Construction of unnatural heterodimeric receptors based on IL‐2 and IL‐6 receptor subunits

Cells have various receptors on their surface for responding to extracellular signals that involve intercellular communication. Although the mechanism of signal transduction by such wild‐type receptors has been studied intensively, there has been minimal effort in investigating whether such receptors could signal when unnaturally coupled. In this study, we investigated whether unnatural receptor pairs comprising interleukin‐2 (IL‐2) and interleukin‐6 (IL‐6) receptor subunits could transduce a signal through forced dimerization. We replaced the extracellular domain of IL‐2R and IL‐6R signaling subunits (IL‐2Rβ, IL‐2Rγ, and gp130) with the FK506‐binding protein (FKBP) or the FKBP12‐rapamycin binding (FRB) domain, the protein pair known to be heterodimerized by rapamycin. When expressed in a hematopoietic cell line, unnatural heterodimers (IL‐2Rβ‐gp130 and IL‐2Rγ‐gp130 pairs) successfully transduced a signal. While the IL‐2Rγ‐gp130 pair maximally mimicked gp130 signaling, the IL‐2Rβ‐gp130 pair gave weaker gp130 signaling and no significant induction of IL‐2Rβ signaling, indicating a high potential of the IL‐2Rγ chain in terms of activating the coupled partners. This is the first report demonstrating that heterodimeric combinations of IL‐2R and IL‐6R subunits are functional for signaling. Further extension of this approach may attain a creative design of artificial receptor pairs that have distinct signaling properties when compared with natural receptors. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1512–1518, 2013     

Structure-based design of a bicyclic peptide antagonist of the vascular endothelial growth factor receptors.

Dysregulated angiogenesis is implicated in several pathologies, including cancer and age-related macular degeneration. A potential antiangiogenic strategy consists in developing VEGF receptor ligands capable of preventing VEGF binding and the subsequent activation of these receptors. Herein, we describe the structure-based design of a VEGF-mimicking peptide, VG3F. This 25-mer peptide was doubly cyclized, on-resin, by formation of both a disulfide bridge and an intramolecular amide bond to constrain it to adopt a bioactive conformation. Tested on in vitro assays, VG3F was able to prevent VEGF binding to VEGF receptor 1 and inhibit both VEGF-induced signal transduction and cell migration.     

Macrophage inhibitory cytokine‐1 (MIC‐1/GDF15): a new marker of all‐cause mortality

Macrophage inhibitory cytokine‐1 (MIC‐1/GDF15) is a member of the TGF‐b superfamily, previously studied in cancer and inflammation. In addition to regulating body weight, MIC‐1/GDF15 may be used to predict mortality and/or disease course in cancer, cardiovascular disease (CVD), chronic renal and heart failure, as well as pulmonary embolism. These data suggested that MIC‐1/GDF15 may be a marker of all‐cause mortality. To determine whether serum MIC‐1/GDF15 estimation is a predictor of all‐cause mortality, we examined a cohort of 876 male subjects aged 35–80 years, selected from the Swedish Population Registry, and followed them for overall mortality. Serum MIC‐1/GDF15 levels were determined for all subjects from samples taken at study entry. A second (independent) cohort of 324 same‐sex twins (69% female) from the Swedish Twin Registry was similarly examined. All the twins had telomere length measured and 183 had serum levels of interleukin 6 (IL‐6) and C‐reactive protein (CRP) available. Patients were followed for up to 14 years and had cause‐specific and all‐cause mortality determined. Serum MIC‐1/GDF15 levels predicted mortality in the all‐male cohort with an adjusted odds ratio (OR) of death of 3.38 (95%CI 1.38–8.26). This finding was validated in the twin cohort. Serum MIC‐1/GDF15 remained an independent predictor of mortality when further adjusted for telomere length, IL‐6 and CRP. Additionally, serum MIC‐1/GDF15 levels were directly correlated with survival time independently of genetic background. Serum MIC‐1/GDF15 is a novel predictor of all‐cause mortality.     

A structure-activity study on the bradykinin B1 antagonist desArg10-HOE 140: The alanine scan

Summary It has recently been shown that the biological activity of the second generation kinin B₁ receptor selective antagonist, desArg¹⁰ HOE 140, can be improved by specific amino acid substitutions. Starting from this observation, we undertook a systematic structure-activity relationship study of this antagonist, based on the alanine-scan technique, in order to obtain useful information for the rational design of more analogues. Our data indicate that the sequence of desArg¹⁰ HOE 140 does not tolerate the replacement by Ala of most of its residues, with the exception of Ser in position 7 and, to a lesser extent, D-Arg in position 1 and Hyp in position 4. The most critical residues appear to be Pro in position 3 and the C-terminal dipeptide Dtic-Oic; Ala replacement at these positions resultes in a total loss of activity. Moreover, replacement by Ala of Gly in position 5 reverts the activity of desArg¹⁰ HOE 140 to that of an agonist.