Recombinant Interferon Alpha-2a Research Articles

Deoxycoformycin-Induced Immunosuppression in Patients With Hairy Cell Leukemia

Immune function in patients with hairy cell leukemia (HCL) was examined serially during treatment with alternating monthly cycles of recombinant interferon alpha-2a and 2'-deoxycoformycin (dCF). At presentation, most patients had normal numbers of T lymphocytes and their cells had normal proliferative responses to mitogens [phytohemagglutinin (PHA) and concanavalin A (Con A)] and alloantigens. Patients had severe monocytopenia, decreased delayed-type hypersensitivity (DTH) reactions, and decreased peripheral blood natural killer (NK) activity. Treatment caused a profound decrease in all lymphocyte subpopulations. T cells were more affected than B cells or NK cells. Numbers of CD4+ and CD8+ lymphocytes decreased to levels less than 200 cells/microliters in all patients during treatment. This decrease in T cell number was associated with a marked decrease in proliferative responsiveness to PHA, Con A, and alloantigens. These abnormalities persisted throughout the 14 months of treatment and have continued for up to 6 months beyond discontinuation of treatment. NK cell activity increased during treatment, but cycled depending on the phase of treatment; highest activities were observed after interferon (IFN)-alpha and lower levels of activity were observed after dCF. DTH responses generally did not improve during therapy. Levels of IgM, IgG, IgA, and IgD did not change during treatment, but IgE levels rose in most patients. All immunosuppressive effects were attributable to dCF since patients receiving IFN-alpha 2a alone did not exhibit these same immunosuppressive effects, and patients receiving dCF alone after IFN failure exhibited similar abnormalities. Despite this severe immunosuppression from dCF, life-threatening opportunistic infections have not been observed in our patient population. Six patients developed localized Herpes zoster infection among 21 patients who had received dCF. Pending the results of long-term follow-up, we recommend that dCF be reserved for patients who have failed splenectomy and IFN therapy.

Effect of biphasic temperature regime on therapeutic recombinant protein production in the green alga Chlamydomonas reinhardtii

Microalgae are increasingly being considered for recombinant protein production because of low cultivation costs, absence of endotoxins and insusceptibility to human infectious agents. Despite these advantages, the yield of recombinant protein produced in microalgae is still low compared to more established expression systems and optimization at the genetic and cultivation levels is required for this new system to be economically viable. This study investigates the effect of biphasic temperature regimes on the yield of recombinant human interferon alpha 2a (IFN-α2a), a therapeutic protein known for its anti-cancer and anti-viral properties, produced by the model green alga Chlamydomonas reinhardtii (Cr.IFN-α2a). Biphasic growth is commonly employed to increase recombinant protein production in mammalian cell lines used for commercial production of therapeutic proteins, with a lowering of the temperature resulting in higher yields. In this study, lowering the temperature from 25 °C to 15 °C in mid-exponential growth phase increased the accumulation of Cr.IFN-α2a by 3.3-fold while it slowed down the growth of the three C. reinhardtii transgenic lines tested. In contrast, a rise of temperature from 25 °C to 35 °C accelerated cell growth, while negatively impacting the production of Cr.IFN-α2a. After a two-step chromatography purification, the Cr.IFN-α2a produced was estimated to be 53% pure with a yield of 90 μg/L of culture. The amino acid sequence of Cr.IFN-α2a was confirmed by mass spectrometry. However, the anti-viral activity of Cr.IFN-α2a was found to be 10 times lower than the human IFN-α2a standard produced using E. coli when challenged in a cytopathic effect (CPE) assay, likely due to the formation of aggregates. While the molecular mechanisms driving the accumulation of Cr.IFN-α2a at lower temperature remains unclear, our results support that reducing the temperature at the peak of expression is a valid strategy to increase the yield of recombinant Cr.IFN-α2a in C. reinhardtii.

Purification and Stability Expression of Open Reading Frames encoding Fusion and Non Fusion Human Interferon Alpha-2a produced in Methilotropic Yeast Pichia pastoris

Recombinant human interferon alpha-2a (hIFNα-2a) is therapeutic protein that widely used in hepatitis B/C and several cancer treatments. We developed higher molecular weight of hIFNα-2a to improve protein pharmacokinetic profile. The protein was designed as a fusion protein with human serum albumin as protein tag. The protein was produced in Pichia pastoris with 85 kDa in size. This research was aimed to purify, characterize and determine the stability expression of open reading frame (ORF) encoding Fusion and Non fusion forms of hIFNα-2a. The proteins were purified using affinity chromatography and characterized using SDS PAGE and Western Blotting methods. Protein recovery yield was determined by ELISA. Stability expression was applied in generation time until 90th generation. The results showed that the Fusion and Non fusion proteins were successfully purified with 74-79% of protein recovery. The proteins can be recognized by specific monoclonal antibody and verified as hIFNα-2a Fusion and Non fusion with 85 kDa and 19 kDa in size respectively. The expression stability showed that the proteins were still produced in Pichia pastoris until 90th generation time with no significant difference of expression level. To conclude, the expression level of ORFs encoding Fusion and Non fusion hIFNα-2a was stable until 90th generations.

Optimization of Expression Condition, Two Dimensional And Melting Point-Based Characterization of Recombinant Human Interferon Alpha-2a Fusion and Non Fusion Forms

Recombinant Human Interferon Alpha-2a (rhIFNα-2a) is a therapeutic protein that used in hepatitis and cancer treatments. In our previous research, we developed higher molecular weight of the protein through human serum albumin fusion. The fusion and non fusion form of rhIFNα-2a were produced in Pichia pastoriswith 86 kDa and 19 kDa in size respectively. In previous research, protein yield was not reproducible due to unoptimized expression conditions. This reseach was aimed to optimize expression condition process and to characterize the fusion and non fusion forms of rhIFNα-2a. The parameters to observe in overproduction include nutrient (media and methanol concentration) and non nutrient (temperature andincubation period). Affinity and size exclusion cromatographicwere compared in protein purification. BCA assay was used to determine quantity of protein. Protein characterization was conducted using two-dimensional SDS PAGE and denaturation analyses. The optimal condition of expression was achieved using complex media with 1% of methanol for 3 day incubation period at 25°C. The protein yield was reproducible and higher comparing to previous research. Affinity chromatography resulted in higher purity of the proteins comparing to size exclusions. Characterization using two dimensional gel analysis revealed that isoelectric point of rhIFNα-2a is 6.5 for fusion form and 6.0 for non fusion form. The melting points of fusion protein were 56°C and 62°C whilst that of non fusion was 56°C.

Overproduction, characterization and preliminary antiproliferative activity determination of non-tagged recombinant human interferon alpha-2a produced in Pichia pastoris

Herawati N, Wardiana A, Ningrum RA. 2017. Overproduction, characterization and antiproliferative activity determination of non-tagged recombinant human interferon alpha-2a produced in Pichia pastoris. Nusantara Bioscience 9: 97-101. Recombinant human interferon alpha-2a (rhIFNα-2a) has been widely used for antiviral, anticancer as well as immunomodulatory clinical therapy. In our previous research, we constructed the Open Reading Frame (ORF) encoding synthetic rhIFNα-2a to be in framed with N-terminal alpha factor secretion system in pPICZαB shuttle vector. This research covered overproduction, characterization of the protein and preliminary determination of biological potency assay by using the estrogen positive cell line MCF-7. To overproduce the protein, cultivation was performed in Buffered complex medium containing glycerol (BMGY) for 24 h and production was performed in Buffered complex medium containing methanol (BMMY) during 48 hours at 30°C. The recombinant protein was purified by affinity chromatography using blue sepharose resin. Analysis of amino acid sequence by using MS/MS2 mass spectrometry covered 21% of the total amino acid residues. To determine the initial biological activity, the effect of rhIFNα-2a on MCF-7 breast cancer cell line was studied in vitro. The anti-proliferative activity of hIFNα-2a was performed by 3-[4.5-dimethyltiazol-2il]-2.5 diphenylltetrazolium bromide (MTT) assay. The result showed that the protein is able to inhibit MCF-7 proliferation and the reduction of cell growth was dose-dependently. To monitor the protein production, we performed stability expression assay as well. The result informed that the expression of ORF still stable until 60th generation

No Development of Neutralizing Antibodies Against Recombinant Interferon-Alpha in Ph-Negative Myeloproliferative Neoplasms - a Prospective Study

BackgroundTreatment of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPNs) with recombinant pegylated interferon alpha2a/b (rIFN-alpha) has proven effective. It is well known that prolonged therapy with recombinant type 1 interferons (IFN-alpha and IFN-beta) may induce neutralizing antibodies (nAbs) against the drug leading to treatment failure. Most data on type 1 IFN immunogenicity are available from studies of patients with multiple sclerosis treated with rIFN-beta, and patients with hepatitis C treated with rIFN-alpha. A few reports have demonstrated nAbs in MPN patients not responding adequately to rIFN-alpha treatment, but the phenomenon has not been thoroughly investigated in MPNs.Patients and MethodsNewly diagnosed patients with MPNs enrolled in the Danish multicenter trial - DALIAH (Low-dose rIFN-alpha versus Hydroxyurea in The Treatment of Ph-Negative MPNs) were included. Patients were randomized to treatment with either rIFN-alpha 2a or 2b at a starting dose of 45 and 35 mikrograms once weekly, respectively. The occurrence of neutralizing Abs (nAbs) against rIFN-alpha was investigated at baseline, month 12 and month 24 by reporter gene assays (iLiteTM alphabeta and iLiteTM antialpha, Biomonitor A/S, Copenhagen, Denmark). JAK2 V617F quantitative mutation analyses were performed as previously described (Larsen TS, BJH 2007). Statistical analyses were performed using STATA version 9.0.ResultsNinety-two patients on sustained treatment with rIFN-alpha2a (n=48) and rIFN-alpha2b (n=44) for 12 months were analyzed for this study. Forty-five patients had ET, 39 patients had PV and 8 patients had proliferative PMF. Thirty-six out of 39 (92%) PV patients, 22 out of 45 (49%) ET patients and 4 out of 8 (50%) PMF patients were JAK2V617F mutated. Hematological responses at 12 months: ET: 67% CR, 29 % PR; PV: 64% CR, 31% PR (ELN 2009 criteria); PMF: 50% had at least a minor response (EUMNET).The median serum concentration of bioactive IFN-alpha at 12 months was 12,4 (range <2,4-86,4) and 2,6 (range <2,4-12,8) IU/mL serum, for patients treated with rIFN-alpha 2a and -2b respectively. No significant association between hematological or molecular response and serum IFN-alpha activity was found. Serum from 92 patients was analyzed at 12 months and 33 patients were analyzed at both 12 and 24 months and no occurrence of nAbs was seen during treatment. Twenty-four patients had pre-treatment levels of IFN nAbs measured. Notably, one patient was tested positive for the presence of nAbs before rIFN-alpha exposure. This autoAb-positive patient remained positive throughout the study and has shown low IFN serum activity (< 2,4 IU/mL) and only partial hematological and molecular response after 24 months of treatment.ConclusionsDevelopment of nAbs in MPN patients completing treatment for 12 months with rIFN-alpha seems exceedingly rare as no patients, neither complete responders nor patients not meeting criteria for complete hematological remission developed nAbs after 12 (24) months of therapy. Its apparent rarity does not justify a routine investigation of nAbs in patients not responding to rIFN-alpha treatment. There was no significant correlation between serum concentration of rIFN-alpha 2a and -2b and clinical or molecular responses. The intriguing finding that one of 24 patients had pre-existing cross reacting nAbs against rIFN-alpha 2a and 2b before commencing rIFN-alpha treatment is interesting and was associated with an insufficient response. DisclosuresOff Label Use: Recombinant interferon-alpha 2a and -2b in the treatment of chronic Philadelphia-negative myeloproliferative neoplasms.. El Fassi:Novartis Denmark: Honoraria, Other: Have conducted an educational session for Novartis Denmark, regarding MPNs and ruxolitinib, for this a honorarium was received.. Bjerrum:Bristoll Myers Squibb, Novartis and Pfizer: Other: educational activities. Hasselbalch:Novartis: Research Funding. Bendtzen:Pfizer: Honoraria; Eurodiagnostica AB: Equity Ownership; Novo-Nordisk: Equity Ownership.

AOP2014/P1101, a Novel Peg-Proline-Interferon Alpha (IFNa) 2b, Specifically Targets JAK2V617F-Positive Polycythemia Vera (PV) Cells

BackgroundPegylated Interferon alpha (PEG-IFNa) has proven clinical and molecular efficacy in the treatment of Bcr-Abl negative myeloproliferative neoplasms. Indeed PEG-IFNa induces complete hematological remissions but also molecular responses demonstrated by the reduction of the JAK2V617F (JAK2VF) allele burden. AOP2014/P1101 is a next generation long-acting PEG-IFNa-2b, recently shown to be safe and well tolerated in phase I-II studies in PV patients (pts) (Gisslinger at al, 2012). In addition, molecular responses (MR) were reported in these early phase trials with 50% of pts achieving partial MR at month 18. Furthermore, one pt with a baseline JAK2VF allelic burden of 22% achieved complete MR after 36 weeks of therapy suggesting a specific effect of AOP2014/P1101 on JAK2VF-positive cells. AimTo study the differential impact of AOP2014/P1101 on the proliferation and differentiation of wild type versus JAK2VF hematopoietic cells. Materials and methodsWe studied the effects of AOP2014/P1101 at different concentrations consistent with assumed exposure in patients (0.5 and 2µg/ml), compared with standard recombinant interferon alpha-2a (rIFNa-2a) at 700 U/ml in human cell lines in liquid suspension assay, and in normal and PV samples in methylcellulose assays. A) Two different human JAK2VF cell lines were studied: HEL characterized by the presence of multiple copies of JAK2VF, and UKE-1 which harbors 2 copies of JAK2VF. Cell lines were grown in presence or absence of drugs and living cells were counted each day during 3 days. B) Normal hematopoietic progenitors derived from cord blood and primary cells from 4 PV patients were studied in clonogenic assays (Methocult, Stemcell technologies©). In PV pts samples the presence of endogenous erythroid colonies (EECs) was determined in cultures without erythropoietin (EPO). Genotyping of the colonies was performed by picking the colonies, extracting the DNA and testing for the presence of JAK2VF using the JAK2 Mutascreen kit (Qiagen©). ResultsIn both HEL and UKE-1 JAK2 mutated cell lines AOP2014/P1101 exhibited a dose-dependent anti-proliferative effect which was comparable to that of rIFNa-2a. AOP2014/P1101 at 0.5µg/ml and 2µg/ml induced a 9%, and 41% inhibition of HEL cells proliferation, respectively (resp). Same doses induced an 18% and 35% inhibition of UKE-1 cells proliferation, resp. A similar antiproliferative effect was observed with rIFNa-2a with 38%, and 36% inhibition of HEL, and UKE-1 cells proliferation, resp.In normal CD34+ hematopoietic progenitors derived from cord blood, AOP2014/P1101 induced a small decrease in the number of erythroid colonies (median reduction by 7%, 15% and 14% with AOP2014/P1101 0.5 µg/ml, 2 µg/ml, and rIFNa-2a, resp). Myeloid colonies were almost unaffected by AOP2014/P1101 (no modification at 0.5µg/ml, and 9% reduction at 2µg/ml) while a reduction by 18% was obtained using rIFNa-2a, confirming the low level of toxicity of AOP2014/P1101 on normal hematopoietic progenitors.In contrast, AOP2014/P1101 drastically reduced the growth of JAK2VF erythroïd progenitors in all the PV samples tested. A 41%, and 62% median reduction of the number of EPO-stimulated colonies was observed with AOP2014/P1101 0.5µg/ml, and 2µg/ml, respectively. A more striking effect was observed on colonies grown in the absence of EPO: the number of EECs was reduced by 82% with AOP2014/P1101 2µg/ml compared to untreated cells. This result suggested that AOP2014/P1101 inhibited more efficiently JAK2-mutant hematopoietic progenitors. This hypothesis was confirmed by the study of the JAK2 genotype at the clonogenic level: a median 2-fold increase in the ratio of wild type to mutant colonies with 2µg/ml AOP2014/P1101 was observed, suggesting that AOP2014/P1101 does specifically target PV erythroid progenitors harboring the JAK2VF mutation. In addition, in one patient with both homozygous and heterozygous JAK2VF colonies, eradication of homozygous colonies was achieved with AOP2014/P1101, suggesting that JAK2VF homozygous progenitors are even more sensitive to AOP2014/P1101. ConclusionOur results show that AOP2014/P1101, a novel form of PEG-IFNa-2b, can specifically target PV JAK2VF hematopoietic progenitors while sparing wild type cells. These results are in line with the clinical efficacy with low hematologic toxicity reported in early phase trials with AOP2014/P1101. Disclosures:Cassinat:AOP Orphan: Research Funding. Klade:AOP Orphan Pharmaceuticals AG: Employment. Zahriychuk:AOP Orphan Pharmaceuticals AG: Employment. Kiladjian:AOP Orphan: Honoraria, Research Funding.

Improved detection of variants in recombinant human interferon alpha-2a products by reverse-phase high-performance liquid chromatography on a core–shell stationary phase

The detection of variants is one of the important aspects in quality control of recombinant DNA drugs. In this study, a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method with fluorescence detection is described for the separation of interferon alpha-2a (rhIFN α-2a) from several product related variants. The methodology employed a core–shell C18 column with a linear gradient elution of 0.2% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 1.0mL/min, and the temperature of the column was maintained at 60°C. The method was validated in terms of linearity, sensitivity, intra- and inter-day variations. Compared to the European Pharmacopeia RP-HPLC method of rhIFN α-2a analysis, this new method can separate N-methionylated variant in both drug substance and finished product, and analyze the variants in untreated, oxidized sample and slightly degraded samples more efficiently. In conclusion the method has an improved capability to detect variants in rhIFN α-2a products.

The ETR (End Treatment Response) of Chinese Brand Human recombinant interferon alpha 2a plus Ribavirin in chronic hepatitis C patients selected from Combined Military Hospitals in Pakistan

This Study was carried out to determine the End of Treatment Response (ETR) of a Chinese Source Human Recombinant Interferon Alpha 2a and Ribavirin in chronic hepatitis C affected patients, selected from different combined military hospitals in Pakistan. In this study hepatitis affected noncirrhotic patients from different combined military hospitals in Pakistan were selected from September 2010 to April 2011. Sixty seven patients were selected based on the inclusion exclusion criteria. Fifty seven patients completed the 24 weeks therapy for Hepatitis C. The Patients (n=57) had persistently raised serum aminotransferase (ALT), positive HCV antibodies by 3rd generation ELISA, positive HCV RNA by polymerase chain reaction. Study group patients underwent 24 weeks therapy with a Chinese source Interferon and Ribavirin and subsequently followed up for the End of Treatment Response by the assay of HCV RNA by polymerase chain reaction at 24 weeks. Patients of chronic hepatitis C (44 males and 13 females) had age range 20-56 years. After 24 weeks of therapy with a Chinese source Interferon and Ribavirin, 73.7% patients showed a favourable ETR manifested by Negative HCV RNA polymerase chain reaction.Treatment with a Chinese source Interferon and Ribavirin combination therapy for 24 weeks produces a favourable End of Treatment Response in patients of chronic hepatitis C from different military hospitals in Pakistan.

Refolding of Recombinant Human Interferon α-2a fromEscherichia coliby Urea Gradient Size Exclusion Chromatography

Protein refolding is still a puzzle in the production of recombinant proteins expressed as inclusion bodies (IBs) in Escherichia coli. Gradient size exclusion chromatography (SEC) is a recently developed method for refolding of recombinant proteins in IBs. In this study, we used a decreasing urea gradient SEC for the refolding of recombinant human interferon alpha-2a (rhLFNalpha-2a) which was overexpressed as IBs in E. coli. In chromatographic process, the denatured rhLFNalpha-2a would pass along the 8.0-3.0 M urea gradient and refold gradually. Several operating conditions, such as final concentration of urea along the column, gradient length, the ratio of reduced to oxidized glutathione and flow rate were investigated, respectively. Under the optimum conditions, 1.2 x 10(8) IU/mg of specific activity and 82% mass recovery were obtained from the loaded 10 ml of 1.75 mg/ml denatured protein, and rhLFNalpha-2a was also purified during this process with the purity of higher than 92%. Compared with dilution method, urea gradient SEC was more efficient for the rhl FNalpha-2a refolding in terms of specific activity and mass recovery.

Pegylated human interferon alpha 2a does not induce depression-associated changes in mice

Interferon (IFN) alpha proteins are proinflammatory cytokines having immunomodulating and antiviral properties. States during which cytokine systems are activated (e.g., during viral infection or during treatment of chronic hepatitis C and various malignancies with IFN alpha, etc.) can be associated with depression-like syndromes or even full-blown depressive episodes. Therefore, the role of IFN alpha and other cytokines in the pathogenesis of depressive disorder (“cytokine hypothesis of depression”) has been assessed for many years with contradictory results. We have investigated whether intraperitoneal administration of high doses (up to 600 µg/kg body weight) of pegylated, recombinant human IFN alpha 2a in mice induces changes known to be associated with depression using three different readouts: behavior in a model of despair (Porsolt swim test), presence of anhedonia (sucrose preference test), and sensitivity of the hypothalamic–pituitary–adrenal system (dexamethasone suppression test). We also assessed potential IFN-induced changes in gene expression in the liver. In none of the performed experiments, depression-associated effects could be found despite very high serum levels of IFN-induced antiviral activity compared to levels measured in hepatitis C virus (HCV) patients treated routinely with pegylated recombinant human IFN alpha 2a. The lack of such expected effects is probably due to the fact that pegylated human recombinant IFN alpha 2a does not activate the murine class I IFN receptor. Our results do not support the hypothesis that administration of recombinant pegylated human IFN alpha to mice produces a robust model of depression.

Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-interferon antibody fragment and human interferon alpha-2A.

Recombinant human interferon alpha-2A (rhIFN-alpha-2A) has been crystallized in complex with the recombinantly produced Fab fragment of a therapeutic monoclonal antibody (MEDI545; IgG1/kappa) which targets several human interferon alpha subtypes. This constitutes the first reported crystals of a human type I interferon bound to an antibody. The orthorhombic crystals belonged to either space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 134.82, b = 153.26, c = 163.49 A. The diffraction of the crystals extended to 3.0 A resolution. The asymmetric unit contained two Fab-rhIFN-alpha-2A complexes. This corresponded to a crystal volume per protein weight (V(M)) of 3.02 A(3) Da(-1) and a solvent content of 59.3%. The corresponding three-dimensional structure is expected to shed light on the mechanism of action of MEDI545 and the molecular basis of its specificity.

Use of Recombinant Human Interferon Alpha-2a in the Management of a Dog With Epitheliotropic Lymphoma

An 8-year-old, mixed-breed dog with preputial epitheliotropic lymphoma was initially treated with cyclophosphamide, vincristine, and prednisolone. A short-term partial response was followed by disease progression after 4 weeks. Recombinant human interferon alpha-2a was administered starting at week 7. The interferon therapy resulted in rapid resolution of clinical signs and a 10-week disease-free interval. The lymphoma recurred at 17 weeks and did not respond to rescue chemotherapy. Additional oral lesions were treated with localized radiotherapy followed by increased dosages of interferon. This additional interferon treatment resulted in another 12 weeks of stable disease.

Characterization of anti-interferon-alpha antibodies appearing during recombinant interferon-alpha 2a treatment.

Patients with malignant midgut carcinoid tumours received recombinant interferon-alpha 2a (rIFN-alpha 2a) or rIFN-alpha 2a and chemotherapy (streptozocin and doxorubicin) for 6 months, and then rIFN-alpha 2a alone. Antibodies, mainly of IgG type, binding to rIFN-alpha 2a developed in nine of 22 patients (41%), as determined by immunoassay. In seven patients, antibodies also neutralized the biologic (anti-viral) activity of rIFN-alpha 2a. Anti-IFN-alpha 2a antibodies were equally frequent in both sexes and treatment groups, but were not observed in those patients (n = 8) that had previously received other types of IFN. Antibodies appeared after a median of 6 months of rIFN-alpha 2a treatment and had a median duration of 6 months. The anti-IFN-alpha 2a antibody titres declined with time with no obvious relation to change of therapy, also during continued IFN-alpha 2a treatment. High titres of neutralizing antibodies appeared to impair anti-tumoural effects in individual potential responders. Anti-IFN-alpha 2a antibodies further examined in six patients bound to native IFN-alpha subtypes present in both allogenic and autologous leucocyte IFN-alpha. Such autoantibodies neutralized the biologic activity of autologous IFN-alpha in two patients, and in a third were partially neutralizing.

Solid-Phase PEGylation of Recombinant Interferon α-2a for Site-Specific Modification: Process Performance, Characterization, andin VitroBioactivity

'Solid-phase' PEGylation, in which a conjugation reaction attaches proteins to a solid matrix, has distinct advantages over the conventional, solution-phase process. We report a case study in which recombinant interferon (rhIFN) alpha-2a was adsorbed to a cation-exchange resin and PEGylated at the N-terminus by 5, 10, and 20 kDa mPEG aldehydes through reductive alkylation. After PEGylation, a salt gradient elution efficiently purified the mono-PEGylate of unwanted species such as unmodified IFN and unreacted PEG. Mono-PEGylation and purification were integrated into a single, chromatographic step. Depending on the molecular weight of the mPEG aldehyde, the mono-PEGylation yield ranged from 50 to 65%. Major problems associated with the solution-phase process such as random or uncontrollable multi-PEGylation and post-PEGylation purification difficulties were overcome. N-terminus sequencing and MALDI-TOF mass spectrophometry confirmed that the PEG molecule was conjugated only to the N-terminus. A cell proliferation study indicated reduced antiviral activity of the mono-PEGylate compared to that of the unmodified IFN. As higher molecular weight PEG was conjugated, in vitro bioactivity and antibody binding activity, as measured by a surface plasmon resonance biosensor, decreased. Nevertheless, trypsin resistance and thermal stability were considerably improved .

P004 Changes in thymic recent output function in patients with acute myeloid leukemia

The influence of recombinant human interferon alpha 2a (rIFN alpha), recombinant human interferon beta 1 (rIFN beta), and recombinant human interferon gamma (rIFN gamma) on human dermal microvascular endothelial cells (HDMEC) cultured in vitro was studied in various rIFN concentrations (0.1 lU/ml-104 IU/ml) over 2,3,4,6,8, and 10 d. Cell morphology and ultrastructure, cell proliferation, expression of class II alloantigens (HLA-DR and HLA-DQ), and intercellular adhesion molecule-1 (ICAM-1) were investigated using an in vitro technique established in our laboratory. All rIFN tested induced alterations of typical HDMEC morphology; the cells became spindle-shaped and fibroblastoid, although they maintained their endothelial cell marker expression. Also, all IFN dose- and time-dependently inhibited the proliferation of HDMEC in vitro (rIFN alpha> beta> gamma), whereby rIFN alpha exerted the strongest growth-inhibitory effect. Alkaline phosphatase anti-alkaline phosphatase (APAAP) immunocytochemistry of the cultured cells showed dose- and time-dependent stimulation of ICAM-1 and class II antigen expression only by rIFN gamma (HLA-DR> HLA-DQ), rIFN alpha and beta did not exert any immunomodulatory activity on HDMEC in vitro. These results indicate that HDMEC are an important target for the action of IFN. Besides growth inhibition, it seems that rIFN gamma in particular may be involved in the modulation of leucocyte adhesion and trafficking by altering the immunophenotype of the endothelial cell population.

A retrospective analysis of malignant pleural mesothelioma patients treated either with chemotherapy or best supportive care between 1990 and 2005: A single institution experience

The aim of this study was to investigate the efficacy and safety profile of chemotherapy (CT) compared to best supportive care (BSC) in patients with histopathologically confirmed diffuse malignant pleural mesothelioma (DMPM). A total of 161 patients between 1990 and 2004 treated either with CT (109 patients) or BSC (52 patients) depending on patients choice were evaluated in this analyses. Chemotherapy protocols included a combination of cisplatin, mitomycin C and recombinant interferon alpha 2a (CM-In), or cisplatin, mitomycin C and ifosfamide (CMI), or cisplatin and gemcitabine (CG). We found a significant difference in the median survivals of the patients with CT compared to BSC, 11.3 months versus 8.0. Objective response rate was 28/109 (25.7%) with 3.7% of complete response rate. Stable disease rate was 39/109 (35.8%). There was a significant difference between median survivals of patients with objective response (17 months) and median survivals of patients with progressive diseases (6 months) and also with stable diseases (16 months). There was a significant difference between the stable disease and the progressive disease. Stages 3 and 4 patients of epithelial cell type having received chemotherapy live longer than those not having received chemotherapy (12 months versus 4). There was no significant difference between the survivals of the different chemotherapy regimens. The toxicity with CT regimens were mild and well-tolerated. We conclude that CT prolongs survival compared to BSC in patients with DMPM. Survivals of patients with objective response prolong considerably with CT compared BSC. We observed that stages 3 and 4 patients with epithelial cell type got benefit from CT. Especially, of epithelial cell type stages 1 and 2 should receive multimodal treatment.

Structural, Kinetic, and Thermodynamic Analysis of the Binding of the 40 kDa PEG−Interferon-α2a and Its Individual Positional Isomers to the Extracellular Domain of the Receptor IFNAR2

Type-I Interferons exert antiviral and antiproliferative activities through the binding to a common cell surface receptor comprising two subunits, IFNAR1 and IFNAR2. Human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) used to treat various cancers and viral diseases including Hepatitis B/C infections. To significantly improve the pharmacological properties of the drug, a pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side-chain of IFNalpha(2a). Here, we report the detailed structural, kinetic, and thermodynamic analysis of the binding to the extracellular domain of the receptor IFNAR2 of (40)PEG-IFNalpha(2a) and its isolated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively, in comparison with unmodified IFNalpha(2a). Our binding studies, using the surface plasmon resonance technique, show that the pegylation does not abolish the binding to the receptor, but significantly reduces the affinity mainly due to a change of the association rate. The results are supported by modeling and simulation of the binding, using Self-Avoiding-Walk calculations for the polymer conformations. A correlation between the structural parameters and the kinetic and thermodynamic parameters of the binding of the positional isomers could be established. For the Isomer-K31 and -K164, the PEG-polymer attachment point is located in proximity to the binding interface, and the isomers display affinity in the range 150-520 nM in an enthalpy-driven binding process. In contrast for the Isomer-K134, -K131, -K121, and -K70, the PEG-polymer is attached remotely from the binding interface, and the isomers exhibit a higher affinity (32-76 nM) in an entropy-driven binding process. This study constitutes an essential collection of knowledge on which the interaction of (40)PEG-IFNalpha(2a) and its positional isomers with its cellular receptors can be better understood.

Viral kinetics of HCV during daily-dose treatment with interferon alfacon-1 vs recombinant interferon alpha-2a: Gaetano Scotto, Infectious Diseases University, Foggia Italy; Fazio Vincenzina, II Laboratory OORR, Foggia Italy

Viral kinetics of HCV during daily-dose treatment with interferon alfacon-1 vs recombinant interferon alpha-2a: Gaetano Scotto, Infectious Diseases University, Foggia Italy; Fazio Vincenzina, II Laboratory OORR, Foggia Italy

Influence of human recombinant interferon-alpha2a (rhIFN-alpha2a) on altered lymphocyte subpopulations and monocytes in Behcet's disease.

In Behçet's disease (BD), several abnormalities of lymphocyte subpopulations have been described. Standard treatment comprises immunosuppressive drugs. We successfully treated 50 patients with ocular BD with interferon-alpha2a (IFN-alpha2a) (response rate 92%), although this is counterintuitive because IFN-alpha is immunostimulatory and can sometimes even induce autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. The aim of the present study was to elucidate the immunomodulatory effects that IFN-alpha might exert on peripheral blood mononuclear cells (PBMC) in BD by examining changes in the distribution of lymphocyte subpopulations under IFN-alpha2a treatment. Fourteen patients with ocular BD were evaluated before and at weeks 4 and 24 of IFN-alpha treatment and compared with 10 healthy controls. PBMC were stained with monoclonal antibodies and measured by flow cytometry. Compared with the controls there is a significant elevation of monocytes (CD14(+)), CD8(+)/gammadelta T cells, CD3(+)/gammadelta T cells, natural killer (NK) cells (CD56(+)/CD16(+)) and activated/regulatory T cells (CD4(+)/CD25(+) and CD8(+)/CD25(+)) in patients with active BD before treatment with IFN-alpha2a. Numbers of naïve T cells (CD8(+)/CD45(+)RA(+)/RO(-), CD4(+)/CD45(+)RA(+)/RO(-)) were significantly lower. Under therapy, NK cells, CD8(+)/gammadelta T cells and CD3(+)/gammadelta T cells decreased significantly, whereas B cells increased. The previously reduced expression of HLA class I on monocytes in HLA-B51-positive patients rose to levels comparable to HLA-B51-negative patients. These results implicate the participation of NK cells and gammadelta T cells, especially CD8(+)/gammadelta T cells, in the pathogenesis of BD and may explain one mechanism by which IFN-alpha2a exerts therapeutic effects. Alternatively, they may result indirectly from remission induction by IFN-alpha2a. The reduced expression of HLA class I on monocytes in HLA-B*51-positive patients might reflect an impaired expression of and antigen presentation by HLA-B*51.