IL-15:IL-15 receptor alpha superagonist complex: High-level co-expression in recombinant mammalian cells, purification and characterization

• Published in: Cytokine (Philadelphia, Pa.) Vol. 56; no. 3; pp. 804 - 810
• Main Authors: Han, Kai-ping, Zhu, Xiaoyun, Liu, Bai, Jeng, Emily, Kong, Lin, Yovandich, Jason L., Vyas, Vinay V., Marcus, Warren D., Chavaillaz, Pierre-Andre, Romero, Christian A., Rhode, Peter R., Wong, Hing C.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2011
• Subjects: Animals; Body Weight - drug effects; CD8+ T lymphocytes; Cell Separation; CHO cell expression; CHO Cells; Chromatography, Gel; Cricetinae; Cricetulus; Electrophoresis, Polyacrylamide Gel; Humans; IL-15 receptor α fusion protein; Interleukin-15 - agonists; Interleukin-15 - isolation & purification; Interleukin-15 Receptor alpha Subunit - agonists; Interleukin-15 Receptor alpha Subunit - isolation & purification; Interleukin-15 superagonist; Lymphocytes - drug effects; Lymphocytes - metabolism; Male; Mammals - metabolism; Mice; Mice, Inbred C57BL; Mutant Proteins - metabolism; NK cells; Organ Size - drug effects; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacokinetics; Recombinant Fusion Proteins - pharmacology; Recombination, Genetic - genetics; NK cells; IL-15 receptor α fusion protein; Interleukin-15 superagonist; IL-15N72D; CHO cell expression; CD8+ T lymphocytes; IL-15RαSu
• ISSN: 1043-4666; 1096-0023
• DOI: 10.1016/j.cyto.2011.09.028

► Soluble IL-15:IL-15Rα superagonist complex was produced at high levels in CHO cells. ► The highly purified superagonist complex has long serum half-life in mice. ► This complex exhibits potent immunomodulatory activities on NK and CD8+ T cells. IL-15, a promising cytokine for treating cancer and viral diseases, is presented in trans by the IL-15 receptor (IL-15R) alpha-chain to the IL-15Rβγc complex displayed on the surface of T cells and natural killer (NK) cells. We previously reported that an asparagine to aspartic acid substitution at amino acid 72 (N72D) of IL-15 provides a 4–5-fold increase in biological activity compared to the native molecule. In this report, we describe Chinese hamster ovary (CHO) cell expression of a soluble complex (IL-15 N72D:IL-15RαSu/Fc) consisting of the IL-15 N72D superagonist and a dimeric IL-15Rα sushi domain-IgG1 Fc fusion protein. A simple but readily scalable affinity and ion exchange chromatography method was developed to highly purify the complex having both IL-15 binding sites fully occupied. The immunostimulatory effects of this complex were confirmed using cell proliferation assays. Treatment of mice with a single intravenous dose of IL-15N72D:IL-15RαSu/Fc resulted in a significant increase in CD8+ T cells and NK cells that was not observed following IL-15 treatment. Pharmacokinetic analysis indicated that the complex has a 25-h half-life in mice which is considerably longer than <40-min half-life of IL-15. Thus, the enhanced activity of the IL-15N72D:IL-15RαSu/Fc complex is likely the result of the increased binding activity of IL-15N72D to IL-15Rβγc, optimized cytokine trans-presentation by the IL-15RαSu domain, the dimeric nature of the cytokine domain and its increased in vivo half-life compared to IL-15. These findings indicate that this IL-15 superagonist complex could serve as a superior immunostimulatory therapeutic agent.