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Thyroid cancer is a rapidly increasing endocrine cancer. Since interleukin-4 receptor (IL-4R) is overexpressed in human solid cancer, we examined expression of IL-4R in 50 cases of anaplastic thyroid cancer (ATC), 37 well-differentiated papillary cancer (WDPC), 35 well-differentiated follicular cancer of thyroid (WDFC), and 37 normal thyroid specimens by immunohistochemistry (IHC) and in-situ hybridization (ISH) techniques. We demonstrated that IL-4Rα was overexpressed in 36/50 (72%) ATC, 20/35 (57%) WDFC, and 11/37 (30%) WDPC tumors. Other two subunits of IL-4R, interleukin-13 receptor α1 (IL-13Rα1) and interleukin-2 receptor gamma (IL-2RγC), were either weakly expressed or absent. As ATC is a highly aggressive cancer with higher incidence of IL-4Rα expression, we characterized IL-4R in 3 ATC cell lines. RT-qPCR and IFA results showed that IL-4Rα is overexpressed while IL-13Rα1 is weakly expressed. Control human umbilical vein endothelial cell line (HUVEC) showed weak expression of IL-4Rα. Binding and competition studies with 125I-IL-4 in ATC cell lines demonstrated that IL-4 specifically bound to IL-4Rα on cell surface. ATC cell lines were highly sensitive to a chimeric fusion cytotoxin consisting of circularly permuted IL-4 and truncated Pseudomonas exotoxin (IL-4-PE), which killed them in a concentration dependent manner. IL-4-PE also blocked colony formation of ATC cell lines in clonogenic assays. IL-4-PE mediated a significant antitumor activity in mouse models of ATC. Intratumoral administration of IL-4-PE caused significant regression of established tumors in a dose dependent manner and increased the overall survival without any visible toxicity. Thus, IL-4Rα in ATC may represent a novel therapeutic target and IL-4-PE may serve as an investigational therapeutic option for ATC.

Adrenocortical carcinoma (ACC) is a rare but lethal malignancy without effective current therapy for metastatic disease. IL-13-PE is a recombinant cytotoxin consisting of human interleukin-13 (IL-13) and a truncated form of Pseudomonas exotoxin A (PE). The main objectives of this Phase I dose-escalation trial were to assess the maximum-tolerated dose (MTD), safety, and pharmacokinetics (PK) of IL-13-PE in patients with metastatic ACC. Eligible patients had confirmed IL-13 receptor alpha 2 (IL-13Rα2) expressions in their tumors. IL-13-PE at dose of 1-2 μg/kg was administered intravenously (IV) on day 1, 3, and 5 in a 4-week cycle. Six patients received 1 μg/kg and two patients received 2 μg/kg of IL-13-PE. Dose-limiting toxicity was observed at 2 μg/kg, at which patients exhibited thrombocytopenia and renal insufficiency without requiring dialysis. PK analysis demonstrated that at MTD, the mean maximum serum concentration (Cmax ) of IL-13-PE was 21.0 ng/mL, and the terminal half-life of IL-13-PE was 30-39 min. Two (25%) of the eight patients had baseline neutralizing antibodies against PE. Three (75%) of the remaining four tested patients developed neutralizing antibodies against IL-13-PE within 14-28 days of initial treatment. Of the five patients treated at MTD and assessed for response, one patient had stable disease for 5.5 months before disease progression; the others progressed within 1-2 months. In conclusion, systemic IV administration of IL-13-PE is safe at 1 μg/kg. All tested patients developed high levels of neutralizing antibodies during IL-13-PE treatment. Use of strategies for immunodepletion before IL-13-PE treatment should be considered in future trials., (© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Convection-enhanced delivery (CED) of cintredekin besudotox (CB) was compared with Gliadel wafers (GW) in adult patients with glioblastoma multiforme (GBM) at first recurrence. Patients were randomized 2:1 to receive CB or GW. CB (0.5 microg/mL; total flow rate 0.75 mL/h) was administered over 96 hours via 2-4 intraparenchymal catheters placed after tumor resection. GW (3.85%/7.7 mg carmustine per wafer; maximum 8 wafers) were placed immediately after tumor resection. The primary endpoint was overall survival from the time of randomization. Prestated interim analyses were built into the study design. Secondary and tertiary endpoints were safety and health-related quality-of-life assessments. From March 2004 to December 2005, 296 patients were enrolled at 52 centers. Demographic and baseline characteristics were balanced between the 2 treatment arms. Median survival was 36.4 weeks (9.1 months) for CB and 35.3 weeks (8.8 months) for GW (P = .476). For the efficacy evaluable population, the median survival was 45.3 weeks (11.3 months) for CB and 39.8 weeks (10 months) for GW (P = .310). The adverse-events profile was similar in both arms, except that pulmonary embolism was higher in the CB arm (8% vs 1%, P = .014). This is the first randomized phase III evaluation of an agent administered via CED and the first with an active comparator in GBM patients. There was no survival difference between CB administered via CED and GW. Drug distribution was not assessed and may be crucial for evaluating future CED-based therapeutics.

Interleukin-13 (IL-13) receptor alpha2 (IL-13Ralpha2), a high-affinity IL-13 binding subunit and a tumor antigen, is amplified in a variety of human tumor cell lines and tumors in vivo. By cDNA microarray, we have shown that gene transfer of human and rat adrenomedullin (AM) up-regulates IL-13Ralpha2 in a human prostate tumor cell line. Here, we show that IL-13Ralpha2 mRNA and protein are also up-regulated in PC-3 prostate tumor cells by recombinant AM (rAM) and human synthetic AM peptide in a dose-dependent manner in vitro and in vivo in mouse prostate tumor model. The 8- to 10-fold up-regulation of IL-13Ralpha2 by rAM or AM peptide in prostate tumor cells in vitro and in vivo increased their sensitivity to IL-13PE cytotoxin consisting of IL-13 and a truncated form of Pseudomonas exotoxin. Immunodeficient mice with established prostate tumors transfected with AM or treated with AM peptide showed reduction in tumor size by intratumoral administration of IL-13PE in a dose-dependent manner. At the highest dose (three 100 mug/kg/d every alternate day), >70% reduction of tumor size was observed compared with controls (P

Object: Convection-enhanced delivery (CED) is an increasingly used novel local/regional delivery method targeted directly to tissue. It relies on a continuous pressure gradient for distribution of therapeutic agents into the interstitial space, with administration of the infusate over a few days. Cintredekin besudotox (also known as IL13- PE38QQR) is a recombinant chimeric cytotoxin consisting of interleukin-13 and a truncated exotoxin produced by the Pseudomonas aeruginosa bacterium, which targets malignant glioma cells., Methods: Cintredekin besudotox was administered via intraparenchymal CED after resection of supratentorial recurrent malignant glioma. The safety and toxicity profile was reviewed for 53 patients in whom infusion catheters had been placed; 51 of them received CED of the study drug. Adverse events were categorized based on time of onset in relation to CED, and the causal relationship with catheter placement or delivery of cintredekin besudotox. Catheters were placed in 53 patients, although only 51 of them received cintredekin besudotox. Most adverse events related to catheter placement or the study drug originated from the central nervous system. Three symptomatic windows were defined: the first one was between surgical procedure and CED; the second was during CED and up to 1 week after its completion; and the third window was 2 to 10 weeks after treatment. Those windows generally reflected adverse events related to surgical procedures, mass effect from infusate, and drug effect on tumor-infiltrated and normal brain parenchyma, respectively., Conclusions: The symptomatic windows identified in this study apply to any CED clinical trials, particularly those in which chimeric cytotoxins are used, and will help to determine the most likely underlying pathophysiological process causing symptoms. This information, in turn, will help to prevent adverse events or minimize their severity. Those events also have implications for dose escalation and outcome measures.

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.

Tumor cells may become resistant to conventional anticancer drugs through the occurrence of transmembrane transporter proteins such as P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), or members of the multidrug resistance-associated protein family (MRP1-MRP5; ABCC1-ABCC5). In this report, we studied whether tumor cells that are cytostatic drug resistant because of overexpression of one of the above mentioned proteins are sensitive to a new anticancer agent, interleukin-4 toxin (IL-4 toxin). IL-4 toxin is a fusion protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin (PE) [IL-4(38-37)-PE38KDEL]. Ninety-six-h cytotoxicity assays and 10-day clonogenic assays showed that drug-selected multidrug resistant (MDR) tumor cells that overexpress P-glycoprotein or breast cancer resistance proteins are still sensitive to IL-4 toxin. Also, tumor cells transfected with cDNA for MRP2-5 showed no resistance, or marginal resistance, only to the toxin as compared with the parent cells. In contrast, MRP1-overexpressing cells, both drug selected and MRP1 transfected, are clearly resistant to IL-4 toxin with resistance factors of 4.3 to 8.4. MRP1-overexpressing cells were not resistant to PE itself. IL-4 toxin resistance in MRP1-overexpressing cells could be reversed by the MRP1 inhibitors probenecid or MK571 and were not affected by glutathione depletion by DL-buthionine-S,R-sulfoximine. In a transport assay using plasma membrane vesicles prepared from MRP1-overexpressing cells, IL-4 toxin and IL-4, but not PE, inhibited the translocation of the known MRP1 substrate 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). These data suggest that MRP1-overexpressing cells are resistant to IL-4 toxin because of extrusion of this agent by MRP1. Still, the results of this study demonstrate that IL-4 toxin effectively kills most MDR tumor cells and, therefore, represents a promising anticancer drug.

Squamous cell carcinoma of the head and neck (SCCHN) is characterized by a high proliferation index and marked propensity for local invasion resulting in poor prognosis for these patients. To develop tumor-targeted novel therapeutic agents, here we demonstrate that SCCHN cell lines express receptors for an immune regulatory cytokine, interleukin (IL) 13. By reverse transcription-PCR (RT-PCR), we found that 16 SCCHN cell lines express equally strong RT-PCR positive bands for mRNA of IL-13Ralpha1 and IL-4Ralpha chains. However, only three cell lines, HN12, YCUM911, and KCCT873, expressed a strong band for transcripts for IL-13Ralpha2 chain and five cell lines, YCUL891, KCCTC871, KCCL871, KCCTCM901, and RPMI 2650 expressed faint bands. Transcripts for IL-2Rgamma(c) chain were absent in all of the cell lines tested. Indirect immunofluorescence analysis for four different receptor chains confirmed RT-PCR results and showed pronounced expression of IL-13Ralpha2 protein in three high IL-13R expressing cell lines. All of the cell lines were equally positive for IL-13Ralpha1 and IL-4Ralpha chains. Receptor-binding studies demonstrated that IL-13Ralpha2-positive cell lines expressed a high density of IL-13 receptors. Using two chimeric proteins composed of IL-13 and mutated forms of Pseudomonas exotoxin (IL-13-PE38 or IL-13-PE38QQR), we found that these two fusion toxins were highly and equally cytotoxic to IL-13Ralpha2-positive SCCHN, whereas IL-13Ralpha2-negative cell lines showed low or no sensitivity to IL-13 toxins. To additionally substantiate the critical role of the IL-13Ralpha2 chain in IL-13R-mediated cytotoxicity, two head and neck tumor cell lines (YCUMS861 and KB), devoid of the transcripts of this chain, were transfected with IL-13Ralpha2 cDNA and then tested for cytotoxicity. Transient transfection of the IL-13Ralpha2 chain highly sensitized these cells to IL-13 toxin as compared with mock-transfected control cells. Thus, our results indicate that IL-13Ralpha2 is present in 50% SCCHN tumor cell lines; of these, 19% are high expresser for this chain and respond to IL-13 cytotoxin. Thus, IL-13 cytotoxin may be a useful agent for high IL-13R-expressing SCCHN.