Immunogenicity as a critical property of therapeutics: Ex vivo immunogenicity analysis of a long-lasting hyperglycosylated derivative of rhIFN-alpha-2b

EDUARDO MUFARREGE; GIORGETTI SOFÍA; RICARDO KRATJE; MARINA ECHEVERRIGARAY

Barcelona

Congreso; 24th ESACT Meeting; 2015

Background and Novelty: Immunogenicity is the ability of a drug to provoke an immune response and must be analyzed for the approval of biosimilars and new therapeutics. There is growing evidence that repeated dosing of rhIFN-alpha-2b over several months induces neutralizing antibodies (NAb) against the therapeutic in up to 80% of patients, depending on the indication. Moreover, type I IFNs can both induce and unmask sub-clinical autoimmune diseases. In an attempt to prolong its plasma half-life, a hyperglycosylated derivative (rhIFN-alpha-2b-4N) with remarkable pharmacokinetic profile was developed. The aim of this study was to investigate the immunogenicity of rhIFN-alpha-2b-4N through a comparative ex vivo study that also included the endogenous O-glycosylated rhIFN-alpha-2b (rhIFN-alpha-2b-wt) and two commercial versions: rhIFN-alpha-2b and pegylated rhIFN-alpha-2b (rhIFN-alpha-2b-PEG).Experimental Approach: Blood samples were taken from 26 healthy donors. Peripheral blood mononuclear cells (PBMC) were isolated from each donor by Ficoll-Hypaque density centrifugation. HLA-DR1 allotypes were determined by Luminex technology.Monocyte derived-dendritic cells (DCs) were generated and used to endocyte and process the IFN variants. Then, antigen-pulsed DCs were incubated with autologous T-cells. After 2 days of incubation, supernatants were collected and analyzed for IFN-gamma and IL-4 productions by sandwich ELISA. A stimulation index (SI) criteria was defined as the ratio of cytokine concentrations from protein challenged treated PBMCs and unchallenged PMBCs (excipients). A geometric mean (GM) was then calculated. Positive responses were defined by donors who produced a SI higher than GM. Results and discussion: Comparison of allotypes expressed in the cohort against those expressed in the world population revealed that all major HLA-DR1 alleles were well represented. T-cell proliferation assays showed that all tested proteins induced both IFN-gamma and IL-4 secretion, but in different levels depending on the protein and the donor. IFN-gamma secretion by T-cells is characteristic for a Th1 profile and it is associated with an inflammatory response, whereas IL-4 production defines a Th2 phenotype and is associated with antibody secretion. Also, a comparative analysis revealed that 85% of the population developed a Th1 profile against rhIFN-alpha-2b-4N. A similar proportion of responders was observed for rhIFN-alpha-2b-wt. This result demonstrates that those glycosylation sites introduced into the molecule to generate rhIFN-alpha-2b-4N did not increase the rhIFN-alpha-2b immunogenicity in comparison with the cytokine produced by the human body. In addition, rhIFN-alpha-2b and rhIFN-alpha-2b-PEG induced a Th1 response in 45% and 28 % of the population, respectively. Interestingly, a different behavior was observed when the Th2 profile was analyzed. In this case, rhIFN-alpha-2b-4N induced the lowest response among all rhIFN-alpha-2b variants (28%). Moreover, the commercial alternatives produced a positive response in a great proportion of the population (72% and 61% for rhIFN-alpha-2b and rhIFN-alpha-2b-PEG, respectively). Taking into account that a Th2 response is strongly correlated with in vivo Ab production, these results are in excellent agreement with the evidence that link anti-IFN-alpha-2b antibody induction and autoimmune disease development with rhIFN-alpha-2b or rhIFN-alpha-2b-PEG therapy, highlighting rhIFN-alpha-2b-4N as a promising candidate for clinical use.