Recombinant Interferon-beta Research Articles

Immunogenic effects of recombinant interferon-beta therapy disrupt the JAK/STAT pathway in primary immune cells from patients with multiple sclerosis

Background: Immunogenicity of recombinant interferon-β (IFN-β) is a known complication in the therapy of relapsing–remitting multiple sclerosis (RRMS). Neutralizing antibodies (NAbs) that can interfere with efficacy are quantified using in vitro bioassays; however, these assays do not reveal the immunogenic state of the patient and are not predictive of treatment outcome. Objective: Assessment of the impact of NAbs on IFN-β responsive cells and signalling pathways in peripheral blood mononuclear cells (PBMCs) with phospho-specific flow cytometry. Method: PBMCs from 10 IFN-β-treated patients with RRMS, two untreated patients, and two healthy controls were re-stimulated in autologous sera and media with a serial dilution of IFN-β (0–8000 U/ml) and levels of phosphorylation of STAT1/3/4/5/6 transcription factors were quantified in PBMC subtypes (NAb titres 0 to > 6000 neutralizing units). Data was subjected to principal component analysis, Hotelling’s T2, and partial least squares analysis. Results: Three significantly distinct clusters of individuals were revealed in autologous sera: therapy-naïve and healthy, treated NAb-negative, and treated NAb-positive. Compared with controls STATs signalling patterns were modulated in treated NAb-negative patients and inhibited in all treated NAb-positive patients independently of NAb titres. In media no clustering of patients could be found. The predictability of NAb titres based on the phospho-flow data was 74%. Conclusion: Phospho-specific flow cytometry can delineate subset-specific cell responses that can act as surrogates for NAb exposure in blood. Immunogenic effects alter the response in primary cells even at low NAb levels. Cell line-based immunogenicity testing is not readily transferable to the immunogenic response in patients.

Clustering samples characterized by time course gene expression profiles using the mixture of state space models.

We propose a novel method to classify samples where each sample is characterized by a time course gene expression profile. By exploiting the mixture of state space model, the proposed method addresses the following tasks: (1) clustering samples according to temporal patterns of gene expressions, (2) automatic detection of genes that discriminate identified clusters, (3) estimation of a restricted autoregressive coefficient for each cluster. We demonstrate the proposed method along with the cluster analysis of 53 multiple sclerosis patients under recombinant interferon beta therapy with the longitudinal time course expression profiles.

Interferon beta for secondary progressive multiple sclerosis

Therapy with either recombinant beta-1a or beta-1b interferons (IFNs) is worldwide approved for Relapsing Remitting Multiple Sclerosis (RRMS). A major unanswered question is whether this treatment is able to safely reverse or retard the progressive phase of the disease. The main objective was to verify whether IFNs treatment in Secondary Progressive Multiple Sclerosis (SPMS) is more effective than placebo in reducing the number of patients who experience disability progression. We searched the Cochrane Multiple Sclerosis Group's Trials Register (1995 to 15 February 2011), the reference lists of relevant articles and conference proceedings. Regulatory agencies were used as additional sources of information. We included all randomised, double or single blind, placebo-controlled trials (RCTs) evaluating the efficacy of IFNs versus placebo in SPMS patients. Two review authors independently assessed all reports retrieved from the search. They independently extracted clinical, safety and MRI data, using a predefined data extraction form, resolving disagreements after discussion with a third reviewer. Risk of bias was evaluated to assess the quality of the studies. Treatment effect was measured using Risk Ratio (RR) with 95% confidence intervals (CI) for the binary outcomes and Standard Mean Difference with 95% CI for the continuous outcomes. Five RCTs met the inclusion criteria, from which 3122 (1829 IFN and 1293 placebo) treated patients contributed to the analysis. Included population was heterogeneous in terms of baseline clinical characteristics of the disease, in particular the percentage of patients affected by secondary progression with superimposed relapse ranging from 72% to 44%. IFN beta 1a and 1b did not decrease the risk of progression sustained at 6 months (RR, 95% CI: 0.98, [0.82-1.16]) after three years of treatment. A significant decrease of the risk of progression sustained at 3 months (RR, 95% CI: 0.88 [0.80, 0.97]) and of the risk of developing new relapses at three years (RR 0.91, [0.84-0.97]) were found. The risk of developing new active brain lesions decreased over time but this data was obtained from single studies on Magnetic Resonance Imaging (MRI), performed in subgroups of patients; in spite of no effect on progression, the radiological data supported an effect on MRI parameters. The safety profile reflects what is commonly reported in MS IFN-treated patients. Well designed RCTs, evaluating a high number of patients were included in the review. Recombinant IFN beta does not prevent the development of permanent physical disability in SPMS. We were unable to verify the effect on cognitive function for the lack of comparable data. This treatment significantly reduces the risk of relapse and of short -term relapse-related disability.Overall, these results show that IFNs' anti-inflammatory effect is unable to retard progression, when established. In the future, no new RCTs for IFNs versus placebo in SPMS will probably be undertaken, because research is now focusing on innovative drugs. We believe that this review gives conclusive evidence on the clinical efficacy of IFNs versus placebo in SPMS.

Analyzing the metabolic stress response of recombinant Escherichia coli cultures expressing human interferon-beta in high cell density fed batch cultures using time course transcriptomic data

Fed batch cultures expressing recombinant interferon beta under the T7 promoter were run with different exponential feeding rates of a complex substrate and induced at varying cell densities. Post-induction profiles of the specific product formation rates showed a strong dependence on the specific growth rate with the maximum product yield obtained at 0.2 h(-1). A study of the relative transcriptomic profiles as a function of pre-induction μ was therefore done to provide insight into the role of cellular physiology in enhancing recombinant protein expression. Hierarchical clustering analysis of the significantly regulated genes allowed us to identify biologically important groups of genes which fall under specific master regulators. The groups were: rpoH, ArcB, CreB, Lrp, RelA, Fis and Hfq. The response of these regulators, which exert a feedback control on the growth and product formation rates correlated well with the expression levels obtained. Thus at the optimum pre-induction μ, the alternative sigma factors and ribosomal machinery genes did not get depressed till the 6th hour post-induction unlike at other specific growth rates, demonstrating a critical role for the genes in sustaining recombinant protein expression.

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.

Inhibition of Endogenous Interferon Beta by Neutralizing Antibodies Against Recombinant Interferon Beta

To determine if neutralizing antibodies (NAbs) against interferon beta from patients with multiple sclerosis (MS) cross-react with other type 1 interferons, especially endogenous interferon beta, and thus might impede the immune systems of affected patients. Masked serum samples from MS patients were challenged in vitro against recombinant interferon beta-1a and interferon beta-1b, as well as human leukocyte interferon and fibroblast interferon, the latter representing endogenous interferon. The neutralizing capacity of serum samples on these type 1 interferons was assessed using a luciferase reporter gene assay. Randomly selected samples were titrated to further delineate the cross-reactivity of antibodies. University medical center in Düsseldorf, Germany. We randomly selected 150 samples from interferon beta-treated MS patients who had previously been tested for the presence of binding antibodies and NAbs. Neutralization of interferon beta bioactivity and cross-reactivity of anti-interferon beta antibodies. Antibody-mediated neutralization of interferon beta bioactivity in vitro against recombinant interferon beta was observed in all serum samples that had previously tested positive for binding antibodies and NAbs. A neutralizing pattern comparable to that of recombinant interferon beta was observed when endogenous interferon was assessed, reflecting cross-reactivity of NAbs. No differences in neutralization between recombinant and endogenous interferon were observed with respect to the interferon beta preparation used for treatment. Furthermore, no neutralization of other type 1interferons by NAbs could be detected. A proportion of MS patients who are treated with recombinant interferon beta develop NAbs that also neutralize endogenous interferon. Because NAbs at high titers can persist for years, these antibodies may impede the immune system in affected MS patients regardless of their current treatment regimen.

Marked differences between MARC-145 cells and swine alveolar macrophages in IFNβ-induced activation of antiviral state against PRRSV

The activation of antiviral activity induced by recombinant swine interferon beta (rswIFNβ) against PRRSV was comparatively examined in MARC-145 cells and porcine alveolar macrophages (PAMs). A dose–response analysis showed, in MARC-145 cells, that isolate Mo25544 was highly sensitive to rswIFNβ while a vaccine strain and isolate PDV130-9301 were resistant to different extents. In contrast, all three viruses were equally sensitive to rswIFNβ in PAMs even at the lowest dose of IFN utilized in the bioassays. To analyze potential differences in mechanisms of antiviral activation between these cells, treatment with 2-aminopurine (2-AP), an inhibitor of double-stranded RNA-dependent protein kinase (PKR), was performed in rswIFNβ-treated cells. Addition of 2-AP to rswIFNβ-primed MARC-145 cells restored replication of the Mo25544 isolate, and to some extent that of vaccine virus and PDV130-9301. In contrast, virus replication could not be rescued for any of the three viruses with 2-AP in rswIFNβ-treated PAMs. The differences in sensitivity of PRRSV to rswIFNβ as well as the effects of 2-AP strongly suggest that MARC-145 cells and PAMs utilize different rswIFNβ-associated antiviral pathways. Therefore, studies to understand virus-host cell interactions performed in MARC-145 cells require additional scrutiny when utilized as a host cell model for immunologic responses to PRRSV.

Richard Rudick: multiple sclerosis missionary

Richard Rudick: multiple sclerosis missionary

Effect of antisense nucleotide against acetate production on recombinant beta interferon production by E. coli

Effect of antisense nucleotide against acetate production on recombinant beta interferon production by E. coli

Generation of Monoclonal Antibodies Against Human Recombinant Interferon Beta Using Genetic Immunization with Simultaneous Expression of IgM and IgG Isotypes

Monoclonal antibodies (MAbs) against human recombinant interferon beta (hrIFNbeta) were generated by genetic immunization (GI). In order to test two viral promoters frequently used in mammalian expression plasmid vectors, mice were inoculated four times by intramuscular injection, without adjuvant, with 100 microg of either pcDNA 3.1hrIFNbeta or pZeoSV2IFNbeta containing the entire human interferon beta gene and under the control of, respectively, human cytomegalovirus (HCMV) immediate-early promoter or early SV-40 enhancer/promoter. Only serum samples from mice immunized with pZeoSV2IFNbeta were positive to anti-hrIFNbeta. The spleens of the immunized mice were fused with myeloma Sp2/0 cells and the hybridoma clones generated screened by an in house enzyme-linked immunosorbent assay (ELISA). Fourteen MAbs were selected as reactive with hrIFNbeta. Western blot analysis was performed and only one recognized the 18 kDa isoform (non-glycosylated) of hrIFNbeta. All MAbs were subjected to antibody isotype characterization with a commercial ELISA and showed unusual profile with simultaneous expression of both IgM and IgG2a isotypes. This observation is further supported by RT-PCR amplification of the IgM CH4 domain using total RNA from hybridomas.

Interferon‐induced Mx proteins in brain tissue of multiple sclerosis patients

Recombinant interferon-beta is proven as an effective long-term treatment in patients with multiple sclerosis (MS). Unlike in other chronic inflammatory diseases, endogenous synthesis of type I interferons (IFN-alpha and IFN-beta) has not been studied extensively in MS. Mx proteins A and B (MxA and MxB) are intracellular proteins that are induced exclusively by type I IFNs. We investigated the expression of Mx proteins in post-mortem brain tissue of IFN-beta-naïve MS patients as a marker for endogenous synthesis of type I IFNs. By employing monoclonal antibodies specific for MxA and MxB positive staining was detectable predominantly in reactive astrocytes within the MS plaques but also in endothelial and ependymal cells as well as in lymphocytic infiltrates. This is of interest in view of results previously published by our group and others that Mx protein concentrations measured by ELISA increase in blood samples from MS patients after IFN-beta therapy. In MS, Mx proteins are detectable in plaques suggesting endogenous synthesis of type I IFNs as part of the acute inflammatory process.

Clinical trials in multiple sclerosis: Current and future requirements – potential pitfalls

Several lines of evidence support early immunomodulatory treatment of relapsing multiple sclerosis with either recombinant beta-interferons or glatiramer acetate and positive results from phase III trials encourage start of treatment even in patients with clinical isolated syndrome (CIS). However, currently available drugs for basic therapy are only partially effective and patients may still encounter relapses or disease progression. As treatment-refractory, clinically active MS can quickly lead to irreversible neurological disability there is an urgent need for more effective therapeutic strategies.The major goal is to reduce the damage, protect the tissue and enhance repair. In order to achieve these goals we not only need effective and safe drugs but also the appropriate study designs to really detect the most relevant outcomes [2, 7].

Review: Escalating immunotherapy of multiple sclerosis

Basic disease-modifying treatment for relapsing forms of active multiple sclerosis (MS) is now available in many countries with high prevalence rates, for this chronic inflammatory disease of the central nervous system. Several lines of evidence support early immunomodulatory treatment with either recombinant interferon-beta or glatiramer acetate, and positive results from phase III trials encourage start of treatment even in patients with clinically isolated syndromes (CIS). However, currently available drugs for basic therapy are only partially effective and patients may still encounter relapses or disease progression. As treatment-refractory, clinically active MS can quickly lead to irreversible neurological disability there is an urgent need for effective escalating strategies. Patients with suboptimal treatment response to basic therapy have been treated with combination therapies, cytotoxic drugs (such as mitoxantrone and cyclophosphamide) or autologous hematopoietic stem cell transplantation. Recently, the monoclonal antibody, natalizumab, was added to this armamentarium. None of these strategies have been vigorously evaluated in large randomized, controlled phase III trials with patients who failed basic therapy. Therefore, the decision to escalate immunotherapy is still based on limited evidence. This article will review potential candidates for intensified immunosuppression and call for innovative study designs to better evaluate escalating immunotherapy in MS.

Interferon-beta injection site reaction: Review of the histology and report of a lupus-like pattern

To the Editor: A 34-year old female with a history of multiple sclerosis developed erythematous plaques on the thighs and hips at the site of subcutaneous injections of recombinant interferon-beta (IFN-β)-1b (Figs 1 and 2). She was self-administering 8 million international units every other day for 7 months. The reaction began a few months after the first injection. Each lesion resolved in approximately 1 month, without ulceration. Overall, the reaction was worsening with time. Topical steroids provided no relief.

CHO cells adapted to hypothermic growth produce high yields of recombinant β‐interferon

Mild hypothermic conditions (30-33 degrees C) have previously been shown to increase cell-specific productivity (Q(p)) of recombinant proteins from mammalian cells. However, this is often associated with a lower growth rate which off-sets any potential advantage of higher product titers. We report the isolation of a population of Chinese Hamster Ovary (CHO) cells that have been adapted to low-temperature growth by continuous subculture at low temperature for up to 300 days. This adapted cell population achieved a growth rate twofold greater than nonadapted cells under low-temperature conditions (32 degrees C) while maintaining an elevated level of cell-specific expression of recombinant beta-interferon. The volumetric titer of beta-interferon was enhanced by 70% in stationary cultures and by more than twofold by application of a temperature-shift strategy involving a growth to production phase. However, the low-temperature-adapted cells were fragile and demonstrated an increased sensitivity to hydrodynamic stress in agitated cultures. This problem was resolved by the use of macroporous microcarriers which protected the cells and allowed growth of high-density cultures under hypothermic conditions. This eventually resulted in a threefold enhancement of volumetric titer of monomeric beta-interferon compared to the original control culture at 37 degrees C.

Recombinant interferon beta or glatiramer acetate for delaying conversion of the first demyelinating event to multiple sclerosis

Immunomodulatory drugs have been shown to be only modestly effective in clinically definite relapsing remitting multiple sclerosis (RRMS). It has been hypothesized that their efficacy could be higher if used at the first appearance of symptoms, that is in the clinically isolated syndromes (CIS) suggestive of demyelinating events, a pathology which carries a high risk to convert to clinically definite MS (CDMS). The objective of this review was to assess the effects of immunomodulatory drugs compared to placebo in adults in preventing conversion from CIS to CDMS which means the prevention of a second attack. We searched the Cochrane MS Group Trials Register (June 2007), Cochrane Central Register of Controlled Trials (CENTRAL)The Cochrane Library Issue 3, 2007, MEDLINE (January 1966 to June 2007), EMBASE (January 1974 to June 2007) and reference lists of articles. We also contacted manufacturers and researchers in the field. The trials selected were double-blind, placebo-controlled, randomised trials of CIS patients treated with immunomodulatory drugs. Study selection have been independently done by two reviewers. Two further reviewers independently assessed trial quality and extracted and analysed data. Study authors were contacted for additional informations. Adverse effects information was collected from the trials. Only three trials tested the efficacy of interferon (IFN) beta including a total of 1160 participants (639 treatment, 521 placebo); no trial tested the efficacy of glatiramer acetate (GA). The metanalyses showed that the proportion of patients converting to CDMS was significantly lower in IFN beta-treated than in placebo-treated patients both after one year (pooled OR 0.53; 95% CI, 0.40 to 0.71; p <0.0001) as well as after two years of follow-up (pooled OR 0.52; 95% CI, 0.38 to 0.70; p <0.0001). Early treatment with IFN beta was associated with the side effect profile reported by the randomised controlled trials with this drug. Since side effects were reported with some heterogeneity in the three studies the metanalysis was possible only for the frequency of serious adverse events, not significantly different in IFN beta-treated or placebo-treated patients. The efficacy of IFN beta treatment on preventing the conversion from CIS to CDMS was confirmed over two years of follow-up. Since patients had some clinical heterogeneity (length of follow-up, clinical findings of initial attack), it could be useful for the clinical practice to further analyse the efficacy of IFN beta treatment in different patient subgroups.

Growth-inhibitory activity of human recombinant beta-interferon (GKT-beta) in vitro.

Growth-inhibitory activity of human recombinant beta-interferon (GKT-beta) against 20 human cultured cell lines derived from leukemias and lymphomas was measured quantitatively by regrowth assay. Daudi cells were the most sensitive to GKT-beta. Two T-cell lines (RPMI-8402, HUT78), three B-cell lines (Raji, P3HR-1, A3/Kawakami), one non-T, non-B acute lymphoblastic leukemia (ALL) cell line (KOPN-1) and one monocytoid cell line (U937) were moderately sensitive to GKT-beta. Although the levels of sensitivity of these cell lines to GKT-beta were different, the cells could be killed by GKT-beta. Morphological changes of the sensitive cells treated with GKT-beta were decrease in mitosis, pyknosis and segmentation of cells. Twelve other cultured cell lines, comprising four T-cell lines, four B-cell lines, one non-T, non-B ALL cell line and three myelomonocytoid cell lines, were not sensitive to GKT-beta. The results indicated that the growth-inhibitory activity of GKT-beta was not always cell lineage-specific or differentiative stage-specific. GKT-beta was instable in vitro and its antiviral activity was reduced to about 10% during the first 24 hr of incubation in culture medium with or without cells. This instability was reflected in a similar reduction of its growth-inhibitory activity. It was demonstrated that GKT-beta had a time-dependent, but not a concentration-denpendent antiproliferative action. This suggests that, in the clinical use of the interferon, direct antiproliferative activity of GKT-beta may be expected only through the use of therapeutic schedules which are suitable for its time-dependent action, such as through daily long-term treatment, but not through a single large-dose therapy.

Response surface methodology for optimizing the induction conditions of recombinant interferon beta during high cell density culture

A human interferon beta expressed from a synthetic gene was produced in high cell density culture by recombinant Escherichia coli using an optimized linear feeding strategy. The optimal induction conditions to be determined consisted of inducer concentration and dry cell weight at the time of induction. For this purpose, the response surface methodology was applied. Under optimal conditions, the maximum interferon beta concentration and overall productivity of 2.2 g/l and 0.151 g/l h were obtained, respectively, as the highest amounts ever reported for this protein. Two optimal ranges of dry cell weight and IPTG concentration consisting of 50 g/l and 2.54 mM, and 70 g/l and 1.29 mM were predicted, respectively, at which maximum productivity was achieved. By using a novel feeding strategy with linear variation of specific growth rate during high cell density fermentation, the maximum biomass productivity of 5.037 g/l h was obtained in a defined medium during 16 h. Then, by applying the optimum induction conditions, we accomplished an increase in overall productivity by more than three-fold over the central point. This is the first report showing the high production of human interferon beta by a synthetic gene in a simple fed-batch high cell density culture of recombinant E. coli in a defined medium.

Genome-Wide Pharmacogenomic Analysis of the Response to Interferon Beta Therapy in Multiple Sclerosis

To identify promising candidate genes linked to interindividual differences in the efficacy of interferon beta therapy. Recombinant interferon beta therapy is widely used to reduce disease activity in multiple sclerosis (MS). However, up to 50% of patients continue to have relapses and worsening disability despite therapy. We used a genome-wide pharmacogenomic approach to identify single-nucleotide polymorphism (SNP) allelic differences associated with interferon beta therapy response. Four collaborating centers in the Mediterranean Basin. Data Coordination Center at the University of California, San Francisco. A cohort of 206 patients with relapsing-remitting MS followed up prospectively for 2 years after initiation of treatment. DNA was pooled and hybridized to Affymetrix 100K GeneChips. Pooling schemes were designed to minimize confounding batch effects and increase confidence by technical replication. Single-nucleotide polymorphism detection. Comparison of allelic frequencies between good responders and nonresponders to interferon beta therapy. A multianalytical approach detected significant associations between several SNPs and treatment response, which were validated by individual DNA genotyping on an independent platform. After the validation stage was complete, 81 additional individuals were added to the analysis to increase power. We found that responders and nonresponders had significantly different genotype frequencies for SNPs located in many genes, including glypican 5, collagen type XXV alpha1, hyaluronan proteoglycan link protein, calpastatin, and neuronal PAS domain protein 3. The reported results address the question of genetic heterogeneity in MS and the response to immunotherapy by analysis of the correlation between different genotypes and clinical response to interferon beta therapy. Many of the detected differences between responders and nonresponders were genes associated with ion channels and signal transduction pathways. The study also suggests that genetic variants in heparan sulfate proteoglycan genes may be of clinical interest in MS as predictors of the response to therapy. In addition to new insights into the mechanistic biology of interferon beta, these results help define the molecular basis of interferon beta therapy response heterogeneity.

Panniculites secondaires à la toxicité vasculaire de l’interféron béta-1a

Interferon-beta-1b is a valuable first-line therapy for patients with relapsing-remitting multiple sclerosis. Many non-severe cutaneous reactions to recombinant interferon beta are described at injection sites. Panniculitis after subcutaneous injection of beta interferon is a rare adverse event; we describe two such cases at beta interferon injection sites. Two women aged 22 years and 45 years with severe multiple sclerosis receiving immunotherapy with beta interferon were admitted to an emergency department following the appearance of extremely painful induration at injection sites rendering walking impossible after several months of interferon injections. One of the patients had fever. Histology tests showed vasculitis and capillary thrombosis in one-woman and dermal oedema in the other. MRI scanners showed extensive avascular necrosis of soft tissue without fasciitis in both patients. Interferon withdrawal and surgical debridement was carried out in one case and beta interferon was successfully reintroduced in both cases. Only two cases have been reported of panniculitis induced by subcutaneous beta interferon injection. Clinically, such cases may mimic infectious processes. The present cases show that MRI may be useful in diagnosis and that the vascular toxicity of interferon beta probably plays a role in panniculitis. Temporary withdrawal of treatment, rotation of several injection sites and alternative routes of administration may all be proposed.