IN VIVO IMMUNOGENICITY ANALYSIS AND IN VITRO STABILITY CHARACTERIZATION OF NEW DE-IMMUNIZED RECOMBINANT HUMAN IFN-ALPHA (RHIFN-α) VERSIONS FOR ANTIVIRAL THERAPY.

GIORGETTI SOFÍA; RICOTTI, SONIA; EDUARDO MUFARREGE; ETCHEVERRIGARAY, MARINA; ANNE DEGROOT

Buenos Aires

Congreso; Reunión Conjunta 2017 entre la Sociedad Argentina de Investigación Clínica (SAIC), la Sociedad Argentina de Inmunología (SAI) y la Sociedad Argentina de Farmacología Experimental (SAFE); 2017

SAIC, SAI, SAFE

rhIFN-α is widely used for the treatment of viral diseases such as chronic Hepatitis B and C (CHC). Although rhIFN-α is a "self" derived protein therapeutic, anti-IFN-α immune responses have been reported in treated patients.Previously, we used an in silico approach to identify immunodominant T-cell epitopes in a hyperglycosylated IFN-alpha2b (4N-IFN) and to modify them so as to generate functional de-immunized variants. Two functional 4N-IFN variants were successfully produced in CHO cells and designated as 4N-IFN(VAR1) and 4N-IFN(VAR3). These de-immunized 4N-IFN variants had significantly reduced ex vivo immunogenicity in human PBMC samples.In this work we deepen our analysis comparing the immunogenicity of these proteins in transgenic mice. After subcutaneous injection, we isolated blood and serum and quantified binding and neutralizing antibody titers by ELISA and antiviral activity assays. Interestingly, no differences in binding antibody titers were detected in serum from mice inoculated with 4N-IFN, 4N-IFN(VAR1) and 4N-IFN(VAR3) (p≤0.05). However, marked reductions in neutralizing antibody titers were observed for the de-immunized variants, highlighting the success of the de-immunization process.Along with immunogenicity, protein stability constitutes a major concern in biotherapeutic manufacturing processes. For this, we characterized the in vitro protein stability of the new 4N-IFN variants against a heat treatment ranging from 25 °C to 95 °C. Both de-immunized variants exhibited enhanced stability (p≤0.05) in comparison with a commercial non-glycosylated protein, highlighting an additional advantage of these muteins.To summarize, the functional de-immunized 4N-IFN proteins exhibit significantly reduced in vivo immunogenicity in transgenic mice and increased in vitro thermal stability when compared with a commercial nonglycosylated protein and the original molecule.