Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues.

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.

Therapeutic antibody expression technology.

With the technological advances made during the past decade, antibodies now represent an important and growing class of biotherapeutics. With the potential new targets resulting from genomics and with methods now in place to make fully human antibodies, the potential of antibodies as valuable therapeutics in oncology, inflammation and cardiovascular disease can be fully realised. Systems to produce these antibodies as full-length molecules and as fragments include expression in both mammalian and bacterial cells grown in bioreactors and in transgenic organisms. Factors including molecular fidelity and the cost of goods are critical in evaluating expression systems. Mammalian cell culture and transgenic organisms show the greatest promise for the expression of full-length, recombinant human antibodies, and bacterial fermentation seems most favorable for the expression of antibody fragments.

Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

Secretion of glycosylation site mutants can be rescued by the signal/pro sequence of tissue plasminogen activator.

Strategies that prevent the attachment of N-linked carbohydrates to nascent glycoproteins often impair intracellular transport and secretion. In the present study, we describe a method to rescue the intracellular transport and secretion of glycoproteins mutagenized to delete N-linked glycosylation sites. Site-directed mutagenesis was used to delete N-linked glycosylation sites from a chimeric protein, TNFR-IgG1. Deletion of any of the three glycosylation sites in the TNFR portion of the molecule, alone or in combination, resulted in a moderate or near total blockade of TNFR-IgG1 intracellular transport and secretion. Pulse chase experiments suggested that the glycosylation site mutants accumulated in the endoplasmic reticulum (ER) and were inefficiently exported to the Golgi apparatus (GA). Replacement of the TNFR signal sequence with the signal/pro sequence of human tissue plasminogen activator (tPA) overcame the blockade to intracellular transport, and restored secretion to levels comparable to those achieved with the fully glycosylated molecule. Ligand binding studies suggested that the secreted glycosylation variants possessed binding characteristics similar to the fully glycosylated protein. This study demonstrates that N-terminal sequences of tPA are unexpectedly efficient in facilitating transport from the ER to the GA and suggests that these sequences contain a previously unrecognized structural element that promotes intracellular transport.

A simple, two-component buffer enhances use of chromatofocusing for processing of therapeutic proteins.

To extend the feasibility of chromatofocusing to industrial use, we have developed a simple chromatofocusing buffer system capable of generating a smooth pH gradient without the use of an external gradient maker. Using two cationic buffering components, an internal pH gradient is produced on appropriate chromatography media over a broad pH range (9.5 to 5.0). The utility of this buffer system is demonstrated with PBE94 and DEAE Sepharose fast flow ion-exchangers, as well as with experimental fast flow chromatofocusing gels. Using a rapid flow rate, we evaluated this buffer system for recovery of a therapeutic protein from a bacterial cell extract. The simplicity of the buffer system requiring no external gradient maker, coupled with the use of fast flow chromatographic media to produce broad-range pH gradients, improves the scalability of chromatofocusing for processing of therapeutic proteins.

A p55 TNF receptor immunoadhesin prevents T cell-mediated intestinal injury by inhibiting matrix metalloproteinase production.

Anti-TNF-alpha Ab therapy has been shown to be of benefit in the treatment of active Crohn's disease, but the tissue-injuring processes in the gut mediated by TNF-alpha that might be inhibited by neutralizing Ab are unknown. In this work, we have used a p55 TNF receptor-human IgG fusion protein (TNFR-IgG) to prevent the severe mucosal injury that ensues when lamina propria T cells in explant cultures of human fetal small intestine are directly activated with the lectin PWM. Following T cell activation and associated with mucosal injury, there is a marked elevation of soluble TNF-alpha in organ culture supernatants and a large increase in TNF-alpha mRNA transcripts. The addition of TNFR-IgG at the onset of cultures greatly reduced PWM-induced tissue injury, without inhibiting the increase in TNF-alpha and IFN-gamma transcripts seen following T cell activation. Mucosal injury in this model is mediated by endogenously-produced matrix metalloproteinases (MMPs). When TNFR-IgG was added to PWM-stimulated explants, there was a reduction in MMPs in the explant culture supernatants, especially stromelysin-1. Recombinant TNF-alpha and IL-1beta added directly to mucosal mesenchymal cell lines also caused an increase in MMP production, but only the former was inhibited by the TNFR-IgG. These results suggest that one of the ways in which TNF-alpha causes tissue injury in the gut is by stimulating mucosal mesenchymal cell to secrete matrix-degrading metalloproteinases. Neutralization of this activity should help maintain tissue integrity.

Immunoadhesins as research tools and therapeutic agents.

An immunoadhesin is a fusion protein that combines the target-binding region of a receptor, an adhesion molecule, a ligand, or an enzyme, with the Fc region of an Ig. Immunoadhesins provide a unique tool for identifying unknown binding targets and for elucidating molecular interactions that control biological function. Recent studies suggest that immunoadhesins also may be useful therapeutically, as inhibitors of autoimmune and inflammatory diseases.

Protection against lethal Escherichia coli bacteremia in baboons (Papio anubis) by pretreatment with a 55-kDa TNF receptor (CD120a)-Ig fusion protein, Ro 45-2081.

Fusion proteins of the human 55-kDa TNF receptor extracellular domain with hinge and C2/C3 constant domains of human IgG1 or IgG3 heavy chains were tested in a primate sepsis model. Twenty-four baboons received 4.6, or 0.2 mg/kg of TNFR5-G1,3, or placebo, before the administration of a lethal dose of live Escherichia coli. Treatment with TNFR5-G1,3 decreased 5-day mortality from 88% in the placebo group to 12% in the TNFR5-G1,3-treated animals (p < 0.01 by Fisher's exact test). Treatments with TNR5-G1 and TNFR5-G3 in doses from 0.2 to 4.6 mg/kg were efficacious. Free plasma TNF was neutralized by all treatments, but inactive TNF/TNFR5-G1,3 complexes remained in circulation for prolonged periods. TNFR5-1,3 treatments attenuated the hemodynamic disturbances, reduced fluid requirements, and decreased the systemic IL-1 beta, IL-6, and IL-8 responses. In addition, TNFR5-G1,3 treatment shortened the granulocytopenia and reduced the loss of cellular TNF receptors from granulocytes. The decrease in fibrinogen concentrations and increase in prothrombin and partial thromboplastin times were significantly attenuated by TNFR5-G1,3 treatment. TNFR5-G1,3 treatment markedly attenuated the rise in plasma lactate concentration. Histologic studies of TNFR5-G1,3 revealed dose-dependent protection against tissue injury by Escherichia coli administration.

Immunoadhesins: principles and applications.

The prototypic immunoadhesin is an antibody-like molecule that fuses the Fc region of an immunoglobulin and the ligand-binding region of a receptor or adhesion molecule. In this article, we review some important structural and functional principles of immunoadhesins. In addition, we highlight some unique advantages of immunoadhesins as experimental tools in biology, as well as some of their exciting potential applications in medicine.

A humanized, bispecific immunoadhesin-antibody that retargets CD3+ effectors to kill HIV-1-infected cells.

We have developed a humanized, bispecific immunoadhesin-antibody (BsIAb) that targets and kills HIV-infected cells. Comprised of CD4-IgG and humanized anti-CD3-IgG, this BsIAb is bifunctional. First, in targeting, it exploits the natural affinity of CD4 for gp120 to target the BsIAb to HIV-infected cells, and second, it recruits and activates, through its anti-CD3 moiety, cytotoxic T lymphocytes (CTL) to lyse target cells in a non-MHC restricted manner. To produce purified BsIAb from supernantants of transfected mammalian cells, we designed a three-step recovery scheme based on the structural elements of this heterotrimeric protein. The ability of purified BsIAb to specifically lyse HIV-infected target cells was demonstrated using CTL from two different sources: whole peripheral blood lymphocyte (PBL) fractions and pure CTL preparations. In contrast, a human anti-gp120 antibody mediated lysis of HIV-infected target cells only with PBL fractions and not with purified CTL. Moreover, lysis observed in the presence of the human anti-gp120 antibody was completely blocked in the presence of human serum (which competes for Fc gamma receptor binding), whereas BsIAb-mediated lysis of target cells was not affected. We measured the monovalent affinities of BsIAb for HIV-gp120 on infected cells and for CD3 epsilon on CTL. Relative to the bivalent parent molecules, CD4/gp120 affinity in the BsIAb is unchanged, whereas anti-CD3/CD3 is substantially decreased. We further demonstrated by fluorescence microscopy that physical association of CD3+ cells with gp120-expressing cells occurs only in the presence of BsIAb. Thus, the cytocidal activity of BsIAb in the presence of serum reflects its unique ability to recruit CTL as effector cells and highlights a potentially important advantage of this type of construct over antibodies for HIV-directed therapy.

Sulfated oligosaccharides promote hepatocyte growth factor association and govern its mitogenic activity.

Hepatocyte growth factor (HGF) is a potent mitogen, motogen, and morphogen for various epithelial cell types. The pleiotropic effects of HGF are mediated by its binding to a specific high affinity receptor, c-Met. In addition, HGF binds to heparan sulfate proteoglycans on cell surfaces and within the extracellular matrix. Incubation of HGF with 0.1, 1.0, and 10 micrograms/ml of heparin, heparan sulfate, or dextran sulfate resulted in a concentration-dependent increase in mitogenic potency in a primary rat hepatocyte bioassay, whereas sodium sulfate or fucoidan did not. Although co-incubation of HGF with sulfated compounds that enhanced HGF-dependent mitogenesis did not alter the binding isotherm of HGF for the c-Met receptor in a solid phase assay, an increase in autophosphorylation of the c-Met receptor in intact A549 cells was observed upon their addition. A series of chemically sulfated malto-oligosaccharides varying in unit size and charge was tested in the bioassay in order to provide additional insights into the nature of the HGF-heparin interaction. While sulfated di-, tri-, tetra-, and pentasaccharides did not significantly potentiate HGF-dependent mitogenesis, larger oligosaccharides such as the sulfated hexa-, hepta-, or a sulfated oligosaccharide mixture containing decasaccharides resulted in an approximate 2-, 4-, and 7-fold enhancement, respectively. We observed a correlation between the sulfated oligosaccharide preparations that enhanced mitogenic potency and those that promoted HGF oligomerization in vitro, as measured by gel filtration and analytical ultracentrifugation. These findings indicate that heparin-like molecules can stabilize HGF oligomers, which may facilitate c-Met receptor dimerization and activation.

Induction of liver growth in normal mice by infusion of hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent stimulator of DNA synthesis in a variety of epithelial cells, including hepatocytes, and has been implicated in liver regeneration. We show here that combining dextran sulfate with HGF/SF markedly increases the plasma concentrations of HGF/SF that are achieved during intraperitoneal infusion. Three days of administration of HGF/SF by this mechanism caused a dose-dependent increase in liver wet weight. Mitotic figures were rarely observed in control livers but were abundant in livers exposed to HGF/SF, and liver DNA content was elevated. Serum levels of triglycerides, cholesterol, total protein, and albumin were also dose dependently increased, whereas alkaline phosphatase was reduced. From these data we conclude 1) that combining HGF/SF with dextran sulfate provides a novel method for delivering HGF/SF in a continuous manner, 2) that HGF/SF can induce liver growth in an intact animal, and 3) that HGF/SF-induced liver enlargement is associated with changes in serum biochemistry.

Generation of soluble interleukin-1 receptor from an immunoadhesin by specific cleavage.

The extracellular portion of the interleukin-1 receptor (IL-1R) is sufficient for high-affinity binding to IL-1; however, the structural basis for binding of the receptor to IL-1 is not known. To produce individual domains of IL-1 receptor for structural studies, we constructed a molecular fusion of IL-1 receptor with immunoglobulin G heavy chain that contains a protease specific sequence joining the two portions of the molecule (IL-1R-G-IgG). We introduced the hexapeptide sequence AAHY:TL (where ":" denotes the scissile bond) at the junction of the IL-1R and IgG regions, for specific cleavage by an H64A variant of subtilisin BPN' (Genenase I), an endoprotease that cleaves selectively at this sequence (Carter et al., (1989) Proteins 6, 240-248). Plasmid DNA encoding the fusion protein was used to transfect human embryonic kidney 293 cells transiently, and secreted IL-1R-G-IgG was purified from cell supernatants by protein A chromatography. The IL-1 receptor's extracellular region was then generated by enzymatic cleavage with Genenase I which was immobilized on controlled-pore glass. Incubation of IL-1R-G-IgG with immobilized Genenase I resulted in specific cleavage at the target site, as confirmed by SDS-PAGE, immunoblotting and direct sequencing of the newly generated termini. The resulting soluble IL-1R was separated from the immunoglobulin Fc cleavage product by re-chromatography on protein A. The purified, soluble IL-1R retained quantitatively the ability to bind to its ligand, IL-1 beta. This approach offers a generic means by which the extracellular region of a given type I transmembrane receptor can be expressed as an immunoadhesin, released enzymatically and then easily purified for crystallographic or ligand binding studies.

Molecular and biological properties of an interleukin-1 receptor immunoadhesin.

Overproduction of the cytokine interleukin 1 (IL-1) is an important factor in the pathogenesis of several autoimmune and inflammatory disease. To develop a recombinant inhibitor of IL-1 with an extended pharmacologic half-life, we constructed an IL-1 receptor immunoadhesin (IL-1R-IgG), by fusing the extracellular domain of the type IL-1 receptor with the hinge and Fc regions of human IgG1 heavy chain. Transfected human 293 cells express IL-1R-IgG as a secreted, disulfide-bonded homodimer. The secreted protein contains an intact antibody Fc region, as indicated by immunoblotting, and a functional IL-1 receptor region, as indicated by ligand-blotting. Saturation binding analysis indicates an equilibrium dissociation constant (KD) of 350 pM for the binding of IL-1R-IgG to its ligand, IL-1 beta. Kinetic analysis of the binding reveals an off rate of 0.1 min-1 and an on rate of 1.5 x 10(8) min-1 M-1, yielding a calculated KD of 770 pM. These binding properties are similar to those of cell-surface type I IL-1 receptor. IL-1R-IgG is capable of inhibiting the biological activity of IL-1 beta in vitro, as evidenced in a thymocyte proliferation assay. Pharmacokinetic analysis in mice indicates that IL-1R-IgG has a terminal half-life of 91 hr in the blood circulation. This half-life is markedly longer than the values reported for other recombinant inhibitors of IL-1 such as the IL-1 receptor antagonist or soluble IL-1 receptor. Thus, IL-1R-IgG may be useful for investigating the interaction of IL-1 with its receptor and the role of IL-1 in disease, as well as for potential intervention in pathological situations involving overproduction of IL-1.

Protection against rat endotoxic shock by p55 tumor necrosis factor (TNF) receptor immunoadhesin: comparison with anti-TNF monoclonal antibody.

The protective efficacy of a p55 tumor necrosis factor receptor immunoadhesin (TNFR-IgG) was compared with that of an anti-TNF monoclonal antibody (MAb) in a rat endotoxic shock model. TNFR-IgG (5 mg/kg), given 30 min before endotoxin (LPS), attenuated LPS induction of hypotension and tachycardia and eliminated LPS induction of serum TNF activity. In contrast, anti-TNF MAb (5 mg/kg) had little effect on LPS-induced hemodynamic changes and neutralized only partially the excessive serum TNF activity. The 6-day survival was 1 of 10 controls; 6 of 11, 5 of 7, and 8 of 9 rats receiving 0.2, 1.0, or 5.0 mg/kg TNFR-IgG, respectively; and 3 of 8 rats receiving 5 mg/kg anti-TNF MAb. These results indicate that TNFR-IgG is more potent than anti-TNF MAb at neutralizing excessive TNF activity in vivo.

A humanized, bispecific immunoadhesin-antibody that retargets CD3+ effectors to kill HIV-1-infected cells.

HIV infection depletes the immune system of the coordinating functions of CD4+ T cells and APCs, whereas the population of CD8+ CTLs remains largely intact: functional but undirected. We have developed a humanized bispecific immunoadhesin-antibody (BIA) that redirects these remaining T cells to kill HIV-infected cells. This BIA expresses effector cell retargeting via a targeting activity that exploits the natural affinity of CD4 for gp120, and a recruiting activity that employs an anti-CD3 moiety to engage CTLs. The resultant molecule is 97% human in origin. In functional tests, this BIA mediated killing of HIV-infected cells using either pure CTL preparations, or whole PBL fractions that additionally include Fc gamma receptor-bearing large granular lymphocyte effectors. In contrast, a human anti-gp120 Ab induced target lysis via Ab-dependent cellular cytotoxicity (ADCC) only with large granular lymphocyte-containing fractions and not with CTLs. ADCC with this Ab was blocked in human serum, whereas BIA-mediated effector cell retargeting lysis of HIV-infected cells by CTLs was preserved. The affinity of the BIA for HIV-gp120 on infected cells and for CD3 epsilon on CTLs was derived in a flow cytometric Scatchard procedure. Relative to the bivalent parent molecules, CD4/gp120 affinity on cells was unchanged in the BIA (Ka 7 x 10(7) M-1), whereas the anti-CD3 affinity was diminished 50-fold (Ka 2 x 10(6) M-1 vs 1 x 10(8) M-1). Physical association of CD3+ effectors and gp120-expressing targets was confirmed by fluorescence microscopy and was dependent upon the presence of BIA and expression of target gp120. The unimpaired cytocidal activity of the BIA in the presence of serum highlights a potentially important advantage of this type of construct over native Abs for HIV-directed therapy.

Liposome targeting to human immunodeficiency virus type 1-infected cells via recombinant soluble CD4 and CD4 immunoadhesin (CD4-IgG).

HIV-infected cells producing virions express the viral envelope glycoprotein gp120/gp41 on their surface. We examined whether liposomes coupled to recombinant soluble CD4 (sCD4, the ectodomain of CD4 which binds gp120 with high affinity) could specifically bind to HIV-infected cells. sCD4 was chemically coupled by 2 different methods to liposomes containing rhodamine-phosphatidylethanolamine in their membrane as a fluorescent marker. In one method, sCD4 was thiolated with N-succinimidyl acetylthioacetate (SATA) and coupled to liposomes via a maleimide-derivatised phospholipid. In the other method, the oligosaccharides on sCD4 were coupled to a sulfhydryl-derivatised phospholipid, utilizing the bifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH). The association of the liposomes with HIV-1-infected or uninfected cells was examined by flow cytometry. CD4-coupled liposomes associated specifically to chronically infected H9/HTLV-IIIB cells, but not to uninfected H9 cells. CD4-coupled liposomes also associated specifically with monocytic THP-1 cells chronically infected with HIV-1 (THP-1/HIV-1IIIB). Control liposomes without coupled CD4 did not associate significantly with any of the cells, while free sCD4 could competitively inhibit the association of the CD4-coupled liposomes with the infected cells. The chimeric molecule CD4-immunoadhesin (CD4-IgG) could also be used as a ligand to target liposomes with covalently coupled Protein A (which binds the Fc region of the CD4-IgG) to H9/HTLV-IIIB cells. The CD4-liposomes inhibited the infectivity of HIV-1 in A3.01 cells, and also bound rgp120. Our results suggest that liposomes containing antiviral or cytotoxic agents may be targeted specifically to HIV-infected cells.

Modification of CD4 immunoadhesin with monomethoxypoly(ethylene glycol) aldehyde via reductive alkylation.

CD4 immunoadhesin (CD4-IgG) is a chimeric glycoprotein molecule comprised of the gp120-binding portion of human CD4 fused to the hinge and Fc portions of human IgG. As a candidate for human therapeutic use, CD4-IgG represents an important advance over soluble CD4, insofar as the systemic clearance in humans of CD4-IgG is significantly slower. In an effort to prolong its in vivo residence time even further, we have modified CD4-IgG chemically by attaching monomethoxypoly(ethylene glycol) (MePEG) moieties to lysine residues via reductive alkylation. We synthesized MePEG aldehyde and investigated reaction conditions for adding a range of MePEG moieties per protein molecule. At neutral pH in the presence of sodium cyanoborohydride, the reaction was sufficiently slow to allow for significant control over the extent of MePEGylation. Addition of 7.7 or 14.4 MePEG moieties to CD4-IgG resulted in an approximately 4- or 5-fold increase, respectively, in the persistence of the protein in rats, as compared with unmodified CD4-IgG. These results suggest that the therapeutic utility of a human receptor IgG chimera can be improved by MePEGylation technology, provided that the modified immunoadhesin retains its biological activity in vivo. Such modification can lead to a significant additional increase in the in vivo residence time of the protein.

Inhibition of interferon-gamma by an interferon-gamma receptor immunoadhesin.

Interferon-gamma (IFN-gamma) is an important cytokine which regulates inflammatory and immune response mechanisms. IFN-gamma enhances the presentation and recognition of antigens by inducing the expression of major histocompatibility complex (MHC) proteins, by activating effector T cells and mononuclear phagocytes, and by modulating immunoglobulin production and class selection in B cells. Inappropriate production of IFN-gamma has been implicated in the pathogenesis of several autoimmune and inflammatory diseases and in graft rejection. Here, we describe a recombinant inhibitor of IFN-gamma, termed murine IFN-gamma receptor immunoadhesin (mIFN-gamma R-IgG). We constructed this immunoadhesin by linking the extracellular portion of the mouse IFN-gamma R to the hinge and Fc region of an IgG1 heavy chain. Murine IFN-gamma R-IgG is secreted by transfected cells as a disulphide-bonded homodimer which binds IFN-gamma bivalently, with high affinity and in a species-specific manner. In vitro, mIFN-gamma R-IgG can block mIFN-gamma-induced antiviral activity and expression of the class I MHC antigen H-2Kk in cultured cells. In vivo, mIFN-gamma R-IgG can block the function of endogenous mIFN-gamma in mouse models of infection with Listeria monocytogenes and of contact sensitivity. These results show that mIFN-gamma R-IgG is an effective and specific inhibitor of mIFN-gamma both in vitro and in vivo. Thus, in general, IFN-gamma receptor immunoadhesins may be useful for investigating the biological functions of IFN-gamma as well as for preventing deleterious effects of IFN-gamma in human disease.

Reduction of alpha-naphthylisothiocyanate-induced hepatotoxicity by recombinant human hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent stimulator of DNA synthesis in cultured hepatocytes. To determine whether HGF has any activity in vivo, we have tested HGF in rats in which intrahepatic cholestasis was induced by acute administration of alpha-naphthylisothiocyanate (ANIT). The hepatotoxic effects of a single injection of ANIT were manifested 48 h later as large increases in serum bilirubin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. These biochemical changes were accompanied by widespread periportal edema, hypertrophy of bile duct epithelium, and randomly scattered areas of liquifaction necrosis in the hepatic parenchyma. The increases in bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were markedly attenuated when HGF was administered 30 min before ANIT and again at 6, 12, 24, 30, and 36 h after ANIT. In addition, this HGF dosing regimen completely prevented the occurrence of parenchymal lesions, although it had no effect on periportal histopathology. The effect of ANIT was dose dependent; a maximal response was observed at 320 micrograms/kg per injection, with an intermediate response at 105 micrograms/kg. Delaying the administration of HGF until 12 h after ANIT was as effective as when administration was begun 30 min before ANIT. Taken together these results show that HGF can prevent some aspects of ANIT hepatotoxicity.