Search

IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)-based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

Our previous big data analyses showed a high level of association between chitinase 3 like1 (CHI3L1) expression and lung tumor development. In the present study, we investigated whether a CHI3L1-inhibiting chemical, 2-({3-[2-(1-cyclohexen-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}sulfanyl)-N-(4-ethylphenyl)butanamide (K284), could inhibit lung metastasis and studied its mechanism of action. We investigated the antitumor effect of K284 both in vitro and in vivo. K284 (0.5 mg·kg -1 body weight) significantly inhibited lung metastasis in in vivo models after injection of murine melanoma cells (B16F10) or adenocarcinomic human alveolar basal epithelial cells (A549). K284 significantly and concentration-dependently also inhibited cancer cell proliferation and migration in the A549 and H460 lung cancer cell lines. We found that the binding of K284 to the chitin-binding domain (CBD) of CHI3L1 prevented the binding of CHI3L1 to its receptor, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), thereby suppressing the CHI3L1 signal. This blocking of the CHI3L1-IL-13Rα2 signal caused the inhibition of c-Jun N-terminal kinase (JNK)-activator protein 1 (AP-1) signals, resulting in the prevention of lung metastasis and cancer cell growth. Our data demonstrate that K284 may serve as a potential candidate anticancer compound targeting CHI3L1., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Glioblastoma multiforme (GBM) is the most common lethal primary brain malignancy without reliable therapeutic drugs. IL-13Rα2 is frequently expressed in GBMs as a molecular marker. Resveratrol (Res) effectively inhibits GBM cell growth but has not been applied in vivo because of its low brain bioavailability when administered systemically. A sustained-release and GBM-targeting resveratrol form may overcome this therapeutic dilemma. To achieve this goal, encapsulated Res 30 ± 4.8 nm IL-13Rα2-targeting nanoparticles (Pep-PP@Res) were constructed. Ultraviolet spectrophotometry revealed prolonged Res release (about 25%) from Pep-PP@Res in 48 h and fluorescent confocal microscopy showed the prolonged intracellular Res retention time of Pep-PP@Res (>24 h) in comparison with that of free Res (<4 h) and PP@Res (<4 h). MTT and EdU cell proliferation assays showed stronger suppressive effects of Pep-PP@Res on rat C6 GBM cells than that of PP@Res ( p = 0.024) and Res ( p = 0.009) when used twice for 4 h/day. Pep-PP@Res had little toxic effect on normal rat brain cells. The in vivo anti-glioblastoma effects of Res can be distinctly improved in the form of Pep-PP@Res nanoparticles via activating JNK signaling, upregulating proapoptosis gene expression and, finally, resulting in extensive apoptosis. Pep-PP@Res with sustained release and GBM-targeting properties would be suitable for in vivo management of GBMs.

Diffuse intrinsic pontine glioma (DIPG), a rare pediatric brain tumor, afflicts approximately 350 new patients each year in the United States. DIPG is noted for its lethality, as fewer than 1% of patients survive to five years. Multiple clinical trials involving chemotherapy, radiotherapy, and/or targeted therapy have all failed to improve clinical outcomes. Recently, high-throughput sequencing of a cohort of DIPG samples identified potential therapeutic targets, including interleukin 13 receptor subunit alpha 2 (IL13Rα2) which was expressed in multiple tumor samples and comparably absent in normal brain tissue, identifying IL13Rα2 as a potential therapeutic target in DIPG. In this work, we investigated the role of IL13Rα2 signaling in progression and invasion of DIPG and viability of IL13Rα2 as a therapeutic target through the use of immunoconjugate agents. We discovered that IL13Rα2 stimulation via canonical ligands demonstrates minimal impact on both the cellular proliferation and cellular invasion of DIPG cells, suggesting IL13Rα2 signaling is non-essential for DIPG progression in vitro. However, exposure to an anti-IL13Rα2 antibody-drug conjugate demonstrated potent pharmacological response in DIPG cell models both in vitro and ex ovo in a manner strongly associated with IL13Rα2 expression, supporting the potential use of targeting IL13Rα2 as a DIPG therapy. However, the tested ADC was effective in most but not all cell models, thus selection of the optimal payload will be essential for clinical translation of an anti-IL13Rα2 ADC for DIPG.

IL13Rα2 is a cell surface tumor antigen that is overexpressed in multiple tumor types. Here, we studied biodistribution and targeting potential of an anti-IL13Rα2 antibody (Ab) and anti-tumor activity of anti-IL13Rα2-antibody-drug conjugate (ADC). The anti-IL13Rα2 Ab was labeled with fluorophore AF680 or radioisotope 89 Zr for in vivo tracking using fluorescence molecular tomography (FMT) or positron emission tomography (PET) imaging, respectively. Both imaging modalities showed that the tumor was the major uptake site for anti-IL13Rα2-Ab, with peak uptake of 5-8% ID and 10% ID/g as quantified from FMT and PET, respectively. Pharmacological in vivo competition with excess of unlabeled anti-IL13Rα2-Ab significantly reduced the tumor uptake, indicative of antigen-specific tumor accumulation. Further, FMT imaging demonstrated similar biodistribution and pharmacokinetic profiles of an auristatin-conjugated anti-IL13Rα2-ADC as compared to the parental Ab. Finally, the anti-IL13Rα2-ADC exhibited a dose-dependent anti-tumor effect on A375 xenografts, with 90% complete responders at a dose of 3 mg/kg. Taken together, both FMT and PET showed a favorable biodistribution profile for anti-IL13Rα2-Ab/ADC, along with antigen-specific tumor targeting and excellent therapeutic efficacy in the A375 xenograft model. This work shows the great potential of this anti-IL13Rα2-ADC as a targeted anti-cancer agent.

There continues to be a major need for more effective inflammatory bowel disease (IBD) therapies. IL-13Rα2 is a decoy receptor that binds the cytokine IL-13 with high affinity and diminishes its STAT6-mediated effector functions. Previously, we found that IL-13Rα2 was necessary for IBD in mice deficient in the anti-inflammatory cytokine IL-10. Here, we tested for the first time a therapeutic antibody specifically targeting IL-13Rα2. We also used the antibody and Il13ra2 -/- mice to dissect the role of IL-13Rα2 in IBD pathogenesis and recovery. Il13ra2 - /- mice were modestly protected from induction of dextran sodium sulfate (DSS)-induced colitis. Following a 7-day recovery period, Il13ra2 -/- mice or wild-type mice administered the IL-13Rα2-neutralizing antibody had significantly improved colon health compared to control mice. Neutralizing IL-13Rα2 to increase IL-13 bioavailability promoted resolution of IBD even if neutralization occurred only during recovery. To link our observations in mice to a large human cohort, we conducted a phenome-wide association study of a more active variant of IL-13 (R130Q) that has reduced affinity for IL-13Rα2. Human subjects carrying R130Q reported a lower risk for Crohn's disease. Our findings endorse moving anti-IL-13Rα2 into preclinical drug development with the goal of accelerating recovery and maintaining remission in Crohn's disease patients.

We previously designed and reported a novel class of drugs, namely hybrid peptides, which are chemically synthesized and composed of a targeted binding peptide and a lytic-type peptide containing cationic amino acid residues that cause cancer cell death. In the present study, we screened for peptides that bind to interleukin-13 receptor alpha 2 (IL-13Rα2) by using a T7 random peptide phage display library system and isolated several positive phage clones. The A2b11 peptide, which was one of the positive clones, was shown to bind to IL-13Rα2 protein by Biacore analysis and a binding assay using glioblastoma (GB) cell lines. This peptide was linked with a lytic peptide containing a linker sequence to form the IL-13Rα2-lytic hybrid peptide. The IL-13Rα2-lytic hybrid peptide showed cytotoxic activity against GB cell lines in vitro. The IL-13Rα2-lytic hybrid peptide also affected Akt and Erk1/2 activation following treatment with interleukin-13 and induced rapid ATP dynamics in GB cells. Anti-tumor activity of the IL-13Rα2-lytic hybrid peptide was observed in vivo after intratumoral injection in a mouse xenograft model of human GB cells. These results suggest that the IL-13Rα2-lytic hybrid peptide might be a potent therapeutic option for patients with GB., (© 2019 John Wiley & Sons A/S.)

In order to fully harness the potential of immunotherapy with chimeric antigen receptor (CAR)-modified T cells, pre-clinical studies must be conducted in immunocompetent animal models that closely mimic the immunosuppressive malignant glioma (MG) microenvironment. Thus, the goal of this project was to study the in vivo fate of T cells expressing CARs specific for the MG antigen IL13Rα2 (IL13Rα2-CARs) in immunocompetent MG models. Murine T cells expressing IL13Rα2-CARs with a CD28.ζ (IL13Rα2-CAR.CD28.ζ) or truncated signaling domain (IL13Rα2-CAR.Δ) were generated by retroviral transduction, and their effector function was evaluated both in vitro and in vivo. IL13Rα2-CAR.CD28.ζ T cells' specificity toward IL13Rα2 was confirmed through cytokine production and cytolytic activity. In vivo, a single intratumoral injection of IL13Rα2-CAR.CD28.ζ T cells significantly extended the survival of IL13Rα2-expressing GL261 and SMA560 glioma-bearing mice; long-term survivors were resistant to re-challenge with IL13Rα2-negative and IL13Rα2-positive tumors. IL13Rα2-CAR.CD28.ζ T cells proliferated, produced cytokines (IFNγ, TNF-α), and promoted a phenotypically pro-inflammatory glioma microenvironment by inducing a significant increase in the number of CD4 + and CD8 + T cells and CD8α + dendritic cells and a decrease in Ly6G + myeloid-derived suppressor cells (MDSCs). Our data underline the significance of CAR T cell studies in immunocompetent hosts and further validate IL13Rα2-CAR T cells as an efficacious therapeutic strategy for MG., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

T cell immunotherapy is emerging as a powerful strategy to treat cancer and may improve outcomes for patients with glioblastoma (GBM). We have developed a chimeric antigen receptor (CAR) T cell immunotherapy targeting IL-13 receptor α2 (IL13Rα2) for the treatment of GBM. Here, we describe the optimization of IL13Rα2-targeted CAR T cells, including the design of a 4-1BB (CD137) co-stimulatory CAR (IL13BBζ) and a manufacturing platform using enriched central memory T cells. Utilizing orthotopic human GBM models with patient-derived tumor sphere lines in NSG mice, we found that IL13BBζ-CAR T cells improved anti-tumor activity and T cell persistence as compared to first-generation IL13ζ-CAR CD8 + T cells that had shown evidence for bioactivity in patients. Investigating the impact of corticosteroids, given their frequent use in the clinical management of GBM, we demonstrate that low-dose dexamethasone does not diminish CAR T cell anti-tumor activity in vivo. Furthermore, we found that local intracranial delivery of CAR T cells elicits superior anti-tumor efficacy as compared to intravenous administration, with intraventricular infusions exhibiting possible benefit over intracranial tumor infusions in a multifocal disease model. Overall, these findings help define parameters for the clinical translation of CAR T cell therapy for the treatment of brain tumors., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Glioblastoma (GBM) is the most aggressive primary malignant brain cancer that invariably results in a dismal prognosis. Chemotherapy and radiotherapy have not been completely effective as standard treatment options for patients due to recurrent disease. We and others have therefore developed molecular strategies to specifically target interleukin 13 receptor alpha 2 (IL13RA2), a GBM restricted receptor expressed abundantly on over 75% of GBM patients. In this work, we evaluated the potential of Pep-1L, a novel IL13RA2 targeted peptide, as a platform to deliver targeted lethal therapies to GBM. To demonstrate GBM-specificity, we radiolabeled Pep-1L with Copper-64 and performed in vitro cell binding studies, which demonstrated specific binding that was blocked by unlabeled Pep-1L. Furthermore, we demonstrated real-time GBM localization of [64Cu]Pep-1L to orthotopic GBMs using small animal PET imaging. Based on these targeting data, we performed an initial in vivo safety and therapeutic study using Pep-1L conjugated to Actinium-225, an alpha particle emitter that has been shown to potently and irreversibly kill targeted cells. We infused [225Ac]Pep-1L into orthotopic GBMs using convection-enhanced delivery and found no significant adverse events at injected doses. Furthermore, our initial data also demonstrated significantly greater overall, median and mean survival in treated mice when compared to those in control groups (p < 0.05). GBM tissue extracted from mice treated with [225Ac]Pep-1L showed double stranded DNA breaks, lower Ki67 expression and greater propidium iodide internalization, indicating anti-GBM therapeutic effects of [225Ac]Pep-1L. Based on our results, Pep-1L warrants further investigation as a potential targeted platform to deliver anti-cancer agents.

The generation of a targeting agent that strictly binds to IL13Rα2 will significantly expand the therapeutic potential for the treatment of IL13Rα2-expressing cancers. In order to fulfill this goal, we generated a single-chain antibody (scFv47) from our parental IL13Rα2 monoclonal antibody and tested its binding properties. Furthermore, to demonstrate the potential therapeutic applicability of scFv47, we engineered an adenovirus by incorporating scFv47 as the targeting moiety in the viral fiber and characterized its properties in vitro and in vivo. The scFv47 binds to human recombinant IL13Rα2, but not to IL13Rα1 with a high affinity of 0.9 · 10(-9) M, similar to that of the parental antibody. Moreover, the scFv47 successfully redirects adenovirus to IL13Rα2 expressing glioma cells both in vitro and in vivo. Our data validate scFv47 as a highly selective IL13Rα2 targeting agent and justify further development of scFv47-modified oncolytic adenovirus and other therapeutics for the treatment of IL13Rα2-expressing glioma and other malignancies.

Superinfection in mice at day 7 postinfluenza infection exacerbates bacterial pneumonia at least in part via downstream effects of increased IFN-γ signaling. Here we show that up to 3 days postinfluenza infection, mice have reduced susceptibility to superinfection with methicillin-resistant Staphylococcus aureus (MRSA), but that superinfection during that time exacerbated influenza disease. This was due to IL-13 signaling that was advantageous for resolving MRSA infection via inhibition of IFN-γ, but was detrimental to the clearance of influenza virus. However, if superinfection did not occur until the near resolution of influenza infection (day 7), IL-13 signaling was inhibited, at least in part by upregulation of IL-13 decoy receptor (IL-13Rα2), which in turn caused increases in IFN-γ signaling and exacerbation of bacterial infection. Understanding these cytokine sequelae is critical to development of immunotherapies for influenza-MRSA coinfection since perturbations of these sequelae at the wrong time could increase susceptibility to MRSA and/or influenza., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Interleukin-4 (IL-4) and IL-13 are critical drivers of immune activation and inflammation in ulcerative colitis, asthma and other diseases. Because these cytokines may have redundant function, dual targeting holds promise for achieving greater efficacy. We have recently described a bifunctional therapeutic targeting IL-4 and IL-13 developed on a novel protein scaffold, generated by combining specific binding domains in an optimal configuration using appropriate linker regions. In the current study, the bifunctional IL-4/IL-13 antagonist was evaluated in the murine oxazolone-induced colitis model, which produces disease with features of ulcerative colitis. The bifunctional IL-4/IL-13 antagonist reduced body weight loss throughout the 7-day course of the model, and ameliorated the increased colon weight and decreased colon length that accompany disease. Colon tissue gene expression was modulated in accordance with the treatment effect. Concentrations of serum amyloid P were elevated in proportion to disease severity, making it an effective biomarker. Serum concentrations of the bifunctional IL-4/IL-13 antagonist were inversely proportional to disease severity, colon tissue expression of pro-inflammatory genes, and serum amyloid P concentration. Taken together, these results define a panel of biomarkers signifying engagement of the IL-4/IL-13 pathway, confirm the T helper type 2 nature of disease in this model, and demonstrate the effectiveness of dual cytokine blockade., (© 2014 John Wiley & Sons Ltd.)

As the most common malignant brain tumors, glioblastoma multiforme (GBM) was characterized by angiogenesis and tumor cells proliferation. Dual targeting to neovasculature and GBM cells could deliver cargoes to these two kinds of cells, leading to a combination treatment. In this study, polymeric nanoparticles were functionalized with RGD and interleukin-13 peptide (IRNPs) to construct a neovasculature and tumor cell dual targeting delivery system in which RGD could target αvβ3 on neovasculature and interleukin-13 peptide could target IL13Rα2 on GBM cells. In vitro, interleukin-13 peptide and RGD could enhance the uptake by corresponding cells (C6 and human umbilical vein endothelial cells). Due to the expression of both receptors on C6 cells, RGD also could enhance the uptake by C6 cells. Through receptor labeling, it clearly showed that αvβ3 could mediate the internalization of RGD modified nanoparticles and IL13Rα2 could mediate the internalization of interleukin-13 peptide modified nanoparticles. The ligand functionalization also resulted in a modification on endocytosis pathways, which changed the main endocytosis pathways from macropinocytosis for unmodified nanoparticles to clathrin-mediated endocytosis for IRNPs. IRNPs also displayed the strongest penetration ability according to tumor spheroid analysis. In vivo, IRNPs could effectively deliver cargoes to GBM with higher intensity than monomodified nanoparticles. After CD31-staining, it demonstrated IRNPs could target both neovasculature and GBM cells. In conclusion, IRNPs showed promising ability in dual targeting both neovasculature and GBM cells.

Glioblastoma (GBM) is the most lethal primary brain tumor, and despite several refinements in its multimodal management, generally has very poor prognosis. Targeted immunotherapy is an emerging field of research that shows great promise in the treatment of GBM. One of the most extensively studied targets is the interleukin-13 receptor alpha chain variant 2 (IL13Rα2). Its selective expression on GBM, discovered almost two decades ago, has been a target for therapy ever since. Immunotherapeutic strategies have been developed targeting IL13Rα2, including monoclonal antibodies as well as cell-based strategies such as IL13Rα2-pulsed dendritic cells and IL13Rα2-targeted chimeric antigen receptor-expressing T cells. Advanced therapeutic development has led to the completion of several clinical trials with promising outcomes. In this review, we will discuss the recent advances in the IL13Rα2-targeted immunotherapy and evaluate the most promising strategy for targeted GBM immunotherapy.

Interleukin 13 receptor alpha 2 (IL-13RA2) is over-expressed in a vast majority of human patients with high-grade astrocytomas like glioblastoma. Spontaneous astrocytomas in dogs resemble human disease and have been proposed as translational model system for investigation of novel therapeutic strategies for brain tumors. We have generated reagents for both detection and therapeutic targeting of IL-13RA2 in human and canine brain tumors. Peptides from three different regions of IL-13RA2 with 100% sequence identity between human and canine receptors were used as immunogens for generation of monoclonal antibodies. Recombinant canine mutant IL-13 (canIL-13.E13K) and canIL-13.E13K based cytotoxin were also produced. The antibodies were examined for their immunoreactivities in western blots, immunohistochemistry, immunofluorescence and cell binding assays using human and canine tumor specimen sections, tissue lysates and established cell lines; the cytotoxin was tested for specific cell killing. Several isolated MAbs were immunoreactive to IL-13RA2 in western blots of cell and tissue lysates from glioblastomas from both human and canine patients. Human and canine astrocytomas and oligodendrogliomas were also positive for IL-13RA2 to various degrees. Interestingly, both human and canine meningiomas also exhibited strong reactivity. Normal human and canine brain samples were virtually negative for IL-13RA2 using the newly generated MAbs. MAb 1E10B9 uniquely worked on tissue specimens and western blots, bound live cells and was internalized in GBM cells over-expressing IL-13RA2. The canIL-13.E13K cytotoxin was very potent and specific in killing canine GBM cell lines. Thus, we have obtained several monoclonal antibodies against IL-13RA2 cross-reacting with human and canine receptors. In addition to GBM, other brain tumors, such as high grade oligodendrogliomas, meningiomas and canine choroid plexus papillomas, appear to express the receptor at high levels and thus may be appropriate candidates for IL-13RA2-targeted imaging/therapies. Canine spontaneous primary brain tumors represent an excellent translational model for human counterparts.

Background: Adrenocortical carcinoma (ACC) is a relatively rare but aggressive malignancy with limited therapeutic options. Previous genome-wide expression studies have demonstrated the overexpression of interleukin-13 receptor alpha2 (IL13Rα2) in some human malignancies., Methods: The authors evaluated IL13Rα2 mRNA and protein expression in 21 normal samples, 78 benign samples, 10 primary malignant samples, and 25 metastatic/recurrent samples and performed functional analyses with IL13 ligand and IL13 Rα2 knockdown in vitro. The sensitivity of 2 ACC cell lines (NCI-H295R [high IL13Rα2 expression] and SW13 [low IL13Rα2 expression]) to a highly specific IL-13 conjugated with Pseudomonas exotoxin (IL-13-PE) also was evaluated in both in vitro and in vivo models., Results: IL13Rα2 was overexpressed in malignant tumors compared with benign and normal samples (15-fold higher; P < .05). Immunohistochemistry also confirmed higher protein expression in malignant and benign tumors than in normal adrenocortical tissues (P < .05). The half-maximal inhibitory concentration for IL-13-PE was 1.3 ng/mL in the NCI-H295R cell line and 1000 ng/mL in the SW13 cell line. Mice that received intratumoral or intraperitoneal IL-13-PE injection had a significant reduction in tumor size and increased tumor necrosis compared with control groups (P < .05) and also had prolonged survival (P < .05). IL13Rα2 protein expression increased in cells that were treated with IL-13 ligand along with cell invasion (P < .05). Direct IL13Rα2 knockdown decreased cellular proliferation and invasion (P < .05)., Conclusions: The current results indicated that IL13Rα2 is overexpressed in ACC and regulates cell invasion and proliferation. IL13Rα2 is a novel therapeutic target for the treatment of human ACC., (Copyright © 2012 American Cancer Society.)

Human studies using Abs to two different, nonoverlapping epitopes of IL-13 suggested that epitope specificity can have a clinically significant impact on clearance of IL-13. We propose that Ab modulation of IL-13 interaction with IL-13Rα2 underlies this effect. Two Abs were administered to healthy subjects and mild asthmatics in separate dose-ranging studies and allergen-challenge studies. IMA-638 allows IL-13 interaction with IL-13Rα1 or IL-13Rα2 but blocks recruitment of IL-4Rα to the IL-13/IL-13Rα1 complex, whereas IMA-026 competes with IL-13 interaction with IL-13Rα1 and IL-13Rα2. We found ∼10-fold higher circulating titer of captured IL-13 in subjects treated with IMA-026 compared with those administered IMA-638. To understand how this difference could be related to epitope, we asked whether either Ab affects IL-13 internalization through cell surface IL-13Rα2. Humans inducibly express cell surface IL-13Rα2 but lack the soluble form that regulates IL-13 responses in mice. Cells with high IL-13Rα2 expression rapidly and efficiently depleted extracellular IL-13, and this activity persisted in the presence of IMA-638 but not IMA-026. The potency and efficiency of this clearance pathway suggest that cell surface IL-13Rα2 acts as a scavenger for IL-13. These findings could have important implications for the design and characterization of IL-13 antagonists.

Purpose: The interleukin-13 receptor α2 (IL-13Rα2) is expressed by a variety of human malignant cells. Here, we have examined the constitutive surface expression and the epigenetic regulation of IL-13Rα2 by human mesothelioma. We have also investigated the therapeutic effect of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and anti-IL-13Rα2 monoclonal antibody on mesothelioma xenografts., Experimental Design: Cell surface expression of IL-13Rα2 by various lung carcinomas was analyzed using flow cytometry. Therapeutic effects of anti-IL-13Rα2 and 5-aza-dC were investigated using antibody-dependent cellular cytotoxicity and proliferation assays and by monitoring the survival of mesothelioma-bearing mice., Results: We found that human malignant mesotheliomas expressed surface IL-13Rα2 on their surface and that it was upregulated by treatment with 5-aza-dC. This augmented expression of IL-13Rα2 resulted in growth inhibition of the mesothelioma cells when cocultured with anti-IL-13Rα2 and effector cells, such as splenocytes and peritoneal exudate cells. The growth inhibition of mesothelioma cells was mediated by IFN-γ that was only detected in the supernatant when effector cells were exposed to 5-aza-dC-treated tumors in the presence of anti-IL-13Rα2. Compared with the control or either regimen alone, in vivo administration of anti-IL-13Rα2 in combination with 5-aza-dC significantly prolonged the survival of mice with mesothelioma xenografts., Conclusions: These observations indicate a promising role for IL-13Rα2 as a target for antibody treatment in malignant mesothelioma, and, in combination with epigenetic regulation by a DNA methylation inhibitor, suggest the potential for a novel strategy to enhance therapeutic potency., (©2011 AACR.)

Eosinophilic esophagitis (EE) is an emerging disease associated with both food and respiratory allergy characterized by extensive esophageal tissue remodeling and abnormal esophageal gene expression, including increased IL-13. We investigated the ability of increased airway IL-13 to induce EE-like changes. Mice with pulmonary (but not esophageal) overexpression of IL-13 evidenced esophageal IL-13 accumulation and developed prominent esophageal remodeling with epithelial hyperplasia, angiogenesis, collagen deposition, and increased circumference. IL-13 induced notable changes in esophageal transcripts that overlapped with the human EE esophageal transcriptome. IL-13-induced esophageal eosinophilia was dependent on eotaxin-1 (but not eotaxin-2). However, remodeling occurred independent of eosinophils as demonstrated by eosinophil lineage-deficient, IL-13 transgenic mice. IL-13-induced remodeling was significantly enhanced by IL-13Ralpha2 deletion, indicating an inhibitory effect of IL-13Ralpha2. In the murine system, there was partial overlap between IL-13-induced genes in the lung and esophagus, yet the transcriptomes were divergent at the tissue level. In human esophagus, IL-13 levels correlated with the magnitude of the EE transcriptome. In conclusion, inducible airway expression of IL-13 results in a pattern of esophageal gene expression and extensive tissue remodeling that resembles human EE. Notably, we identified a pathway that induces EE-like changes and is IL-13-driven, eosinophil-independent, and suppressed by IL-13Ralpha2.

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

Background & Aims: Previous studies have shown that fibrosis developing in chronic experimental colitis is driven by interleukin (IL)-13 signaling via IL-13R alpha(2) and the production of transforming growth factor (TGF)-beta1. In the present study, we sought to determine the fibrogenic downstream events set in motion by such signaling., Methods: Experimental colitis with late-onset intestinal fibrosis was induced by weekly intrarectal administration of trinitrobenzene sulfonic acid (TNBS) to BALB/c mice. Blockade of IL-13 signaling via IL-13R alpha(2) and TGF-beta1 signaling was achieved by the administration of small interfering RNA or decoy oligonucleotides that target promoter sequences of signaling components of these receptors. Effects of blockade were determined by enzyme-linked immunosorbent assay or Western blotting detecting specific key fibrogenic factors and by measurement of collagen production., Results: Initially, we showed that abrogation of IL-13 activity via blockade of IL-13R alpha(2) and TGF-beta1 signaling results in severe inhibition of expression of colonic insulin-like growth factor (IGF)-I and early growth response gene (Egr)-1, factors known to initiate and sustain fibrosis. We then showed that Egr-1 was necessary early in the fibrotic process for caspase-mediated apoptosis of myofibroblasts and the production of urokinase plasminogen activator, a protein that enhances TGF-beta1 activation. Finally, we showed that IGF-I (together with TGF-beta1) acts later in the process to stimulate myofibroblasts to deposit collagen in the colon., Conclusions: These studies establish that IL-13 signaling via the IL-13R alpha(2) is a key initiation point for a complex fibrotic program in the colon consisting of TGF-beta1 activation, IGF-I and Egr-1 expression, myofibroblast apoptosis, and myofibroblast production of collagen.

In previous studies, we described a "counter-immunosurveillance" mechanism initiated by tumor-activated, interleukin-13 (IL-13)-producing natural killer T cells that signal Gr-1(+) cells to produce transforming growth factor-beta(1) (TGF-beta(1)), a cytokine that suppresses the activity of tumor-inhibiting cytolytic CD8(+) T cells. Here, we show that in two tumor models (the CT-26 metastatic colon cancer and the 15-12RM fibrosarcoma regressor models), this counter-surveillance mechanism requires the expression of a novel IL-13 receptor, IL-13R alpha(2), on Gr-1(intermediate) cells, because down-regulation of IL-13R alpha(2) expression or the activator protein-1 signal generated by the receptor via in vivo administration of specific small interfering RNA or decoy oligonucleotides leads to loss of TGF-beta(1) production. Furthermore, acting on prior studies showing that IL-13R alpha(2) expression is induced (in part) by tumor necrosis factor-alpha (TNF-alpha), we show that receptor expression and TGF-beta(1) production is inhibited by administration of a TNF-alpha-neutralizing substance, TNF-alpha R-Fc (etanercept). Taking advantage of this latter fact, we then show in the CT-26 model that counter-immunosurveillance can be inhibited, anti-CT-26-specific CD8(+) cytolytic activity can be restored, and CT-26 metastatic tumor nodules can be greatly decreased by administration of TNF-alpha R-Fc. Corroborative data were obtained using the 15-12RM fibrosarcoma model. These studies point to the prevention of metastatic cancer with an available agent with already known clinically acceptable adverse effects and toxicity.

Background: The high affinity receptor for interleukin (IL)-13, IL-13 receptor alpha2 (IL-13Ralpha2), acts as a decoy receptor for IL-13, modulates fibrosis and has an anti-tumor effect. Recently, IL-13Ralpha2 has been considered as a therapeutic target for fibrosis and tumor growth. However, the mechanism of IL-13Ralpha2 expression in cardiomyocytes is unclear., Methods and Results: The mechanism of IL-13Ralpha2 expression was examined using cultured rat neonatal cardiomyocytes. Cyclical mechanical stretch induced IL-13Ralpha2 mRNA expression in rat cardiomyocytes. Treatment with angiotensin II, which plays a pivotal role in mechanical stretch-induced cardiomyocyte hypertrophy, upregulated IL-13Ralpha2 mRNA expression in rat cardiomyocytes. IL-13Ralpha2 mRNA expression was also upregulated through IL-13 treatment. Furthermore, mechanical stretch and angiotensin II treatment caused IL-13 secretion from rat cardiomyocytes, which was suppressed by angiotensin type1 receptor (AT1R) RNA interference. Upregulation of IL-13Ralpha2 mRNA expression through mechanical stretch, angiotensin II treatment and IL-13 treatment was inhibited by anti-IL-13Ralpha1 antibody and STAT6 depletion through RNA interference. Positive immunohistochemical staining for IL-13Ralpha2 was observed in the myocardium of endomyocardial biopsy specimens from the failing human heart, but not in autopsy specimens from control subjects., Conclusion: IL-13 might act in an autocrine and paracrine fashion to upregulate IL-13Ralpha2 expression in cardiomyocytes.

To investigate the immunopathogenesis of inflammation-associated fibrosis, we analyzed the chronic colitis and late-developing fibrosis occurring in BALB/c mice administered weekly doses of intrarectal 2,4,6-trinitrobenzene sulfonic acid. We showed first in this model that an initial Th1 response involving IL-12p70 and IFN-gamma subsides after 3 wk to be supplanted by an IL-23/IL-25 response beginning after 4-5 wk. This evolution is followed by gradually increasing production of IL-17 and cytokines ordinarily seen in a Th2 response, particularly IL-13, which reaches a plateau at 8-9 wk. In vitro stimulation studies suggest that this IL-13 production is dependent on IL-23 and IL-25, but not on IL-12p70. We then show that IL-13 production results in the induction of an IL-13R formerly thought to function only as a decoy receptor, IL-13Ralpha(2), and this receptor is critical to the production of TGF-beta(1) and the onset of fibrosis. Thus, if IL-13 signaling through this receptor is blocked by administration of soluble IL-13Ralpha(2)-Fc, or by administration of IL-13Ralpha(2)-specific small interfering RNA, TGF-beta(1) is not produced and fibrosis does not occur. These studies show that in chronic 2,4,6-trinitrobenzene sulfonic acid colitis, fibrosis is dependent on the development of an IL-13 response that acts through a novel cell surface-expressed IL-13R to induce TGF-beta(1). A similar mechanism may obtain in certain forms of human inflammatory bowel disease.

Purpose: IL13-PE38, a targeted cytotoxin comprised of interleukin 13 (IL-13) and a mutated form of Pseudomonas exotoxin, induces specific killing of tumor cells expressing abundant levels of the IL-13Ralpha2 chain. We hypothesized that tumor cells killed by the cytotoxin may release antigens and/or apoptotic bodies when cells are dying, which then induce adoptive immunity, and that the PE38 portion of IL13-PE38 may act as a stimulant for the induction of a CTL response., Experimental Design: To test this hypothesis, we established D5 melanoma tumors with or without expression of the IL-13Ralpha2 chain in both flanks of C57BL/6 mice, and then IL13-PE38 was injected in the right flank tumors only., Results and Conclusions: When animals with IL-13Ralpha2-expressing D5 tumor (right) were injected with IL13-PE38, right flank tumors expressing the IL-13Ralpha2 chain not only showed dramatic regression but contralateral tumors (left flank) also showed tumor regression. Cell depletion experiments in tumor-bearing animals indicated that both CD8(+) and CD4(+) T cells contribute to the regression of contralateral tumors through CTL activation in the periphery and cellular infiltration into tumors. In addition, intratumoral treatment into s.c. tumors of mice bearing metastatic lung tumors with IL13-PE38 showed not only the reduction of treated s.c. tumor but also the reduction of lung metastasis. Thus, IL13-PE38 mediates an antitumor effect not only directly but also indirectly by inducing a host CD8(+) T cell immune response. Accordingly, targeted cytotoxins may be used to treat local disease even if they cannot be administered systemically, and yet may still induce a reasonable systemic antitumor response.