PMID: 26563299

 

    Legend: Sugar

Title : Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation

Abstract :
  1. Human interleukin 15 ( IL-15 ) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha ( sIL-15Rα)
  2. This heterodimeric IL-15 : sIL-15Rα complex (het IL-15 ) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials
  3. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of het IL-15 , which, however, remains largely uncharacterized
  4. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human het IL-15
  5. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies
  6. 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.
  7. The two potential IL-15 N-glycosylation sites ( Asn71 and Asn112 ) located at the IL-2 receptor interface were unoccupied
  8. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation
  9. 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
  10. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation.
  11. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by het IL-15
  12. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of het IL-15 demonstrated consistent manufacturing and purification
  13. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate het IL-15
  14. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of het IL-15