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 the treatment of rheumatoid arthritis, bronchial asthma, and cancer. It is expected that IL-13, its mutants and soluble receptors 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) Previous reconstitution studies have demonstrated that IL-13R alpha1 chain, but not alpha2 chain, is a novel component of the IL-4R system. Thus, IL-4R and IL-13R share two chains (IL4R alpha and IL-13R alpha1) with each other that may help explain similar biological activities of IL-4 and IL-13 on many different cell types. 2) To determine the subunit structure of IL-13 receptor RT-PCR and immunofluorescence studies were performed in primary brain tumor cells. These studies established that IL-13R alpha2 chain is over expressed on glioma cells, which may serve as a marker, or a target for receptor directed cancer therapy. Later studies demonstrated that IL-13R alpha2 chain is internalized after binding to the ligand and that a dileucine motif in the transmembrane domain of IL-13R alpha2 chain is responsible for this internalization. 3) To create a mutant of IL-13 that is cancer IL-13R selective, we created a novel IL-13 mutant by site directed mutagenesis. This molecule termed IL-13R112D in which arginine (R) at position 112 was substituted to aspartic acid (D). This mutant was a 10x better agonist than wild type (wt) IL-13. We then generated another mutation in the alpha helix A of the IL-13 molecule. In this molecule E was changed to K at aa 13. This molecule turned out to be a powerful antagonist. IL-13E13K bound to IL-13R with 4 fold higher affinity than wtIL-13 and it neutralized the biological activity of wtIL-13 in EBV B cells, monocytic cell line and cancer cells. In addition, IL13E13K inhibited IL-13 induced STAT6 activation in cancer cells. Furthermore, IL-13E13K neutralized the cytotoxic activity of a fusion protein composed of wtIL-13 and a mutated form of Pseudomonas exotoxin (IL-13-PE38QQR). Based on these results, we concluded that IL-13E13K interacts with much stronger affinity than wtIL-13 and that Glu13 plays an important role in the interaction with its receptors. IL13E13K may be a useful agent for conditions where neutralization of IL-13 would be desirable e.g., inflammatory diseases such as asthma, allergic diseases and perhaps in cancer in which IL-13 acts as an autocrine growth factor. 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 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. 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. 4) 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 trial for brain tumor therapy at many clinical sites. Abstracts of these studies will be presented at the Congress of Neurological Surgeons in San Diego and Society of Neuro-Oncology in Washington, DC this fall. These clinical studies will help elucidate the safety and efficacy of these and other recombinant chimeric toxins being tested in clinic under various INDs.
