Functional Deimmunization of Interferon Beta1b by Identifying and Silencing Human T Cells Epitopes

Interferonbeta1b (IFN #x3B2;1b) developed as therapeutic protein for the treatment of multiple sclerosis (MS). Studies have been shown that Longterm usage of this protein can lead to the development of antidrug antibodies (ADAs) and this phenomenon cause total loss or reduced efficacy of IFN #x3B2;1b. The aim of this study was to predict and silence IFN #x3B2;1b Tcells epitopes by in silico methods and genetic engineering. Based on bioinformatics studies we identified optimal sets of conservative point mutations for eliminating Tcells epitopes in IFN #x3B2;1b protein. Four synthetic genes with desirable mutation constructed and PET26b+ was used as an expression vector in E. coli. The expression of this proteins confirmed by SDSPAGE and Western blotting, consequently, IFN #x3B2;1b proteins was purified by Histag chromatography. To determined activity of mutants #x2019; variants antiproliferative and antiviral activity compared to wild form was evaluated using MTT assay in A549 and Vero cells lines respectively. Also the immunogenicity of mutant proteins compared with Betaseron measured in BALB/c mice. The in vitro bioactivity analysis demonstrated that functional activities of all mutant proteins were maintained and is the same as biological activity of Betaseron. Pharmacokinetic studies suggest that, in engineered proteins that contain substitution of Histidine to Glutamic Acid at position 131 (mut 2 and mut 1+2) antibodies response reduced by about 50%, as compared to that for Betaseron. Computational analysis expedites identification and prediction of epitopes in therapeutic protein, therefore, we used immunoinformatic tools for modification of dominant Tcell epitope in IFN #x3B2;1b protein, and this strategy has capacity to create proteins which have naturally reduced immunogenicity.