IFN a 2b Human
Description
IFN-a 2b Human Recombinant produced in E.Coli is a single, non-glycosylated, polypeptide chain containing 166 amino acids and having a molecular mass of 19400 Dalton. The difference between IFNA2A and IFNA2B is in the amino acid present at position 23. IFN-alpha 2a has a lysine at that position 23 while IFN-alpha 2b has arginine.
The IFN-alpha 2b gene was obtained from human leukocytes.
The IFN-a 2b is purified by proprietary chromatographic techniques.
Product Specs
IFN-alpha, produced by macrophages, possesses antiviral properties. It stimulates the production of protein kinase and oligoadenylate synthetase enzymes.
Recombinant Human IFN-a 2b, produced in E. coli, is a non-glycosylated polypeptide chain consisting of 166 amino acids. With a molecular weight of 19400 Daltons, it differs from IFN-alpha 2a by a single amino acid at position 23 (arginine in IFN-alpha 2b instead of lysine in IFN-alpha 2a). The IFN-alpha 2b gene originates from human leukocytes, and the protein is purified using proprietary chromatographic techniques.
Reconstitute the lyophilized IFN-alpha 2b in sterile 18 MΩ-cm H2O to a concentration of at least 100 µg/ml. Further dilutions can be made in other aqueous solutions.
Lyophilized IFN-alpha 2b remains stable at room temperature for 3 weeks but should be stored desiccated below -18°C. Upon reconstitution, store at 4°C for 2-7 days. For long-term storage, freeze at -18°C after adding a carrier protein (0.1% HSA or BSA). Avoid freeze-thaw cycles.
Greater than 98.0% purity as determined by:
(a) RP-HPLC analysis
(b) SDS-PAGE analysis
Protein content was quantified using two independent methods:
1. UV spectroscopy at 280 nm employing an absorbance value of 0.924 as the extinction coefficient for a 0.1% (1 mg/ml) solution. This value is calculated using the PC GENE computer analysis program for protein sequences (IntelliGenetics).
2. RP-HPLC analysis using a calibrated IFN-alpha 2b solution as a reference standard.
IFN alpha 2b, IFNA, INFA2, MGC125764, MGC125765.
MCDLPQTHSL GSRRTLMLLA QMRRISLFSC LKDRHDFGFP QEEFGNQFQK AETIPVLHEM IQQIFNLFST KDSSAAWDET LLDKFYTELY QQLNDLEACV IQGVGVTETP LMKEDSILAV RKYFQRITLY LKEKKYSPCA WEVVRAEIMR SFSLSTNLQE SLRSKE
Q&A
What are the fundamental molecular properties of human interferon alpha-2b?
Human interferon alpha-2b belongs to the type I interferon family, which includes IFNα, IFNβ, IFNω, and IFNτ. These cytokines share 30-80% amino acid sequence similarity. hIFNα-2b specifically is a polypeptide chain of 165-166 amino acids encoded by genes located on human chromosome 9. These genes have no introns and produce proteins with varying degrees of glycosylation. Some IFNα subtypes are unglycosylated while others feature different glycosylation patterns .
The protein functions through specific receptors grouped into two subunits: IFNAR-1 and IFNAR-2. The IFNAR-2 subunit exists in three forms (IFNAR-2a, IFNAR-2b, and IFNAR-2c), with IFNAR-2c playing the critical role in ligand binding and signal transduction. Interestingly, IFNAR-2a and IFNAR-2b act as competitive inhibitors that prevent IFN from binding to the signaling-competent IFNAR-2c receptor .
How can researchers synthesize hIFNα-2b for experimental studies?
Researchers can obtain hIFNα-2b through total chemical synthesis via native chemical ligation. This approach produces a 165-amino-acid protein that exhibits biological properties comparable to recombinant versions. The synthetic process represents an important advancement toward developing D-peptide antagonists with potential therapeutic applications .
When evaluating synthetic versus recombinant hIFNα-2b, comparative biological testing has confirmed equivalent functionality. Both forms demonstrate similar dose-dependent antiviral protection in A549 cells challenged with encephalomyocarditis virus (ECMV) and comparable antiproliferative effects on human T cells following stimulation with SEB and IL-2 .
What reference standards exist for hIFNα-2b research?
The World Health Organization has established an International Standard for human interferon alpha-2b (IFN-alpha 2b). The current preparation is the 2nd WHO International Standard (NIBSC code: 95/566), which replaced the 1st International Standard (code 82/576). This reference material serves as the primary biological standard for bioassays measuring IFNα-2b activity .
How does hIFNα-2b activate cellular signaling pathways?
Human interferon alpha-2b initiates signaling through the JAK-STAT pathway following receptor binding on the cell surface. The binding activates Janus kinase (JAK) family proteins, particularly JAK-1 and tyrosine kinase-2 (TYK-2), which phosphorylate STAT proteins. The STAT family comprises seven proteins (STAT-1, STAT-2, STAT-3, STAT-4, STAT-5a, STAT-5b, and STAT-6), while the JAK family includes four members (JAK-1, JAK-2, JAK-3, and TYK-2) .
Once activated, these pathways regulate more than 300 genes involved in antiviral and antiproliferative activities. Studies using various cell lines including melanoma (WM9), fibrosarcoma (HT1080), embryonic fibroblasts, and human dendritic cells have documented this extensive gene regulation capability .
What mechanisms underlie the antiproliferative effects of hIFNα-2b?
The antiproliferative activity of hIFNα-2b occurs through both direct and indirect mechanisms:
Direct mechanisms include:
-
Cell cycle arrest
-
Induction of apoptosis
-
Promotion of cell differentiation
Indirect mechanisms involve:
-
Activation of immune cells (T cells and natural killer cells)
-
Inhibition of vascularization (antiangiogenesis)
The apoptotic pathway activated by hIFNα-2b involves regulation of caspase proteins (caspase-8, caspase-9, caspase-3, caspase-6, and caspase-7). DNA damage signaling initiates this cascade, leading to BID protein cleavage, altered mitochondrial membrane permeability, and cytochrome c release. Released cytochrome c activates Apaf-1 and caspase-9, ultimately resulting in cell death. Additionally, hIFNα-2b increases the expression of caspase-3 and caspase-7, as well as caspase-8, producing DNA fragmentation .
Another pathway involves stimulation of insulin receptor subunits (IRS1 and IRS2), which activate phosphatidylinositol3-kinase (PI3K). While PI3K can function both as a cell survival factor and an inducer of apoptosis, in tumor cells, the PI3K/mTOR pathway is necessary for apoptosis following IFNα treatment and leads to DNA fragmentation .
What standardized assays exist for measuring hIFNα-2b biological activity?
Researchers employ several well-established assays to evaluate hIFNα-2b activity:
-
Antiviral protection assay: Measures the ability of IFNα-2b to protect A549 cells from cytopathic effects following challenge with encephalomyocarditis virus (ECMV). The protection occurs in a dose-dependent manner and is quantified by measuring cell viability .
-
Antiproliferative assay: Assesses the suppression of human T cell proliferation after stimulation with SEB and IL-2 in the presence of IFNα-2b. This effect can be measured through either:
-
Cytotoxicity assay: Evaluates IFNα-2b-induced cytotoxicity in specific cell lines, such as TF-1 cells, with typical ED50 values of 20-50 pg/mL .
How effective is hIFNα-2b in hematological malignancies?
Human interferon alpha-2b was first approved for treatment of hairy cell leukemia in 1986. The standard therapeutic regimen involves subcutaneous administration at 2 million U/m² three times weekly for 12 months .
Clinical studies have demonstrated significant efficacy:
| Clinical Setting | Treatment Protocol | Results |
|---|---|---|
| Seven patients with hairy cell leukemia | 3 million U of partially purified human IFN intramuscularly daily | Three patients achieved complete remission; four achieved partial remission |
| 50 patients with hairy cell leukemia | 2.0 × 10⁶ IU/m² subcutaneously three times weekly | At 24 months, none of the 38 remaining patients showed signs of relapse; mild toxicity observed in 76% of patients |
| 69 patients (including hairy cell leukemia cases) | 2.0 × 10⁶ IU/m² for 12-18 months | 13 patients developed second neoplasms (6 of hematopoietic origin, remainder were adenocarcinomas) |
These studies highlight both the therapeutic efficacy and potential long-term risks associated with interferon treatment, emphasizing the importance of monitoring patients for secondary malignancies.
What evidence supports hIFNα-2b use in solid tumors like melanoma?
The FDA approved high-dose IFNα-2b for adjuvant therapy of stage IIB and III melanoma in 1995. Multiple clinical trials have evaluated its efficacy:
A landmark study by Kirkwood et al. involving 287 patients compared:
The Scottish melanoma group conducted a randomized trial comparing:
-
Observation alone
-
6 months of subcutaneous low-dose interferon (3 MU/day, three times weekly)
This study demonstrated statistically significant improvement in disease-free survival for up to 24 months .
How does hIFNα-2b affect pulmonary hypertension models?
Recombinant human interferon alpha-2b has demonstrated remarkable effects in experimental pulmonary hypertension models:
In both rat SU5416/hypoxia (SUH) and mouse hypoxia models, IFNα-2b:
-
Attenuated the development of pulmonary hypertension
-
Reversed established pulmonary hypertension
-
Reduced right ventricular systolic pressure and right ventricular hypertrophy
These effects were mediated through the type I interferon receptor (IFNAR), as mice lacking a subunit of IFNAR showed no protection from IFNα-2b treatment. Morphometric analysis revealed that IFNα-2b inhibited pulmonary vascular remodeling in both models and reversed remodeling in rats with established disease .
At the cellular level, immunohistochemical staining demonstrated decreased numbers of PCNA (proliferating cell nuclear antigen) and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positive cells in pulmonary arteriole walls. In vitro experiments confirmed that IFNα-2b inhibited proliferation of human pulmonary artery smooth muscle cells and affected both proliferation and apoptosis of human pulmonary artery endothelial cells .
What is the efficacy of hIFNα-2b in experimental endometriosis models?
Human interferon alpha-2b has shown promising results in experimental endometriosis:
A randomized, placebo-controlled, single-blind study in 30 Wistar female rats evaluated the effect of interferon alpha-2b (100,000 IU subcutaneously per rat, administered three times at 48-hour intervals) compared to saline controls. After measuring initial implant volumes (volume-1) and following 8 weeks of treatment, researchers found that:
-
In the interferon group, the final implant volume (volume-2) was significantly reduced compared to initial measurements
-
No significant volume change occurred in the control group
-
Both stromal and glandular tissues were significantly decreased in the interferon group compared to controls
Additional research has compared short versus long-term treatment protocols, concluding that longer treatment duration with human interferon-alpha-2b proves more effective in reducing surgically induced endometriosis in rats .
How is hIFNα-2b implicated in autoimmune diseases?
Human interferon alpha-2b has complex roles in autoimmune conditions. Chronic IFNα production (often associated with Coxsackie B virus infection) has been linked to insulin-dependent diabetes mellitus (IDDM) in both humans and animal models. Additionally, IFNα is involved in other autoimmune diseases including multiple sclerosis, rheumatoid arthritis, myasthenia gravis, and autoimmune hemolytic anemia .
The mechanism involves IFNα's effects on immune cells. IFNα cooperates with IL-6 to promote activation and maturation of antibody-secreting B cells. It also enhances cross-presentation of viral and self-antigens to CD4+ and CD8+ T cells, including auto-reactive T cells that may have escaped central and peripheral tolerance mechanisms .
These findings highlight the importance of proper spatial and temporal regulation of IFNα expression, as dysregulation can contribute to autoimmune pathologies. Understanding these mechanisms provides rationale for developing therapeutic strategies to suppress the detrimental effects of chronic exacerbated IFNα expression .
What role does hIFNα-2b play in chronic viral infections?
In chronic viral infections, human interferon alpha-2b functions as a key mediator of immunosuppression. Research has demonstrated that:
-
During HIV-1 infection, IFNα expression is markedly upregulated and contributes to disease progression
-
Over-expression of IFNα distinguishes pathogenic SIV infection of rhesus macaques from non-pathogenic infection of natural hosts (sooty mangabeys)
-
In human cytomegalovirus (HCMV) infection, chronic IFNα production:
These findings suggest that while acute IFNα responses are beneficial for controlling initial viral infections, persistent IFNα signaling may contribute to immune dysfunction and disease progression in chronic infections .
How do mutations in the hIFNα-2b gene impact immune function in cancer patients?
Research examining the impact of ionizing radiation on the hIFNα-2b gene in radiotherapy-treated cancer patients has revealed important insights into interferon signaling and cancer immunology. Studies show that IFN-γ signaling is reduced in B cells of patients with various cancers including breast cancer, melanoma, and gastrointestinal cancer .
This impaired interferon signaling represents an initial and constant mechanism of immune dysfunction in cancer patients. The findings suggest that compromised IFN response pathways may contribute to immune evasion by tumors and potentially impact the efficacy of immunotherapeutic strategies .
To study these mutations, researchers isolate genomic DNA from blood samples and amplify the hIFNα-2b gene (accession number NM-00605) using specific primers. A typical approach involves using primer sets (such as IFN1F and IFN2R) to amplify the gene with signal peptide, yielding approximately 700 bp fragments containing the 625 bp encoding region .
What methodological approaches are used to compare synthetic and recombinant hIFNα-2b?
Comparative analysis of synthetic and recombinant hIFNα-2b utilizes several standardized biological assays to ensure functional equivalence:
-
Antiviral activity assessment:
-
A549 cells are treated with varying concentrations of either synthetic or recombinant hIFNα-2b
-
Cells are challenged with encephalomyocarditis virus (ECMV)
-
Protection against cytopathic effect is measured in a dose-dependent manner
-
Statistical analysis is performed to detect significant differences in protective capacity
-
-
Antiproliferative effect evaluation:
-
Human T cells are stimulated with SEB and IL-2 in the presence of different concentrations of synthetic or recombinant hIFNα-2b
-
Growth inhibition is quantified through:
a) Incorporation of radioactive thymidine into genomic DNA
b) Direct cell counting after trypan blue staining
-
These methodologies have confirmed that synthetic hIFNα-2b exhibits the same biological properties and similar potency as commercial recombinant versions, validating its use in research applications including phage display library screening to identify peptide antagonists .
What are the emerging therapeutic applications of hIFNα-2b beyond oncology?
While human interferon alpha-2b has established applications in cancer treatment, recent experimental findings suggest potential therapeutic value in other disease areas:
-
Pulmonary hypertension: Evidence from animal models indicates that hIFNα-2b can both prevent and reverse pulmonary hypertension, suggesting potential application in this currently incurable disease .
-
Endometriosis: Multiple studies demonstrate efficacy in reducing endometriotic implants, pointing to possible therapeutic value for this common gynecological condition .
-
Autoimmune disease modulation: Understanding the dual role of IFNα in immune function suggests that targeted manipulation of IFNα signaling could provide novel treatment approaches for autoimmune conditions .
Future research should focus on optimizing dosing regimens, delivery methods, and combination therapies to maximize therapeutic efficacy while minimizing side effects in these emerging applications.
How might advanced genetic and proteomic approaches enhance hIFNα-2b research?
Advanced molecular techniques offer opportunities to deepen our understanding of hIFNα-2b function and improve its therapeutic applications:
-
CRISPR/Cas9 gene editing could enable precise manipulation of the hIFNα-2b gene and its regulatory elements to study structure-function relationships and develop optimized variants with enhanced therapeutic properties.
-
Single-cell RNA sequencing could provide detailed insights into cell-specific responses to hIFNα-2b treatment, enabling more targeted therapeutic approaches.
-
Proteomics approaches can identify the complete set of proteins regulated by hIFNα-2b signaling beyond the currently known 300+ genes, potentially revealing new therapeutic targets .
-
Development of biomarkers predicting response to hIFNα-2b therapy could enable personalized treatment approaches, improving efficacy and reducing unnecessary exposure in non-responders.
These advanced methodologies will facilitate more comprehensive understanding of hIFNα-2b biology and accelerate translation of research findings into clinical applications.
Product Science Overview
Interferon-alpha 2b (IFN-α2b) is a type I interferon, a group of cytokines known for their antiviral, antitumor, and immunomodulatory properties. IFN-α2b is produced by recombinant DNA technology, making it a valuable therapeutic agent in the treatment of various viral infections and cancers.
IFN-α2b is a protein consisting of 165 amino acids. It is produced by recombinant DNA technology, where the human IFN-α2b gene is inserted into bacterial or mammalian cells, which then express the protein. The recombinant protein is purified and formulated for therapeutic use.
- Antiviral Activity: IFN-α2b inhibits viral replication by inducing the expression of antiviral proteins in host cells. It enhances the immune response against viral infections by activating natural killer (NK) cells and macrophages.
- Antitumor Activity: IFN-α2b has antiproliferative effects on tumor cells. It inhibits cell division and induces apoptosis (programmed cell death) in cancer cells. It also enhances the immune system’s ability to recognize and destroy tumor cells.
- Immunomodulatory Effects: IFN-α2b modulates the immune response by increasing the expression of major histocompatibility complex (MHC) molecules, enhancing antigen presentation, and promoting the differentiation of T-helper cells.
- Chronic Hepatitis B and C: IFN-α2b is used to treat chronic hepatitis B and C infections. It reduces viral load and improves liver function by enhancing the immune response against the hepatitis viruses.
- Cancer Therapy: IFN-α2b is used in the treatment of certain cancers, including melanoma, hairy cell leukemia, and renal cell carcinoma. It helps to slow the growth of cancer cells and boosts the immune system’s ability to fight cancer.
- Other Viral Infections: IFN-α2b is also used to treat other viral infections, such as human papillomavirus (HPV) and respiratory syncytial virus (RSV).
IFN-α2b binds to specific receptors on the surface of target cells, triggering a signaling cascade that leads to the activation of various genes involved in antiviral, antiproliferative, and immunomodulatory responses. The binding of IFN-α2b to its receptor activates the JAK-STAT signaling pathway, which results in the transcription of interferon-stimulated genes (ISGs) that mediate its biological effects.
Recent advancements in the development of IFN-α2b include the creation of long-acting formulations to improve its half-life and therapeutic efficacy. These formulations involve modifications such as the addition of polyethylene glycol (PEG) or fusion with other proteins to extend the duration of action and reduce the frequency of administration .
