Koji Kawakami

IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis.

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-β1 in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Rα2. Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-α. Second, it involves IL-13 signaling through IL-13Rα2 to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Rα2 expression reduced production of TGF-β1 in oxazolone-induced colitis and that prevention of IL-13Rα2 expression, Il13ra2 gene silencing or blockade of IL-13Rα2 signaling led to marked downregulation of TGF-β1 production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Rα2 signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-β1–mediated fibrosis.

Interleukin-4-Pseudomonas exotoxin chimeric fusion protein for malignant glioma therapy

Human malignant glioma cell lines, primary cell cultures, and tumor specimens derived from surgical samples have been shown to overexpress high-affinity receptors (R) for interleukin-4 (IL-4) in vitro and in situ. The significance of IL-4R expression on malignant glioma cells is still unclear. However, IL-4 has been reported to mediate functional effects in several solid tumor cell lines. These activities include inhibition of cell proliferation, regulation of adhesion molecules, and induction of signal transduction through the JAK/STAT pathway. To target IL-4Rs on tumor cells, we have produced a chimeric recombinant fusion protein consisting of a binding ligand, circularly permuted IL-4 and a mutated form of Pseudomonas exotoxin. This molecule is termed IL4(38–37)-PE38KDEL, cpIL4-PE, or IL-4 cytotoxin. Recombinant cpIL4-PE is highly and specifically cytotoxic to glioma cell lines in vitro, while it is not cytotoxic or less cytotoxic to hematopoietic and normal brain cells. In a nude mouse model, cpIL4-PE showed significant antitumor activity and partial or complete regression of small or large established human glioblastoma tumors. Encouraging preclinical efficacy, safety, and tolerability studies lead to testing of this agent in patients with recurrent glioblastoma. Based on these pilot studies, an extended Phase I/II clinical trial is currently ongoing to determine safety, tolerability, and efficacy of cpIL4-PE when injected stereotactically directly into the recurrent glioma by convection enhanced delivery. Preliminary clinical results suggest that cpIL4-PE can cause pronounced necrosis of recurrent glioma tumors without systemic toxicity. The central nervous system toxicities observed were attributed to the volume of infusion and/or nonspecific toxicity. Ongoing clinical trials will reveal antitumor activities of IL-4 cytotoxin in recurrent malignant glioma.

Analysis of interleukin-13 receptor α2 expression in human pediatric brain tumors†‡§

Compared with normal brain tissue cells, human malignant glioma cells express higher levels of interleukin‐13 receptor (IL‐13R). However, whether this receptor is expressed in situ has not been carefully examined. With IL‐13R–targeted cytotoxin (IL13‐PE38QQR, comprising IL‐13 and a mutated form of Pseudomonas exotoxin [PE]) being tested in three Phase I/II clinical trials for the treatment of adult human glioma, and with pediatric studies being planned, the authors set out to analyze pediatric brain tumor tissue specimens for the expression of IL‐13R.

Identification of Distinct Roles for a Dileucine and a Tyrosine Internalization Motif in the Interleukin (IL)-13 Binding Component IL-13 Receptor α2 Chain

Interleukin (IL)-13 receptor α2 (IL-13Rα2) chain is an essential binding component for IL-13-mediated ligand binding. Recently, we have demonstrated that this receptor chain also plays an important role in the internalization of IL-13. To study the mechanism of IL-13 internalization, we generated mutated IL-13Rα2 chains that targeted trileucine residues (Leu335, Leu336, and Leu337) in the transmembrane domain and a tyrosine motif (Tyr343) in the intracellular domain and transfected these cDNAs in COS-7 cells. Cells that expressed a C-terminally truncated IL-13Rα2 chain (Δ335) did not bind IL-13, suggesting that the trileucine region modulates IL-13 binding. Truncation of IL-13Rα2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13Rα2 chain transfected cells. COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13Rα2 chain transfected cells; however, dissociation of cell surface IL-13 was faster compared with wild type IL-13Rα2 transfectants. These results were further confirmed by determining the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonasexotoxin (IL13-PE38QQR) to cells that expressed IL-13Rα2 chain mutants. We further demonstrate that the IL-13Rα2 chain is not ubiquitinated and that internalization of IL-13Rα2 did not depend on ubiquitination. Together, our findings suggest that the dileucine motif in the trileucine region and tyrosine motif participate in IL-13Rα2 internalization in distinct manners.

Apoptotic pathways of cell death induced by an interleukin-13 receptor-targeted recombinant cytotoxin in head and neck cancer cells

Abstract. Interleukin 13 receptor (IL-13R)-targeted cytotoxin, IL13-PE38QQR, composed of IL-13 and a mutated form of Pseudomonas exotoxin (PE), is found to be highly and specifically cytotoxic to human solid cancer cell lines. However, the mechanism of tumor cell death mediated by IL-13 toxin is still not known. To elucidate the mechanism, we utilized four head and neck cancer cell lines (SCC-25, HN12, KCCT873, and YCUM911), which express high levels of IL-13R, and IL-13 toxin is highly cytotoxic to these cells. We observed chromatin condensation and DNA fragmentation, indicating apoptotic cell death, after treatment with IL-13 toxin, as determined by bis-benzimide staining and DNA ladder assays. However, IL-13 did not induce cell death. Flow cytometric analysis suggested that these cancer cell lines increased the sub-G1/G0 phase DNA population in a dose- and time-dependent manner (ranged between 10 and 30%) after treatment with IL-13 toxin. By Western blot analysis, cleavage of caspase-3 and PARP was observed after treatment with a high concentration of IL-13 toxin, also suggesting apoptotic cell death. In addition, the results of immunofluorescence and RT-PCR assays showed that the apoptosis-regulator, Bcl-2 was downregulated after treatment with IL-13 toxin, while Bax was upregulated. Moreover, significant nitrite production was detected in the HN12 cell line after treatment with IL-13 toxin for 48–96xa0h. Taken together, our results suggest that IL-13 toxin-induced cytotoxicity is at least partially mediated by the apoptosis and nitric oxide pathways. This information may be useful in developing specific approaches where apoptotic bodies from tumor cells may be used to pulse antigen-presenting cells for immunotherapy of cancer.

Improved Anti-tumor Activity and Safety of Interleukin-13 Receptor Targeted Cytotoxin by Systemic Continuous Administration in Head and Neck Cancer Xenograft Model

BackgroundIL-13 receptor (IL-13R) targeted cytotoxin, IL13-PE38QQR, has been shown to have very potent anti-tumor activity to IL-13R-expressing head and neck tumor cells in vitro and in vivo. However, its effect is limited in aggressive tumors. To further improve the anti-tumor activity and safety of IL-13 cytotoxin, we employed continuous infusion technique in animal model of head and neck cancer.Materials and MethodsWe surgically implanted continuous infusion (CI) pump intraperitoneally that released drug for 7 days, and its anti-tumor effect was evaluated. A comparison was made for antitumor activity and safety with intravenously (IV) administered IL-13 cytotoxin in a head and neck (KCCT873 and HN12) subcutaneous (SC) xenograft tumor models in nude mice. Vital organ toxicities were assessed by histologic examinations and blood serum chemistry analyses.ResultsThe 50 or 75 µg/kg/day for 7 days of IL-13 cytotoxin either by IV or CI administration did not show any difference in safety or anti-tumor activity. IV administration of 150 or 200 µg/kg/day of IL-13 cytotoxin for 7 days was lethal to nude mice, whereas 200 µg/kg/day X 7 days of CI administration was highly effective in the regression of established tumors without any toxicities. Additionally, CI administration of IL-13 cytotoxin (200 µg/kg/day) showed growth inhibition of larger HN12 tumors in nude mice.ConclusionWith a CI schedule, IL-13 cytotoxin can be systemically administrated at approximately twice the dose otherwise given by daily IV bolus administration.

IL-13 Receptor-Targeted Cytotoxin Cancer Therapy Leads to Complete Eradication of Tumors with the Aid of Phagocytic Cells in Nude Mice Model of Human Cancer

Tumor-directed therapeutic approaches require unique or overexpressed specific Ag or receptor as a target to achieve selective tumor killing. However, heterogeneous expression of these targets on tumor cells limits the efficacy of this form of therapy. In this study, we forced abundant expression of IL-13Rα2 chain by plasmid-mediated gene transfer in head and neck, as well as prostate tumors to provide a potential target. This was followed by successfully treating xenograft tumor-bearing nude mice with IL-13R-directed cytotoxin (IL13-PE38QQR). Although we did not observe an indirect cytotoxic bystander effect conveyed to nontransduced tumor cells in vitro, our approach in vivo led to a complete regression of established tumors transfected with IL-13Rα2 chain in most animals. We found that the tumor eradication was achieved in part by infiltration of macrophages and NK cells, assessed by immunohistochemistry. Moreover, head and neck tumors xenografted in macrophage-depleted nude mice were less sensitive to the antitumor effect of IL-13 cytotoxin. Because we did not observe vector-related toxicity in any vital organs, our novel combination strategy of gene transfer of IL-13Rα2 chain and receptor-directed cytotoxin therapy may be a useful approach for the treatment of localized cancer.

Tumor regression mechanisms by IL‐13 receptor–targeted cancer therapy involve apoptotic pathways

IL‐13 cytotoxin, composed of IL‐13 and a truncated form of Pseudomonas exotoxin, targets IL‐13R‐overexpressing tumor cell lines in vitro and in vivo. To reveal the molecular mechanism of IL‐13 cytotoxin–induced cell death in vivo, we demonstrate activation of apoptotic pathways in 2 s.c. growing human SCCHN tumor models in immunodeficient mice after i.t. administration of IL‐13 cytotoxin. Treatment of HN12 tumor bearing mice with i.p. or i.t. administration of IL‐13 cytotoxin mediated marked regression of established tumors with complete remission. Interestingly, after a single i.t. administration, IL‐13 cytotoxin disappeared within 6 hr but accumulation of caspase‐3, ‐8 and –9 and cleavage of procaspase‐3 and PARP continued within the tumors for a prolonged period. We further demonstrate that IL‐13 cytotoxin also utilizes an alternate pathway of cell death via the release of cytochrome c from mitochondria to the cytosol. Our results indicate that IL‐13 cytotoxin induces 2 major pathways of apoptosis, which may play a role in tumor regression. In addition, apoptotic molecules may serve as surrogate molecular markers of tumor response to IL‐13R‐directed cytotoxin therapy.

Adenoviral vector–mediated gene transfer of IL-13Rα2 chain followed by IL-13 cytotoxin treatment offers potent targeted therapy for cytotoxin-resistant cancers

Previous studies demonstrated that IL‐13Rα2 chain–overexpressing cancer cells were highly sensitive to IL‐13 cytotoxin (IL13‐PE38QQR) and could be targeted by cytotoxin treatment. However, the majority of human tumors do not express high levels of IL‐13Rα2 chain. To expand the IL‐13 cytotoxin–mediated cancer targeting therapy, we combined cytotoxin treatment with gene transfer of IL‐13Rα2 chain. We constructed a recombinant adenoviral vector carrying the human IL‐13Rα2 gene (Ad‐IL‐13Rα2), which expresses high levels of IL‐13Rα2 chain on infected cells. Human cancer cell lines A549 and HOS, which originally show no IL‐13Rα2 expression and little sensitivity to IL‐13 cytotoxin, were effectively converted to become sensitive to this cytotoxin after Ad‐IL‐13Rα2 infection. The CC50 of IL‐13 cytotoxin for Ad‐IL‐13Rα2‐infected A549 cells was <10 ng/ml, whereas the CC50 for uninfected or control vector‐infected cells was >500 ng/ml. We also examined the antitumor activity of IL‐13 cytotoxin in an established xenograft model of cytotoxin‐resistant human lung tumor. Only a single i.t. injection of Ad‐IL‐13Rα2 markedly enhanced the sensitivity of established tumors to IL‐13 cytotoxin treatment; furthermore, this antitumor effect was significantly sustained for more than 1 month after the last treatment with IL‐13 cytotoxin. Taken together, these results suggest the combination of adenoviral vector–mediated IL‐13Rα2 gene transfer and IL‐13 cytotoxin administration can be an effective targeting approach for several types of IL‐13 cytotoxin–resistant cancers which show no or little expression of IL‐13Rα2 chain.

MIP-T3 associates with IL-13Rα1 and suppresses STAT6 activation in response to IL-13 stimulation

To unravel the mechanism of interleukin‐13 (IL‐13)‐specific functions, we sought to identify IL‐13 receptor (IL‐13R) binding molecules. A novel human IL‐13Rα1 binding protein (IL13RBP1) has been identified using yeast tri‐hybrid system, which was found to encode the same protein as MIP‐T3 (microtubule interacting protein that associates with tumor necrosis factor (TNF) receptor associating factor‐3 (TRAF3)). It constitutively associates with IL‐13Rα1 and suppresses IL‐4/13‐induced signal transducer and activator of transcription‐6 (STAT6) phosphorylation. IL‐13‐induced STAT6 activation was also inhibited as determined by dual luciferase assay and electrophoretic mobility shift assay (EMSA). These results suggest that MIP‐T3 is a novel inhibitor of IL‐13 signaling and may be a useful molecule in ameliorating various conditions in which IL‐13 plays a central role.