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A novel interleukin 13 (IL-13) antagonist that blocks the biological activity of human IL-13 in immune and nonimmune cells¹

Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA

²Correspondence: Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Dr., NIH Bldg. 29B, Room 2NN10, Bethesda, MD 20892, USA. E-mail: puri{at}cber.fda.gov

SPECIFIC AIMS

The aim of this study was to develop novel agonists or antagonists of human interleukin 13 (IL-13) based on the homology of IL-13 between species and the homology between the IL-4 family of lymphokines and the known 3-dimensional structure of four helical bundle cytokines. We hypothesized and tested whether arginine (R) at position 112 and glutamic acid (E) at position 13 of human IL-13 molecule might be hot residues with significant biological importance in the ligand–receptor interaction in various cell types.

PRINCIPAL FINDINGS

1. Recombinant protein isolation and purification
Recombinant wild-type IL-13 (wtIL-13), IL-13R112D in which R at position 112 was substituted to aspartic acid (D) and IL-13 DM, IL-13E13KR112D in which E at position 13 was substituted to lysine (K) and R to D at position 112 were expressed in Escherichia coli and purified from inclusion bodies. Visual inspection of bands suggested purity of all proteins to be more than 95%.

2. IL-13E13KR112D competes for the binding of radiolabeled IL-13
Binding studies were performed on the PM-RCC renal carcinoma cell line and U251 glioblastoma cell line; both express the type I IL-13 receptor (IL-13R). As expected, wtIL-13 displaced specific binding of radiolabeled IL-13 (¹²⁵I-IL-13) in both cell lines. IL-13 DM also inhibited binding of ¹²⁵I-IL-13. Binding affinity of IL-13 DM was similar to wtIL-13 (Fig. 1 ).

View larger version (27K): [in this window] [in a new window]   Figure 1. Competition for the binding of 125I-IL-13 by wtIL-13 and DM. PM-RCC and U251 cells were incubated with 125I-IL-13 with various concentrations of unlabeled wtIL-13 or DM. The cell-bound radioactivity was determined with a gamma counter. The error bars represent the SD of duplicate determination. The experiments were repeated twice with identical results.

3. IL-13E13KR112D blocks cytotoxicity mediated by IL-13PE38QQR
To support the findings that IL-13 DM binds to IL-13R at physiological temperatures, we tested whether IL-13E13KR112D can block the cytotoxicity mediated by IL-13PE38QQR. Since IL-13PE38QQR mediates cytotoxicity through binding to IL-13R, its cytotoxicity is correlated with the receptor interaction. As expected, IL-13PE38QQR mediated cytotoxicity in a concentration-dependent manner. Consistent with their ability to compete for ¹²⁵I-IL-13 binding, IL-13, IL-13R112D, or IL-13 DM blocked this cytotoxicity in a concentration-dependent manner in both cell lines studied.

4. IL-13 DM blocks proliferative activity of IL-13
We measured the proliferative responses of wtIL-13, IL-13R112D, and IL-13 DM alone or in combination with wtIL-13 and IL-13E13KR112D in a TF-1 cell line that expresses type III IL-13R. As expected, IL-13 and IL-13R112D stimulated the growth of TF-1 cells in a concentration-dependent manner. In contrast, IL-13E13KR112D showed no proliferative activity (Fig. 2A ). To determine the effect of IL-13E13KR112D on IL-13-induced proliferation of TF-1 and L1236 hematopoietic cell lines, we cultured cells in the presence of IL-13E13KR112D and wtIL-13 under various conditions. IL-13 DM significantly blocked the mitogenic activity of wtIL-13 in a concentration-dependent manner (Fig. 2B , C , D ).

View larger version (40K): [in this window] [in a new window]   Figure 2. IL-13E13KR112D inhibits wtIL-13 induced cell proliferation. A) 10,000–20,000 TF-1 cells per well were cultured in the presence of various concentrations of wtIL-13 or mutein for 52–54 h. B) TF-1 cells were cultured in the presence or absence of 1 µg/ml DM and various concentrations of wtIL-13 for 57 h. C) TF-1 cells were cultured in the presence of 1 ng/ml wtIL-13 and various concentrations of DM for 48 h. Baseline count was defined as the mean count obtained without stimulation by wtIL-13 in the same assay. D) L1236 cells were cultured in the presence of 1 ng/ml wtIL-13 and various concentrations of DM for 48 h. After pulsing with 3H-thymidine for 9 h, the cells were harvested. Experiments were repeated at least twice with similar results.

5. IL-13 DM can neutralize the effect of wtIL-13 in down-regulation of CD14 expression on monocytes
Since IL-13 has been shown to down-regulate CD14 expression on monocytes, we investigated whether IL-13 DM can nullify the down-regulating activity induced by wtIL-13. wtIL-13 suppressed CD14 expression, although modestly, on monocytes and IL-13E13KR112D neutralized the effect of wtIL-13.

6. IL-13 DM blocks signal transduction induced by wtIL-13
STAT6 activation is an early cellular response induced by IL-13 and is responsible for the biological effect mediated by IL-13. We therefore tested whether IL-13 DM can generate signaling through the STAT6 pathway. Tory Epstein-Barr virus immortalized B cells and THP-1 monocytic cells that express type II/III IL-13R and KSY-1 AIDS-associated Kaposi’s sarcoma cells that express type I IL-13R were used for this purpose. In all cell types tested, both wtIL-13 and IL-13R112D induced STAT6 activation in a concentration-dependent manner. In sharp contrast, IL-13E13KR112D did not stimulate STAT6 activation even at a high concentration. In addition, IL-13 DM blocked wtIL-13-induced STAT6 activation in THP-1 and KSY-1 cell lines in a concentration-dependent manner.

CONCLUSIONS AND SIGNIFICANCE
In this report, we describe the production and characterization of an antagonist of IL-13 that was produced by site-directed mutagenesis of two amino acids in the predicted helix A and D of IL-13 molecule. To be an effective antagonist, the agent must be able to reverse or reduce the effect of an agonist and to bind and block cell receptors that normally bind naturally occurring ligand; therefore, we determined whether IL-13 mutein IL-13E13KR112D mediated these activities. We report that IL-13 DM inhibited wtIL-13 induced proliferation of TF-1 and L1236 cells, reversed the wtIL-13-induced down-modulation of CD14 expression, and inhibited activation of STAT6 induced by wtIL-13. The binding activity of IL-13 DM to IL-13R was confirmed by¹²⁵I-IL-13 binding and neutralization of cytotoxicity mediated by IL13-PE38QQR in cancer cells. Thus, the antagonistic activities of IL-13E13KR112D were evident in cells that expressed type I and type II/III IL-13R (Fig. 3 ).

View larger version (130K): [in this window] [in a new window]   Figure 3. Schematic diagram of the experimental findings. As DM binds to IL-13 receptor with similar affinity, it effectively blocks IL-13 toxin and wtIL-13-induced effects. IL-13 DM suppresses IL-13-induced STAT-6 activation, cell proliferation, and the down-modulatory effect of wtIL-13 on CD14 expression. *Signal transduction for the suppression of CD14 expression has not been shown directly via the STAT-6 pathway. Magenta, yellow, and pale blue in each structural cartoon represent -helix, ß-sheet, and turn, respectively. GPI represents glycosylphosphatidylinositol (GPI) anchorage.

Previously we had produced a mutation in IL-13 molecule by a single amino acid substitution in helix D of IL-13 molecule, IL-13R112D. Because the position of amino acid 112 in IL-13 was predicted to be localized in the corresponding amino acid at position 124 in the IL-4 molecule and the mutation in this amino acid resulted into an IL-4 antagonist (IL-4Y124D), it was predicted that IL-13R112D would be an IL-13 antagonist. However, the reverse was observed. IL-13R112D turned out to be a powerful IL-13 agonist with 5- to 10-fold improved binding affinity to IL-13 receptors. We have also produced a mutation at position 13 of IL-13, which is predicted to be located in helix A of IL-13 molecule. In this IL-13 mutein, E at position 13 was changed to K. This mutant protein (IL-13E13K) reported a 50-fold higher binding affinity to IL-13 receptors than wtIL-13 on U251 cell lines. To further improve the binding affinity of IL-13, we decided to produce a IL-13 DM combining the features of two previously produced mutations. It was predicted that this molecule would have a much higher binding affinity to IL-13 receptor vs. wtIL-13. This molecule was expressed in E. coli and homogeneously purified material was found to bind IL-13R. However, we did not observe a dramatic increase in binding affinity. When tested in various biological assays, this IL-13 DM turned out to be an antagonist of IL-13.

The mechanisms underlying the antagonistic action of IL-13E13KR112D are not known. It is possible that a double mutation in IL-13 molecule causes inappropriate aggregation of receptor subunits resulting in defective coupling of intracellular messenger molecules. Alternatively, these mutations may have created a novel conformation of IL-13 that binds IL-13R, but alters conformation, which is incapable with signaling. These possibilities are subject of our ongoing studies.

Since IL-13 has been shown to be involved in many inflammatory diseases including bronchial asthma, allergic rhinitis, and atopic dermatitis, it is expected that IL-13E13KR112D will be able to neutralize the effect of IL-13, eventually ameliorating the disease phenotype. Finally, since IL-13 is an autocrine growth factor for Hodgkin/Reed-Sternberg tumor cells, IL-13 DM may be useful in the therapy of Hodgkin’s disease. Future studies will examine these possibilities.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0711fje ; to cite this article, use FASEB J. (April 27, 2001) 10.1096/fj.00-0711fje

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