Expression analysis and specific blockade of the receptor for human thymic stromal lymphopoietin (TSLP) by novel antibodies to the human TSLPRα receptor chain.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine with a pivotal role in development and maintenance of atopic diseases such as allergic asthma and atopic dermatitis. Moreover, recent studies show an involvement of TSLP in the progression of various cancers. TSLP signaling is mediated by the TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor. It consists of the IL-7 receptor alpha chain (IL-7Rα), which is shared with the IL-7 receptor, and the TSLPRα chain as a specific subunit. Blocking signal release by TSLP without affecting IL-7 function is a potentially interesting option for the treatment of atopic diseases or certain tumors. By employing the extracellular domain of human TSLPRα chain (hTSLPRα(ex)) as an antigen, we generated a set of monoclonal antibodies. Several binders to native and/or denatured receptor protein were identified and characterized by cytometry and Western blot analysis. A screen based on a STAT3-driven reporter gene assay in murine pro-B cells expressing a functional hTSLPR yielded two hybridoma clones with specific antagonistic properties towards hTSLP, but not IL-7. Kinetic studies measuring blockade of hTSLP-dependent STAT phosphorylation in a TSLP-responsive cell line revealed an inhibitory constant in the nanomolar range.

Blockade of interleukin-13-mediated cell activation by a novel inhibitory antibody to human IL-13 receptor alpha1.

Interleukin-13 (IL-13) is a cytokine with a crucial role in the development of allergic asthma. The IL-13 receptor shares the IL-4Ralpha subunit with the IL-4R system, but contains as a specific component the IL-13Ralpha1 chain. Blocking signal release by IL-13 without affecting IL-4 function is a potentially interesting therapeutical option for the treatment of asthma. Employing genetic immunization, we generated a set of novel monoclonal antibodies to the IL-13Ralpha1 receptor that proved very specific and efficient inhibitors of human IL-13 activity. Receptor binding antibodies were identified by their specific reactivity with both human monocytes and a murine pro-B cell line overexpressing human IL-13Ralpha1 by flow cytometry and cell ELISA. A luciferase reporter cell system based on STAT6-mediated promoter activation in murine Ba/F3 cells was employed to screen the antibodies for IL-13 antagonistic properties. Inhibitory antibody effects were quantified by interference with IL-13-dependent proliferation of TF-1 cells. The capability of blocking IL-13-driven responses of primary, inflammation-relevant cells was tested by Western blot analysis of STAT6 tyrosine phosphorylation and expression of 15-lipoxygenase in monocytes from fresh blood. The most potent inhibitory antibody identified, GM1E7, inhibited IL-13-driven gene activation and cell proliferation in immune cell lines with IC(50) values in the low nanomolar range. Both short-term (STAT6 activation) and long-term (15-LO induction) responses of primary human blood cells to IL-13 were almost entirely blocked, whereas IL-4 effects remained virtually unaffected. GM1E7 is superior to available agents interfering with IL-13 activity in terms of specificity and efficiency and offers potential novel therapeutic perspectives for the treatment of allergic asthma.