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Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and β-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.

Interleukin-15 (IL-15) is a common γ-chain cytokine that has a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha (IL-15Rα) chain, which together form the IL-15 heterodimer. We have generated DNA vectors expressing the heterodimeric IL-15 by optimizing mRNA expression and protein trafficking. Repeated administration of these DNA plasmids by intramuscular injection followed by in vivo electroporation in rhesus macaques resulted in sustained high levels of IL-15 in plasma, with no significant toxicity. Administration of DNAs expressing heterodimeric IL-15 also resulted in an increased frequency of NK and T cells undergoing proliferation in peripheral blood. Heterodimeric IL-15 led to preferential expansion of CD8(+)NK cells, all memory CD8(+) T-cell subsets and effector memory CD4(+) T cells. Expression of heterodimeric IL-15 by DNA delivery to the muscle is an efficient procedure to obtain high systemic levels of bioactive cytokine, without the toxicity linked to the high transient cytokine peak associated with protein injection.

The aim of the present study was to examine the participation of the rostral ventrolateral medulla (RVLM) in the maintenance of hypertension in rats submitted to the renovascular Goldblatt (two-kidney, one clip) procedure. We inhibited or stimulated this area with the use of drugs such as glycine, L-glutamate, or kynurenic acid. (1) Bilateral microinjection of glycine (100 nmol, 200 nL, n = 13) into the RVLM of hypertensive rats produced a decrease in mean arterial blood pressure (MAP) from 177.2 +/- 29.3 to 102.3 +/- 20.9 mm Hg (P < .05), which was similar to the decrease produced by intravenous administration of hexamethonium. The inhibition of RVLM with glycine in normotensive rats produced a decrease in MAP from 106 +/- 17.1 to 59.7 +/- 7.3 mm Hg (P < .05, n = 9). (2) An impressive increase in MAP from 153.3 +/- 16.3 to 228 +/- 34.9 mm Hg (P < .05) occurred in hypertensive rats after microinjection of L-glutamate (50 nmol, 200 nL, n = 6) into the RVLM. The same procedure caused a significant but less intense increase in MAP from 105 +/- 13.8 to 148.3 +/- 24.9 mm Hg in normotensive rats (P < .05, n = 6). (3) A decrease in MAP from 151.6 +/- 25.3 to 96.8 +/- 22.5 mm Hg occurred in hypertensive rats after microinjection of the broad-spectrum glutamate antagonist kynurenic acid (4 nmol, 200 nL, n = 6) into the RVLM, whereas the same procedure did not change MAP in normotensive animals (n = 6). Heart rate was not significantly affected in any group.(ABSTRACT TRUNCATED AT 250 WORDS)