Functional Deimmunization of Interferon Beta-1b by Identifying and Silencing Human T Cells Epitopes.

Interferonbeta-1b (IFNß-1b) developed as therapeutic protein for the treatment of multiple sclerosis (MS). Studies have been shown that Long-term usage of this protein can lead to the development of anti-drug antibodies (ADAs) and this phenomenon cause total loss or reduced efficacy of IFNß-1b. The aim of this study was to predict and silence IFNß-1b T-cells epitopes by in silico methods and genetic engineering. Based on bioinformatics studies we identified optimal sets of conservative point mutations for eliminating T-cells epitopes in IFNß-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 SDS-PAGE and Western blotting, consequently, IFNß-1b proteins was purified by His-tag chromatography. To determined activity of mutants' variants anti-proliferative and anti-viral 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 T-cell epitope in IFNß-1b protein, and this strategy has capacity to create proteins which have naturally reduced immunogenicity.

Effect of HLA-DRB1 alleles and genetic variants on the development of neutralizing antibodies to interferon beta in the BEYOND and BENEFIT trials.

BACKGROUND: Treatment of multiple sclerosis (MS) with interferon ß can lead to the development of antibodies directed against interferon ß that interfere with treatment efficacy. Several observational studies have proposed different HLA alleles and genetic variants associated with the development of antibodies against interferon ß. OBJECTIVE: To validate the proposed genetic markers and to identify new markers. METHODS: Associations of genetic candidate markers with antibody presence and development were examined in a post hoc analysis in 941 patients treated with interferon ß-1b in the Betaferon® Efficacy Yielding Outcomes of a New Dose (BEYOND) and BEtaseron®/BEtaferon® in Newly Emerging multiple sclerosis For Initial Treatment (BENEFIT) prospective phase III trials. All patients were treated with interferon ß-1b for at least 6 months. In addition, a genome-wide association study was conducted to identify new genetic variants. RESULTS: We confirmed an increased risk for carriers of HLA-DRB1*04:01 (odds ratio (OR) = 3.3, p = 6.9 × 10-4) and HLA-DRB1*07:01 (OR = 1.8, p = 3.5 × 10-3) for developing neutralizing antibodies (NAbs). Several additional, previously proposed HLA alleles and genetic variants showed nominally significant associations. In the exploratory analysis, variants in the HLA region were associated with NAb development at genome-wide significance (OR = 2.6, p = 2.30 × 10-15). CONCLUSION: The contribution of HLA alleles and HLA-associated single-nucleotide polymorphisms (SNPs) to the development and titer of antibodies against interferon ß was confirmed in the combined analysis of two multi-national, multi-center studies.

The cross-reactivity of binding antibodies with different interferon beta formulations used as disease-modifying drugs in multiple sclerosis patients.

Interferon beta (IFNb) preparations are commonly used as first-line therapy in relapsing-remitting multiple sclerosis (RRMS). They are, however, characterized by limited efficacy, partly due to the formation of anti-IFNb antibodies in patients.In this pilot study, we assessed with the ELISA method the presence of the binding antibodies (BAbs) against interferon beta after 2 years of therapy with subcutaneous interferon beta 1a (Rebif) in 49 RRMS patients. Antibody levels were established again within 1 year after treatment withdrawal. We used 3 interferons that are commercially available for MS therapy, namely Avonex (Biogen Idec Limited), Rebif (Merck Serono), and Betaferon (Bayer Pharma AG), as antigens.BAbs reacting with Rebif were found in 24.4% to 55% of patients, depending on the units of their expression. The levels of anti-Rebif antibodies remained high in 8 patients and in 4 patients they dropped significantly. Strong correlations were obtained in all assays (anti-Rebif-anti-Avonex, anti-Rebif-anti-Betaferon, and anti-Betaferon-anti-Avonex) and the existence of cross-reactivity in the formation of antibodies against all the tested formulations of interferon beta was confirmed. The levels of BAbs remain significant in the clinical context, and their assessment is the first choice screening; however, methods of BAbs evaluation can be crucial for further decisions. More studies are needed to confirm our results; specifically it would be of interest to evaluate methods of neutralizing antibodies identification, as we only assessed the binding antibodies. Nevertheless, our results support the concept that in interferon nonresponders, that are positive for binding antibodies, switching the therapy to alternative disease-modifying agent (for example glatiramer acetate, fingolimod, or natalizumab) is justified, whereas the switch to another interferon formulation will probably be of no benefit.

Immunogenicity of Recombinant Human Interferon Beta-1b in Immune-Tolerant Transgenic Mice Corresponds with the Biophysical Characteristics of Aggregates.

Determining to what extent biophysical characteristics of aggregates affect immunogenicity of therapeutic interferon beta-1b. Three recombinant human interferon beta-1b (rhIFNß-1b) samples with different levels of aggregates generated by copper oxidation, thermal stress, or left untreated, as well as Avonex(®) drug substance and Betaferon(®) drug product, were injected intraperitoneally in nontransgenic and interferon beta transgenic FVB/N mice 5 times per week for 3 weeks. Antibodies against interferon beta were measured using enzyme-linked immunosorbent assay. UV and fluorescence spectroscopy, dynamic light scattering, size exclusion chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), fluid imaging microscopy, and resonant mass measurement, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, were used to characterize and quantitate aggregates in the 3 rhIFNß preparations, to correlate biophysical characteristics with immunogenicity. In immune-tolerant interferon beta transgenic FVB/N mice, Betaferon drug product showed the highest immunogenicity, while Avonex drug substance showed the lowest level of immunogenicity. Of the 3 forms of rhIFNß-1b, copper-oxidized rhIFNß-1b showed lower immunogenicity than thermally stressed rhIFNß-1b, despite containing larger aggregates. Both copper-oxidized rhIFNß-1b and thermally stressed rhIFNß-1b exhibited changes in protein structure as shown using fluorescence spectroscopy and RP-HPLC. Nontransgenic, nonimmune-tolerant FVB/N mice generated high antibody titers against all interferon beta samples tested. The level of immunogenicity and the breaking of tolerance in FVB/N transgenic mice are not only related to the level of aggregation but also depend on the size and structure of the aggregates.