Interleukin-4 (IL-4), its mutants and soluble receptors are being tested in the clinic by systemic injection or by gene transfer (using viral or plasmid vectors) for treatment of rheumatoid arthritis, bronchial asthma, and cancer. It is expected that IL-13, its mutants, soluble receptors and gene transfer of receptor chain will also be tested in the clinic in the near future. Studies are underway to characterize the structure, function, signal transduction and targeting of receptors (R) for IL-4 and IL-13 to immune and cancer cells. 1) Reconstitution studies have demonstrated that IL-13R alpha2 chain is not shared with IL-4 receptor system while IL-13Ralpha1 chain is shared with the IL-4 receptor system and with IL-4Ralpha chain IL-13Ralpha1 forms a signaling complex for the IL-13 receptor system. In addition, new studies have demonstrated that IL-13Ralpha2 chain is internalized after binding to ligand but it does not mediate signal transduction through STAT6 pathway. 2) To determine the function, IL-13Ralpha2 chain was stably transfected in human breast cancer and pancreatic cancer cell lines and in vivo tumorigenecity experiements were performed in immunodeficient animals. Although there was no difference in growth between control vector transfected and IL-13Ralpha2 chain transfected tumor cells in vitro, surprisingly, in immunodeficient mice, tumorigenicity was profoundly inhibited in IL-13Ralpha2 chain over-expressing tumors. Because breast tumors that grew later showed loss of IL-13Ralpha2 gene expression, lack of tumorigenicity correlated positively with IL-13Ralpha2 chain expression. Inflammatory cells including neutrophils and macrophages were identified to play a role in this antitumor activity of IL-13Ralpha2 chain. Additional studies also showed the role of IL-8 produced by tumor and inflammatory cells in anti-tumor effect. These results establish a novel function of a cytokine receptor chain and further suggest that the presence of this chain on tumor cells by itself may play a key role in tumorigenicity. 3) The IL-4 and IL-13R directed targeting of a Pseudomonas exotoxin, Diphtheria toxin, or alternatively receptor directed gene transfer is also being investigated. The receptors for these two interleukins are expressed in abundance on many human tumor cell lines and offer an attractive target for toxin therapy or gene therapy. In vivo experiments in immunodeficient mice with human medulloblastoma, pancreatic cancer and head and neck tumors have demonstrated complete responses in a dose-dependent and route of administration-dependent manner in response to IL-4 toxin administration. Additional studies have shown that gene transfer of IL-4Ralpha chain further sensitizes tumors to IL-4 cytotoxin in vitro and in vivo in human breast cancer models. Similarly, IL-13-toxin has shown remarkable antitumor activity in animal models of prostate cancer and head and neck cancer particularly when tumor cells are forced to express high levels of IL-13R alpah2 chain in vitro and in vivo. 4) The mechanism of antitumor effect of IL-13 cytotoxin and cell death is also being investigated. Our initial studies have shown that IL-13 cytotoxin but not IL-13 can induce apopotosis in four different head and neck cancer cell lines 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. Flow cytometric and Western blot analysis supported the morphological changes and demonstrated that caspases were involved in apoptosis. The apoptosis-regulator, Bcl-2 was downregulated after treatment with IL-13 toxin, while Bax was upregulated. In addition, significant nitrite production was detected in cancer cell line after treatment with IL-13 toxin. Taken together, our results suggest that IL-13 toxin-induced cytotoxicity is at least partially mediated by 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 and 5)Our previous studies on IL-4R targeting resulted in a Phase I clinical trial for malignant glioma. This trial is currently ongoing at many major medical Centers in the USA and in Germany. IL-13-toxin is also in clinical trials for brain tumor therapy at many national and international clinical sites. Abstracts of these studies have been presented at various scientific meetings including Society of Neuro-Oncology and Amer Soc. of Clinical Oncology. These clinical studies will help elucidate the safety and efficacy of these and other recombinant chimeric toxins being tested in clinic under various INDs.
