Immunogenicity of an interferon-beta1a product.

In order to determine whether Blastoferon®, a biosimilar interferon (IFN)- beta 1a formulation, shares epitopes with other known IFN-beta products, a series of neutralization bioassays were performed with a set of well-characterized anti-IFN- beta monoclonal antibodies and human sera (World Health Organization Reference Reagents). The bioassay was the interferon-induced inhibition of virus cytopathic effect on human cells in culture (EMC virus and A-549 cells). Computer-calculated results were reported as Tenfold Reduction Units (TRU)/ml. To further assess Blastoferon® immunogenicity, in vivo production of anti-IFN beta antibodies was determined in sera of patients included in the pharmacovigilance plan of Blastoferon® by the level of IFN- beta 1a binding antibodies (by enzyme immunoassay -EIA) and neutralizing antibodies (in the Wish-VSV system). The highly characterized neutralizing monoclonal antibodies A1 and A5 that bind to specific regions of the IFN- beta molecule reacted positively with the three beta 1a IFNs: Blastoferon®, Rebif®, and the IFN- beta WHO Second International Standard 00/572. As expected, the non-neutralizing monoclonal antibodies B4 and B7 did not neutralize any of the IFN- beta preparations. The commercially available monoclonal antibody B-02 reacted essentially equally with Rebif® and Blastoferon®. The WHO Reference Reagent human serum anti-IFN- beta polyclonal antibody neutralized all the IFN- beta products, whereas the WHO Reference Reagent human serum anti-IFN-alpha polyclonal antibody G037-501-572 appropriately failed to react with any of the IFN- beta products. On the basis of in vitro reactivity with known, well-characterized monoclonal and polyclonal antibody preparations, Blastoferon® shares immunological determinants with other human interferon- beta products, especially IFN- beta 1a. In vivo antibodies were detected by EIA in 72.9% of 37 chronically treated multiple sclerosis patients, whereas neutralizing antibodies were found in 8.1% of them. Blastoferon® appears to have immunological characteristics comparable to other IFN- beta 1a products.

Repression of interferon action: induced dedifferentiation of embryonic cells.

Cells cultured from young (6-day) chicken embryos differ from those of older (13-day) embryos in having a greater susceptibility to infection by certain viruses and a considerably lesser sensitivity to the action of interferon. These circumstances parallel those observed in the intact embryo. The addition of a small percentage of cells from young embryos alters the response of cells cultured from older embryos by increasing viral susceptibility sevenfold and decreasing sensitivity to interferon 25-fold. We postulate that a repressor which inhibits the expression of interferon in older embryonic cells is elaborated by cells from young embryos.

Cytotoxicity of human beta-interferon for differentiating leukemic HL-60 cells.

We examined the effect of human interferon (HuIFN) -alpha and -beta on the proliferation and differentiation induced by dimethyl sulfoxide (DMSO) of HL-60 human promyelocytic leukemia cells into mature granulocytes. Neither HuIFN-alpha nor -beta, alone, from 1 to 1000 IU/ml, nor the homologous mock HuIFN preparations affected HL-60 cell differentiation or proliferation. Whereas the combination of HuIFN-alpha (10 to 1000 IU/ml) with DMSO also did not affect the proliferation or differentiation of HL-60 cells, the addition of HuIFN-beta (1000 IU/ml) and DMSO (1.25%) to growing cultures reduced cell viability as much as 14% of that observed for cells treated with DMSO alone or to 4% of that observed for either untreated cells or those treated with HuIFN-beta alone. The cytotoxic effect declined with decreasing concentrations of HuIFN-beta. The cytotoxic effect of DMSO and HuIFN-beta was exerted only as cells began to differentiate. Removal of HuIFN-beta at Day 2 did not reverse the cytotoxic effect, and addition of HuIFN-beta at Day 2 did not inhibit cell proliferation. Addition of HuIFN-beta to postmitotic cells on Day 4 after DMSO treatment did not affect proliferation but did slow differentiation. The cytotoxic and antidifferentiative effects of naturally produced HuIFN-beta were confirmed with highly purified recombinant HuIFN-beta. Undifferentiated HL-60 cells were resistant to the antiviral effects of HuIFN-beta, requiring 4 to 6 times the concentration to protect 50% of the cells against vesicular stomatitis virus as that needed to produce a cytotoxic or antidifferentiative effect. The profoundly cytotoxic effects of HuIFN-beta reported here may provide a model to study this interferon in combination with inducers of leukemic cell differentiation as a possible strategy in cancer therapy.

Phase I trial of an oral immunomodulator and interferon inducer in cancer patients.

Imiquimod [1-(2-methylpropyl)-1H-imidazo[4,5c]quinolin-4-amine] is a compound of low molecular weight that, when administered p.o., induces interferon-alpha in several animal species and inhibits tumor growth in mice. To determine maximum tolerated dose, toxicity, and biological response in humans, a phase I clinical trial was conducted with 14 eligible cancer patients who received 100-500 mg imiquimod p.o. either once or twice weekly. Imiquimod induced interferon-alpha in serum in 10 of 19 doses of 200-300 mg. Interferon serum levels peaked 8-24 h after treatment and reached a maximum of 23,000 IU/ml in one patient. Significant mean increases (P < 0.01) in serum beta 2-microglobulin (1.5-fold), serum neopterin (3.5-fold), and 2-5A synthetase activity in peripheral blood mononuclear cells (7.9-fold) indicated that 200-300 mg imiquimod had biological and immunological activity in all evaluable patients. Increases in serum interferon, beta 2-microglobulin, and neopterin correlated significantly with dose (P < 0.001). No patient developed measurable antibody to interferon-alpha. Dose-limiting side effects included fatigue, malaise, fever, headache, and lymphocytopenia; no hepatic or renal toxicity or other hematological changes exceeded the normal range. Patients tolerated weekly doses of up to 500 mg, with the longest treatment lasting 4 weeks at 200 mg weekly. Twice-weekly doses up to to 300 mg were tolerated, with the longest twice-weekly treatments being 200 mg for 9 weeks and 100 mg for 25 weeks. No clinical responses were observed. Imiquimod, as an oral inducer of interferon, may have therapeutic usefulness in human cancers, viral infections, and other diseases. However, before initiation of phase II trials, additional work will be required to establish a tolerated dose and schedule for continued administration.

Cytotoxicity of Clostridium difficile toxin A for human colonic and pancreatic carcinoma cell lines.

The use of bacterial exotoxins may constitute novel adjuncts to treatment of gastrointestinal tract malignancies. Clostridium difficile toxin A was evaluated for its cytotoxic effect in vitro on 24 human cell lines and strains including carcinomas of the colon, pancreas, prostate, lung, breast, and lymphoid malignancies, as well as nonmalignant tissues. All nine colon and five pancreas cell lines were extraordinarily sensitive to the cytotoxic effect of Clostridium difficile toxin A at very low concentrations. This effect, which occurred rapidly and was dose dependent, was observed in all cells of seven colon and two pancreas cell lines at concentrations as low as 1-5 ng/ml (10(-12) to 10(-11) M), whereas cells derived from other sites required 60 to greater than 500 ng/ml to achieve an equivalent effect. The data suggest that Clostridium difficile toxin A may have potential therapeutic value in the treatment of some gastrointestinal tract cancers.

The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems.

The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of expression of neutralizing potency and substantiate the earlier operational 10/1 LU/ml approach recommended by the World Health Organization. The accompanying paper relates neutralization results to the sensitivity of the bioassay to IFN and describes the rationale for a recommended unit of antibody neutralization.

The neutralization of interferons by antibody. II. Neutralizing antibody unitage and its relationship to bioassay sensitivity: the tenfold reduction unit.

The importance of establishing a common method of reporting neutralizing antibody levels is emphasized by the fact that patients injected repeatedly with a human interferon (HuIFN) may develop such antibodies that can abrogate the beneficial effects of the treatment. The earlier experimental and theoretical constructs of Kawade led to certain recommendations by the World Health Organization (WHO) concerning the methodology of neutralization tests and how to report the resultant data. A WHO international collaborative study on two human sera with antibodies against HuIFN-alpha and HuIFN-beta provided the opportunity not only to test the theoretical concepts concerning the neutralization reaction with data obtained in different bioassay systems in different laboratories but also to obtain enough data points for statistical evaluation with bioassays having a great range of sensitivity to IFN. The analyses substantiate and extend the original conclusions of Kawade that the neutralization follows the reaction mode of low-affinity antibody, in accord with the constant proportion hypothesis by which antibody reduces IFN activity in a set ratio of added/residual biologically active IFN, a consequence of the low molar concentration of free IFN at the neutralization end point. The present results support the recommendation that the preferred way to state the index of neutralization of antibodies is a titer (t), calculated by the formula t = f(n - 1)/9, where f is the reciprocal of the antibody dilution achieving the end point, and n is the IFN concentration measured in that day's titration. The tenfold reduction unit (TRU) of neutralization is proposed for use in expressing the quantity, or unitage, of IFN neutralizing antibody. The utility of its application is explained. The use of the index of neutralization described and the proposed derivative term of antibody unitage, TRU, should help make the results from different laboratories employing different bioassay systems more readily comparable and interpretable, provided the bioassays are sufficiently sensitive to IFN.

Quantitative liquid-phase chemiluminescence ELISA: detection of subtle epitope differences in HuIFN-beta.

We have developed a new liquid-phase, chemiluminescence-enhanced, inhibition ELISA (LP-CEI-ELISA) to explore the binding sites recognized by two neutralizing monoclonal antibodies (mAb) against recombinant human IFN-(beta)ser (rHuIFN-(beta)ser). In this assay, the initial antigen-antibody reaction occurs in solution under more physiologic conditions than in a standard solid-phase ELISA. Subsequently, the reaction mixture is applied to a membrane that is exposed to a second, peroxidase-labeled mAb, chemiluminescent reagents are added, and the membrane is photographically recorded. Competitive inhibition of binding of a second, labeled mAb by the first mAb decreases the signal detected. Two well-characterized mAb A1 and A7, have been shown to recognize distinct epitopes on rHuIFN-(beta)ser and to neutralize its antiviral and antiproliferative activity (Proc. Natl. Acad. Sci. USA 88, 4040-4044, 1991). In conventional solid-phase ELISA, mAb A1 does not inhibit the binding of A7 to rHuIFN-(beta)ser, but we observed partial inhibition in the new liquid-phase assay. In contrast, A7 did not inhibit the binding of A1, consistent with the solid-phase ELISA results. This observation suggests that in the LP-CEI-ELISA, A1 and A7 may recognize epitopes differently than in solid-phase assays. Thus, the LP-CEI-ELISA, which is simple, sensitive, and quantifiable, appears also to be able to detect subtle, conformational differences of epitopes not evident in a standard solid-phase ELISA.

Mapping of IFN-beta epitopes important for receptor binding and biologic activation: comparison of results achieved using antibody-based methods and alanine substitution mutagenesis.

The epitopes important for receptor binding and activation of human interferon-beta1a (IFN-beta1a) were mapped with monoclonal antibodies (mAb), grouped on the basis of their specificity and ability to neutralize biologic activity, and alanine scanning mutagenesis (ASM). The binding properties of nine mAb were defined, using ASM-IFN-beta mutants having alanine substituted at targeted, surface-exposed residues. The results were correlated with the mAb neutralizing potency. Of six mAb that bound either at or adjacent to the IFNAR-2 receptor chain binding site defined by the ASM epitopes, only three had measurable neutralizing activity. Two of these inhibited IFN-beta/IFNAR-2 complex formation, suggesting that steric hindrance of receptor binding constitutes their mechanism of neutralization. However, two mAb that bound to sites remote from the IFNAR-2 binding site on IFN-beta also inhibited IFN-beta/IFNAR-2 complex formation and demonstrated potent neutralizing activity. Thus, neutralizing mAb may employ mechanisms other than steric blockade to inhibit directly the binding of receptor by cytokine, limiting their usefulness as tools to define precise receptor-ligand interaction sites.

A novel bioassay for the determination of neutralizing antibodies to IFN-beta1b.

We have adapted the new MxA gene-induction bioassay to measure neutralizing antibodies to interferon-beta1b (IFN-beta1b, the active ingredient in Betaseron) in sera from patients treated with Betaseron. This antibody assay has been validated to quantify neutralizing titers of 1:20 and above, with a precision of +/- 0.20 in log10. We have used this MxA gene-induction antibody assay to reinvestigate serum samples from multiple sclerosis (MS) patients treated with Betaseron. The titers measured were closely comparable to those obtained in antiviral assays. Data obtained by both methods show that neutralizing antibodies may appear and subsequently disappear over time in the sera of some patients treated with Betaseron. Sera from some patients contain binding antibodies to IFN-beta1b. It was shown that binding antibody titers do not correlate quantitatively or qualitatively with neutralizing antibody titers, and indeed, a number of patients develop high levels of binding antibodies but never form measurable levels of neutralizing antibodies.

Synergy, activity and tolerability of zidovudine and interferon-alpha in patients with symptomatic HIV-1 infection: AIDS Clincal Trial Group 068.

Thirty-four subjects with symptomatic HIV-1 infection, p24 antigenaemia, and CD4 cell counts > 200/mm3 were randomly assigned to receive treatment with either zidovudine (ZDV) orally, interferon-alpha (IFN-alpha) subcutaneously, or both at respective low (200 mg ZDV/ 2 million international units IFN-alpha (MIU)), middle (400 mg/4 MIU) or high (600 mg/6 MIU) daily dose levels for 12 weeks. Thereafter, all patients received combination therapy at the initially assigned dose level to a total of 96 weeks. This design permitted analysis by the combination index (CI) method, which demonstrated antiretroviral synergy between ZDV and IFN-alpha with respect to p24 antigen suppression. Over the first 12 weeks, combination therapy was acceptably tolerated, more so than IFN-alpha monotherapy, and it was significantly more active in suppressing antigenaemia than either of the monotherapies. Similarly, the high-dose combination was the most active dose level over weeks 12 to 96. Combination ZDV/IFN-alpha at the optimal dose level defined by this trial merits further study. In addition, the CI design strategy employed here may be useful for the investigation of new antiretroviral combinations.

The effects of interferon-alpha on the production and action of other cytokines.

Various interferon-alpha (IFN-alpha) preparations, either as individual subtypes or natural mixtures, induce or inhibit expression of several other cytokines, as well as cytokine receptors and chemokines. The cytokines and receptors reportedly affected by IFN-alpha include interleukin-1 (IL-1), IL-2, IL-6, IL-8, IL-1 receptor, IL-1 receptor antagonist, tumor necrosis factor, tumor necrosis factor receptor, and IFN-gamma, all of which may amplify the effects of IFN-alpha treatment. The mechanism by which IFN-alpha induces expression of these cytokines is not clear. Some of the therapeutic and toxic effects associated with IFN-alpha therapy may be caused by the induction or inhibition of other cytokines and their respective cellular effects. Side effects including fever, anorexia, and fatigue can be caused by one or more of the cytokines induced by IFN-alpha. The response of different cell types, normal or malignant, to cytokines can vary. Such variation in cell type-specific responses may contribute to the diverse array of physiologic effects associated with IFN-alpha therapy. Further research is required to systematically uncover how other cytokines, receptors, or cellular factors contribute to the therapeutic and toxic effects of IFN-alpha.

Molecular and biologic characterization of recombinant interferon-alpha2b.

Recombinant IFN-alpha2b (Intron A, Schering-Plough Corp, Kenilworth, NJ), derived from Escherichia coli, is currently approved worldwide for the therapy of various cancers and chronic hepatitis B and C. We describe here the purity and identity tests for both physicochemical properties and bioactivity that have been developed for the characterization of highly purified IFN-alpha2b with a specific activity of 2.5 x 10(8) IU/mg protein. The data indicate that a product of high purity can be consistently produced without DNA contamination. The antiviral bioassay chosen to measure potency is based on the ability of IFN-alpha2b to protect human foreskin fibroblast FS-71 cells from the cytopathic effects of encephalomyocarditis virus. Various immunoassays are described that have been used to quantitate the presence of binding and neutralizing antibodies in patients treated with IFN-alpha2b. Overall, the available data indicate a very low incidence of neutralizing antibody formation. We also summarize the current state of knowledge with respect to IFN reference standards for the biologic assay as well as recommendations for the calculation of antibody neutralization titers.

Recent progress in interferon research: molecular mechanisms of regulation, action, and virus circumvention.

A complex system of cis regulatory elements exists by which induction of IFN gene expression is initiated in response to a variety of inducers; cis elements also appear to be involved in the down-regulation of IFN production. IFN gene activation or inhibition of expression may be tightly regulated by the specific binding of newly synthesized or modified proteins to be regulatory regions of the IFN genes. IFN itself acts as a potent modulator of multiple cellular activities. By binding to specific cell surface receptors and probable internalization via receptor-mediated endocytosis and transport into the dense chromatin, IFN treatment leads to activation of numerous genes, some of which possess known antiviral or immunoregulatory functions, whereas the function of others remains to be identified. As with the IFN genes themselves, many of the IFN-inducible genes appear to possess complex regulatory mechanisms, including domains for binding of specific trans-acting proteins. To add to this molecular complexity some viruses have successfully developed methods to circumvent, among other mechanisms, the 2',5'-A-mediated system and the P1 protein kinase system.

Characterization of genetically engineered mengoviruses in mice.

We have shown that genetically engineered mengoviruses containing artificially shortened 5' noncoding poly(C) tracts (e.g., C0 or C13UC10) are dramatically attenuated in adult Swiss/ICR mice when compared to wild-type virus or to a genetically engineered virus containing a wild-type length poly(C) tract (C44UC10). To explore further the relationship between poly(C) tracts and virulence, we have conducted more extensive characterizations of several engineered viruses in the murine model. Both short and long poly(C) tract viruses were highly virulent in newborn mice, underscoring the importance of age in poly(C)-mediated attenuation. Virus vMC24, with a tract sequence of C13UC10, was as attenuated in 4-week-old BALB/c, C.C3-H2k/LiMcdJ, and DBA/2 mice as in Swiss/ICR mice. But it was more pathogenic for C57BL/6 mice, and highly virulent for C3H/Hej and C3H/Hen mice, demonstrating the importance of murine genotype. As expected from its virulence in all mouse strains, vMwt, with a poly(C) of C44UC10, induced higher levels of viremia than vMC24. The vMwt also induced higher levels of circulating interferon and had reduced pathogenicity in chemically immunosuppressed Swiss/ICR mice. Similar immunosuppression did not increase the virulence of vMC24. Collectively, the data suggest that endogenous immune components and the immune competence of the host play significant roles in determining the susceptibility of mice to mengovirus infection.

The effects of interferon on 3T3-L1 cell differentiation.

Mouse 3T3-L1 fibroblasts, after attaining confluence, slowly differentiate functionally and morphologically into adipose cells. This conversion, characterized by a great accumulation of lipids, mostly triglycerides, is hastened by added insulin. The cells are relatively sensitive to the antiviral action of mouse fibroblast interferon (IF). Most cells treated with insulin in the presence of partially or highly purified interferon fail to differentiate. IF-treated cell cultures have far fewer adipose cells and much less lipid that replicate control cultures likewise stimulated to differentiate. The greatest differences are noted in the intracellular levels of triglycerides, cholesterol, and cholesterol esters, as well as in the incorporation of [14C]acetates into Folch-extractable lipids. Variously inactivated or mock IF preparations as well as several heterologous species interferons fail to inhibit 3T3-L1 differentiation. Thus, interferon appears to alter the program of events involved in the conversion of 3T3-L1 fibroblasts into adipose cells.

Interferon and antibody titrations using haemagglutinating Togaviridae and trypsinized human erythrocytes.

Several Togaviridae of the alphavirus and flavivirus genera agglutinate trypsinized human group O erythrocytes (THOE) (Shortridge and Hu, 1976). Haemagglutinin titers of Semliki Forest virus (SFV) and Japanese encephalitis virus (JEV) measured with THOE were equivalent to, if not higher than, those obtained with Embden gander erythrocytes, even with unextracted haemagglutinin. Results obtained with THOE in JEV haemagglutination-inhibition tests on sera taken from a previously infected individual over a 20-yr period were similar to those measured during the initial JEV infection. The inhibition of SFV haemagglutinin production as measured with THOE was a very sensitive bioassay for chicken interferon: interferon titers were 6- to 10-fold higher than those obtained with the vesicular stomatitis virus plaque-reduction method. The generally greater availability of human erythrocytes (including those stabilized with glutaraldehyde), the simplicity of the trypsin treatment, and the possibility of using unextracted haemagglutinin recommend this technique for use with haemagglutinating Togaviridae.

Inhibition of the differentiation of 3T3-L1 cells by interferon-beta and difluoromethyl ornithine.

Both mouse interferon-beta (MuIFN-beta) and the inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethyl ornithine (DFMO), inhibited the differentiation of mouse 3T3-L1 fibroblasts into adipocytes in a dose-dependent manner. DFMO and MuIFN-beta added together to cultures that were induced to differentiate produced an additive anti-differentiation effect. In contrast to this additive cellular effect, DFMO reduced the antiviral activity of MuIFN-beta in both undifferentiated and differentiated cells; DFMO alone had no detectable effect on replication of encephalomyocarditis virus. Putrescine, the product of ornithine decarboxylation, when added to 3T3-L1 cultures (i) enhanced differentiation, (ii) reversed completely the inhibition of differentiation by DFMO, but (iii) had little effect on the anti-differentiation effect of MuIFN-beta. Polyamine content changed four-fold or less in cultures treated with 0.5 mM DFMO and less than two-fold in cultures treated with 100 IU/ml MuIFN-beta for seven days. Thus, it appears not only that MuIFN-beta and DFMO inhibit differentiation of 3T3-L1 cells by different mechanisms but also that the antiviral action of IFN does not involve the regulation of polyamine metabolism by ornithine decarboxylase.

Inhibition of the differentiation of 3T3-L1 cells by interferon-beta and difluoromethyl ornithine.

Both mouse interferon-beta (MuIFN-beta) and the inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethyl ornithine (DFMO), inhibited the differentiation of mouse 3T3-L1 fibroblasts into adipocytes in a dose-dependent manner. DFMO and MuIFN-beta added together to cultures that were induced to differentiate produced an additive anti-differentiation effect. In contrast to this additive cellular effect, DFMO reduced the antiviral activity of MuIFN-beta in both undifferentiated and differentiated cells; DFMO alone had no detectable effect on replication of encephalomyocarditis virus. Putrescine, the product of ornithine decarboxylation, when added to 3T3-L1 cultures (i) enhanced differentiation, (ii) reversed completely the inhibition of differentiation by DFMO, but (iii) had little effect on the antidifferentiation effect of MuIFN-beta. Polyamine content changed four-fold or less in cultures treated with 0.5 mM DFMO and less than two-fold in cultures treated with 100 IU/ml MuIFN-beta for seven days. Thus, it appears not only that MuIFN-beta and DFMO inhibit differentiation of 3T3-L1 cells by different mechanisms but also that the antiviral action of IFN does not involve the regulation of polyamine metabolism by ornithine decarboxylase.