Immunogenicity of biotherapeutics. Effect of N-glycosylation on human interferon alpha 2b ex- vivo immunogenicity.

SOFÍA I. GIORGETTI; EDUARDO MUFARREGE; MARINA ETCHEVERRIGARAY

Buenos Aires

Congreso; IV LASID Meeting LXIII Argentinean Society for Immunology Meeting II French-Argentinean. Immunology Meeting. Sociedad Argentina de Inmunología.; 2015

BackgroundThere is growing evidence that repeated dosing of rhIFN-alpha2b over several months induces neutralizing antibodies against the therapeutic in up to 80% of patients. The aim of this study was to investigate the immunogenicity of a long lasting hyperglycosylated rhIFN-alpha2b variant developed in our lab, known as rhIFN-alpha2b-4N, through a comparative ex vivo study that also included two commercial versions: rhIFN-alpha2b and rhIFN-alpha2b-PEG. Methods: Monocyte derived-dendritic cells (DCs) were generated and used as antigen presenting cells. Then, IFN derived peptides-pulsed DCs were incubated with autologous T-cells. Finally, culture supernatants were collected and analyzed for IFN-gamma and IL-4 levels by sandwich ELISA. Results: All tested proteins induced both IFN-gamma (Th1 profile) and IL-4 (Th2 phenotype) secretion by T-cells,but in different levels. rhIFN-alpha2b-PEG showed reduced Th1 immunogenicity in comparison with rhIFN-alpha2b, suggesting that pegylation may affect its antigenicity and/or immunogenicity. The unmodified cytokine (rhIFN-alpha2b-WT) and rhIFN-alpha2b-4N mainly showed Th1 responses, being the amount of positive responses triggered by the first one significantly higher. When Th2 profiles were analyzed, rhIFN-alpha2b-4N induced the lowest response among all rhIFN-alpha2b variants.ConclusionsThe results obtained with rhIFN-α2b-WT and rhIFN-alpha2b-4N demonstrate that the addition of 4 N-glycosylation sites to the cytokine sequence produced a marked reduction of its ex vivo immunogenicity. Also, the analysis of the global percentage of T-cell effector responses induced by each version of rhIFN-alpha2b revealed that rhIFN-alpha2b-4N showed the lowest ex vivo immunogenicity. This fact highlights the hyperglycosylated cytokine as a promising candidate for clinical use.