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

Abstract

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-15N72D: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-hour half-life in mice which is considerably longer than <40-minute 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.

The tumor immunosuppressive microenvironment impairs the therapy of anti-HER2/neu antibody

“…1A and 1B, the percentage of DX5 + NK cells and CD3 + CD8 + T cells increased 2-3 folds in the spleens of IL-15R/IL-15 plasmid-injected mice compared with that of control mice three days after treatment. As reported (Han et al, 2011), IL-15R/IL-15 stimulated the expansion of the memory phenotype CD44 + CD8 + T cells (Fig. 1C).…”

“…It is reported that the complex of IL-15Rα and IL-15 is a strong stimulator for the responses of NK cells and CD8 + T cells (Dubois et al, 2008;Epardaud et al, 2008;Han et al, 2011). We prepared IL-15R/IL-15 fusion protein by fusing IL-15 receptor α sushi domain with IL-15 (Mortier et al, 2006).…”

A Novel Fusion of ALT-803 (Interleukin (IL)-15 Superagonist) with an Antibody Demonstrates Antigen-specific Antitumor Responses

“…ALT-803 exhibits superior activity in vitro and in vivo. The N72D mutation increases the biological activity of IL-15 ϳ5-fold, and the IL-15N72D⅐IL-15R␣SuFc complex further enhances the activity of IL-15 ϳ25-fold compared with native IL-15 in vivo (3,4). Also, ALT-803 has greater binding activity with the IL-2R␤␥ C complex displayed on the surface of immune cells, a substantially longer serum half-life, and better biodistribution and retention in lymphoid tissues than native IL-15 (3,4,12).…”

“…The N72D mutation increases the biological activity of IL-15 ϳ5-fold, and the IL-15N72D⅐IL-15R␣SuFc complex further enhances the activity of IL-15 ϳ25-fold compared with native IL-15 in vivo (3,4). Also, ALT-803 has greater binding activity with the IL-2R␤␥ C complex displayed on the surface of immune cells, a substantially longer serum half-life, and better biodistribution and retention in lymphoid tissues than native IL-15 (3,4,12). Exhibiting potent immunostimulatory and antitumor properties, ALT-803 is an effective agent against various tumors in animal models either as a single agent or in combination with other therapies (5,6).…”

“…We identified a novel IL-15 variant carrying an asparagineto-aspartic acid mutation at amino acid 72 (N72D) that exhibits superior binding to IL-2R␤␥ C on immune cells and increased immunostimulatory activity (3). Our previous studies have demonstrated that this IL-15 variant, when associated with a soluble IL-15R␣ sushi domain fusion to IgG1 Fc (IL15R␣SuFc), could form a heterodimeric complex, IL-15N72D⅐ IL-15R␣SuFc (designated , that also exhibits increased binding activity to the IL-2R␤␥ C complex, enhanced capacity to stimulate NK and T cells, and has a longer biological half-life compared with native IL-15 (4). In various animal models, ALT-803 acts as a potent immunostimulant that is capable of simultaneously activating the innate and adaptive arms of the immune system to elicit both rapid and long-lasting protective responses against neoplastic challenges (5).…”