Recombinant Human Interferon-beta Research Articles

Enhanced production of recombinant proteins in Corynebacterium glutamicum using a molecular chaperone.

Protein synthesis in Corynebacterium glutamicum is critical for applications in biotechnology and medicine. However, the use of C. glutamicum for protein production is limited by its low expression and aggregation. To overcome these limitations, a molecular chaperone plasmid system was developed in this study to improve the efficiency of recombinant protein synthesis in C. glutamicum. The effect of molecular chaperones on target protein synthesis (Single-chain variable fragment, Scfv) under three different promoter strengths was tested. In addition, the plasmid containing the molecular chaperone and target protein was verified for growth stability and plasmid stability. This expression model was further validated using two recombinant proteins, human interferon-beta (Hifn) and hirudin variant III (Rhv3). Finally, the Rhv3 protein was purified, and analysis of Rhv3 activity confirmed that the use of a molecular chaperone led to an improvement in test protein synthesis. Thus, the use of molecular chaperones is believed to will improve recombinant proteins synthesis in C. glutamicum.

Validation of a test procedure for determination of specific activity of human recombinant interferon beta (Infibeta®) using various cell/virus combinations

Interferon beta preparations have demonstrated efficacy in the treatment of multiple sclerosis. One of the most important quality attributes that support efficacy and safety of interferon beta preparations is specific antiviral activity. Interferon beta activity is determined by the biological test method which can lead to an erroneous final result due to the system uncertainties that contribute to the overall uncertainty. Accurate assessment of specific activity plays an important role in adequate determination of the product’s therapeutic dose, therefore the test method standardisation and validation are of particular relevance. The aim of the study was to validate a test procedure for assessing specific activity of human recombinant interferon beta preparations, using various cell/virus combinations, as illustrated by the example of Infibeta®. Materials and methods: WISH, Vero, A-549, and MDBK cells in combination with mouse encephalomyocarditis virus were used in the study. The testing was performed using the biological test method based on the interferon ability to suppress virus-induced cytopathic effects in cell cultures. The results were processed using methods of mathematical statistics and the GraphPadPrism, Statistical 2/0 software package. Results: the paper compares the results of specific activity determination of Infibeta® (interferon beta-1b) using various cell/virus combinations. The dose-response curves were used to compare the test results. It was demonstrated that all the tested cell lines could be used in the biological test procedure for determination of interferon beta specific activity. However, the best results were obtained with A-549, WISH cells in combination with mouse encephalomyocarditis virus. The following validation characteristics were determined in the cell/virus systems: specificity, linearity, precision, and robustness. Conclusions: the study validated the test procedure that enables measurement of interferon beta specific activity in the range of 4.8–11.2 IU/mL at a satisfactory accuracy level, which guarantees reliable test results. The study demonstrated robustness, intermediate precision, and reproducibility of the test procedure.

The Effects of Self-cleavage Intein-ELK16 Tag in the Transcript Steric Hindrance of IFN.

Intervening proteins (Inteins) are identified as protein domains in a precursor protein structure. Inteins can excise itself from precursor protein and join the remaining portions which result in forming an active protein. In this study, the transcript expression level of recombinant human Interferon beta (rhIFNβ) connected to the self-cleavage Intein-ELK16 (LELELKLKLELELKLK) tag was measured by real-time PCR in HEK293T cell line. First, the sequence of Mycobacterium tuberculosis RecA (Mtu recA) was obtained from the InBase database to do appropriate changes including adding the restriction sites, kozak sequence, signal peptide and ELK16 sequence by SnapGene software. The RNA secondary structure were also examined using the online RNA Fold 2.2 web server. Next, the construct was inserted into pUC19 plasmid. The sequence of rhIFNβ was also cloned into pBudCE4.1 vector. In the next step, the rhIFNβ was ligated into the construct (self-cleavage tag of ELK16) using T4 DNA ligase and the recombinant construct was transfected into HEK293T cell line. Finally, expression of the cassette was evaluated by real-time PCR. The analysis of secondary RNA structure indicates a minimum free energy of MEF - 261.10kcal/mol. Our results indicate that IFNβ was upregulated (37.8-fold, p < 0.0001) in cells which transfected by rhIFNβ-ELK16 compared to the mock and un-transfected conditions. Altogether, our results show that the presence of mini self-cleavage Intein-ELK16 tag along with the rhIFNβ had no interference in transcription of rhIFNβ in the HEK293T cell line.

Design, construction, and expression of recombinant human interferon beta gene in CHO-s cell line using EBV-based expression system.

Background and purpose:Codon optimization has been considered as a powerful strategy to increase the expression level of protein therapeutics in mammalian cells. As an empirical approach to study the effects of the codon usage and GC content on heterologous gene expression in suspension adapted Chinese hamster ovary (CHO-s) cells, we redesigned the recombinant human interferon beta (rhIFN- β) gene based on the codon preference of the CHO cell in a way to increase the GC content in the third position of each codon.Experimental approach:The nucleotide sequence of the codon-optimized rhIFN-β was synthesized in parallel with the wild-type and expressed transiently in CHO-s cells using Epstein-Bar virus (EBV)-based expression system. The protein expression of the rhIFN-β by codon-optimized and wild-type genes were quantified using ELISA test.Findings / Results:The results indicated a 2.8-fold increase in the expression level of the biologically active form of the rhIFN-β by codon-optimized sequence.Conclusion and implications:These results shed light on the capability of codon optimization to create a stable CHO cell for scaling up the production of recombinant therapeutics such as rhIFN-β.

Intact bioactivities and improved pharmacokinetic of the SL335-IFN-β-1a fusion protein that created by genetic fusion of SL335, a human anti-serum albumin fab, and human interferon-β

Recombinant human interferon beta (rIFN-β) has long been used as a first-line treatment for multiple sclerosis (MS), and any attempt to develop a long-acting rIFN-β is desirable since only one pegylated version of long-acting rIFN-β-1a (Plegridy) is currently available in clinics. Previously, we reported that SL335, a human Fab molecule specific to serum albumin, exhibits an extended serum half-life via utilizing the FcRn recycling mechanism. With the ultimate goal of developing a long-acting rIFN-®, we generated a fusion construct by linking human IFN-β cDNA to the C-terminus of the SL335 H chain at the DNA level followed by expression of the fusion protein, referred to as SL335-IFN-β-1a, in Chinese hamster ovary-S (CHO-S) cells. In its N-linked glycosylated form, the resulting fusion protein was easily purified from the culture supernatant via a three-step chromatography process. In vitro functional assays revealed that the fusion protein retained its intrinsic binding capabilities to human serum albumin (HSA) and interferon α/β receptor (IFNAR) that were almost identical to those of parental SL335 and rIFN-β-1a (Rebif). In addition, the fusion protein possessed an antiviral potency and anti-proliferation activity comparable to those of Rebif. In pharmacokinetic (PK) analyses using Lewis rats and cynomolgus monkeys, SL335-IFN-β-1a exhibited at least a two-fold longer serum half-life and a significantly reduced renal clearance rate compared to those of Rebif. Finally, a four-week repeated dose toxicity study revealed no abnormal toxicological signs. In conclusion, our results clearly demonstrated that SL335-IFN-β-1a is worthy of further development as an alternative long-acting IFN-β therapeutic.

Evaluation of recombinant human interferon beta 1b by liquid chromatography methods and bioassay

Recombinant human interferon beta 1b (rhIFNβ-1b) is clinically used to treat multiple sclerosis. A reversed-phase liquid chromatography (RP-LC) method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was acetonitrile with 0.1% TFA run at a flow rate of 1.0 mL/min. A size exclusion liquid chromatography (SE-LC) method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.). The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Retention times were 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1-200 µg/mL (r2 = 0.9998) and 0.50-200 µg/mL (r2 = 0.9999), respectively, for RP-LC and SE-LC, with detection at 214 nm. Liquid chromatography (LC) methods were validated and employed in conjunction with the in vitro bioassay to assess the content/potency of rhIFNβ-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic.

Effects of recombinant human interferon β1a on the chondrogenic differentiation of human bone marrow mesenchymal stem cell

Recombinant human interferon beta (rhIFN-β) is a glycoprotein produced by genetically engineered cells and has anti-virus, anti-tumor and immunoregulation functions. Although studies have shown that other subtypes of IFN such as IFN-γ affects cell proliferation and differentiation to some extent, the effect of rhIFN-β on chondrogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) is less known. In this study we studied the effect of rhIFN-β on the chondrogenic differentiation of hMSCs by inducing hMSCs into cartilage pellet via adding IFN-β1a into regular TGF-β3 chondrogenic differentiation medium. We collected the induced pellets and then detected GAG content, assessed pellets size, observed agreecan using alcian blue staining, and analyzed the expression of Sox and CollangenⅡusing real-time PCR and Western blotting. Addition of 100 ng/mL IFN-β1a to regular TGF-β3 chondrogenic differentiation medium could improve the concentration of GAG, increase the size of pellets, promote the formation of aggrecan and up-regulate the expression of CollangenII and Sox9. IFN-β1a combined with TGF-β3 could promote chondrogenic differentiation of hMSCs.

Antidrug Antibodies: B Cell Immunity Against Therapy.

Chronic inflammatory diseases are now treated with a range of different biopharmaceuticals, often requiring lifelong parenteral administrations. This exposure to drugs is unnatural and can trigger the immune system and result in the formation of antidrug antibodies. Drug-specific antibodies will, if of sufficiently high titre and affinity, block the intended effect of the drug, increase its clearance and make continued treatment worthless. We expect the immune system to react towards therapies against which tolerance has never been established, which is the case for factor VIII treatment in patients with haemophilia A. However, even biopharmaceutical molecules that we should be tolerant against can elicit antidrug antibodies, for instance in treatment of multiple sclerosis patients with recombinant human interferon-beta. Possible immunological mechanisms behind the breaking of tolerance against drugs, the impact this has on continuous treatment success, clinical practice and drug development, will be discussed in this review.

Optimization of Fermentation Conditions for Recombinant Human Interferon Beta Production by Escherichia coli Using the Response Surface Methodology.

Background:The periplasmic overexpression of recombinant human interferon beta (rhIFN-β)-1b using a synthetic gene in Escherichia coli BL21 (DE3) was optimized in shake flasks using Response Surface Methodology (RSM) based on the Box-Behnken Design (BBD).Objectives:This study aimed to predict and develop the optimal fermentation conditions for periplasmic expression of rhIFN-β-1b in shake flasks whilst keeping the acetate excretion as the lowest amount and exploit the best results condition for rhIFN-β in a bench top bioreactor.Materials and Methods:The process variables studied were the concentration of glucose as carbon source, cell density prior the induction (OD 600 nm) and induction temperature. Ultimately, a three-factor three-level BBD was employed during the optimization process. The rhIFN-β production and the acetate excretion served as the evaluated responses.Results:The proposed optimum fermentation condition consisted of 7.81 g L-1 glucose, OD 600 nm prior induction 1.66 and induction temperature of 30.27°C. The model prediction of 0.267 g L-1 of rhIFN-β and 0.961 g L-1 of acetate at the optimum conditions was verified experimentally as 0.255 g L-1 and 0.981 g L-1 of acetate. This agreement between the predicted and observed values confirmed the precision of the applied method to predict the optimum conditions.Conclusions:It can be concluded that the RSM is an effective method for the optimization of recombinant protein expression using synthetic genes in E. coli.

Development of ADA Against Recombinant Human Interferon Beta in Immune Tolerant Mice Requires Rapid Recruitment of CD4+ T Cells, Induces Formation of Germinal Centers but Lacks Susceptibility for (Most) Adjuvants

Immunological processes leading to formation of antidrug antibodies (Abs) against recombinant human proteins remain poorly understood. Animal and clinical studies revealed that immunogenicity shares both T-cell-dependent (requirement of CD4(+) T cells, isotype switching) and T-cell-independent (involvement of Marginal Zone B cells, apparent lack of memory) characteristics. We used immune tolerant mice to study the mechanism underlying immunogenicity in more detail. We found that CD4(+) T cells were crucial at early stages of Ab responses against rhIFNβ. In addition, we found a similar number of germinal centers (GCs) in spleen after rhIFNβ treatment as after treatment with a foreign protein. However, neither Ab titers nor the number of GCs was increased by adsorption of rhIFNβ on aluminum hydroxide. Therefore, we tested the effect of several immune adjuvants in a follow-up study. We found that only conjugation of rhIFNβ to a carrier protein (cholera toxin subunit B) was effective in boosting Ab titers. However, these conjugates failed to trigger rhIFNβ specific memory formation. Our findings show that early events of the immunogenicity reaction to self-proteins are CD4(+) T-cell dependent. Nevertheless, despite those similarities, immunogenicity of human proteins is clearly not a classical CD4(+) T-cell-dependent response.

A modified immune tolerant mouse model to study the immunogenicity of recombinant human interferon beta

Interferon beta may induce antibodies in multiple sclerosis patients and the incidence of immunogenicity depends on the type of product. These antibodies can reduce the efficacy of interferon beta. Two transgenic immune tolerant mouse models for human interferon beta (hIFNβ) (C57Bl/6, and C57Bl/6×FVB/N F1 hybrid mice) have been developed previously for studying immunogenicity. These models, however, may not be used for every interferon beta product because of the lack of immunogenicity in the wildtype genetic background. We therefore developed a modified transgenic mouse model by backcrossing the F1 hybrid C57Bl/6×FVB/N transgenic mice with wildtype FVB/N for 10 generations. These F10 offspring (referred to hear as FVB/N) have a genetic background consisting of mostly FVB/N (99.9%) and very little C57Bl/6 (0.1%), and are expected to have the more sensitive antibody producing phenotype of the parental FVB/N strain. The newly generated “FVB/N” strain was assessed for antibody formation against different rhIFNβ formulations compared to the C57Bl/6, and C57Bl/6×FVB/N transgenic mouse models.The new FVB/N transgenic mouse model was more sensitive for all tested rhIFNβ products, and the difference in antibody titers between the transgenic and non-transgenic mice of the FVB/N strain was much bigger compared to the antibody levels of the C57Bl/6, and C57Bl/6×FVB/N strains.

Metabolic changes of recombinant Escherichia coli BL21 (DE3) during overexpression of recombinant human interferon beta in HCDC

The influence of the overexpression of recombinant human interferon beta (rhINF-β) during high cell density cultivation (HCDC) on metabolic characteristics of recombinant E. coli BL21 (DE3) was studied. Fed-batch culture technique was employed to grow the host cell for the production of human interferon beta in pET expression system in a 5L bench-top bioreactor. The final cell density of 42.15 g L-1 and cell growth (OD600nm) of 103.5 were obtained respectively in fed-batch culture experiment. Four hours after the induction, the specific growth rate was decreased. The initial concentration of the glucose, ammonium and phosphate in the TB culture medium were 5.0, 12.0 and 3.12 g L-1 respectively. The maximum amount of the produced rhINF-β was 2.8 g L-1 after applying the fed-batch cultivation technique. Besides, time profiles of the acetate and lactate production were similar, the acetate concentration (4.91 g L -1 ) was lower than that of lactate (10.08 g L -1 ) and the

Antibody Response Against Betaferon® in Immune Tolerant Mice: Involvement of Marginal Zone B-cells and CD4+ T-cells and Apparent Lack of Immunological Memory

The immunological processes underlying immunogenicity of recombinant human therapeutics are poorly understood. Using an immune tolerant mouse model we previously demonstrated that aggregates are a major trigger of the antidrug antibody (ADA) response against recombinant human interferon beta (rhIFNβ) products including Betaferon®, and that immunological memory seems to be lacking after a rechallenge with non-aggregated rhIFNβ. The apparent absence of immunological memory indicates a CD4+ T-cell independent (Tind) immune response underlying ADA formation against Betaferon®. This hypothesis was tested. Using the immune tolerant mouse model we first validated that rechallenge with highly aggregated rhIFNβ (Betaferon®) does not lead to a subsequent fast increase in ADA titers, suggesting a lack of immunological memory. Next we assessed whether Betaferon® could act as Tind antigen by inactivation of marginal zone (MZ) B-cells during treatment. MZ B-cells are major effector cells involved in a Tind immune response. In a following experiment we depleted the mice from CD4+ T-cells to test their involvement in the ADA response against Betaferon®. Inactivation of MZ B-cells at the start of Betaferon® treatment drastically lowered ADA levels, suggesting a Tind immune response. However, persistent depletion of CD4+ T-cells before and during Betaferon® treatment abolished the ADA response in almost all mice. The immune response against rhIFNβ in immune tolerant mice is neither a T-cell independent nor a classical T-cell dependent immune response. Further studies are needed to confirm absence of immunological memory (cells).

Immunogenicity of Recombinant Human Interferon Beta Interacting with Particles of Glass, Metal, and Polystyrene

Aggregates play a major role in the immunogenicity of recombinant human interferon beta (rhIFNβ), a protein used to treat multiple sclerosis. A possible cause of aggregation is interaction between therapeutic protein and surfaces encountered during processing, storage, and administration. Moreover, proteins may adsorb to particles shed from these surfaces. In this work, we studied the immunogenicity of recombinant human interferon beta-1a (rhIFNβ-1a) interacting with glass microparticles, stainless steel microparticles, and polystyrene nanoparticles. At physiological pH, rhIFNβ-1a readily adsorbed to the particles, while the degree of adsorption was influenced by the ionic strength of the phosphate buffer. Front-face fluorescence showed that the tertiary structure of rhIFNβ-1a slightly changed upon adsorption to glass. The interaction with stainless steel microparticles resulted in increased levels of aggregates in the free protein fraction. Furthermore, protein adsorbed to stainless steel microparticles was more difficult to desorb than protein adsorbed to glass. Incubation with stainless steel considerably enhanced the immunogenicity of rhIFNβ-1a in transgenic mice immune tolerant for human interferon beta. The protein fraction adsorbed on stainless steel particles was responsible for this. In conclusion, rhIFNβ-1a adsorbs to common hydrophilic surface materials, possibly increasing the immunogenicity of the protein.

Comparative transcriptomic profile analysis of fed-batch cultures expressing different recombinant proteins in Escherichia coli

There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-β), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-β and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression.

Oxidized and Aggregated Recombinant Human Interferon Beta is Immunogenic in Human Interferon Beta Transgenic Mice

PurposeTo study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNβ-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice.MethodsUntreated rhIFNβ-1a was degraded by metal-catalyzed oxidation, H2O2-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNβ-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured.ResultsAll rhIFNβ-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNβ-1a contained high levels of covalent aggregates as compared with untreated rhIFNβ-1a. H2O2-treated rhIFNβ-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNβ-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNβ-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H2O2-treated rhIFNβ-1a as compared to untreated and guanidine-treated rhIFNβ-1a.ConclusionsOxidation-mediated aggregation increased the immunogenicity of rhIFNβ-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic.

High productivity of human recombinant beta-interferon from a low-temperature perfusion culture

Recombinant human interferon-beta (β-IFN), used in the therapeutic treatment of multiple sclerosis (MS), can be produced on a large-scale from genetically engineered Chinese hamster ovary (CHO) cells. However, its hydrophobicity causes non-reversible, molecular aggregation in culture. The parameters affecting aggregation were determined to be concentration, culture residence time, temperature and glycosylation. Although the protein can be produced in Escherichia coli in a non-glycosylated form, the addition of glycans confers a reduced rate of aggregation as well as a 10-fold higher bioactivity. We report on the application of a low temperature perfusion culture designed to control the parameters that cause aggregation. In this three-phase culture system there is a transition to a low temperature (32 °C) in a batch mode prior to implementing perfusion at 1 volume/day using an acoustic cell separator. Perfusion at the low temperature resulted in a 3.5-fold increase in specific productivity and a 7-fold increase in volumetric productivity compared to the batch culture at 37 °C. The percentage aggregation of β-IFN was reduced from a maximum of 43% in batch culture to a minimum of 5% toward the end of the perfusion phase. The glycosylation profile of all samples showed predominantly sialylated biantennary fucosylated structures. The extent of sialylation, which is important for bioactivity, was enhanced significantly in the perfusion culture, compared to the batch culture.

On the Role of Aggregates in the Immunogenicity of Recombinant Human Interferon Beta in Patients with Multiple Sclerosis

Like many other therapeutic proteins, recombinant human interferon beta (rhIFN-&#x03B2;) elicits undesirable immune responses. rhIFN-&#x03B2;-treated multiple sclerosis patients may form binding antibodies and neutralizing antibodies (NAbs), with the latter being responsible for inhibition of the therapeutic effect of the protein. The incidence of binding antibodies and NAbs against rhIFN-&#x03B2; as well as the titer and persistence of NAbs differ among the marketed products. The proportion of patients forming antibodies against rhIFN-&#x03B2;-1b is higher than that against rhIFN-&#x03B2;-1a, which is likely explained by the differences in protein structure and aggregation behavior between the 2 types of rhIFN-&#x03B2;. Here, we summarize the different factors influencing the immunogenicity of rhIFN-&#x03B2; in patients with multiple sclerosis and discuss the role played by rhIFN-&#x03B2; aggregates.

Aggregated Recombinant Human Interferon Beta Induces Antibodies but No Memory in Immune-Tolerant Transgenic Mice

PurposeTo study the influence of protein aggregation on the immunogenicity of recombinant human interferon beta (rhIFNβ) in wild-type mice and transgenic, immune-tolerant mice, and to evaluate the induction of immunological memory.MethodsRhIFNβ-1b and three rhIFNβ-1a preparations with different aggregate levels were injected intraperitoneally in mice 15× during 3 weeks, and the mice were rechallenged with rhIFNβ-1a. The formation of binding (BABs) and neutralizing antibodies (NABs) was monitored.ResultsBulk rhIFNβ-1a contained large, mainly non-covalent aggregates and stressed rhIFNβ-1a mainly covalent, homogeneous (ca. 100 nm) aggregates. Reformulated rhIFNβ-1a was essentially aggregate-free. All products induced BABs and NABs in wild-type mice. Immunogenicity in the transgenic mice was product dependent. RhIFNβ-1b showed the highest and reformulated rhIFNβ-1a the lowest immunogenicity. In contrast with wild-type mice, transgenic mice did not show NABs, nor did they respond to the rechallenge.ConclusionsThe immunogenicity of the products in transgenic mice, unlike in wild-type mice, varied. In the transgenic mice, neither NABs nor immunological memory developed. The immunogenicity of rhIFNβ in a model reflecting the human immune system depends on the presence and the characteristics of aggregates.

Hybrid transgenic immune tolerant mouse model for assessing the breaking of B cell tolerance by human interferon beta

To date, the therapeutic efficacy of recombinant human proteins is limited by their potential to break B cell tolerance in patients. The formation of neutralising antibodies (NABs) directed against recombinant human interferon beta (rhIFNβ) is associated with a decrease in the therapeutic effect of the protein. For this reason, there is a need to study factors that can cause the immunogenicity of rhIFNβ. Transgenic C57Bl/6 mice that are immune tolerant for human interferon beta (hIFNβ) have been employed in a mouse model for assessing the breaking of immune tolerance by rhIFNβ. In this study, we used the original C57Bl/6 mouse model as well as the hybrid offspring from crossings of transgenic C57Bl/6 mice with wildtype FVB/N mice to study the immunogenicity of three commercial rhIFNβ products, Rebif ®, Avonex ® and Betaferon ®. As determined by ELISA, wildtype C57Bl/6 mice failed to form binding antibodies (BABs) against Rebif ® and Avonex ® formulated with human serum albumin. Because not all interferon beta products induce antibodies in wildtype C57Bl/6 mice, the transgenic C57Bl/6 mice cannot be used to study the breaking of tolerance by these products. However, the crossing of transgenic C57Bl/6 mice with FVB/N mice resulted in wildtype hybrid offspring in which all products were immunogenic and transgenic hybrid offspring that showed immune tolerance for hIFNβ. Thus, these C57Bl/6 × FVB/N hybrid transgenic mice can be used to study the breaking of immune tolerance for all rhIFNβ products. Of the three products, only Betaferon ® was able to break immune tolerance in the transgenic hybrids. With an MxA gene expression inhibition assay, NABs were detected in Betaferon ® treated wildtype hybrid mice, but not in transgenic hybrid mice, indicating a distinct immune mechanism in wildtype and transgenic mice. A pegylated rhIFNβ-1a variant, PEG-rhIFNβ-1a, induced antibodies in wildtype hybrid mice, but did not break the immune tolerance of transgenic hybrid mice. This suggests that pegylation did not affect the potential of rhIFNβ-1a to break B cell tolerance.