Resistin-like molecule (RELM)-beta is a cysteine-rich cytokine implicated in insulin resistance and asthmatic responses, but its function remains an enigma. We now report that RELM-beta has a role in ...promoting airway inflammation and lung remodeling in the mouse lung. RELM-beta is strongly induced by diverse allergens and T helper type 2 (Th2) cytokines by an IL-13- and STAT6-dependent mechanism. To understand the in vivo role of RELM-beta, we delivered recombinant murine RELM-beta intratracheally to naïve mice. RELM-beta induced dose-dependent leukocyte accumulation (most prominently involving macrophages) and goblet cell hyperplasia. The most prominent effect induced by RELM-beta was increased perivascular and peribronchial collagen deposition. Mice genetically deficient in RELM-beta had reduced accumulation of collagen and goblet cell hyperplasia in an experimental model of allergic airway inflammation. In vitro experiments demonstrated that RELM-beta had fibroblast motogenic activity. These results identify RELM-beta as a Th2-associated cytokine with potent inflammatory and remodeling activity.
Angiopoietins are a recently discovered family of angiogenic factors that interact with the endothelial receptor tyrosine kinase Tie2, either as agonists (angiopoietin-1) or as context-dependent ...agonists/antagonists (angiopoietin-2). Here we show that angiopoietin-1 has a modular structure unlike any previously characterized growth factor. This modular structure consists of a receptor-binding domain, a dimerization motif and a superclustering motif that forms variable-sized multimers. Genetic engineering of precise multimers of the receptor-binding domain of angiopoietin-1, using surrogate multimerization motifs, reveals that tetramers are the minimal size required for activating endothelial Tie2 receptors. In contrast, engineered dimers can antagonize endothelial Tie2 receptors. Surprisingly, angiopoietin-2 has a modular structure and multimerization state similar to that of angiopoietin-1, and its antagonist activity seems to be a subtle property encoded in its receptor-binding domain.
BackgroundThe clinical benefit of CTLA-4 blockade to cancer patients has been well established. However, the promising antitumor activity shown by anti-CTLA-4 monoclonal antibodies (mAb) has been ...limited by the occurrence of immune-mediated adverse reactions, especially when CTLA-4 inhibition is used in combination with anti-PD-1 therapy. These dose-limiting toxicities restrict the therapeutic use of CTLA-4 blockade. To overcome these limitations, we have developed a potent anti-CTLA-4 antibody that is selectively active in the tumor microenvironment (TME). This antibody is engineered with an Fc region for enhanced FcγR binding and peptides that mask antigen-binding regions. The masking peptides are designed to be selectively cleaved and released by proteases that are more active in the TME, resulting in restoration of full activity of the antibody in the TME.MethodsA novel, fully-humanized anti-huCTLA-4 mAb was shown to bind human CTLA-4 with improved affinity compared to ipilimumab, as measured by SPR. Engineering of the Fc region enhanced FcγR binding and ADCC function. In addition, CDR-binding peptides identified by phage display were covalently linked to the antibody using a protease-sensitive polypeptide linker. This engineered anti-CTLA-4 antibody (XTX101) showed protease-dependent binding to CTLA-4 both with recombinant and tumor tissue derived proteases.ResultsXTX101 demonstrated a 100-fold reduction in binding to human CTLA-4 by ELISA, compared to the non-masked antibody. Incubation with recombinant protease led to cleavage and release of the masking peptides and restored full binding to CTLA-4. Similarly, in vitro ADCC activity was impaired by masking and restored in a protease-dependent manner. SEB-stimulated human PMBCs were minimally responsive in vitro to XTX101, whereas PBMCs treated with proteolytically-activated XTX101 exhibited robust activation of T cell function. In human CTLA-4 knock-in mice with syngeneic MB49 tumors, XTX101 treatment led to complete tumor regression, enhanced CD8+ T cell proliferation, and depletion of tumor Tregs in the TME. By contrast, XTX101 had minimal pharmacodynamic effects in the periphery. In addition, XTX101 is effectively activated in culture supernatants from human solid tumor explants obtained from a broad range of tumor types.ConclusionsXTX101 is a tumor-selective anti-CTLA-4 mAb capable of: 1) effective CTLA-4 blockade, 2) depletion of intratumoral Tregs through enhanced antibody-dependent cellular cytotoxicity (ADCC) function, 3) minimization of systemic immune cell activation, and 4) potent anti-tumor activity. These pre-clinical data support the further evaluation of XTX101 in clinical studies.
Leptin is an adipocyte-derived cytokine that regulates food intake and body weight via interaction with its Ob receptor (ObR). Serum leptin levels are chronically elevated in obese humans, suggesting ...that obesity may be associated with leptin resistance and the inability to generate an adequate ObR response. Evidence suggests that transcriptional activation of target genes by STAT3 (signal transducer and activator of transcription) in the hypothalamus is a critical pathway that mediates leptin's action. Herein we report that activation of ObR induces the tyrosine phosphorylation of the tyrosine phosphatase SH2-containing phosphatase 2 (SHP-2) and demonstrate that Tyr986 within the ObR cytoplasmic domain is essential to mediate phosphorylation of SHP-2 and binding of SHP-2 to ObR. Surprisingly, mutation of Tyr986 to Phe, which abrogates SHP-2 phosphorylatlon and binding to the receptor, dramatically increases gene induction mediated by STAT3. Our findings indicate that SHP-2 is a negative regulator of STAT3-mediated gene induction after activation of ObR and raise the possibility that blocking the interaction of SHP-2 with ObR could overcome leptin resistance by boosting leptin's weight-reducing effects in obese individuals
BackgroundHigh-dose recombinant human interleukin-2 (aldesleukin) elicits durable anti-tumor immunity and gained FDA approval two decades prior to checkpoint blockers. However, use of aldesleukin is ...limited due to treatment-related life-threatening toxicities. Second generation efforts to alleviate toxicities have largely focused on eliminating binding to IL-2Rα, often with half-life extension. We have determined that mice and non-human primates (NHPs) treated with a 2nd generation IL-2 surrogate that does not bind IL-2Rα still experience characteristic dose-limiting toxicities, including vascular leak syndrome (VLS), and exhibit dysregulated peripheral immune function due to reduced Treg activation. To overcome these toxicities and improve the therapeutic index of IL-2 as an anti-tumor immunotherapy, we employed protein engineering to generate XTX201, a highly potent 3rd generation IL-2 that is designed to be selectively active in tumors, stimulating cytolytic responses against tumor cells while sparing systemic immune activation.MethodsXTX201 binding interactions were measured with SPR, and bioactivity was measured using STAT-5 phosphorylation in human PBMCs and reporter cell lines. Anti-tumor efficacy and immune activation was evaluated in tumors compared to peripheral organs in syngeneic tumor mouse models. Safety and pharmacokinetics were evaluated in rodents and NHPs.ResultsNon-activated XTX201 showed no detectable binding to IL-2Rα or IL-2Rβ, and limited IL-2R-dependent STAT-5 signaling in vitro. Activation of XTX201 resulted in high-affinity binding to IL-2Rβ and no binding to IL-2Rα, leading to a ~1000-fold reduction in Treg activation as compared to WT IL-2, while retaining CD8+ T and NK cell activation. Mice and NHPs treated with a 2nd generation IL-2 surrogate experienced toxicities that are commonly observed in patients treated with aldesleukin, including pulmonary edema, VLS, fever and lethality. However, XTX201 did not induce toxicities at exposures 100-fold higher than the MTD of the activated version, and achieved similar anti-tumor efficacy in mice. Experiments in primary human solid tumors and human plasma indicated that XTX201 is preferentially activated in the tumor microenvironment.ConclusionsOur data demonstrate that 2nd generation IL-2s that are systemically active and lack binding to IL-2Rα exhibit dose-limiting toxicities unless further engineered for selective activity in tumors. XTX201, a 3rd generation, tumor-selective IL-2, exhibits a long half-life and is innocuous outside of tumors. XTX201 is activated within tumors to release an IL-2Rβ/γ biased cytokine that inhibits tumor growth in syngeneic models, and exhibits tumor-specific pharmacodynamic effects without peripheral toxicities. XTX201 has the potential to be a best-in-class IL-2 immunotherapy by expanding the curative anti-tumor activity of aldesleukin while minimizing dose-limiting toxicities.
Previous experiments using enzyme inhibitors, cell lysates, and purified enzyme have suggested that puromycin-sensitive aminopeptidase (PSA) plays a role in creating and destroying MHC class ...I-presented peptides although its precise contribution to these processes is unknown. To examine the importance of this enzyme in MHC class I Ag presentation, we have generated PSA-deficient mice and cell lines from these animals. PSA-deficient mice are smaller and do not reproduce as well as wild type mice. In addition, dendritic cells from PSA-deficient mice display more MHC class I molecules on the cell surface, suggesting that PSA normally limits Ag presentation by destroying certain peptides in these key APCs. Surprisingly, MHC class I levels are not altered on other PSA-deficient cells and the processing and presentation of peptide precursors in PSA-deficient fibroblasts is normal. Moreover, PSA-deficient mice have normal numbers of T cells in the periphery, and respond as well as wild type mice to eight epitopes from three viruses. These data indicate that PSA may play a role in limiting MHC class I Ag presentation in dendritic cells in vivo but that it is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several Ags.
To detect viral infections and tumors, CD8+ T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are ...generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Leucine aminopeptidase (LAP) is an IFN-inducible cytosolic aminopeptidase that can trim precursor peptides to mature epitopes and has been thought to play an important role in Ag presentation. To examine the role of LAP in generating MHC class I peptides in vivo, we generated LAP-deficient mice and LAP-deficient cell lines. These mutant mice and cells are viable and grow normally. The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full-length Ag constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. These data demonstrate that LAP is not an essential enzyme for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases.
The cytokine interleukin-22 (IL-22) is primarily expressed by T helper 17 (Th17) CD4
+ T cells and is highly upregulated during chronic inflammatory diseases. IL-22 receptor expression is absent on ...immune cells, but is instead restricted to the tissues, providing signaling directionality from the immune system to the tissues. However, the role of IL-22 in inflammatory responses has been confounded by data suggesting both pro- and anti-inflammatory functions. Herein, we provide evidence that during inflammation, IL-22 played a protective role in preventing tissue injury. Hepatocytes from mice deficient in IL-22 were highly sensitive to the detrimental immune response associated with hepatitis. Additionally, IL-22-expressing Th17 cells provided protection during hepatitis in IL-22-deficient mice. On the other hand, interleukin-17 (IL-17), which is coexpressed with IL-22 and can induce similar cellular responses, had no observable role in liver inflammation. Our data suggest that IL-22 serves as a protective molecule to counteract the destructive nature of the immune response to limit tissue damage.
We report the characterization of the msbA gene, isolated as a multicopy suppressor of the HtrB temperature-sensitive phenotype. The msbA gene maps to 20.5 min on the Escherichia coli genetic map and ...encodes a protein with an estimated molecular mass of 64,460 Da, with the properties of an integral membrane protein. The amino acid sequence of MsbA is very similar to those of the family of ATP-dependent translocators, which includes the haemolysin B protein of E. coli and the mammalian multidrug resistance (MDR) proteins. Mutational analysis of msbA indicates that it may form an operon with a downstream gene, orfE, and that both of these genes are essential for bacterial viability under all growth conditions tested.
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