title: De-immunized and Functional Therapeutic (DeFT) versions of a long lasting recombinant alpha interferon for antiviral therapy.
abstract: Interferon α (IFN-α) exerts potent antiviral, immunomodulatory, and antiproliferative activity and have proven clinical utility in chronic hepatitis B and C virus infections. However, repeated IFN-α administration induces neutralizing antibodies (NAb) against the therapeutic in a significant number of patients. Associations between IFN-α immunogenicity and loss of efficacy have been described. So as to improve the in vivo biological efficacy of IFN-α, a long lasting hyperglycosylated protein (4N-IFN) derived from IFN-α2b wild type (WT-IFN) was developed. However, in silico analysis performed using established in silico methods revealed that 4N-IFN had more T cell epitopes than WT-IFN. In order to develop a safer and more efficient IFN therapy, we applied the DeFT (De-immunization of Functional Therapeutics) approach to producing functional, de-immunized versions of 4N-IFN. Using the OptiMatrix in silico tool in ISPRI, the 4N-IFN sequence was modified to reduce HLA binding potential of specific T cell epitopes. Following verification of predictions by HLA binding assays, eight modifications were selected and integrated in three variants: 4N-IFN(VAR1), (VAR2) and (VAR3). Two of the three variants (VAR1 and VAR3) retained anti-viral function and demonstrated reduced T-cell immunogenicity in terms of T-cell proliferation and Th1 and Th2 cytokine levels, when compared to controls (commercial NG-IFN (non-glycosylated), PEG-IFN, WT-IFN and 4N-IFN). It was previously demonstrated that N-glycosylation improved IFN-α pharmacokinetic properties. Here, we further reduce immunogenicity as measured in vitro using T cell assays and cytokine profiling by modifying the T cell epitope content of a protein (de-immunizing). Taking into consideration the present results and previously reported immunogenicity data for commercial IFN-α2b variants, 4N-IFN(VAR1) and 4N-IFN-4N(VAR3) appear to be promising candidates for improved IFN-α therapy of HCV and HBV.
authors.affiliation: Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Laboratorio de Cultivos Celulares, FBCB, UNL, Santa Fe, Argentina. Electronic address: mufarrege@fbcb.unl.edu.ar.
authors.forename: Eduardo F
authors.initials: EF
authors.lastname: Mufarrege
authors.affiliation: Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Laboratorio de Cultivos Celulares, FBCB, UNL, Santa Fe, Argentina.
authors.forename: Sofía
authors.initials: S
authors.lastname: Giorgetti
authors.affiliation: Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Laboratorio de Cultivos Celulares, FBCB, UNL, Santa Fe, Argentina.
authors.forename: Marina
authors.initials: M
authors.lastname: Etcheverrigaray
authors.affiliation: EpiVax, Inc., Providence, RI, USA.
authors.forename: Frances
authors.initials: F
authors.lastname: Terry
authors.affiliation: EpiVax, Inc., Providence, RI, USA.
authors.forename: William
authors.initials: W
authors.lastname: Martin
authors.affiliation: EpiVax, Inc., Providence, RI, USA; Institute for Immunology and Informatics, University of Rhode Island, RI, USA.
authors.forename: Anne S
authors.initials: AS
authors.lastname: De Groot
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000998:Antiviral Agents / Q000276:immunology* / Q000627:therapeutic use*; D016466:CHO Cells; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003412:Cricetulus; D016207:Cytokines / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000276:immunology / Q000627:therapeutic use; D005260:Female; D006031:Glycosylation / Q000187:drug effects; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000276:immunology; D006526:Hepatitis C / Q000188:drug therapy / Q000276:immunology; D006801:Humans; D016898:Interferon-alpha / Q000276:immunology* / Q000627:therapeutic use*; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D008297:Male; D008875:Middle Aged; D011994:Recombinant Proteins / Q000276:immunology* / Q000627:therapeutic use*; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D018417:Th1 Cells / Q000187:drug effects / Q000276:immunology; D018418:Th2 Cells / Q000187:drug effects / Q000276:immunology; D055815:Young Adult
chemical_list: D057134:Antibodies, Neutralizing; D000998:Antiviral Agents; D016207:Cytokines; D018984:Epitopes, T-Lymphocyte; D016898:Interferon-alpha; D011994:Recombinant Proteins
keywords: De-immunization; Hepatitis therapy; IFN alpha; IFN-α; Immunogenicity; In silico prediction; T cell epitope; T-cell proliferation assay