EFFECT OF N-GLYCOSYLATION ON RECOMBINANT HUMAN INTERFERON ALPHA IMMUNOGENICITY

MARÍA JESÚS LEOPOLD; EDUARDO MUFARREGE; MARINA ECHEVERRIGARAY

Río de Janeiro

Simposio; VIII Simposio Latinoamericano de Tecnología de Cultivos Celulares (SLATCC); 2018

Recombinant human Interferon alpha 2b (rhIFN-α2b) is a therapeutic protein used for treatment of a variety of human viral diseases and cancers. Repeated dosing of IFN over several months induces neutralizing antibodies (NAb) against the therapeutic in up to 80% of patients. Moreover, type I IFNs can both induce and unmask sub-clinical autoimmune diseases. This undesired immune response may impose a limitation for its clinical use.In addition, its short circulating half-life constitutes another limiting factor for IFN-α therapy. To circumvent this inconvenient, highly glycosylated IFN variants were developed and designated as: IFN-2NM47/95, IFN-3NM47, IFN-3NM47/95 and IFN3NM47 Nter. These new molecules showed different glycosylation patterns and improved pharmacokinetic properties compared with the original molecule. However, no immunogenicity analysis was carried out to evaluate product safety.For this, the aim of this study was to investigate the immunogenicity of these new IFN variants through a comparative ex vivo study that also included the unmodified IFN version (IFN-WT).IFN variants were produced in CHO-K1 cell supernatants and purified by immunoaffinity chromatography. For ex vivo assays, blood samples were collected from 12 healthy donors and peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque. HLA-DR1 allotypes were determined by Luminex technology. Monocytes were isolated by plastic adherence, differentiated into immature dendritic cells (iDCs) and incubated with each IFN variant. Upon maturation with recombinant TNF-α, antigen-pulsed DCs were incubated with autologous T-cells. After 48-72 h, supernatants were collected and assayed for IFN-gamma and IL-4 production by sandwich ELISA. A stimulation index (SI) criteria was defined as the ratio of cytokine concentrations from protein challenged PBMCs and unchallenged PMBCs (culture media). A response was considered positive when SI ≥ 2.T-cell proliferation assays showed that all tested proteins exclusively induced IFN-gamma production, but in different levels in a protein and donor-depending manner. In particular, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*13 and HLA-DRB1*16 alleles were directly involved in IFN-derived peptide presentation. Also, a comparative analysis revealed that 3NM47/95 was the variant which exhibited the higher immunogenicity with 42% of positive responses. In contrast, IFN-3NM47-Nter was less immunogenic than the other IFN variants (25% of responders). In addition, a similar proportion of responders (33%) was observed for IFN-2NM47/95, IFN-3NM47 and IFN-WT. It is important to highlight that IFN-3NM47-Nter is the most glycosylated IFN version. Therefore, this suggests that higher glycan contents played a role in antigen recognition, processing and/or presentation. Considering the reduced immunogenicity for IFN-3NM47-Nter observed here and its superior pharmacokinetic properties, altogether these results highlight IFN-3NM47-Nter as a promising candidate for clinical use in antiviral therapy.