Human Interferon-α2b Research Articles

Purification and conformational properties of a human interferon alpha2b produced in Escherichia coli.

Recombinant human interferon alpha2b was expressed intracellularly in Escherichia coli as insoluble aggregates using a new expression vector, and was purified to homogeneity using essentially two-step chromatographic procedures, i.e. immobilized metal-ion-affinity chromatography and reversed-phase HPLC. The established purification process is highly reproducible and leads to a total recovery of approx. 12% with a specific biological activity of higher than 1x10(8) i.u./mg of protein, which is comparable with the international requirement for interferon alpha2b. For purified protein we report conformational stability as a function of pH and temperature using differential scanning calorimetry and CD. Thermal unfolding as a function of pH showed only one endotherm at a temperature higher than 45 degrees C, and was reversible at pH 2-3.75 and irreversible at pH 4-10. At pH 7.0, the most stable condition, the conformational stability depends on protein concentration and ionic strength. The highly helical secondary structure is very conserved over the whole pH range studied, including at high temperatures.

Selective expression of nonsecreted interferon by an adenoviral vector confers antiproliferative and antiviral properties and causes reduction of tumor growth in nude mice.

Replication-deficient adenoviruses expressing human interferon-alpha2b (HuIFN-alpha2b) or the hybrid IFN-alpha2alpha1 or those with the secretory signal deleted, whose express is driven by the alpha-fetoprotein (AFP) promoter, were constructed and characterized. Synthesis of IFN protein and secretion or intracellular retention were tested by Western blotting and immunoassay. Expression of IFN by the recombinant adenoviruses was restricted to cells that constitutively express AFP. In these cells, expression of both secreted and nonsecreted recombinant IFN resulted in inhibition of cell proliferation, resistance to viral infection, induction of major histocompatibility complex (MHC) class I expression, increased apoptosis, and activation of an IFN-stimulated response element (ISRE)-containing promoter. Also, the induction of protein kinase R (PKR), increased phosphorylation of Stat1, and accumulation of hypophosphorylated pRb were observed for both the secreted and nonsecreted IFN, suggesting that the nonsecreted IFN may act through a similar pathway. Hep3B cells, an AFP-positive line derived from a patient with hepatocellular carcinoma (HCC), were injected subcutaneously (s.c.) into athymic nude mice to generate established tumors. Intratumoral injection of recombinant adenoviruses expressing secreted as well as the nonsecreted IFN caused suppression of tumor growth. As the AFP promoter is activated in many HCC cells but is silent in normal cells, these constructs may be useful in restricting IFN effects to the tumor cells while reducing toxicity to the neighboring tissues.

Laparoscopic intraperitoneal injection of human interferon-α2b in the treatment of pelvic endometriosis: a new modality

Background: Some reports have stated that an immunologic alteration plays a role in the development and progression of endometriosis. Once endometrial cells are implanted ectopically, they may produce additional changes through a defect in cellular or humoral immunity. Natural killer cell, cytokines, and peritoneal macrophages could determine the role of progression of the disease. Therapeutic manipulation of the immune system in patients with endometriosis may be worth mention. Braum et al (1992) suggested that macrophage activators such as interferons (IFNs) could be useful in addition to hormone suppression therapy, to control and eliminate the growth of endometrial ectopic tissue.Objective: To use for the first time laparoscopic intraperitonal injection of human IFN-α2b in the treatment of pelvic endometriosis.Methods: Twenty-five infertile women were enrolled in the study. They had pelvic endometriosis. The diagnosis was based on laparoscopy, and CA 125 was measured in all cases. They were classified as the following: Stage II—mild endometriosis (5 cases), Stage III—moderate endometriosis (10 cases), and Stage IV—severe endometriosis. We proposed a protocol of dosage system. For Stage I—we used 3 million IU of IFN-α2b, for Stage II—6 million units, for Stage IV—12 million IU. We directly injected the dosage through laparoscopy. Objective measurement for evaluating the implant growth depends on the measurement of the largest diameter and change of the degree of the stage of endometriosis. Second-look laparoscopy was done 3 months later in all patients and CA 125 measurement was also done. All the patients were evaluated clinically for dyspareunia, painful defecation, and progressive dysmenorrhea. Vaginal examination was done for cul-de-sac induration, fixed ovarian masses, and uterosacral ligament nodularity.Results: There was a statistically significant (P <0.05) decrease in all symptoms and signs after treatment; the decrease was proportional to the size and diameter of large implant and the degree of endometriosis. There was a statistically significant decrease in the level of CA 125 after treatment (P <0.001). On laparoscopy, there was diminution of the stage of disease. Grade II changed to grade I, grade III changed to grade II, and grade IV changed to grade III. Complete disappearance of disease was noted in 2 cases in grade II (20%) and in 2 cases in grade III (10%). In grade IV, there was no reported case of complete disappearance of endometriosis. Cumulative pregnancy rate in all three stages after treatment was 33.3%. No reported side effects of treatment were noted.Conclusions: Human IFN-α2b immune stimulatory therapy significantly reduces the symptoms of endometriosis. Reduce the stage of the disease, reduce CA 125 level, reduce the size of implant, and improve the pregnancy rate, so we can say that this line is a break treatment in the field of endometriosis. More studies are needed; closed, double-blind, controlled, randomized studies are needed for better evaluation of the results of treatment.

Zinc mediated dimer of human interferon-α 2b revealed by X-ray crystallography

Background The human α-interferon (huIFN-α) family displays broad spectrum antiviral, antiproliferative and immunomodulatory activities on a variety of cell types. The diverse biological activities of the IFN-α's are conveyed to cells through specific interactions with cell-surface receptors. Despite considerable effort, no crystal structure of a member of this family has yet been reported, because the quality of the protein crystals have been unsuitable for crystallographic studies. Until now, structural models of the IFN-α's have been based on the structure of murine IFN-β(muIFN-β). These models are likely to be inaccurate, as the amino acid sequence of muIFN-β differs significantly from the IFN-α's at proposed receptor-binding sites. Structural information on a huIFN-α subtype would provide an improved basis for modeling the structures of the entire IFN-α family. Results The crystal structure of recombinant human interferon-α 2b (huIFN-α 2b) has been determined at 2.9 å resolution. HuIFN-α 2b exists in the crystal as a noncovalent dimer, which associates in a novel manner. Unlike other structurally characterized cytokines, extensive interactions in the dimer interface are mediated by a zinc ion (Zn 2+). The overall fold of huIFN-α 2b is most similar to the structure of muIFN-β. Unique to huIFN-α 2b is a 3 10 helix in the AB loop which is held to the core of the molecule by a disulfide bond. Conclusions The structure of huIFN-α 2b provides an accurate model for analysis of the >15 related type I interferon molecules. HuIFN-α 2b displays considerable structural similarity with muIFN-β, interleukin-10 and interferon- γ, which also bind related class 2 cytokine receptors. From these structural comparisons and numerous studies on the effects of mutations on biological activity, we have identified protein surfaces that appear to be important in receptor activation. This study also reveals the potential biological importance of the huIFN-α 2b dimer.

Expression and Purification of Recombinant, Glycosylated Human Interferon Alpha 2b in Murine Myeloma NSo Cells

We have expressed recombinant human interferon-α2b in mammalian cells and isolated cell lines constitutively secreting very high levels of biologically active protein. The expression system takes advantage of the strong human cytomegalovirus immediate early promoter in mouse myeloma NSo cells and glutamine synthetase as a selectable marker; spontaneous mutants with amplified gene copy numbers were selected by growth of primary transfectants in the presence of methionine sulfoximine. Using this procedure, we have isolated a recombinant NSo cell line which secretes human interferon at the rate of 20 μg/106cells/24 h and accumulates up to 120 μg/ml (∼2.4 × 107U/ml) following prolonged undiluted culture. The interferon (IFN) could be efficiently purified on a polyclonal bovine anti-human IFNα specific antibody column and the glycosylation pattern was found to be similar to that of nonrecombinant IFNα2b purified from virus-induced human Namalwa cells. The biological activity of the recombinant material was indistinguishable from that of natural IFN from Namalwa cells, and the specific antiviral activity, as assayed on human HeLa cells challenged with encephalomyocarditis virus, was 2 × 108IU/mg, similar to that of nonrecombinant IFN preparations. This represents the highest reported level of glycosylated, recombinant IFN expression in a stable mammalian system and is a significant advance in the large-scale production of these clinically important cytokines.

Inhibitory Effect of Interferon-α on Respiratory Burst and Glucose Metabolism in Phagocytic Cells

Anion superoxide (O2-) production and glucose metabolism was studied in murine macrophages following in vivo or in vitro treatment with human recombinant interferon-alpha 2b (IFN-alpha 2b). The PMA-dependent O2- production was inhibited by IFN-alpha 2b in a concentration- and time-dependent manner. NO2- production by macrophages in culture was slightly inhibited (about 16%) at 30 nM IFN-alpha and a clear decrease (35%) was obtained with 150 nM IFN-alpha. Low doses (0.3 and 3 nM IFN-alpha) had no effect. Also, IFN-alpha 2b inhibited lactate release and 3H2O production from [2-3H] and [3-3H]glucose in macrophages isolated after in vivo treatment for 24 h. The data support an inhibitory role of IFN-alpha in the metabolic activation of macrophages and suggest a putative mechanism for the inhibition of some macrophage functions as previously reported.

Lack of effect of recombinant human interferon-alpha 2b on expression of 17-1A antigen on human colon cancer cells.

The effects of recombinant human interferon alpha (rHuIFN-alpha 2b) on cell growth, expression of antigenic receptor sites (r) and the affinity constant (Ka) of monoclonal antibody CO 17-1A IgG were studied on two human colorectal cancer cell lines, CX-1 and SW 1116. Cells were incubated with varying concentrations of rHuIFN-alpha 2b prior to exposure to 125I-labeled 17-1A IgG at 37 degrees C following which r and Ka were determined by means of Scatchard plots. Varying concentrations of rHuIFN-alpha 2b had significant growth inhibitory effects on CX-1 and SW 1116 cells, which were time and concentration dependent, but no effects on expression of r and Ka compared to controls. Our data indicate that rHuIFN-alpha 2b does not invariably increase the expression of tumor-associated antigens and that this effect may be independent of its antiproliferative activity. The in vitro response or lack thereof of neoplastic cells to rHuIFN-alpha 2b may be useful to identify those patients who potentially might gain from a clinical course of rHuIFN-alpha 2b for either therapeutic or diagnostic purposes.

Home therapy with recombinant interleukin-2 and interferon-α2b in advanced human malignancies

The safety, tolerance, and clinical effects of a home therapy regimen of recombinant human interleukin-2 (rIL-2) and interferon-α2b (IFN-α2b) self injected subcutaneously have been assessed in 35 patients with advanced cancer refractory to standard therapy. 52 treatment cycles were given, each consisting of a 2-day rlL-2 pulse of 9·0 million IU/m 2 every 12 h, followed by 6 weeks of rIL-2 1·8 million IU/m 2 twice daily for 5 days per week and of IFN-α2b 5·0 million U/m 2 thrice a week. The main adverse effects were fever, chills, nausea, anorexia, and hypotension and were limited to WHO grades of severity I and II in 29 of 35 patients. No treatment-related deaths occurred. The response rates among patients with renal-cell carcinoma were similar to those reported for high-dose intravenous regimens of interleukin-2 that are toxic and have to be given in hospital.