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Reactivity of Interleukin 13.E13C Mutant toward Interleukin 13 Receptors Shana Kondor and Dr. Jeffrey Thompson, Biology Department, York College of PA RESEARCH DESIGN PROJECT SUMMARY Create IL13.E13C mutant through point mutation of WT IL13 Although fairly extensive and ongoing research is being conducted regarding human Interleukin-13 (hIL-13) and its various mutants, the region of uncharted territory is vast. Understanding completely how hIL-13 interacts with its receptors on both normal and malignant cells could be a key component to developing a cancer-targeting drug. Only by continuing in the efforts of understanding the functionality of the protein can any knowledge be gained towards the subject. The proposed research involves a mutant of hIL-13 not yet studied, that being hIL13.E13C. This mutation resides on a protein domain that is involved with normal cell binding but not cancer cell binding. With this in mind, mutating at this position will hopefully eliminate binding affinity towards the normal cells while retaining its ability to bind with cancer cells, therefore making it able to target cancer cells only. RESEARCH OBJECTIVES 1) Determine ability of IL13.E13C to target cancer cells 2) Determine effectiveness of IL13.E13C as cytotoxin delivery complex 3) Determine the difference, if any, between eukaryotic and prokaryotic protein expression 4) Gain insight into IL13 structure and function to be related to other applications of IL13 Express Samples 1 and 2 by plating transformed E. coli on Ampicillin agar plates Solution Structure of Human IL13 www.cytok.com/structure.php?start=80 Express WT (Sample 3) and mutant (Sample 4) with COS7 cell cultures for eukaryotic assay LITERATURE REVIEW Thompson and Debinski, 1997 • -helix A mutants both caused and failed to cause TF-1 cell proliferation • All position 13 mutants except E13R and E13D failed to cause TF-1 proliferation • Position 13 mutants that failed to cause TF-1 proliferation were most effective at blocking hIL13-CTX action on U-251 MG and SNB-19 • Interaction with the hIL13 shared receptor depends on multiple factors including amino acid size, electrostatics, and polarity Purify all samples Test TF-1 proliferation (all samples) Cytotoxicity assay (all samples) Competition assay (samples 2 + 4) EXPECTED RESULTS Figure 2 Figure 1 Cytotoxicity Blocking Assay with hIL13-CTX TF-1 Cell Proliferation IL13.E13C hIL13 (WT) IL13.E13C eukaryote 100 80 60 40 20 0 0.1 1 10 Interleukin, (ng/ml) A490, percent of control Mintz et al., 2002 • High-grade astrocytomas bind IL13 and are sensitive to IL13 cytotoxins • IL13.E13K binds to HGA IL13 site 50X better than wild type IL13 • Identified transcript for hIL13R 2 in HGA’s • HGA’s bind IL13 but not IL4 • IL13.E13K completely blocked killing of HGA’s by cytotoxin 120 120 IL13.E13C hIL13 (WT) IL13.E13C (eukaryote) 100 80 60 40 20 0 0.1 100 1 10 100 hIL13-CTX, (ng/ml) Figure 3 Competition Assay with hIL13.E13C-CTX 120 Kruse et al., 2002 • IL4 and IL13 have individual signaling processes • Substrate binding and signaling strength vary depending on the variant of IL4R protein • IL13 acts independently of IL4 in inducing pathophysiological features of asthma Wäldele et al., 2004 • IL13 stimulates human T-cell leukemia virus type 1 (HTLV-1) through up-regulation of IL13 by Tax, a viral transactivator • IL13 inhibits apoptosis and stimulates the cell cycle • Various cytokines that induce cell death are inhibited by IL13 • IL13 linked to leukemogenesis as found through Hodgkin lymphoma cell stimulation Kuznetsov and Puri, 1999 • IL13 inhibits B cell proliferation • Kinetic studies show heterogeneity in IL13 binding, possibly explaining cytotoxicity of IL13 toxin in renal cell carcinomas Aversa et al., 1993 • IL13 and IL4 work independently to induce IgG4 and IgE synthesis • IL4 receptor complex and IL13 receptor complex are distinct receptors that may have common components • B cell proliferation studies suggest that IL4 and IL13 bind the same receptor • IL13 failed to bind the IL4 receptor on mouse BaF3 cells or monkey COS-7 cells, but blocked IL4 binding on TF-1 cells Vladich et al., 2005 • Demonstrated that eukaryotic IL13 was much more active than prokaryotic IL13 • Used COS-7 cell lines as eukaryotic model to compare with E. coli prokaryotic model • COS-7 cells were transfected and assessed for IL13 concentrations by ELISA, with correction factors to detect WT versus mutant A490, percent of control INTRODUCTION Transform E. coli with WT (Sample 1) and mutant (Sample 2) by transforming E. coli with respective samples + AmpR A490, percent of control Interleukin 13 (IL-13) is a pleiotropic cytokine comprised of four -helical regions (A-D) expressed in Th1 and Th2 lymphocytes, stimulated keratinocytes, activated mast cells, and transformed B lymphocytes (Kuznetsov and Puri 1999). Helices A and C bind to the shared receptor, while B and D bind to IL13 exclusive sites on cancer cells. Previous studies have shown success in decreasing normal cell affinity while maintaining cancer cell affinity (Thompson and Debinski 1999). The objectives are to create an IL13.E13C mutant, express the protein in Escherichia coli and human monocytes, and assess binding affinity and cytotoxicity through TF-1 proliferation and a cytotoxicity blocking assay with Glioblastoma cells. By mutating at position 13 on -helix A, affinity for normal cells should decrease while maintaining affinity for cancer cells. Replacing glutamic acid with cysteine creates a free sulfhydryl group for use as a cytotoxin attachment point. This mutated form of IL13 could be used as a chemotherapy aid that will allow it to target only cancer cells, leaving normal cells unharmed. 100 80 60 40 20 0 0.1 1 10 100 hIL13.E13C-CTX, (ng/ml) • Figure 1 shows hypothetical TF-1 cell proliferation of hIL13 (WT), IL13.E13C (prokaryotic expression) and IL13.E13C (eukaryotic expression). It is suspected that both mutants of IL13 will have decreased TF-1 cell proliferation due to lack of binding on the shared IL13/4 receptor. With large enough concentrations, however, cell proliferation will occur. • Figure 2 shows a hypothetical cytotoxicity blocking assay using hIL13-CTX. When competing with WT hIL13, blocking will occur approximately 50% because the cytotoxin bound hIL13 and the WT hIL13 should have similar binding affinities. When in competition with the mutants, however, blocking should occur, because the mutants are hoped to have better binding affinity for the IL13 specific receptor. It is hypothesized that the eukaryote expressed mutant will block slightly better because it has been found that eukaryote expressed IL13 is more active than the prokaryote expression. • Figure 3 shows a cytotoxicity blocking assay using IL13.E13C-CTX. It is suspected that the eukaryote expressed protein will effectively block the prokaryote expressed protein linked with the cytotoxin, because of findings that eukaryote expression is more effective. LITERATURE CITED Aversa, G., Punnonen, J., Cocks, B., de Waal Malefyt, R., Vega, F., Zurawski, S., Zurawski, G. and deVries, J. 1993. An Interleukin 4 (IL-4) Mutant Protein Inhibits both IL-4 and IL13-induced Human Immunoglobulin G4 (IgG4) and IgE Synthesis and B Cell Proliferation: Support for a Common Component Shared by IL-4 and IL-13 Receptors. Journal of Experimental Medicine 178:2213-2218. Kruse, S., Braun, S. and Deichmann, K. 2002. Distinct signal transduction processes by IL4 and IL-13 and influences from the Q551R variant of the human IL-4 receptor alpha chain. Respiratory Research 3:24. Kuznetsov, V. and Puri, R. 1999. Kinetic Analysis of High Affinity Forms of Interleukin (IL)13 Receptors: Suppression of IL-13 Binding by IL-2 Receptor Chain. Biophysical Journal 77: 154-172. Mintz, A., Gibo, D., Slagel-Webb, B., Christensen, N. and Debinski, W. 2002. IL-13R2 is a Glioma-Restricted Receptor for Interleukin-13. Neoplasia 4:388-399. Thompson, J. and Debinski, W. 1999. Mutants of Interleukin 13 with Altered Reactivity toward Interleukin 13 Receptors. Journal of Biological Chemistry 247:29944-29950. Vladich, F., Brazille, S., Stern, D., Peck, M., Ghittoni, R. and Vercelli, D. 2005. IL-13 R130Q, a common variant associated with allergy and asthma, enhances effector mechanisms essential for human allergic inflammation. Journal of Clinical Investigation 115:747-754. Wäldele, K., Schneider, G., Ruckes, T. and Grassmann, R. 2004. Interleukin-13 Overexpression by Tax Transactivation: a Potential Autocrine Stimulus in Human T-Cell Leukemia Virus-Infected Lymphocytes. Journal of Virology 78:6081-6090.