Conversion of interleukin-13 into a high affinity agonist by a single amino acid substitution

We created a novel mutated form of human interleukin-13 (IL-13) in which a positively charged arginine (R) at position 112 was substituted to a negati vely charged aspartic acid (D). This mutant, termed IL-13R112D, was express ed in Escherichia coli and purified to near homogeneity. IL-13R112D was fou nd to be a potent IL-13 agonist with 5-10-fold improved binding affinity to IL-13 receptors compared with wild-type IL-13 (wtIL-13). The conclusion of IL-13 agonist activity was drawn on the basis of approximately 10-fold imp roved activity over wtIL-13 in several assays: (a) inhibition of CD14 expre ssion in primary monocytes; (b) proliferation of TF-1 and B9 cell lines; an d (c) activation of STAT6 in Epstein-Barr virus-immortalized B cells, prima ry monocytes, and THP-1 monocytic cell line. Furthermore, mutant IL-13R112D neutralized the cytotoxic activity of a chimeric fusion protein composed o f wtIL-13 and a Pseudomonas exotoxin A (IL-13-PE38) approximately 10 times better than wtIL-13. Based on these results, it was concluded that IL-13R11 2D interacts with much stronger affinity than wtIL-13 on all cell types tes ted and that Arg-112 plays an important role in the interaction with its re ceptors (IL-13R). Thus, these results suggest that IL-13R112D may be a usef ul ligand for the study of IL-13 interaction with its receptors or, alterna tively, in designing specific targeted agents for IL-13R-positive malignanc ies.