The research in Dr. Puri's laboratory concerns the identification and characterization of over expressed or unique antigens or receptors on human tumor cells. Current projects focus on the investigation of the structure, function, and mechanisms of signal transduction through interleukin (IL)-4 and IL-13 receptors in the context of tumor biology. It has been discovered that IL-4 and IL-13 receptors are over expressed in many human solid tumor cells. To target these receptors, two targeted fusion proteins have been developed. These recombinant fusion proteins are comprised of IL-4 or IL-13 and a powerful bacterial toxin, Pseudomonas exotoxin (PE). These recombinant fusion proteins expressed and purified from E.coli have been found to be highly effective in the elimination of human cancer cells that express these receptors on their cell surface in tissue cultures. In addition, these proteins are highly effective in the regression and complete cure of established human tumors in immunodeficient animal models of human cancer. Based on these and other studies several clinical trials are ongoing where both these agents are being tested for the treatment of malignant brain tumors. Current ongoing research has focused on the development of intracranial glioma model, regulation of IL-13 receptors in tumor cells and identification of new molecular markers and targets on tumor cells by using DNA micorrary technology. Interleukins, Receptors, and Targeting in Tumor Biology and AIDS. 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) Previous studies have demonstrated that IL-13R is comprised of two major chains (IL-13Ralpha1 and IL-13R alpha2) and IL-13Ralpha1 chain forms a signaling complex with IL-4Ralpha chain. In addition, we have demonstrated that IL-13Ralpha2 chain is internalized after binding to ligand but it does not mediate signal transduction through STAT6 pathway. In a collaborative study, we have recently identified a IL13R binding protein termed IL-13RBP1, which acts as a novel inhibitor of IL13 signaling. Since IL-13 plays a major role in various conditions including allergy, pulmonary asthma, parasitic infections and cancer, this molecule may have therapeutic benefit. 2) To determine the significance of IL-13Ralpha2 chain, various transfection and in vivo growth studies were performed in various human tumor models. While IL-13Ralpha2 transfected human breast cancer and pancreatic cancer cell lines loose tumorigenicity in nude mice, other tumors (e.g., prostate) do not show this tendency. 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 of some tumors. Future studies are focused on the mechanism of differential response and on the role of alpha2 chain in the context of tumor biology. 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 various human tumor models have demonstrated remarkable responses in a dose-dependent and route of administration-dependent manner in response to IL-4 and IL-13 cytotoxin administration. Additional studies have shown that gene transfer of IL-4Ralpha chain or IL-13Ralpha2 chain further sensitizes tumors to IL-4 or IL-13 cytotoxin, respectively, in vitro and in vivo in human breast cancer, head and neck cancer, prostate cancer, and lung cancer models. 4) The mechanism of antitumor effect of IL-13 cytotoxin and cell death is also being investigated. We have demonstrated that IL-13 cyototoxin not only causes necrosis but also apoptosis of tumor cells for cytotoxicity. These phenomena are observed in vitro and in vivo. Two major pathways of apopotosis including cytochrome C release and caspase activation were activated in response to IL-13-PE. These informations are useful in developing specific approaches where apoptotic bodies from tumor cells may be used to pulse antigen presenting cells for immunotherapy of cancer. 5)Our previous studies on IL-4R targeting resulted in a Phase I clinical trial for malignant glioma. This trial was undertaken at many major medical Centers in the USA and in Germany and have now completed. Similarly, IL-13-toxin is also being tested in three different clinical trials for brain tumor at many national and international clinical sites. Abstracts of these studies have been presented at various scientific meetings including Society of Neuro-Oncology, American Association of Neurological Surgeons, 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. Applications of DNA Microarray Technology in Product development and Counter Bioterrorism. Unlike conventional biochemical and molecular approaches, DNA microarray technology represents new era in biological science that brings along a new set of interesting paradigms. This technology can enable discovery of unknown patterns and biological processes without a piori assumptions. One can assess status of thousands of genes at once which was not possible by conventional biochemical/molecular biology means. It is believed that this technology will impact on the development of new sets of biotechnology products. After establishing a CBER/NCI Interagency Agreement (IAG), a microarray laboratory was developed. This laboratory has been renovated, equipments purchased and now operational. Research programs focus on 1) development of oligonucleotide arrays for the identification of potential presence of adventitious agents in cell substrates used to manufacture biological products e.g., vaccines, cell therapy products, gene therapy products, tissues, recombinant proteins and antibodies or presence of these agents in the products themselves 2) development of oligonucleotide chips containing genes from human pathogens to be used for detecting pathogens in affected population. These chips will help identify specific gene expression changes in target tissues by specific pathogens for data base development and future identification of pathogens in infected population. Expression of different genes will help identify protein targets for development of therapeutic agents in countering bioterrorism 3)development of tests for identity of therapeutic tumor vaccines by expression profiling. 4) characterization of identity, differentiation state and quality of human embryonic stem cell and adult stem cell products. 5) determination of quality of cell substrates by gene expression profiling. To undertake above mentioned projects one needs access to good quality chips for hybridization. We have produced more than 350 oligonucleotide chips containing more than 17,000 human genes. These high quality chips have been tested for quality control and being supplied to investigators in FDA and NIH. Generation and testing of protective activity of extracellular domain of anthrax toxin receptor from anthrax in vitro and in vivo. Anthrax toxin (AT) is a tripartite toxin and has three components: edema factor (EF), lethal factor (LF) and protective antigen (PA). Recent studies have demonstrated that AT binds through its PA component to cells through its unique and widely expressed transmembrane receptor termed (ATR). After binding to ATR, PA is cleaved by furin-like proteases and carboxyl (PA63) terminal of this molecule heptamerizes with EF and/or LF. The complex is then taken up by cells by receptor-mediated endocytosis in low pH endosomal compartment. Once inside the cell, EF mediates its enzymatic activity (adenylate cyclase) and inhibits professional phagocytosic activity. LF is a zinc-dependent protease that cleaves MAP kinases and causes lysis of macrophages. In addition, it has been recently shown that the extracellular domain (ec) of ATR is cleaved which may bind and inhibit interaction of PA and thus LF and EF to ATR. In this study we propose to express and purify recombinant extracellular domain of ATR from E. coli and test its protective activity in human and murine cell lines and primary cell cultures. We have completed cloning of ECD of ATR in the expression vector and protein expression is being pursued. Neutralization of both AT and Bacillus anthracis induced cytotoxicity will be tested in vitro and in animal models. For in vivo testing, rat intoxication model (as reported by Mourez et al) will be utilized. This project incorporates FY2002 projects 1Z01BM006001-10, 1Z01BM006009-03, 1Z01BM006013-02, and 1Z01BM006014-01.
