Waldemar Debinski

Expression of a Restrictive Receptor for Interleukin 13 is Associated with Glial Transformation

We have previously documented that the vast majority of high-grade gliomas over-express binding sites for interleukin 13 (IL13) in situ. We now extend this analysis to evaluate the distribution of the binding of IL13 among other brain tumors. Tumor specimens from patients with low-grade gliomas, oligodendrogliomas, ependymomas, pilocytic astrocytomas, gliosarcomas, medulloblastomas, meningiomas, and metastases to the brain were analyzed and compared to a new series of gioblastoma multiforme (GBM) samples. Serial tumor tissue sections were incubated with 125I-labeled (i) IL13, (ii) antibody against transferrin (Tf) receptor, and (iii) epidermal growth factor (EGF). Most (17/18) GBMs stained specifically for IL13 binding sites while sections from 3/11 low-grade gliomas, 5/5 high-grade gliomas (grade III), 3/5 oligodendrogliomas (all three were anaplastic), and 1/2 gliosarcomas also showed specific binding for IL13. We did not detect IL13 binding sites in medulloblastomas (0/4) and found them only in 2/20 meningiomas. Metastases to the brain (4/12, i.e., lung adenocarcinomas and renal cell carcinoma) showed some binding of 125I-IL13. The presence of receptors for Tf was ubiquitous among all studied tumors while EGF receptor expression was much more variable. Since it appears that primarily the least differentiated forms of gliomas possess IL13 binding sites in abundance, it is plausible that IL13 receptor expressed in low-grade gliomas might be a prognostically significant marker associated with their progression to high-grade gliomas. Finally, we demonstrate that the glioma-associated IL13 receptor is truly more restrictive in nature also due to its selective representation among brain tumors of glial origin.

Mutants of Interleukin 13 with Altered Reactivity toward Interleukin 13 Receptors

Interleukin 13 (IL13) belongs to a family of cytokines whose members exhibit structural homology, despite amino acid sequence dissimilarity. For example, while of limited sequence homology, IL13 and IL4 share a signaling receptor, IL13/4 receptor, on a variety of human normal cells. However, a subclass of IL4-independent IL13 receptors is overexpressed on certain transformed cells, including human malignant gliomas. We introduced mutations into human (h) IL13 to determine the site(s) involved in interaction with the shared receptor and/or the glioma-associated receptor. This analysis identified at least three protein regions that are needed for signaling through the shared receptor. These regions were localized to α-helices A, C, and D and were mainly separate from the region(s) needed to interact with the glioma-associated receptor. Glutamic acids at positions 13 and 16 in hIL13 α-helix A, arginine and serine at positions 66 and 69 in helix C, and arginine at position 109 in helix D were found to be important in inducing biological signaling since their specific mutation resulted in loss and/or gain of function phenomena. We demonstrate that the molecular requirements of hIL13 to interact with its respective receptors are generally distinct and can be controlled by mutagenesis of the cytokine.

Molecular targeting with recombinant cytotoxins of interleukin-13 receptor α2-expressing glioma

SummaryA restricted receptor for interleukin 13 (IL-13Rα2) is over-expressed in high-grade astrocytoma (HGA), but not in normal organs. In order to design and examine new anti-HGA therapies, which are molecularly directed against IL-13Rα2, we established an IL-13Rα2-expressing syngeneic immunocompetent murine model of HGA. The model was obtained by transfecting G-26 murine glioma cells with IL-13Rα2. G-26-IL-13Rα2(+) cells, but not mock-transfected cells, became susceptible to IL-13 mutant-based cytotoxic proteins that kill human HGA cells. G-26-IL-13Rα2(+) cells maintained their tumorigenicity in immunocompetent C57BL/J6 mice and preserved their expression of IL-13Rα2in vivo. These characteristics of the G-26-IL-13Rα2(+) tumors allowed us to test molecularly defined anti-glioma passive immunotherapy. A targeted recombinant chimera cytotoxin composed of multiply mutated IL-13 (IL-13.E13Y/R66D/S69D) and a derivative ofPseudomonas exotoxin (PE), PE1E, IL-13.E13Y/R66D/S69D-PE1E, was used in anti-tumor experiments. G-26-IL-13Rα2(+) cells were killed by IL-13.E13Y/R66D/S69D-PE1E in an IL-4-independent fashion. To test the cytotoxinin vivo, G-26-IL-13Rα2(+) tumors were established in C57BL/J6 mice and when the tumors reached a size of at least 50 mm3, the mice were treated with IL-13.E13Y/R66D/S69D-PE1E. In the mice treated with the targeted fusion cytotoxin, the tumors regressed and 80% of the animals were cured. This study documents the establishment of an IL-13Rα2-positive model of HGA in immunocompetent rodents. Furthermore, the effectiveness and safety of the targeted IL-13-based cytotoxin against IL-13Rα2-expressing tumors in a more clinically relevantin vivo HGA model is promising with regard to the future clinical utility of the cytotoxin.

Local Treatment of Brain Tumors with Targeted Chimera Cytotoxic Proteins

High-grade astrocytomas (HGA) are the most prevalent brain tumors that represent a unique pharmaceutical challenge. Their cerebral localization and characteristic features of tumor progression primarily dictate this challenge. Targeted chimera conjugate/fusion cytotoxic proteins have become the newest class of investigative drug candidates for the treatment of HGA due to their inherent properties that are compatible with drug deliveries to brain tumors. A conjugate of a human transferrin with bacterial toxin, Diphtheria toxin, has shown clinical efficacy in Phase I and II trials when administered intratumorally through convection-enhanced delivery. This immunotoxin belongs to the first group of immunotoxins that started to live up to early expectations. Other anti-brain tumor cytotoxins have entered or will enter the clinic shortly. The clinical applicability of targeted bacterial toxin-containing cytotoxins in the treatment of brain tumors warrants further development and careful clinical evaluation.

Novel way to increase targeting specificity to a human glioblastoma‐associated receptor for interleukin 13

Human brain cancers (gliomas) overexpress large numbers of a receptor for interleukin 13 (IL13), making this receptor an attractive target for anti‐glioma therapies. We have recently proposed that the glioma‐associated IL13 receptor is different from the one expressed on some hemopoietic and somatic cells. In an attempt to identify an even more glioma‐specific target, we have used an antagonist of a related cytokine, IL4, which neutralizes the physiological effects of both IL13 and IL4 on normal cells. Here we demonstrate that the IL4 antagonist also counteracts the action of cytotoxins targeted to the IL13 receptor on normal human cells. Importantly, the IL4 antagonist does not inhibit IL13‐based cytotoxins on glioma cells at all. Thus, the IL13 receptor on glioma cells can be categorized as tumor‐specific in the presence of an IL4 antagonist. We conclude that IL13 receptor‐directed cytotoxins can be delivered to glioma cells without being cytotoxic to normal cells. Int. J. Cancer 76:547–551, 1998.© 1998 Wiley‐Liss, Inc.

Molecular targeting of malignant gliomas with novel multiply-mutated interleukin 13-based cytotoxins

A vast majority of high-grade gliomas over-express a receptor for interleukin 13 (IL13). This glioma-associated receptor for IL13 is interleukin 4 (IL4)-independent. This is in contrast to the physiological and IL4-shared receptor for the IL13, IL13/4 receptor, which is found on many normal organs. IL13-based Pseudomonas exotoxin (PE)-containing cytotoxic fusion proteins have been shown to be very potent anti-glioma agents. However, native IL13-based cytotoxins interact with both forms of the IL13 receptor. Therefore, mutations in IL13 were made in order to diminish/eliminate IL13s interaction with the shared IL13/4 receptor of normal tissue. These mutations encompassed amino acids located on alpha-helix A and C of IL13. We have engineered double or triple mutants of IL13 linked to various forms of PE. We found that these mutations could be successfully incorporated into IL13 without the loss of the proteins ability to selectively deliver the toxin to glioma cells while reducing their toxicity.

Epigenetics in High‐Grade Astrocytomas

Abstract: Human high‐grade astrocytomas (HGA) are the most prevalent incurable brain tumors. We found that the vast majority of HGA patients overexpress a restricted receptor for an immune regulatory cytokine, interleukin 13 (IL‐13). Interestingly, the HGA‐associated restricted receptor protein IL‐13Rα2 is expressed in the testes, and its gene is localized to chromosome X. These mirror the expression pattern and genomic localization of cancer/testes tumor antigens (CTA). Hypothetical considerations and now experimental evidence are beginning to point towards epigenetics, and DNA methylation alterations in particular, as being responsible for the appearance in cancer of CTA, including IL‐13Rα2. In line with our interest in the X chromosome and oncogenesis, we have identified a new ubiquitous angiogenic factor in HGA, a vascular endothelial growth factor‐D (VEGF‐D). We have also demonstrated that the activating protein‐1 (AP‐1) family of transcription factors play a potentially critical role in the progression of gliomas by eliciting uncontrolled upregulation of VEGF‐D and other compounds essential for cancer cell proliferation, tumorigenesis, and infiltration. The possibility exists that an unopposed constitutive increase in AP‐1 activity in HGA is related to epigenetic silencing of the inhibitors of AP‐1 activity. These phenomena offer potential targets for exploitation in either prevention or early detection of brain tumors. For example, anticancer vaccines against shared CTA could help in prevention of HGA development. Furthermore, drugs with anti‐AP‐1 activity could be effective in preventing formation/progression of HGA, or progression from less malignant lower grade gliomas to HGA. Also, circulating antibodies against CTA and factors that are AP‐1 regulated may provide a useful tool in early detection of brain tumors or for monitoring their progression following initial treatment.

Opinion and Evidence in Cancer

AbstractOncology is a vast and rapidly evolving area, with many novel approaches to treatment fast becoming a reality in the clinic. Of note, are the exciting results shown recently using targeted molecular therapies. To help you keep up-to-date with the latest advances worldwide on all aspects of drug therapy and management of cancer, this section of the journal brings you information selected from the drug therapy reporting service Inpharma Weekly1. Each issue contains easy-to-read summaries of the most important research and development news, clinical studies, treatment guidelines, pharmacoeconomic and adverse reaction news, and expert opinion pieces published in the world’s top oncology publications.