PEGylated Polyamidoamine dendrimer conjugated with tumor homing peptide as a potential targeted delivery system for glioma.

Glioblastoma multiforme (GBM) is the most common and aggressive primary central nervous system (CNS) tumor with a short survival time. The failure of chemotherapy is ascribed to the low transport of chemotherapeutics across the Blood Brain Tumor Barrier (BBTB) and poor penetration into tumor tissue. In order to overcome the two barriers, small nanoparticles with active targeted capability are urgently needed for GBM drug delivery. In this study, we proposed PEGylated Polyamidoamine (PAMAM) dendrimer nanoparticles conjugated with glioma homing peptides (Pep-1) as potential glioma targeting delivery system (Pep-PEG-PAMAM), where PEGylated PAMAM dendrimer nanoparticle was utilized as carrier due to its small size and perfect penetration into tumor and Pep-1 was used to overcome BBTB via interleukin 13 receptor α2 (IL-13Rα2) mediated endocytosis. The preliminary availability and safety of Pep-PEG-PAMAM as a nanocarrier for glioma was evaluated. In vitro results indicated that a significantly higher amount of Pep-PEG-PAMAM was endocytosed by U87 MG cells. In vivo fluorescence imaging of U87MG tumor-bearing mice confirmed that the fluorescence intensity at glioma site of targeted group was 2.02 folds higher than that of untargeted group (**p<0.01), and glioma distribution experiment further revealed that Pep-PEG-PAMAM exhibited a significantly enhanced accumulation and improved penetration at tumor site. In conclusion, Pep-1 modified PAMAM was a promising nanocarrier for targeted delivery of brain glioma.

The association of the cytoplasmic domains of interleukin 4 receptor alpha and interleukin 13 receptor alpha 2 regulates interleukin 4 signaling.

Interleukin-4 (IL-4) and Interleukin-13 (IL-13), key cytokines in the pathogenesis of allergic inflammatory disease, mediate their effects via a receptor composed of IL-13Rα1 and IL-4Rα. A third (decoy) receptor called IL-13Rα2 regulates interleukin signaling through this receptor complex. We employed a variety of biophysical and cell-based techniques to decipher the role of this decoy receptor in mediating IL-4 signaling though the IL-4Rα-IL-13Rα1 receptor complex. Surface plasmon resonance (SPR) analysis showed that IL-13Rα2 does not bind IL-4, and does not affect binding of IL-4 to IL-4Rα. These results indicate that the extracellular domains of IL-4Rα and IL-13Rα2 are not involved in the regulation of IL-4 signaling by IL-13Rα2. We next used a two-hybrid system to show that the cytoplasmic domains of IL-4Rα and IL-13Rα2 interact, and that the secondary structure of the IL-13Rα2 intracellular domain is critical for this interaction. The cellular relevance of this interaction was next investigated. BEAS-2B bronchial epithelial cells that stably express full length IL-13Rα2, or IL-13Rα2 lacking its cytoplasmic domain, were established. Over expression of IL-13Rα2 attenuated IL-4 and IL-13 mediated STAT6 phosphorylation. IL-13Rα2 lacking its cytoplasmic domain continued to attenuate IL-13-mediated signaling, but had no effect on IL-4-mediated STAT6 signaling. Our results suggest that the physical interaction between the cytoplasmic domains of IL-13Rα2 and IL-4Rα regulates IL-4 signaling through the IL-4Rα-IL-13Rα1 receptor complex.

Equilibrium and kinetic analysis of human interleukin-13 and IL-13 receptor alpha-2 complex formation.

Interleukin 13 (IL-13) is a pleiotropic cytokine secreted by activated T cells. Both IL-13 and its polymorphic variant (IL-13-R110Q) have been shown to be associated with multiple diseases such as asthma and allergy. Two IL-13 receptors have been identified, IL-13R alpha-1 receptor (IL-13Rα1) and IL-13R alpha-2 receptor (IL-13Rα2). It has been well established that IL-13 binds to IL-13Rα1 alone with low nM affinity while binding to the IL-13Rα1/IL-4R receptor complex is significantly tighter (pM). The affinity between IL-13 and IL-13Rα2, however, remains elusive. Several values have been reported in the literature varying from 20 pM to 2.5 nM. The affinities previously reported were obtained using surface plasmon resonance (SPR) or Scatchard analysis of (125) I-IL-13 binding data. This report presents the results for the kinetics and equilibrium binding analysis studies performed using label-free kinetic exclusion assay (KEA) for the interaction of human IL-13 and IL-13Rα2. KEA equilibrium analysis showed that the affinities of IL-13Rα2 are 107 and 56 pM for IL-13 and its variant (IL-13-R110Q), respectively. KEA kinetic analysis showed that a tight and very stable complex is formed between IL-13Rα2 and IL-13, as shown by calculated dissociation rate constants slower than 5 × 10(-5) per second. Kinetic analysis also showed significant differences in the kinetic behavior of wild type (wt) versus IL-13-R110Q. IL-13-R110Q not only associates to IL-13Rα2 slower than wt human IL-13 (wt-IL-13), as previously reported, but IL-13-R110Q also dissociates slower than wt-IL-13. These results show that IL-13Rα2 is a high affinity receptor and provide a new perspective on kinetic behavior that could have significant implications in the understanding of the role of IL-13-R110Q in the disease state.

Mapping mouse IL-13 binding regions using structure modeling, molecular docking, and high-density peptide microarray analysis.

Interleukin-13 is a Th2-associated cytokine responsible for many pathological responses in allergic asthma including mucus production, inflammation, and extracellular matrix remodeling. In addition, IL-13 is required for immunity to many helminth infections. IL-13 signals via the type-II IL-4 receptor, a heterodimeric receptor of IL-13Rα1 and IL-4Rα, which is also used by IL-4. IL-13 also binds to IL-13Rα2, but with much higher affinity than the type-II IL-4 receptor. Binding of IL-13 to IL-13Rα2 has been shown to attenuate IL-13 signaling through the type-II IL-4 receptor. However, molecular determinants that dictate the specificity and affinity of mouse IL-13 for the different receptors are largely unknown. Here, we used high-density overlapping peptide arrays, structural modeling, and molecular docking methods to map IL-13 binding sequences on its receptors. Predicted binding sequences on mouse IL-13Rα1 and IL-13Rα2 were in agreement with the reported human IL-13 receptor complex structures and site-directed mutational analysis. Novel structural differences were identified between IL-13 receptors, particularly at the IL-13 binding interface. Notably, additional binding sites were observed for IL-13 on IL-13Rα2. In addition, the identification of peptide sequences that are unique to IL-13Rα1 allowed us to generate a monoclonal antibody that selectively binds IL-13Rα1. Thus, high-density peptide arrays combined with molecular docking studies provide a novel, rapid, and reliable method to map cytokine-receptor interactions that may be used to generate signaling and decoy receptor-specific antagonists.

Molecular basis for shared cytokine recognition revealed in the structure of an unusually high affinity complex between IL-13 and IL-13Ralpha2.

Interleukin-13 is a cytokine important for development of T helper cell type 2 (Th2) responses and plays a critical role in asthma and allergy. The IL-13 Receptor alpha2 (IL-13Ralpha2) is a receptor for IL-13 lacking canonical Jak/STAT signaling functions. Here we present the crystal structure along with a mutational and biophysical analysis of the IL-13/IL-13Ralpha2 complex. While retaining a similar mode of IL-13 binding to its related signaling receptor, IL-13Ralpha1, IL-13Ralpha2 uses peripheral receptor residues unused in the IL-13/IL-13Ralpha1 complex to generate a larger and more complementary interface for IL-13. This results in a four orders of magnitude increase in affinity, to the femtomolar level, compared to IL-13Ralpha1. Alanine scanning mutagenesis of the IL-13 interface reveals several common "hotspot" residues important for binding to both receptors, but also identifies a prominent IL-13Ralpha2-specific contact. These results provide a framework for development of receptor subtype-selective IL-13 antagonists and indicate a decoy function for IL-13Ralpha2.

Matrix metalloproteinase 8 contributes to solubilization of IL-13 receptor alpha2 in vivo.

BACKGROUND: IL-13 receptor alpha2 (IL-13R alpha 2) is a high-affinity receptor for IL-13, a central mediator of allergic asthma. It acts predominantly as a decoy receptor but can also contribute to IL-13 responses under certain conditions. IL-13R alpha 2 exists in soluble and membrane forms, which can both bind IL-13 and modulate its activity. Yet the proteolytic processes that contribute to the generation of soluble IL-13R alpha 2 are largely unknown. OBJECTIVE: We sought to investigate the role of matrix metalloproteinases (MMPs) in the generation of soluble IL-13R alpha 2. METHODS: Acellular cleavage assays by MMPs were performed by using glutathione-S-transferase fusion proteins of murine or human IL-13R alpha 2. IL-13R alpha 2 stable-transfected cells were used for analysis of surface expression and release of soluble IL-13R alpha 2. Wild-type and MMP-8-deficient mice were used for analysis of allergen-induced airway hyperresponsiveness and solubilization of IL-13R alpha 2. RESULTS: Among several MMPs tested, only MMP-8 cleaved IL-13R alpha 2. Treatment of transfected human or murine cells expressing high levels of surface IL-13R alpha 2 with MMP-8 resulted in release of soluble IL-13R alpha 2 into the supernatants, with a concomitant decrease in surface IL-13R alpha 2 levels. The IL-13R alpha 2 solubilized by MMP-8 retained IL-13 binding activity. In an asthma model MMP-8-deficient mice displayed increased airway hyperresponsiveness and decreased soluble IL-13R alpha 2 protein levels in bronchoalveolar lavage fluid compared with those seen in wild-type mice after house dust mite challenge. CONCLUSION: MMP-8 cleaves IL-13R alpha 2 in vitro and contributes to the solubilization of IL-13R alpha 2 in vivo.

Targeting IL-13Ralpha2-positive cancer with a novel recombinant immunotoxin composed of a single-chain antibody and mutated Pseudomonas exotoxin.

We have shown previously that high-affinity receptors for interleukin-13 (IL-13Ralpha2) are overexpressed on a variety of solid cancer cells, diseased fibroblasts, and other cells, and a chimeric fusion protein composed of human IL-13 and mutated Pseudomonas exotoxin (IL-13-PE38) is highly and specifically cytotoxic to these cells in vitro and in vivo. To improve the specificity for the target, we isolated specific antibodies against IL-13Ralpha2 from human single-chain Fv (scFv) antibody phage library and developed immunotoxin by selecting two high-affinity clones of scFv and fused to PE. The fusion chimeric gene was expressed in Escherichia coli, and highly purified IL-13R-specific immunotoxin, termed anti-IL-13Ralpha2(scFv)-PE38, was tested for its cytotoxicity. This molecule was highly cytotoxic to U251 glioma and PM-RCC renal cell carcinoma cell lines in vitro. The cytotoxic activity was neutralized by purified extracellular domain of IL-13Ralpha2 but not by IL-13, indicating that cytotoxic activity is specific. Anti-IL-13Ralpha2(scFv)-PE38 showed significant antitumor activity in immunodeficient mice with s.c. glioma tumors. Both i.p. and i.t. routes of administration showed antitumor activity in a dose-dependent manner. The maximum tolerated dose of anti-IL-13Ralpha2(scFv)-PE38 was 200 microg/kg i.p. twice daily for 5 days. These results indicate that anti-IL-13Ralpha2(scFv)-PE38 is a highly selective therapeutic agent for cancer therapy and should be further tested in animal models of human cancer.

Endogenous metalloprotease solubilizes IL-13 receptor alpha2 in airway epithelial cells.

IL-13 receptor alpha2 (IL-13Ralpha2) has been postulated to be a decoy receptor. The precise mechanisms for the generation of soluble IL-13Ralpha2 and the biological activity of the endogenous soluble form have not been reported. Hypothesizing that the soluble form of IL-13Ralpha2 is generated by proteolytic cleavage of membrane-bound receptors, we transfected human airway epithelial cells with adenoviral vectors encoding full-length IL-13Ralpha2. Eotaxin production from IL-13Ralpha2-transfected cells was suppressed, and soluble IL-13Ralpha2 in the supernatants was increased time-dependently after the transfection. The transfer of conditioned media from IL-13Ralpha2-transfected cells inhibited IL-13-induced eotaxin production and STAT6 phosphorylation in non-transfected cells. PMA enhanced the release of soluble IL-13Ralpha2, and metalloprotease inhibitors inhibited this release. These findings suggest that airway epithelial cells with upregulation of membrane-bound IL-13Ralpha2 secrete soluble IL-13Ralpha2 into its supernatant, causing the autocrine and paracrine downregulation of the IL-13/STAT6 signal. Metalloprotease(s) are responsible for the proteolytic cleavage of cell surface IL-13Ralpha2.

Expression of human IL-13 receptor alpha2 extracellular domain in Pichia pastoris.

Interleukin-13 receptor alpha2 (IL-13Ralpha2) binds IL-13 with high affinity and plays an important role in IL-13 signaling as a decoy receptor. We expressed the extracellular domain of human IL-13Ralpha2 (1-313) in methylotrophic yeast Pichia pastoris. SDS-PAGE analysis by PAS staining and Western blot analysis detected the product of the extracellular domain of human IL-13Ralpha2 as glycoprotein from P. pastoris. The yield of purified extracellular domain of human IL-13Ralpha2 was 2mg from 1L of culture. From CD analysis, the 2D structure of the purified IL-13Ralpha2 showed the typical beta-sheet. ELISA of the purified IL-13Ralpha2 detected the binding activity for human IL-13. Thus, it was found that the active extracellular domain of human IL-13Ralpha2 was expressed from P. pastoris.

Analysis of internal motions of interleukin-13 variant associated with severe bronchial asthma using (15)N NMR relaxation measurements.

The single nucleotide polymorphism interleukin-13 (IL-13) R110Q is associated with severe bronchial asthma because its lower affinity leads to the augmentation of local IL-13 concentration, resulting in an increase in the signal transduction via IL-13R. Since the mutation site does not directly bind to IL-13Ralpha2, we carried out NMR relaxation analyses of the wild-type IL-13 and IL-13-R110Q in order to examine whether the R110Q mutation affects the internal motions in IL-13 molecules. The results showed that the internal motion in the micro- to millisecond time scale on helix D, which is suggested to be important for the interaction between IL-13 and IL-13Ralpha2, is increased in IL-13-R110Q compared with that in the wild-type IL-13. It therefore appears that the difference in the internal motions on helix D between the wild-type IL-13 and IL-13-R110Q may be involved in their affinity differences with IL-13Ralpha2.