Correlation between the anti-virus-induced cytopathic effect activity of interferon-alpha subtypes and induction of MxA protein in vitro

There are several interferon-alpha (IFN-alpha) subtypes. Mechanism of disparity in biological effects among members of IFN-alpha subtypes remains unexplained. Biological activity of IFN-alpha is mediated in part by induction of intracellular antiviral proteins. We studied whether differences in biologic effects of IFN-alpha subtypes may rely on their antiviral protein inducing effect. Intracellular induction of MxA protein and anti-virus-induced cytopathic effect (CPE) activity of 11 IFN-alpha subtypes in human amnion WISH cells have been studied. MxA protein quantitation in cell lysates was performed by immunochemiluminescence assay and anti-virus-induced CPE activity was assessed by protection against vesicular stomatitis virus (VSV)-induced CPE. Range of MxA values was high when cells were treated with 10 and 100 IU/ml of each IFN-alpha subtype. Levels of MxA correlated with anti-VSV-induced CPE obtained with 10 IU/ml IFN-alpha subtype. Together our data show a disparity in MxA-inducing activity of IFN-alpha subtypes and suggest that differences in anti-VSV-induced CPE of IFN-alpha subtypes in WISH cells can be related to their different ability to induce MxA.     

Characterization of the murine alpha interferon gene family

Mouse and human genomes carry more than a dozen genes coding for closely related alpha interferon (IFN-alpha) subtypes. IFN-alpha, as well as IFN-beta, IFN-kappa, IFN-epsilon, and limitin, are thought to bind the same receptor, raising the question of whether different IFN subtypes possess specific functions. As some confusion existed in the identity and characteristics of mouse IFN-alpha subtypes, the availability of data from the mouse genome sequence prompted us to characterize the murine IFN-alpha family. A total of 14 IFN-alpha genes were detected in the mouse genome, in addition to three IFN-alpha pseudogenes. Four IFN-alpha genes (IFN-alpha1, IFN-alpha7/10, IFN-alpha8/6, and IFN-alpha11) exhibited surprising allelic divergence between 129/Sv and C57BL/6 mice. All IFN-alpha subtypes were found to be stable at pH 2 and to exhibit antiviral activity. Interestingly, some IFN subtypes (IFN-alpha4, IFN-alpha11, IFN-alpha12, IFN-beta, and limitin) showed higher biological activity levels than others, whereas IFN-alpha7/10 exhibited lower activity. Most murine IFN-alpha turned out to be N-glycosylated. However, no correlation was found between N-glycosylation and activity. The various IFN-alpha subtypes displayed a good correlation between their antiviral and antiproliferative potencies, suggesting that IFN-alpha subtypes did not diverge primarily to acquire specific biological activities but probably evolved to acquire specific expression patterns. In L929 cells, IFN genes activated in response to poly(I*C) transfection or to viral infection were, however, similar.     

Mutation of the IFNAR-1 receptor binding site of human IFN-alpha2 generates type I IFN competitive antagonists

Type I interferons (IFNs) are multifunctional cytokines that activate cellular responses by binding a common receptor consisting of two subunits, IFNAR-1 and IFNAR-2. Although the binding of IFNs to IFNAR-2 is well characterized, the binding to the lower affinity IFNAR-1 remains less well understood. Previous reports identified a region of human IFN-alpha2 on the B and C helices ("site 1A": N65, L80, Y85, Y89) that plays a key role in binding IFNAR-1 and contributes strongly to differential activation by various type I IFNs. The current studies demonstrate that residues on the D helix are also involved in IFNAR-1 binding. In particular, residue 120 (Arg in IFN-alpha2; Lys in IFN-alpha2/alpha1) appears to be a "hot-spot" residue: substitution by alanine significantly decreased biological activity, and the charge-reversal mutation of residue 120 to Glu caused drastic loss of antiviral and antiproliferative activity for both IFN-alpha2 and IFN-alpha2/alpha1. Mutations in residues of helix D maintained their affinity for IFNAR-2 but had decreased affinity for IFNAR-1. Single-site or multiple-site mutants in the IFNAR-1 binding site that had little or no detectable in vitro biological activity were capable of blocking in vitro antiviral and antiproliferative activity of native IFN-alpha2; i.e., they are type I IFN antagonists. These prototype IFN antagonists can be developed further for possible therapeutic use in systemic lupus erythematosus, and analogous molecules can be designed for use in animal models.     

Comparison of augmentation of human natural killer cell cytotoxicity by interferon-alpha subtypes

The capacity of 3 interferon-alpha (IFN-alpha) subtypes, alpha 1, alpha 2 and alpha 4, to augment human natural killer cytotoxicity after exposure in vitro was shown to be dose-dependent and to differ according to subtype. With 10(2) IU/ml, the lowest IFN concentration used, stimulation of NK activity by IFN-alpha 2 was consistently and significantly greater than by IFN-alpha 4 or IFN-alpha 1. An IFN-alpha analogue in which arginine and lysine residues 121 and 122 were replaced by 2 leucines was generated by site-directed in vitro mutagenesis of the IFN-alpha 4 gene; at equivalent concentrations of antiviral activity, this analogue was 10-fold less effective in NK stimulation. There was a lack of correlation between NK-stimulatory and other activities of the IFN-alpha subtypes and the mutant, suggesting that different biological activities may be mediated by different regions of the IFN-alpha molecule.     

Binding and activity of all human alpha interferon subtypes

Vertebrates have multiple genes encoding Type I interferons (IFN), for reasons that are not fully understood. The Type I IFN appear to bind to the same heterodimeric receptor and the subtypes have been shown to have different potencies in various experimental systems. To put this concept on a quantitative basis, we have determined the binding affinities and rate constants of 12 human Alpha-IFN subtypes to isolated interferon receptor chains 1 and 2. Alpha-IFNs bind IFNAR1 and IFNAR2 at affinities of 0.5–5 μM and 0.4–5 nM respectively (except for IFN-alpha1 – 220 nM). Additionally we have examined the biological activity of these molecules in several antiviral and antiproliferative models. Particularly for antiproliferative potency, the binding affinity and activity correlate. However, the EC₅₀ values differ significantly (1.5 nM versus 0.1 nM for IFN-alpha2 in WISH versus OVCAR cells). For antiviral potency, there are several instances where the relationship appears to be more complicated than simple binding. These results will serve as a point of reference for further understanding of this multiple ligand/receptor system.     

Characterization and comparison of the properties of sarcoma cell lines in vitro and in vivo

To expand the available tools for investigating human sarcomas, we characterized the primary properties of 22 common, uncommon, and newly characterized sarcoma cell lines representing eight different histological subtypes. Throughout the characterization process we noticed that in vitro markers and assays are poor indicators of tumorigenicity and that generated xenografts often bear little resemblance to the original histopathology. In vitro properties examined included morphology, proliferation rate, cell cycle characteristics, invasiveness, and immunohistochemical expression of p53 and phospho-AKT. In vivo properties examined included days to tumor formation in NOD/SCID mice, xenograft morphology in several locations and immunohistochemical expression of Ki67, p53 and phospho-AKT. We believe that such an in depth comparison of a large cohort of sarcoma cell lines will be useful in both designing and interpreting experiments aimed at elucidating both the molecular biology and efficacy of therapeutic agents in sarcomas. However, that data generated also suggests a small set of sarcoma cell lines may be inappropriate for generalizations regarding biological behavior of specific sarcoma subtypes. Integration of functional genomics or other more sophisticated assays of cell lines may help bridge the differences in vitro and in vivo .     

Effects of interferon-alpha on human B cell responsiveness: biphasic effects in cultures stimulated with Staphylococcus aureus.

Although interferon-alpha (IFN-alpha) has been found to be involved in the immune regulation in vivo, the effects of IFN-alpha on human B cells have not yet been clarified because of conflicting results in the literature. The present study therefore examined the effects of several subtypes of IFN-alpha (natural, alpha 1, alpha 2a, alpha 2b) on B cell responsiveness in detail by comparing different experimental conditions. Highly purified B cells from normal human individuals were cultured with Staphylococcus aureus (SA) + IL-2 or with immobilized anti-CD3-activated T4 cells in the presence or absence of IFN-alpha. IFN-alpha enhanced the immunoglobulin (Ig) production induced by immobilized anti-CD3-activated T4 cells. By contrast, IFN-alpha (5-50,000 IU/ml) suppressed the Ig production induced by SA + IL-2. The suppression by IFN-alpha was dependent on the concentration of SA. The inhibitory effects of IFN-alpha in SA-stimulated cultures were exerted in the first 72 hr of cultures and required the presence of IL-2, whereas IFN-alpha enhanced the maturation of B cells when it was added after 72 hr of cultures. The suppressive effects of IFN-alpha were overcome by addition of immobilized anti-CD3-preactivated T cells that had been treated with mitomycin C, but not by the addition of fresh T cells or soluble factors produced by activated T cells. Of interest, IFN-alpha did not inhibit the expression of IL-2R, but inhibited that of intercellular adhesion molecule-1 (ICAM-1) on B cells after stimulation with SA + IL-2, suggesting that the suppressive effects of IFN-alpha might be related to the regulation of B cell-B cell contacts through ICAM-1. There was no significant difference in effects on B cells among various subtypes of IFN-alpha. These results suggest that the effects of IFN-alpha on human B cell responsiveness may be different depending on the nature of stimulation. Moreover, the data indicate that IFN-alpha enhances the differentiation of activated B cells irrespective of the activation signals.     

Distinction of two subtypes of human leukocyte interferon (IFN-alpha) on B cell activation. B cell proliferation by two subtypes of IFN-alpha

We examined the effect of interferon (IFN), with particular emphasis on the effects of the two subtypes of IFN-alpha (IFN-alpha A and IFN-alpha B) on the B cell proliferation induced by Staphylococcus aureus Cowan I bacterium (SpA Col). An increase of SpA Col-induced proliferation was observed in the presence of 100 to 1000 U/ml of IFN-alpha, but a decrease of SpA Col-induced proliferation was observed in the presence of 1000 to 10,000 U/ml of IFN-beta. The two subtypes of IFN-alpha had different effects on cell proliferation; a significant enhancement was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha A, but inhibition was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha B. In the reconstitution test of the two subtypes of IFN-alpha, the boundary between enhancement and inhibition of SpA Col-induced proliferation was revealed when the proportion of IFN-alpha A and IFN-alpha B (IFN-alpha A:IFN-alpha B) ranged between 8:2 and 9:1. Toward the SpA Col-induced responses, the above IFN were all found to act on B cells directly, independent of the presence of T cells. Proliferative responses by IFN-alpha and IFN-alpha A, however, were shown to be slightly dependent on the presence of monocytes. The lymphocyte proliferation induced by other mitogens (phytohemagglutinin, concanavalin A, pokeweed mitogen, and protein A of S. aureus) were all inhibited by the above IFN.     

Phagocytic activity and protein content of human monocytes cultivated in the presence of various homologous interferon preparations

We have studied the effect of different types of interferons (IFN) on phagocytic activity and protein content when present during in vitro cultivation of human blood monocytes. Recombinant IFN-alpha and partially and highly purified leukocyte IFN preparations blocked the increase in phagocytic activity and protein content that occurs during in vitro cultivation of human monocytes. Fibroblast IFN blocked the increase in protein content, but did not significantly alter the phagocytic activity. IFN-gamma slightly enhanced phagocytic activity and protein content, while lymphoblastoid IFN preparations had no effect. The phagocytic activity and protein content of monocytes matured in vitro without IFN and then treated with IFN for 24 h was also tested. Phagocytosis via the non-specific receptors and the protein content was reduced by treatment of these cells with the IFN-alpha preparations. On the other hand Fc-receptor mediated phagocytosis was stimulated by IFN-gamma. Our data indicate that IFN effects on monocytes in culture varies depending on type and possibly subtypes of IFNs, and also on the timing of the treatment.     

Two interferons alpha influence each other during their interaction with the extracellular domain of human type interferon receptor subunit 2

The interaction between two human interferons alpha (IFN-alphas) and the extracellular (EC) domain of human type I IFN receptor subunit 2 (IFNAR2) was analyzed. Previous experiments using Daudi cells showed that IFN-alpha21b and some IFN-alpha hybrids (made from IFN-alpha2c and 21b) competed poorly for the IFN-alpha2b binding site. This study examined the causes of the poor competition between these IFN-alphas. IFN-alpha2c and the IFN hybrid CM3 {IFN-alpha21b(1-75)(81-95)/IFN-alpha2c(76-80) (96-166), Y86K} were selected for this study based on their cell binding and biological properties. Competitive binding ELISA, native electrophoresis followed by Western blot, electrospray ionization mass spectrometry (ESI-MS), surface plasmon resonance biosensor (SPR) analysis, as well as neutralization of antiproliferative activities on Daudi cells in the presence of soluble IFNAR2-EC show evidence that each of the described IFN-alpha subtypes affected the binding of the other IFN-alpha to IFNAR2-EC by affecting the stability of the complex, i.e., dissociation of the complex. Moreover, native electrophoresis with different IFNAR2-EC mutants showed that IFN-alpha2c and CM3 utilize different amino acids in the binding domain of IFNAR2-EC. In addition to that, analytical ultracentrifugation (AUC) revealed differences in the oligomeric state of the two studied interferons. Our results demonstrated that two individual IFN-alphas interact differentially with IFNAR2-EC and influence each other during this interaction. This study contributes to the understanding of the mutual interaction between multiple IFN-alpha subtypes during the competition for binding to the receptor.     

Enhanced circulating half-life and antitumor activity of a site-specific pegylated interferon-alpha protein therapeutic

Recombinant interferon alpha-2 (IFN-alpha2) has proven useful for treating a variety of human cancers and viral diseases. IFN-alpha2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. It is possible to extend the circulating half-life of IFN-alpha2 by random modification of lysine residues in the protein with polyethylene glycol (PEG); however, such preparations have heterogeneous structures and low specific activities, and may not provide optimal therapeutic benefits to patients. A long-acting, site-specific, monoPEGylated IFN-alpha2 protein has now been created by targeted attachment of a 20 kDa or a 40 kDa maleimide-PEG to a cysteine analogue of IFN-alpha2, M111C. In vitro bioactivities of the purified 20 kDa and 40 kDa PEG-M111C proteins were within 2- to 3-fold of those of wild type IFN-alpha2 and 7- to 10-fold better than that of a 40 kDa PEG IFN-alpha2 protein created using nontargeted, amine-PEGylation methodology. The 20 kDa and 40 kDa PEG-M111C proteins demonstrated 26- to 38-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. The 20 kDa PEG M111C protein inhibited growth of human NIH:OVCAR-3 cells transplanted into nude mice by >90%, as measured by tumor size, tumor weight, and number of animals with detectable tumors at necropsy, and was significantly more effective than a comparable dose of IFN-alpha2. These data extend our previous findings that bioactivity of IFN-alpha2 can be largely preserved by targeted attachment of PEG moieties to nonessential sites in the protein and provide evidence that site-specific PEGylated IFN-alpha2 proteins possess enhanced tumoricidal properties in vivo.     

Biased agonism at chemokine receptors

In the human chemokine system, interactions between the approximately 50 known endogenous chemokine ligands and 20 known chemokine receptors (CKRs) regulate a wide range of cellular functions and biological processes including immune cell activation and homeostasis, development, angiogenesis, and neuromodulation. CKRs are a family of G protein-coupled receptors (GPCR), which represent the most common and versatile class of receptors in the human genome and the targets of approximately one third of all Food and Drug Administration-approved drugs. Chemokines and CKRs bind with significant promiscuity, as most CKRs can be activated by multiple chemokines and most chemokines can activate multiple CKRs. While these ligand-receptor interactions were previously regarded as redundant, it is now appreciated that many chemokine:CKR interactions display biased agonism, the phenomenon in which different ligands binding to the same receptor signal through different pathways with different efficacies, leading to distinct biological effects. Notably, these biased responses can be modulated through changes in ligand, receptor, and or the specific cellular context (system). In this review, we explore the biochemical mechanisms, functional consequences, and therapeutic potential of biased agonism in the chemokine system. An enhanced understanding of biased agonism in the chemokine system may prove transformative in the understanding of the mechanisms and consequences of biased signaling across all GPCR subtypes and aid in the development of biased pharmaceuticals with increased therapeutic efficacy and safer side effect profiles.     

Excipient exchange in the comparison of preparations of the same biologic made by different manufacturing processes: An exploratory study with recombinant human growth hormone (rhGH)

Demonstrations of bio-similarity between subsequent entry (follow-on) biologics and innovator’s formulated drug products may depend upon methods that either remove excipients completely or allow the exchange of excipients to give equivalent formulations. Excipient exchange through dialysis is perhaps the simplest of such methods but its use has been hotly debated. This debate, in the absence of published data, has relied largely on theoretical considerations. This study presents data that indicate that excipient exchange can allow comparisons of different formulations of the same therapeutic protein. The use of excipient exchange to and from one concentration of mannitol to another or to a mixture of glycine and mannitol was reproducibly demonstrated for recombinant human growth hormone (rhGH). We show that marketed rhGH products from several different manufacturers exhibit differences in conformational stability when compared directly. These differences, however, are shown to be the result of differences in formulation rather than in the drug substance itself and were removed through excipient exchange. The data presented, therefore, also indicate that failure to assure a common excipient background can lead to erroneous conclusions about the similarities and differences in the physico-chemical properties of two preparations of the same therapeutic protein made by different manufacturing processes.     

Identification of residues of the IFNAR1 chain of the type I human interferon receptor critical for ligand binding and biological activity

The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.     

Transcriptome analyses reveal molecular mechanisms underlying phenotypic differences among transcriptional subtypes of glioblastoma

Using molecular signatures, previous studies have defined glioblastoma (GBM) subtypes with different phenotypes, such as the proneural (PN), neural (NL), mesenchymal (MES) and classical (CL) subtypes. However, the gene programmes underlying the phenotypes of these subtypes were less known. We applied weighted gene co-expression network analysis to establish gene modules corresponding to various subtypes. RNA-seq and immunohistochemical data were used to validate the expression of identified genes. We identified seven molecular subtype-specific modules and several candidate signature genes for different subtypes. Next, we revealed, for the first time, that radioresistant/chemoresistant gene signatures exist only in the PN subtype, as described by Verhaak et al, but do not exist in the PN subtype described by Phillips et al PN subtype. Moreover, we revealed that the tumour cells in the MES subtype GBMs are under ER stress and that angiogenesis and the immune inflammatory response are both significantly elevated in this subtype. The molecular basis of these biological processes was also uncovered. Genes associated with alternative RNA splicing are up-regulated in the CL subtype GBMs, and genes pertaining to energy synthesis are elevated in the NL subtype GBMs. In addition, we identified several survival-associated genes that positively correlated with glioma grades. The identified intrinsic characteristics of different GBM subtypes can offer a potential clue to the pathogenesis and possible therapeutic targets for various subtypes.     

Regulation of macrophage growth and antiviral activity by interferon-gamma

Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.