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

BACKGROUND: Current 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. METHODS: CD24+/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. RESULTS: Our 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. CONCLUSION: Our 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.

Targeting of interleukin-13 receptor α2 for treatment of head and neck squamous cell carcinoma induced by conditional deletion of TGF-ß and PTEN signaling.

BACKGROUND: The sixth leading class of cancer worldwide is head and neck cancer, which typically arise within the squamous epithelium of the oral mucosa. Human head and neck squamous cell carcinoma (HNSCC) is known to be difficult to treat and has only a 50% five-year survival rate. With HNSCC, novel therapeutics are needed along with a means of rapidly screening anti-cancer agents in vivo, such as mouse models. METHODS: In order to develop new animal models of cancer to test safety and efficacy of novel therapeutic agents for human HNSCC, tumors resembling clinical cases of human HNSCC were induced in the head and neck epithelium of a genetically engineered mouse model. This mouse model was generated by conditional deletion of two tumor suppressors, Transforming Growth Factor-ß Receptor 1 (TGFßRI) and Phosphatase and Tensin homolog (PTEN), in the oral epithelium. We discovered that the tumors derived from these Tgfbr1/Pten double conditional knockout (2cKO) mice over-expressed IL-13Rα2, a high affinity receptor for IL-13 that can function as a tumor antigen. To demonstrate a proof-of-concept that targeted therapy against IL-13Rα2 expression would have any antitumor efficacy in this spontaneous tumor model, these mice were treated systemically with IL-13-PE, a recombinant immunotoxin consisting of IL-13 fused to the Pseudomonas exotoxin A. RESULTS: Tgfbr1/Pten 2cKO mice when treated with IL-13-PE displayed significantly increased survival when compared to the untreated control mice. The untreated mice exhibited weight loss, particularly with the rapid onset of tongue tumors, but the treated mice gained weight while on IL-13-PE therapy and showed no clinical signs of toxicity due to the immunotoxin. Expression of IL-13Rα2 in tumors was significantly decreased with IL-13-PE treatment as compared to the controls and the number of myeloid-derived suppressor cells (MDSC) was also significantly reduced in the spleens of the IL-13-PE treated mice. CONCLUSIONS: Our study demonstrates that the Tgfbr1/Pten 2cKO mouse model of human HNSCC is a useful model for assessing antitumor activity of new cancer therapeutic agents, and that IL-13-PE has therapeutic potential to treat human head and neck cancer.

A novel combination immunotherapy for cancer by IL-13Rα2-targeted DNA vaccine and immunotoxin in murine tumor models.

Optimum efficacy of therapeutic cancer vaccines may require combinations that generate effective antitumor immune responses, as well as overcome immune evasion and tolerance mechanisms mediated by progressing tumor. Previous studies showed that IL-13Rα2, a unique tumor-associated Ag, is a promising target for cancer immunotherapy. A targeted cytotoxin composed of IL-13 and mutated Pseudomonas exotoxin induced specific killing of IL-13Rα2(+) tumor cells. When combined with IL-13Rα2 DNA cancer vaccine, surprisingly, it mediated synergistic antitumor effects on tumor growth and metastasis in established murine breast carcinoma and sarcoma tumor models. The mechanism of synergistic activity involved direct killing of tumor cells and cell-mediated immune responses, as well as elimination of myeloid-derived suppressor cells and, consequently, regulatory T cells. These novel results provide a strong rationale for combining immunotoxins with cancer vaccines for the treatment of patients with advanced cancer.

A Novel Recombinant Modified Vaccinia Ankara Virus expressing Interleukin-13 Receptor α2 Antigen for Potential Cancer Immunotherapy.

BACKGROUND: Genetically altered recombinant poxviruses hold great therapeutic promise in animal models of cancer. Poxviruses can induce effective cell-mediated immune responses against tumor-associated antigens. Preventive and therapeutic vaccination with a DNA vaccine expressing IL-13Rα2 can mediate partial regression of established tumors in vivo, indicating that host immune responses against IL-13Rα2 need further augmentation. OBJECTIVE: The aim of the study is developing a recombinant modified vaccinia Ankara (MVA) expressing IL-13RΑ2 (rMVA-IL13RΑ2) virus and study in vitro infectivity and efficacy against IL-13Rα2 positive cell lines. METHODS: We constructed a recombinant MVA expressing IL-13Rα2 and a green fluorescent protein (GFP) reporter gene. Purified virus titration by infection of target cells and immunostaining using anti-vaccinia and anti-IL-13Rα2 antibodies was used to confirm the identity and purity of the rMVA-IL13Rα2. RESULTS: Western Blot analysis confirmed the presence of IL-13Rα2 protein (~52 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells when infected with rMVA-IL13Rα2 virus demonstrated cell-surface expression of IL-13Rα2, indicating the infectivity of the recombinant virus. Incubation of T98G-IL13α2 cells with varying concentrations (0.1-100 ng/ml) of interleukin-13 fused to truncated Pseudomonas exotoxin (IL13-PE) resulted in depletion of GFP+ fluorescence in T98G-IL13Rα2 cells. IL13-PE (10-1000 ng/ml) at higher concentrations also inhibited the protein synthesis in T98G-IL13Rα2 cells compared to cells infected with the control pLW44-MVA virus. IL13-PE treatment of rMVA-IL13Rα2 infected chicken embryonic fibroblast and DF-1 cell line reduced virus titer compared to untreated cells. CONCLUSION: rMVA-IL13Rα2 virus can successfully infect mammalian cells to express IL-13Rα2 in a biologically active form on the surface of infected cells. To evaluate the efficacy of rMVA-IL13Rα2, immunization studies are planned in murine tumor models.

Potent antitumor effects of combination therapy with IFNs and monocytes in mouse models of established human ovarian and melanoma tumors.

Interferon-activated monocytes are known to exert cytocidal activity against tumor cells in vitro. Here, we have examined whether a combination of IFN-α2a and IFN-γ and human monocytes mediate significant antitumor effects against human ovarian and melanoma tumor xenografts in mouse models. OVCAR-3 tumors were treated i.t. with monocytes alone, IFN-α2a and IFN-γ alone or combination of all three on day 0, 15 or 30 post-tumor implantation. Mice receiving combination therapy beginning day 15 showed significantly reduced tumor growth and prolonged survival including complete regression in 40% mice. Tumor volumes measured on day 80 in mice receiving combination therapy (206 mm(3)) were significantly smaller than those of mice receiving the IFNs alone (1,041 mm(3)), monocytes alone (1,111 mm(3)) or untreated controls (1,728 mm(3)). Similarly, combination therapy with monocytes and IFNs of much larger tumor also inhibited OVCAR-3 tumor growth. Immunohistochemistry studies showed a large number of activated macrophages (CD31(+)/CD68(+)) infiltrating into OVCAR-3 tumors and higher densities of IL-12, IP10 and NOS2, markers of M1 (classical) macrophages in tumors treated with combination therapy compared to the controls. Interestingly, IFNs-activated macrophages induced apoptosis of OVCAR-3 tumor cells as monocytes alone or IFNs alone did not mediate significant apoptosis. Similar antitumor activity was observed in the LOX melanoma mouse model, but not as profound as seen with the OVCAR-3 tumors. Administration of either mixture of monocytes and IFN-α2a or monocytes and IFN-γ did not inhibit Lox melanoma growth; however, a significant inhibition was observed when tumors were treated with a mixture of monocytes, IFN-α2a and IFN-γ. These results indicate that monocytes and both IFN-α2a and IFN-γ may be required to mediate profound antitumor effect against human ovarian and melanoma tumors in mouse models.

Recent Advances in IL-13Rα2-Directed Cancer Immunotherapy.

Interleukin-13 receptor subunit alpha-2 (IL-13Rα2, CD213A), a high-affinity membrane receptor of the anti-inflammatory Th2 cytokine IL-13, is overexpressed in a variety of solid tumors and is correlated with poor prognosis in glioblastoma, colorectal cancer, adrenocortical carcinoma, pancreatic cancer, and breast cancer. While initially hypothesized as a decoy receptor for IL-13-mediated signaling, recent evidence demonstrates IL-13 can signal through IL-13Rα2 in human cells. In addition, expression of IL-13Rα2 and IL-13Rα2-mediated signaling has been shown to promote tumor proliferation, cell survival, tumor progression, invasion, and metastasis. Given its differential expression in tumor versus normal tissue, IL-13Rα2 is an attractive immunotherapy target, as both a targetable receptor and an immunogenic antigen. Multiple promising strategies, including immunotoxins, cancer vaccines, and chimeric antigen receptor (CAR) T cells, have been developed to target IL-13Rα2. In this mini-review, we discuss recent developments surrounding IL-13Rα2-targeted therapies in pre-clinical and clinical study, including potential strategies to improve IL-13Rα2-directed cancer treatment efficacy.

Interleukin-13 receptor α2 DNA prime boost vaccine induces tumor immunity in murine tumor models.

BACKGROUND: DNA vaccines represent an attractive approach for cancer treatment by inducing active T cell and B cell immune responses to tumor antigens. Previous studies have shown that interleukin-13 receptor α2 chain (IL-13Rα2), a tumor-associated antigen is a promising target for cancer immunotherapy as high levels of IL-13Rα2 are expressed on a variety of human tumors. To enhance the effectiveness of DNA vaccine, we used extracellular domain of IL-13Rα2 (ECDα2) as a protein-boost against murine tumor models. METHODS: We have developed murine models of tumors naturally expressing IL-13Rα2 (MCA304 sarcoma, 4T1 breast carcinoma) and D5 melanoma tumors transfected with human IL-13Rα2 in syngeneic mice and examined the antitumor activity of DNA vaccine expressing IL-13Rα2 gene with or without ECDα2 protein mixed with CpG and IFA adjuvants as a boost vaccine. RESULTS: Mice receiving IL-13Rα2 DNA vaccine boosted with ECDα2 protein were superior in exhibiting inhibition of tumor growth, compared to mice receiving DNA vaccine alone, in both prophylactic and therapeutic vaccine settings. In addition, prime-boost vaccination significantly prolonged the survival of mice compared to DNA vaccine alone. Furthermore, ECDα2 booster vaccination increased IFN-γ production and CTL activity against tumor expressing IL-13Rα2. The immunohistochemical analysis showed the infiltration of CD4 and CD8 positive T cells and IFN-γ-induced chemokines (CXCL9 and CXCL10) in regressing tumors of immunized mice. Finally, the prime boost strategy was able to reduce immunosuppressive CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in the spleen and tumor of vaccinated mice. CONCLUSION: These results suggest that immunization with IL-13Rα2 DNA vaccine followed by ECDα2 boost mixed with CpG and IFA adjuvants inhibits tumor growth in T cell dependent manner. Thus our results show an enhancement of efficacy of IL-13Rα2 DNA vaccine with ECDα2 protein boost and offers an exciting approach in the development of new DNA vaccine targeting IL-13Rα2 for cancer immunotherapy.

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-13Ralpha2), which is colocalized with smooth muscle actin, whereas fatty liver and normal liver specimens do not express IL-13Ralpha2. HSCs engineered to overexpress IL-13Ralpha2 respond to IL-13 and induce TGFB1 promoter activity and TGF-beta1 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-13Ralpha2 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.

IL-13 cytotoxin has potent antitumor activity and synergizes with paclitaxel in a mouse model of oral squamous cell carcinoma.

Interleukin-13 receptor-targeted cytotoxin (IL13-PE38) is highly cytotoxic to certain types of human cancers expressing abundant levels of IL-13Ralpha2 chain. Although IL13-PE38 is being tested in a Phase III clinical trial in brain tumors, the activity of IL13-PE38 alone or when combined with taxane, a chemotherapeutic drug for oral squamous cell carcinoma (OSCC), has not been investigated. Here, we show that approximately 40% of OSCCs (n = 50) in a tissue array are strongly positive for IL-13Ralpha2, whereas normal oral mucosa (n = 10) expresses very low or undetectable levels evaluated by immunohistochemistry. IL13-PE38 was highly cytotoxic to OSCC cell lines, but not cytotoxic to normal oral fibroblasts. IL13-PE38 mediated a synergistic antitumor effect with paclitaxel in OSC-19 in vitro and in vivo in the orthotopic OSCC tongue tumor model. Real-time tumor growth was monitored by optical imaging using a Xenogen-IVIS imaging system. Treated animals showed significant (p < 0.05) improvement in survival, which correlated with in vivo imaging of tumor response without evidence of visible toxicity. Gene transfer of IL-13Ralpha2 in oral cancer cells increased sensitivity of OSCC cell line to IL13-PE38 in vitro. Retrovirus-mediated gene-transfer of IL-13Ralpha2 in HSC-3 into tongue tumors in vivo dramatically enhanced the antitumor activity of IL13-PE38, providing complete elimination of established tumors and prolonging survival of these animals. These results indicate that IL13-PE38 in combination with paclitaxel acting via different mechanisms may be a potential treatment option for IL-13Ralpha2 expressing OSCC or for the treatment of non-IL-13Ralpha2 expressing OSCC combined with gene transfer of IL-13Ralpha2.

Interleukin-4 cytotoxin therapy synergizes with gemcitabine in a mouse model of pancreatic ductal adenocarcinoma.

Targeting cell surface receptors with cytotoxins or immunotoxins provides a unique opportunity for tumor therapy. Here, we show the efficacy of the combination therapy of gemcitabine with an interleukin-4 (IL-4) cytotoxin composed of IL-4 and truncated Pseudomonas exotoxin in animal models of pancreatic ductal adenocarcinoma (PDA). We have observed that 42 of 70 (60%) tumor samples from patients with PDA express moderate- to high-density surface IL-4 receptor (IL-4R), whereas normal pancreatic samples express no or low-density IL-4R. IL-4 cytotoxin was specifically and highly cytotoxic [50% protein synthesis inhibition (IC50) ranging from >0.1 to 13 ng/mL] to six of eight pancreatic cancer cell lines, whereas no cytotoxicity (IC50>1,000 ng/mL) was observed in normal human pancreatic duct epithelium cells, fibroblasts, and human umbilical vein endothelial cells (HUVEC). We also showed that IL-4 cytotoxin in combination with gemcitabine exhibited synergistic antitumor activity in vitro. To confirm synergistic antitumor activity in vivo and monitor precise real-time disease progression, we used a novel metastatic and orthotopic mouse model using green fluorescent protein-transfected cancer cells and whole-body imaging system. The combination of both agents caused complete eradication of tumors in 40% of nude mice with small established PDA tumors. In addition, combined treatment significantly prolonged the survival of nude mice bearing day 14 advanced distant metastatic PDA tumors. Similar results were observed in mice xenografted with PDA obtained from a patient undergoing surgical resection. These results indicate that IL-4 cytotoxin combined with gemcitabine may provide effective therapy for the treatment of patients with PDA.

Combination immunotherapy with IL-4 Pseudomonas exotoxin and IFN-α and IFN-γ mediate antitumor effects in vitro and in a mouse model of human ovarian cancer.

AIM: We have shown that IL-4 fused to Pseudomonas exotoxin (IL-4-PE) is cytotoxic to ovarian cancer cell lines. The antineoplastic properties of IFN-α, IFN-γ and IL-4-PE have been studied and showed some promise in the clinical trials. Here, we investigated whether the combination of IL-4-PE, IFN-α and IFN-γ will result in increased ovarian cancer cell death in vitro and in vivo. MATERIALS & METHODS: Ovarian cancer cells were tested in vitro to analyze the cytotoxic effects of IL-4-PE, IFN-α and IFN-γ, and the combination of all three. Tumor-bearing xenograft mice were treated with the combination of IL-4-PE, IFN-α and IFN-γ to monitor their overall survival. The JAK/STAT phosphorylation signaling pathways were studied to delineate the mechanism of synergistic antitumor activity. RESULTS: The combination of IL-4-PE with IFN-α and IFN-γ resulted in increased ovarian cancer cell death in vitro and in vivo. Mechanistically, the synergistic antitumor effect was dependent on interferon signaling, but not IL-4-PE signaling as determined by signaling specific chemical inhibitors. The combination therapy induced the activation of critical mediators of apoptosis. CONCLUSION: The combination of IL-4-PE with interferons increased overall survival of mice with human ovarian cancer xenograft. These data suggest that this novel combination could provide a unique approach to treating ovarian cancer.

Effects of Exoskeleton Training Intervention on Net Loading Force in Chronic Spinal Cord Injury.

The goal of this study was to understand the rehabilitative effects of longitudinal overground exoskeleton training $( >100$ hours) on gait mechanics, especially foot loading, for gains in walking speed in an individual with chronic motorincomplete SCI. Biomechanical measures included: normalized plantar loading forces, walking speed and bilateral weight transfer ratio during walking in the EksoGT $^{\mathrm{ TM}}$ exoskeleton. Longitudinal training with a robotic exoskeleton yielded improvements in clinical outcomes (AIS classification, ISNCSCI motor scores and 10MWT) and provided functional gains in terms of biomechanical outcomes (plantar forces, weight transfer point) to increase overall walking speed.

Mechanisms for improving walking speed after longitudinal powered robotic exoskeleton training for individuals with spinal cord injury.

The goal of this study was to establish strideparameter gait models correlated to speed on individuals with chronic SCI and able-bodied controls walking with a powered robotic exoskeleton (EksoGT $^{\mathrm{ TM}}$). Longitudinal exoskeleton training $( >100$ hours) across eight individuals with SCI resulted in a 30% increase in walking speed. A simple linear regression between step length, stride length for given speed were very tightly correlated along a line of best fit $( \mathrm {p}<$.001). The temporal parameters of stride time, stance time and double support time depicted a non-linear exponentially decaying relationship for given walking speed. The research findings indicate that although longitudinal exoskeleton training reduces the temporal parameters, increases in spatial parameters are only marginal.

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 (IL13-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.

The IL-4 and IL-13 pseudomonas exotoxins: new hope for brain tumor therapy.

Targeting cell surface receptors with cytotoxins or immunotoxins provides a unique opportunity for brain tumor therapy. The authors have discovered that receptors for two cytokines, interleukin (IL)-4 and IL-13, are overexpressed on tumor biopsy samples and on cell lines derived from a variety of human tumors, including brain tumors. These investigators have demonstrated that the structure of these cytokine receptors on tumor cells is different from that found on normal immune cells. In human solid tumor cells, IL-4 binds to two chains (IL-4Ra and IL-13Ra1), whereas IL- 13 binds to three chains in many solid tumor cells, including glioma cells (to IL-4Ra, IL-13Ra1, and IL-13Ra2). To target IL-4Rs and IL-13Rs, the authors generated two recombinant fusion cytotoxins composed of IL-4 or IL-13 and a mutated form of pseudomonas exotoxin (PE), which for simplicity are called IL4-PE and IL13-PE in this paper. These chimeric cytotoxins are highly toxic in vitro to human tumor cell lines and primary cell cultures, including glioma cells, and in vivo to animal models of human tumors, including gliomas. In contrast, normal cells, including immune, endothelial, and brain cells, are spared from their cytotoxic effects. Based on numerous preclinical studies, IL13-PE (also known as IL13-PE38QQR or cintredekin besudotox) has been tested in four Phase I/II clinical trials. The agent IL13-PE was administered intracranially by using convection-enhanced delivery (CED). The drug was delivered through catheters placed either directly into the tumor bed or in the peritumoral region after resection of the lesion. The CED of IL13-PE was fairly well tolerated, with a reasonable benefit/risk profile for treatment of patients with glioma. Based on Phase I/II clinical trials, the Phase III Randomized Evaluation of CED of IL13-PE Compared to Gliadel Wafer with Survival Endpoint Trial (also known as the PRECISE Trial) in patients with initial recurrence of glioblastoma multiforme has recently been completed. Patients are being monitored for safety of the agents, duration of overall survival, and quality of life.

Safety of intraparenchymal convection-enhanced delivery of cintredekin besudotox in early-phase studies.

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.

Targeting interleukin-4 receptors for effective pancreatic cancer therapy.

We demonstrate that pancreatic cancer tissues express receptors for interleukin (IL)-4 in situ at high density. Using the approach of selective receptor targeting, we have tested the efficacy of a recombinant cytotoxin IL4-Pseudomonas exotoxin A, which is composed of a targeting moiety (IL-4) and a mutated form of Pseudomonas exotoxin. Our results demonstrate that this molecule exerts vigorous antitumor activity against human pancreatic tumors implanted s.c. in immunodeficient animals. Sixty percent of animals treated with intratumoral injections of IL4-Pseudomonas exotoxin A experienced complete disappearance of established tumors. Animals with pancreatic tumors implanted orthotopically exhibited prolonged survival that was significantly greater by comparison with untreated animals. Thus, IL-4 receptor-targeted cytotoxin represents a potent agent that may provide an effective therapy for pancreatic cancer.

Effect of interleukin (IL)-4 cytotoxin on breast tumor growth after in vivo gene transfer of IL-4 receptor alpha chain.

Although human breast cancer cells express interleukin-4 receptors (IL-4Rs), a recombinant fusion protein, IL-4 cytotoxin, did not mediate desirable antitumor activity in tumor models of breast cancer. Recent studies have identified that a primary IL-4 binding protein, IL-4Ralpha chain, is internalized after binding to IL-4 in cancer cells. The consequent expression of high-level IL-4Ralpha in tumor cells sensitizes them to the cytotoxic effect of IL-4 cytotoxin in vitro. To assess whether overexpression of IL-4Ralpha chain in vivo by plasmid-mediated gene transfer can enhance antitumor activity of IL-4 cytotoxin in mouse models of breast tumor, we injected MDA-MB-231 human breast cancer cells in both flanks of athymic nude mice. Animals then received three intratumoral (i.t.) injections of either IL-4Ralpha encoding vector (left flank) or vector only (right flank) mixed with liposome followed by IL-4 cytotoxin administration. Both i.p. and i.t. administration of IL-4 cytotoxin profoundly reduced the growth of IL-4Ralpha plasmid-injected MDA-MB-231 tumors, compared with control. Innate immune cells, including macrophages and neutrophils, were found to infiltrate at the regressing tumor site. This study provides proof of principle that i.t. IL-4Ralpha plasmid injection followed by systemic or i.t. IL-4 cytotoxin administration may be a useful strategy for the treatment of breast cancer.

Interleukin 4 receptor on human lung cancer: a molecular target for cytotoxin therapy.

Previous studies have demonstrated that human lung tumor cell lines express interleukin 4 (IL-4) receptors, and IL-4 can mediate modest to moderate antiproliferative activity in vitro and in vivo in animal models of human lung tumors. On the basis of these studies, IL-4 was tested in clinical trials; however, it showed little antitumor activity in lung cancer patients. In the present study, we examined the expression of IL-4 receptors (IL-4Rs) in lung tumor samples and normal lung tissues and tested whether an IL-4R targeted agent will have better antitumor activity in vitro and in vivo compared with IL-4. IL-4R expression was tested by immunohistochemistry in 54 lung tumor samples and normal lung tissues in a tissue array, by reverse-transcription PCR and Northern blot analyses in lung tumor cell lines. Cytotoxic activity of IL-4 cytotoxin [IL-4(38-37)-PE38KDEL], composed of a circular permuted IL-4 and a mutated form of Pseudomonas exotoxin (PE38KDEL) was tested by protein synthesis inhibition and clonogenic assays in seven lung tumor cell lines. Antitumor activity of IL-4 cytotoxin was tested in vitro and in immunodeficient animal models of human lung tumors. We observed that IL-4Rs are expressed at higher levels in situ in lung tumor samples compared with normal lung tissues and IL-4 cytotoxin is highly and specifically cytotoxic to lung tumor cell lines in vitro. Intratumoral and i.p. administration of IL-4 cytotoxin to immunodeficient mice with s.c. established human lung H358 non-small cell lung cancer tumors mediated considerable antitumor activity in a dose-dependent manner with the higher dose producing durable complete responses. On the other hand, H460 non-small cell lung cancer tumors expressing low levels of IL-4R did not respond to IL-4 cytotoxin therapy. Because IL-4 cytotoxin mediates its antitumor activity through IL-4R, and a variety of lung tumors expressed high levels of IL-4R, we propose testing the safety of this agent in patients with lung cancer.

Multidrug-resistant tumor cells remain sensitive to a recombinant interleukin-4-Pseudomonas exotoxin, except when overexpressing the multidrug resistance protein MRP1.

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.