Novel Human Interleukin-15 Agonists

Abstract

IL-15 is an immunostimulatory cytokine trans-presented with the IL-15 receptor α-chain to the shared IL-2/IL-15Rβ and common γ-chains displayed on the surface of T cells and NK cells. To further define the functionally important regions of this cytokine, activity and binding studies were conducted on human IL-15 muteins generated by site-directed mutagenesis. Amino acid substitutions of the asparagine residue at position 72, which is located at the end of helix C, were found to provide both partial agonist and superagonist activity, with various nonconservative substitutions providing enhanced activity. Particularly, the N72D substitution provided a 4–5-fold increase in biological activity of the IL-15 mutein compared with the native molecule based on proliferation assays with cells bearing human IL-15Rβ and common γ-chains. The IL-15N72D mutein exhibited superagonist activity through improved binding ability to the human IL-15Rβ-chain. However, the enhanced potency of IL-15N72D was not observed with cells expressing the mouse IL-15Rα-IL-15Rβ-γc complex, suggesting that this effect is specific to the human IL-15 receptor. The enhanced biological activity of IL-15N72D was associated with more intense phosphorylation of Jak1 and Stat5 and better anti-apoptotic activity compared with the wild-type IL-15. IL-15N72D superagonist activity was also preserved when linked to a single-chain TCR domain to generate a tumor-specific fusion protein. Thus, the human IL-15 superagonist muteins and fusions may create opportunities to construct more efficacious immunotherapeutic agents with clinical utility.

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).…”

“…In our previous reports, the scTCR-IL-15N72D and scTCR-IL-15N72D⅐scTCR-IL-15R␣Su fusion complexes were shown to retain IL-15 biological activity, although at reduced levels compared with IL-15 (3,8). This lower activity is presumably due to steric hindrance between the fused scTCR domain and IL-15N72D-IL-2R␤␥ C interactions.…”

“…IL-15 binds to IL-15R␣ at high affinity, and IL-15R␣ functions as a chaperone and conformational stabilizer to enhance the interaction between IL-15 and IL-2R␤␥ C (2). 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).…”

“…This lower activity is presumably due to steric hindrance between the fused scTCR domain and IL-15N72D-IL-2R␤␥ C interactions. To assess the IL-15 biological activity of 2B8T2M, an IL-15-dependent cell line, 32D␤, was used as described previously (3). The results demonstrate that 2B8T2M supported 32D␤ cell proliferation but exhibited significantly lower activity compared with native IL-15 or ALT-803 (2B8T2M, EC 50 ϭ 889 pM; IL-15, EC 50 ϭ 34 pM; ALT-803, EC 50 ϭ 14 pM).…”