Development of biobetters: Immunogenicity analysis of Deimmunized and Functional Therapeutic (DeFT) versions of a long lasting rhIFN-alfa2b

EDUARDO MUFARREGE; SOFIA GIORGETTI; MARINA ECHEVERRIGARAY; FRANCES TERRY; WILLIAM MARTIN; ANNE DEGROOT

Mar del Plata

Congreso; Reunión Conjunta 2016 entre la Sociedad Argentina de Investigación Clínica (SAIC), la Sociedad Argentina de Inmunología (SAI) y la Sociedad Argentina de Farmacología Experimental (SAFE); 2016

SAIC, SAI, SAFE

Alpha interferons (IFN-α) have proven clinical utility for the treatment of chronic hepatitis B and C virus infections. However, there is growing evidence that repeated dosing of IFN-α2b over several months induces neutralizing antibodies against the therapeutic in a significant number of patients. Associations between IFN immunogenicity and loss of efficacy have been described.In an attempt to improve the in vivo biological efficacy of IFN-α2b wild type (WT-IFN), a hyperglycosylated protein (4N-IFN) was developed. However, in silico analysis revealed that 4N-IFN had more predicted T cell epitopes than WT-IFN. In order to develop a safer and most efficient IFN therapy, we used the DeFT (Deimmunization of Functional Therapeutics) approach to produce functional, deimmunized versions of 4N-IFN.Using the Optimatrix of the in silico toolkit ISPRI, 4N-IFN sequence was optimized to reduce MHC binding of clustered epitopes. Eight modifications were selected and integrated in three variants: 4N-IFN(VAR1) [5]; 4N-IFN(VAR2) [8] and 4N-IFN(VAR3) [3]; the number of modifications is identified by the number in the brackets. Preliminary experiments revealed 4N-IFN(VAR2) did not have any antiviral activity thus was not included in further assays. For 4N-IFN(VAR1) and 4N-IFN(VAR3) T-cell proliferation assay showed reduced immunogenicity and Th1 and Th2 cytokine profile, when compared to controls (commercial NG-IFN (non-glycosylated), PEG-IFN, WT-IFN and 4N-IFN). Immunogenicity ranking in decreasing order for Th1 profile was: WT-IFN > 4N-IFN > NG-IFN > PEG-IFN = 4N-IFN(VAR1) > 4N-IFN(VAR3). For Th2 responses the results were: NG-IFN > PEG-IFN > WT-IFN > 4N-IFN > 4N-IFN(VAR3) > 4N-IFN(VAR1).These results demonstrate that deimmunization of the long lasting IFN variant reduced its immunogenicity as measured in vitro using T cell assays and cytokine profiling. In conclusion, 4N-IFN(VAR1) and 4N-IFN-4N(VAR3) appear to be promising candidates for antiviral therapy of HCV and HBV.