Deimmunized and Functional Therapeutic (DeFT) versions of a long lasting recombinant Alpha Interferon

EDUARDO MUFARREGE; MARINA ECHEVERRIGARAY; WILLIAM MARTIN; ANNE DEGROOT

Baltimore

Congreso; Seventh Annual Immunogenicity and Bioassay Summit 2015; 2015

Cambridge Healthcare Institute

Interferons a (IFN-a)-exert potent antiviral, immunomodulatory, and antiproliferative activity and have proven clinical utility in chronic hepatitis B and C virus infections. Repeated administration induces neutralizing antibodies (NAb) against the therapeutic in up to 80% of patients. An association between anti-drug antibodies and loss of efficacy has been described. Our group has developed a less-immunogenic hyperglycosylated protein (IFN-a2b-4N) derived from IFN-a2b-WT. However, in silico analysis revealed that IFN-a2b-4N had more T cell epitopes than IFN-a2b-WT. In order to develop a safer and most efficient IFN therapy, we applied the DeFT (Deimmunization of Functional Therapeutics) approach to producing functional, deimmunized versions of IFN-a2b-4N. Using the Optimatrix of the in silico toolkit ISPRI, IFN-a2b-4N sequence was optimized to reduce MHC binding of clustered, or promiscuous epitopes. Following verification of binding predictions by HLA assay, eight modifications were selected and integrated in three variants: IFN-a2b-4N (VAR1) [5]; IFN-a2b-4N (VAR2) [8] and IFN-a2b-4N (VAR3) [3]; the number of modifications is identified by the number in the brackets. IFN-a2b-4N (VAR2) did not have any antiviral activity thus was not included in further assays.T-cell proliferation assay and Th1 and Th2 cytokine profiling showed a markedly reduced immunogenicity for IFN-a2b-4N(VAR1) and IFN-a2b-4N(VAR3), when compared to controls (commercial IFN-a2b-NG (non-glycosylated), commercial IFN-a2b-PEG, IFN-a2b WT and IFN-a2b-4N). Immunogenicity ranking in decreasing order for Th1 profile was: IFN-a2b-WT > IFN-a2b-4N > IFN-a2b-NG > IFN-a2b-PEG = IFN-a2b-4N (VAR1) > IFN-a2b-4N (VAR3). For Th2 responses the results were: IFN-a2b-NG > IFN-a2b-PEG > IFN-a2b-WT > IFN-a2b-4N > IFN-a2b-4N(VAR3) > IFN-a2b-4N(VAR1).While N-glycosylation was able to reduce immunogenicity, further deimmunization was able to reduce immunogenicity as measured in vitro using T cell assays and cytokine profiling. Taking into consideration the present results and previously reported immunogenicity data for commercial IFN-a2b variants, IFN-a2b-4N (VAR1) and IFN-a2b-4N (VAR3) appear to be promising candidates for antiviral therapy of HCV and HBV.