Syed R. Husain

Interleukin-13 receptor as a unique target for anti-glioblastoma therapy

Surgery, radiotherapy and chemotherapy have minimally altered survival of glioblastoma patients. We explored a specific approach for glioblastoma therapy in which cellular interleukin‐13 (IL‐13) receptors were targeted by an IL‐13 cytotoxin. A wide array of human glioblastoma cell lines expressing the receptor for IL‐13 were effectively killed by an IL‐13 cytotoxin, a chimeric protein composed of human IL‐13 and a mutated form of Pseudomonas exotoxin (termed IL13‐PE38QQR or IL‐13 toxin). Daily (qd) intratumoral injections of IL‐13 toxin (50 and 100 μg/kg/day) for 5 consecutive days into subcutaneous human U251 glioblastoma tumors (approx. 30 mm2) in nude mice resulted in complete regression of tumors in 4/5 and 5/5 mice, respectively. Tumor regression persisted for at least 221 days postimplantation. Three alternate day injections (qod) of IL‐13 toxin (250 μg/kg/day) into other subcutaneous U87 glioblastoma tumors also produced durable complete responses (CR) in all 5 mice. Twice daily (bid) intraperitoneal injections of IL‐13 toxin at 25 or 50 μg/kg/dose for 5 days (total doses = 10) regressed U251 tumors by 45% and 58% with 1/5 and 2/5 CRs, respectively, on day 54. Intraperitoneal administration of IL‐13 toxin with an identical schedule at a dose of 50 μg/kg injected into mice bearing U87 xenografts reduced tumor burden by one‐half on day 36. Similar doses (25 or 50 μg/kg) with a daily schedule (qd × 5) by the intravenous route also suppressed growth of U251 subcutaneous tumors by 75% and 81% with 1/6 CR in either group by day 34. All mice tolerated therapy well without any visible signs of toxicity. On the basis of these studies, we have initiated a Phase I clinical trial using IL13‐PE38QQR in patients with recurrent glioblastoma. Published 2001 Wiley‐Liss, Inc.

Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside.

Central nervous system malignant neoplasias, in particular, glioblastoma multiforme (GBM) have defied all current therapeutic modalities. New therapies involving tumor targeting approach are being explored. This approach relies on the identification of unique or over-expressed cell surface receptors or antigens on tumor cells. In that regard, we have identified receptor for an immune regulatory cytokine, interleukin-13 (IL-13), which is over-expressed on human malignant glioma cell lines and primary tumor cell cultures. To target IL-13 receptors (IL-13R) for cancer therapy, we have developed a recombinant fusion protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR or IL-13 cytotoxin). The IL-13 cytotoxin was found to be highly selective and potent in killing human GBM cells in vitro while normal cells including immune cells, endothelial cells and normal brain cells were generally spared the cytotoxic effect of IL-13 cytotoxin. This is because these cells either expressed none or expressed low levels of IL-13R.Consistent with in vitro cytotoxic activity, IL-13 cytotoxin mediated remarkable anti-tumor activity to human glioma in animal xenograft models. The direct injection of IL-13 cytotoxin into subcutaneous human GBM tumors grown in nude mice produced complete and durable regression of established tumors. Intravenous and intraperitoneal administration of IL-13 cytotoxin also reduced tumor burden significantly with fewer complete responders. All animals tolerated therapy well with minimal toxicity to vital organs. Pre-clinical safety and toxicity studies were performed in mice, rats and monkeys. Systemic administration of IL-13 cytotoxin appeared to be well tolerated at high doses (up to 50ug/kg). Intrabrain parenchyma administration of IL-13 cytotoxin at doses up to 100ug/ml was very well tolerated without any evidence of gross or microscopic necrosis, whereas at 500ug/ml dose, localized necrosis was observed in normal rat brain. Based on these encouraging pre-clinical studies, three Phase I/II clinical trials in adults with malignant glioma have been initiated. The first clinical trial involves convection-enhanced delivery (CED) of IL-13 cytotoxin into recurrent malignant glioma. This route of IL-13 cytotoxin administration appears to be fairly well tolerated with no neurotoxicity. The second clinical trial involves infusion of IL-13 cytotoxin by CED following tumor resection. The initial stage of the second study assessed histologic effect of drug administered prior to resection. In third one, IL-13 cytotoxin is infused by CED followed by tumor resection. All three clinical trials are currently ongoing.

Convection-enhanced Delivery of Interleukin-13 Receptor-directed Cytotoxin for Malignant Glioma Therapy*:

The treatment of patients with malignant brain tumors, in particular glioblastoma multiforme (GBM) is very challenging because of their diffuse infiltrative nature and the cytological heterogeneity. The median survival of patients with newly diagnosed GBM is only 12–15 months, and only 8–12% of them survive for two years. Novel approaches for brain tumor therapy are needed. Recently, targeted therapies have emerged as promising modality for cancer targeting. We have discovered that high affinity plasma membrane receptor for interleukin-13 (IL-13), an immune regulatory cytokine, is over-expressed in 60–80% of malignant brain tumors. To target these IL-13R, we generated a chimeric fusion protein, composed of human IL-13 and mutated Pseudomonas exotoxin (PE), termed IL-13 cytotoxin (IL 13-PE), and tested its cytotoxicity to IL-13R-expressing GBM cells. IL-13 cytotoxin was highly potent and selective in killing IL-13R-expressing GBM cells. In contrast, normal cells including brain, immune, and endothelial cells were generally not affected by this cytotoxin due to no or low expression of IL-13R. In vivo pre-clinical studies for safety and toxicity were also performed in mice, rats, and monkeys, and IL-13 cytotoxin was found to be well tolerated by both systemic and intracerebral administrations. IL-13 cytotoxin was found to mediate remarkable efficacy in animal models of human brain tumors. Encouraged by these pre-clinical studies, four Phase 1/2 clinical trials in adult patients with recurrent malignant glioma have been completed. These clinical trials involved convection-enhanced delivery (CED) of IL-13 cytotoxin either intratumoral or intraparenchymal after resection of tumor. CED is a novel loco-regional drug delivery method for intracranial tumors that relies on a continuous pressure gradient to distribute drug into interstitial space. This route of IL-13 cytotoxin administration appears to be very well tolerated and have a good risk-benefit profile. Most recently, a randomized controlled Phase 3 clinical trial (PRECISE) with intraparenchymal IL-13 cytotoxin administration was completed and subjects are being monitored for safety and survival.

Interleukin‐13 receptor α2 chain

Epithelial ovarian cancer demonstrates high mortality due to diagnosis at an advanced stage. In the search for a biomarker for early diagnosis and a target for therapy, the issue of whether interleukin‐13 receptor (IL‐13R), shown to be expressed on a variety of human cancers, is expressed in ovarian tumor samples was explored. In addition, whether this receptor serves as a biomarker and can be targeted by IL‐13 cytotoxin was examined.

Interleukin-4 receptor-directed cytotoxin therapy of AIDS- associated Kaposi's sarcoma tumors in xenograft model

The elusive and enigmatic origin of AIDS-associated Kaposis sarcoma (AIDS-KS) makes it a complex tumor and therefore difficult to treat. Here we demonstrate that AIDS-KS cells express surface interleukin-4 (IL-4) receptors, and that IL-4 toxin (IL-4(38-37)-PE38KDEL) is specifically cytotoxic to these cells. Intratumoral, intraperitoneal and intravenous administration of IL-4 toxin in nude mice with established subcutaneous AIDS-KS tumors caused considerable anti-tumor activity in a dose-dependent manner, with highest dose producing durable complete responses. Metabolic changes, including cachexia and lymphopenia, induced by KS tumors were prevented by IL-4 toxin treatment. This report establishes IL-4(38-37)-PE38KDEL as an experimental therapeutic agent for the treatment of AIDS-KS.

Novel Role of IL-13 in Fibrosis Induced by Nonalcoholic Steatohepatitis and Its Amelioration by IL-13R-Directed Cytotoxin in a Rat Model

Nonalcoholic steatohepatitis (NASH), the most common cause of chronic liver fibrosis, progresses to cirrhosis in up to 20% of patients. We report that hepatic stellate cells (HSC) in sinusoidal lesions of liver of patients with NASH express high levels of high-affinity IL-13R (IL-13Rα2), which is colocalized with smooth muscle actin, whereas fatty liver and normal liver specimens do not express IL-13Rα2. HSCs engineered to overexpress IL-13Rα2 respond to IL-13 and induce TGFB1 promoter activity and TGF-β1 production. We also developed NASH in rats by feeding a choline-deficient l-amino acid diet. These rats developed liver fibrosis as assessed by H&E staining, Masson’s trichrome and Sirius red staining, and hydroxyproline assays. Treatment of these rats with IL-13R-directed cytotoxin caused a substantial decline in fibrosis and liver enzymes without organ toxicity. These studies demonstrate that functional IL-13Rα2 are overexpressed in activated HSCs involved in NASH and that IL-13 cytotoxin ameliorates pathological features of NASH in rat liver, indicating a novel role of this cytotoxin in potential therapy.

Role of interleukin-13 in cancer, pulmonary fibrosis, and other TH2-type diseases

Interleukin (IL)-13 plays a major role in various inflammatory diseases including cancer, asthma, and allergy. It mediates a variety of different effects on various cell types including B cells, monocytes, natural killer cells, endothelial cells, and fibroblasts. IL-13 binds to two primary receptor chains IL-13Ralpha1 and IL-13Ralpha2. The IL-13Ralpha2 but not IL-13Ralpha1 chain binds IL-13 with high affinity and is overexpressed in a variety of human cancer cells derived from glioma, squamous cell carcinoma of head and neck, and AIDS-associated Kaposis sarcoma. We have also demonstrated that IL-13Ralpha2 expression is greatly increased in lung cells when mice were challenged intranasally with bleomycin or Aspergillus fumigatus. In addition, IL-13Ralpha2 increased in surgical lung biopsies from patients with usual interstitial pneumonia, nonspecific interstitial pneumonia, and respiratory bronchiolitic interstitial pneumonia of unknown origin. Based on various studies, it is concluded that IL-13Ralpha2-expressing cells are involved in various pulmonary pathological conditions. In contrast, normal tissues such as brain, lung, endothelial cells, and head and neck tissues express IL-13Ralpha1 chain, but show only marginal expression of IL-13Ralpha2 chain. Thus, IL-13Ralpha2 chain may serve as a novel biomarker for diseased cells such as cancer or fibrosis and a target for receptor-directed therapeutic agents. To target IL-13R, a recombinant fusion protein composed of IL-13 and a derivative of Pseudomonas exotoxin (PE) has been produced. This cytotoxin termed as IL-13PE38QQR or IL-13PE38, or IL-13PE is highly and specifically cytotoxic to a variety of human tumor cell lines. In preclinical models of human glioblastoma, head and neck and AIDS-associated Kaposis cancer, IL-13PE has been found to have significant antitumor activity at a tolerated dose. Several phase I clinical trials have been completed in patients with recurrent malignant glioma. Recently a phase III clinical trial (PRECISE) in patients with recurrent malignant glioma has been completed recruiting a total of 294 patients. IL-13PE cytotoxin has also shown a significant therapeutic effect in preclinical bleomycin or A. fumigatus or Schistosoma mansoni-induced pulmonary pathology including granulomatous fibrosis in mouse models. A clinical study in these diseases has yet to be initiated.

Identification and characterization of cancer stem cells in human head and neck squamous cell carcinoma

BackgroundCurrent evidence suggests that initiation, growth, and invasion of cancer are driven by a small population of cancer stem cells (CSC). Previous studies have identified CD44+ cells as cancer stem cells in head and neck squamous cell carcinoma (HNSCC). However, CD44 is widely expressed in most cells in HNSCC tumor samples and several cell lines tested. We previously identified a small population of CD24+/CD44+ cells in HNSCC. In this study, we examined whether this population of cells may represent CSC in HNSCC.MethodsCD24+/CD44+ cells from HNSCC cell lines were sorted by flow cytometry, and their phenotype was confirmed by qRT-PCR. Their self-renewal and differentiation properties, clonogenicity in collagen gels, and response to anticancer drugs were tested in vitro. The tumorigenicity potential of CD24+/CD44+ cells was tested in athymic nude mice in vivo.ResultsOur results show that CD24+/CD44+ cells possessed stemness characteristics of self-renewal and differentiation. CD24+/CD44+ cells showed higher cell invasion in vitro and made higher number of colonies in collagen gels compared to CD24-/CD44+ HNSCC cells. In addition, the CD24+/CD44+ cells were more chemo-resistant to gemcitabine and cisplatin compared to CD24-/CD44+ cells. In vivo, CD24+/CD44+ cells showed a tendency to generate larger tumors in nude mice compared to CD24-/CD44+ cell population.ConclusionOur study clearly demonstrates that a distinct small population of CD24+/CD44+ cells is present in HNSCC that shows stem cell-like properties. This distinct small population of cells should be further characterized and may provide an opportunity to target HNSCC CSC for therapy.

Interleukin 13 Mediates Signal Transduction through Interleukin 13 Receptor α2 in Pancreatic Ductal Adenocarcinoma: Role of IL-13 Pseudomonas Exotoxin in Pancreatic Cancer Therapy

Purpose: Interleukin-13 receptor α2 (IL-13Rα2) is a tumor antigen that is overexpressed in certain human tumors. However, its significance and expression in pancreatic cancer is not known. It is also not known whether IL-13 can signal through IL-13Rα2 in cancer. Experimental Design: The expression of IL-13Rα2 was assessed in pancreatic cancer samples by immunohistochemistry and in cell lines by flow cytometry and reverse transcription-PCR. The role of IL-13Rα2 was examined by IL-13–induced signaling in pancreatic cancer cell lines. IL-13Rα2–positive tumors were targeted by IL-13PE cytotoxin in vitro and in vivo in an orthotopic murine model of human pancreatic cancer. Results: Of the pancreatic tumor samples 71% overexpressed moderate to high-density IL-13Rα2 chain compared with normal pancreatic samples. IL-13 induced transforming growth factor-β1 promoter activity in IL-13Rα2–positive tumor cells and in cells engineered to express IL-13Rα2 but not in IL-13Rα2–negative or RNA interference knockdown cells. c-Jun and c-Fos of the AP-1 family of nuclear factors were activated by IL-13 only in IL-13Rα2–positive cells. In the orthotopic mouse model, IL13-PE significantly decreased tumor growth when assessed by whole-body imaging and prolonged the mean survival time. Similar results were observed in mice xenografted with a surgically resected human pancreatic tumor sample. Conclusions: These results indicate that IL-13Rα2 is a functional receptor as IL-13 mediates signaling in human pancreatic cancer cell lines. IL-13 causes transforming growth factor-β activation via AP-1 pathway, which may cause tumor induced immunosuppression in the host. In addition, IL13-PE cytotoxin may be an effective therapeutic agent for the treatment of pancreatic cancer. Clin Cancer Res; 16(2); 577–86

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.