A Fusion Protein of the gp130 and Interleukin-6Rα Ligand-binding Domains Acts as a Potent Interleukin-6 Inhibitor

Interleukin (IL)-6 is involved in the maintenance and progression of several diseases such as multiple myeloma, rheumatoid arthritis, or osteoporosis. The present work aims at the development of an IL-6 inhibitor for the use in anti-cytokine therapies. The IL-6 receptor is composed of two different subunits, an α-subunit (IL-6Rα) that binds IL-6 with low affinity and a औ-subunit (gp130) that binds the IL-6·IL-6Rα complex with high affinity and as a result triggers intracellular signaling. In its soluble form, gp130 is a natural antagonist that neutralizes IL-6·soluble IL-6Rα complexes. It was our strategy to appropriately fuse the two receptor subunit fragments involved in IL-6 receptor complex formation to bind IL-6 with high affinity and to antagonize its effects. The ligand-binding domains of gp130 (D1-D2-D3) and IL-6Rα (D2-D3) were connected using three different linkers. The resulting constructs were expressed in stably transfected insect cells and tested for their ability to inhibit IL-6 activity in several in vitro systems. All fusion proteins were strong inhibitors of IL-6 signaling and abrogated IL-6-induced phosphorylation of STAT3, proliferation of transfected Ba/F3 cells, and induction of acute-phase protein synthesis. As intended, the fused receptors were much more effective than the separately expressed soluble receptor proteins. The fusion protein strategy presented here can also be applied to other cytokines that signal via receptors composed of two different subunits to design new potent inhibitors for anti-cytokine therapies. Interleukin (IL)-6 is involved in the maintenance and progression of several diseases such as multiple myeloma, rheumatoid arthritis, or osteoporosis. The present work aims at the development of an IL-6 inhibitor for the use in anti-cytokine therapies. The IL-6 receptor is composed of two different subunits, an α-subunit (IL-6Rα) that binds IL-6 with low affinity and a औ-subunit (gp130) that binds the IL-6·IL-6Rα complex with high affinity and as a result triggers intracellular signaling. In its soluble form, gp130 is a natural antagonist that neutralizes IL-6·soluble IL-6Rα complexes. It was our strategy to appropriately fuse the two receptor subunit fragments involved in IL-6 receptor complex formation to bind IL-6 with high affinity and to antagonize its effects. The ligand-binding domains of gp130 (D1-D2-D3) and IL-6Rα (D2-D3) were connected using three different linkers. The resulting constructs were expressed in stably transfected insect cells and tested for their ability to inhibit IL-6 activity in several in vitro systems. All fusion proteins were strong inhibitors of IL-6 signaling and abrogated IL-6-induced phosphorylation of STAT3, proliferation of transfected Ba/F3 cells, and induction of acute-phase protein synthesis. As intended, the fused receptors were much more effective than the separately expressed soluble receptor proteins. The fusion protein strategy presented here can also be applied to other cytokines that signal via receptors composed of two different subunits to design new potent inhibitors for anti-cytokine therapies. interleukin domain fusion protein soluble signal transducer and activator of transcription enzyme-linked immunosorbent assay Anti-cytokine therapies are aimed at the inhibition of a certain cytokine that is responsible for the maintenance of a disease. Different strategies have been used to neutralize cytokines in patients. Most effective has been the application of soluble cytokine receptors that consist solely of the ectodomain but lack the transmembrane and cytoplasmic regions. They bind the respective cytokine with high affinity and specificity as membrane-bound receptors do. In the treatment of chronic inflammatory diseases such as rheumatoid arthritis, the use of dimeric soluble tumor necrosis factor receptors for the neutralization of tumor necrosis factor has been a real breakthrough (1Goldenberg M.M. Clin. Ther. 1999; 21: 75-87Abstract Full Text PDF PubMed Scopus (132) Google Scholar). IL-61 is secreted by several cell types in response to various inflammatory stimuli. It is the major mediator of the acute-phase response of the liver and is involved in the coordination of inflammatory and immune responses at the site of inflammation (2Akira S. Taga T. Kishimoto T. Adv. Immunol. 1993; 54: 1-78Crossref PubMed Google Scholar). In several acute and chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel diseases, in postmenopausal osteoporosis, but also in certain types of cancer, IL-6 levels are elevated and a causal role for IL-6 in disease progression has been suggested. In some cases inhibition of IL-6 activity by receptor antagonists or neutralizing antibodies has beneficial effects (3Wendling D. Racadot E. Wijdenes J. J. Rheumatol. 1993; 20: 259-262PubMed Google Scholar, 4Bataille R. Barlogie B. Lu Z.Y. Rossi J.F. Lavabre-Bertrand T. Beck T. Wijdenes J. Brochier J. Klein B. Blood. 1995; 86: 685-691Crossref PubMed Google Scholar). IL-6 belongs to the family of hematopoietic cytokines (5Wells J.A. de Vos A.M. Annu. Rev. Biochem. 1996; 65: 609-634Crossref PubMed Scopus (255) Google Scholar). It is a member of the subfamily of IL-6-type cytokines (6Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1749) Google Scholar) comprising IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and cardiotrophin-like cytokine. They all use the hematopoietic cytokine receptor gp130 as a common signal-transducing receptor subunit (7Bravo J. Heath J.K. EMBO J. 2000; 19: 2399-2411Crossref PubMed Google Scholar). As a result of receptor activation the transcription factor STAT3 becomes tyrosine-phosphorylated and translocates into the nucleus to induce target gene expression (8Lütticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Kishimoto T. Barbieri G. Pellegrini S. Sendtner M. Heinrich P.C. Horn F. Science. 1994; 263: 89-92Crossref PubMed Scopus (710) Google Scholar, 9Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J.N. Yancopoulus G.D. Science. 1994; 263: 92-95Crossref PubMed Scopus (849) Google Scholar). Expression of gp130 is not sufficient for cells to become responsive to IL-6. They additionally have to express the cytokine specific α-receptor subunit IL-6Rα. This α-receptor is not involved in the initiation of the cytoplasmic signal transduction cascades but is essential for cytokine binding. Thus, activation of the receptor by IL-6 requires two steps: (i) low affinity IL-6 binding to IL-6Rα and (ii) subsequent recruitment of the complex of IL-6 and IL-6Rα to two gp130 molecules leading to the formation of a high affinity ternary complex (10Taga T. Hibi M. Hirata Y. Yamasaki K. Yasukawa K. Matsuda T. Hirano T. Kishimoto T. Cell. 1989; 58: 573-581Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). Cells lacking IL-6Rα can be stimulated with the combination of IL-6 and soluble IL-6Rα (sIL-6Rα) (10Taga T. Hibi M. Hirata Y. Yamasaki K. Yasukawa K. Matsuda T. Hirano T. Kishimoto T. Cell. 1989; 58: 573-581Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). In such a situation, IL-6 binds to sIL-6Rα in solution and the heterodimer of IL-6/sIL-6Rα activates membrane-bound gp130. Soluble gp130 (sgp130) alone acts as a relatively weak IL-6 antagonist (11Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G.D. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar). Most interestingly, the antagonizing activity of sgp130 is substantially increased by the presence of sIL-6Rα (12Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. 1998; 161: 6347-6355PubMed Google Scholar). Both sIL-6R (13Honda M. Yamamoto S. Cheng M. Yasukawa K. Suzuki H. Saito T. Usugi Y. Tokunaga T. Kishimoto T. J. Immunol. 1992; 148: 2175-2180PubMed Google Scholar, 14Müller-Newen G. Köhne C. Keul R. Hemmann U. Müller-Esterl W. Wijdenes J. Brakenhoff J.P.J. Hart M.H.L. Heinrich P.C. Eur. J. Biochem. 1996; 236: 837-842Crossref PubMed Scopus (61) Google Scholar) and sgp130 (11Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G.D. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar, 12Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. 1998; 161: 6347-6355PubMed Google Scholar) are found in high concentrations in human blood (about 50 and 300 ng/ml, respectively). This pair of soluble receptors might act as a natural IL-6 inhibitor to limit systemic IL-6 responses (12Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. 1998; 161: 6347-6355PubMed Google Scholar). Structurally, IL-6 belongs to the family of the α-helix-bundle cytokines. IL-6Rα as well as gp130 belong to the family of class I cytokine receptors (5Wells J.A. de Vos A.M. Annu. Rev. Biochem. 1996; 65: 609-634Crossref PubMed Scopus (255) Google Scholar). The extracellular regions of IL-6Rα and gp130 consist of three (D1–D3) (15Yamasaki K. Taga T. Hirata Y. Yawata H. Kawanishi Y. Seed B. Taniguchi T. Hirano T. Kishimoto T. Science. 1988; 241: 825-828Crossref PubMed Scopus (884) Google Scholar) or six domains (D1–D6) (16Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1100) Google Scholar), respectively. D2 and D3 of IL-6Rα are involved in IL-6 binding (17Yawata H. Yasukawa K. Natsuka S. Murakami M. Yamasaki K. Hibi M. Taga T. Kishimoto T. EMBO J. 1993; 12: 1705-1712Crossref PubMed Scopus (180) Google Scholar). The complex of IL-6 and IL-6Rα is bound by D1–D3 of gp130 (18Horsten U. Schmitz-Van de Leur H. Müllberg J. Heinrich P.C. Rose-John S. FEBS Lett. 1995; 360: 43-46Crossref PubMed Scopus (44) Google Scholar). IL-6 contains three receptor-binding sites. Site I is occupied by D2-D3 of IL-6Rα, and sites II and III bind to D2-D3 and D1 of gp130, respectively (19Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (301) Google Scholar, 20Kurth I. Horsten U. Pflanz S. Dahmen H. Küster A. Grötzinger J. Heinrich P.C. Müller-Newen G. J. Immunol. 1999; 162: 1480-1487PubMed Google Scholar, 21Pflanz S. Kurth I. Grötzinger J. Heinrich P.C. Müller-Newen G. J. Immunol. 2000; 165: 7042-7049Crossref PubMed Scopus (36) Google Scholar). Based on the mutagenesis data and the recently solved structure of D1–D3 of gp130 bound to viral IL-6, which binds gp130 in the absence of any α-receptor, a reliable model of the IL-6·IL-6Rα·gp130 ternary complex has been proposed (22Chow D. He X. Snow A.L. Rose-John S. Garcia K.C. Science. 2001; 291: 2150-2155Crossref PubMed Scopus (226) Google Scholar). Inhibition of IL-6 activity by the use of soluble receptors is challenging because of the bipartite nature of the IL-6 receptor. IL-6 alone does not bind to gp130. To be neutralized by sgp130, IL-6 must first bind to sIL-6Rα. A fusion protein of gp130 and sIL-6Rα would therefore guarantee that the agonistic complex of IL-6·sIL-6Rα is immediately neutralized. Only recently, due to the new structural data on the IL-6·receptor complex (22Chow D. He X. Snow A.L. Rose-John S. Garcia K.C. Science. 2001; 291: 2150-2155Crossref PubMed Scopus (226) Google Scholar), a promising rational approach on how to design an IL-6-antagonist based on a fusion of sgp130 with sIL-6Rα became possible. In this study, we present a highly potent IL-6 antagonist consisting of the ligand-binding moieties of sgp130 and sIL-6Rα. A fragment corresponding to D2-D3 of IL-6Rα (Val110–Lys338) was amplified by PCR introducing a multiple cloning site (SmaI,NotI, MluI, NheI) with the sense primer and aApaI site, a stop codon instead of Met331, and aBamHI site with the antisense primer. The product was cut with SmaI (Roche Diagnostic GmbH, Mannheim, Germany) andBamHI (MBI Fermentas GmbH, St. Leon-Rot, Germany) and cloned into pSVL-gp130 (D1–D3) (Met1–Pro326) digested with the same enzymes. Then three different linkers were added after digestion of the obtained chimeric construct with MluI (Promega, Madison, WI) and NheI (MBI Fermentas GmbH). The first linker (stalk-49) corresponding to the short extracellular membrane proximal part of IL-6Rα (Ala323–Val362) was produced by PCR. Its amino acid sequence is GSAAATRAEN EVSTPMQALT TNKDDDNILF RDSANATSLP VQDSSSVAS. The two other linkers were constructed with hybridized oligonucleotides. The 41 amino acids of AGS-41 are GSAAATRGSA GSGGSATGSG SAAGSGDSVA AGSGGGSGSA S. AGS-33 consists of the sequence GSAAATRGSA GSGGSATGSG SAAGSGDSVR RAS. A FLAG tag was added to the C terminus of all fusion proteins using hybridization of an oligonucleotide pair containing ApaI,XbaI, and BamHI restriction sites and a stop codon. The fusion protein constructs were subcloned into the pIB/v5-his vector (Invitrogen, Groningen, The Netherlands) cut withBamHI and HindIII (Roche Diagnostic GmbH) to express the protein in insect cells. The integrity of all constructs was verified by DNA sequencing. High 5 (H5) cells cultured in Sf-900II medium (Invitrogen, Paisley, Scotland) were stably transfected with the empty pIB/v5-his vector or vectors containing the fusion protein constructs, using the CellFECTIN method (Invitrogen). Cell supernatants were harvested every 3 days, cleared by and at The fusion proteins cell supernatants were at with IL-6 to were by to with the antibodies as in the and for used for protein were as sIL-6Rα STAT3 and STAT3 of cell supernatants containing the respective fusion proteins were applied to an at with proteins were with of The was or cell and the of fusion proteins were by was as (12Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. 1998; 161: 6347-6355PubMed Google Scholar), using of FLAG for and 50 of as The was obtained by of expressed in cells and by sgp130 (12Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. 1998; 161: 6347-6355PubMed Google Scholar). transfected and cells were cultured in medium containing on and stimulated with IL-6 or as H. Horsten U. Küster A. Y. S. G. G. Heinrich P.C. Müller-Newen G. Biochem. J. 1998; PubMed Scopus Google Scholar) in the presence of first or cell of cells were using a assay based on the cell proliferation II assay (Roche Diagnostic GmbH). by cells was by of protein as (12Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. 1998; 161: 6347-6355PubMed Google Scholar). The fusion protein was to the regions of IL-6Rα and gp130 for high affinity IL-6 binding. the terminus of gp130 not be by the is for binding Ward L.D. H. Yasukawa K. R.J. 1999; PubMed Scopus Google Scholar). Thus, the fusion protein consists of domains of gp130 the signal sequence at the terminus that its by a linker and domains D2 and D3 of IL-6Rα The two receptor fragments have to be connected by the linker in a that the fusion protein to the for neutralization of IL-6. to the ternary complex model based on the structure of viral IL-6 bound to D1–D3 of gp130 (22Chow D. He X. Snow A.L. Rose-John S. Garcia K.C. Science. 2001; 291: 2150-2155Crossref PubMed Scopus (226) Google Scholar), the C terminus of and the terminus of are by at This can be by a linker of amino acids The linker be of high of low and to fusion proteins containing different linkers were of AGS-33 and are of and of and 41 amino respectively. In an linkers to The consists of a short fragment of the extracellular part of IL-6Rα J.N. A. H. N. R.J. Sci. U. S. A. PubMed Scopus Google Scholar). its this linker is to be of low is the IL-6Rα. a FLAG tag was added at the C of all constructs of the fusion proteins stably transfected insect cell were The fusion proteins were cell supernatants with and by The of the fusion proteins are for for and for The substantially of is due to an site with the of the affinity of the fusion proteins to IL-6 for their and with The insect cell the and the of IL-6 affinity were for the presence of fusion protein by with the the fusion protein is in the fusion protein is in the The concentrations of fusion protein in the by a well with the of the in the was used for the containing the other fusion proteins and supernatants of insect cells were the same The was used as in the To the IL-6 antagonizing activity of the fusion supernatants of stably transfected insect cells were with IL-6 for to the fusion protein to bind to IL-6. Ba/F3 cells stably transfected with gp130 and IL-6Rα were stimulated with the cells were and STAT3 phosphorylation was In the presence of insect cells, of cells with IL-6 is sufficient to induce phosphorylation of STAT3 and of cells with IL-6 that was with supernatants cells the fusion proteins not result in phosphorylation of STAT3 Thus, all three fusion proteins in the supernatants inhibit IL-6 STAT3 phosphorylation is in response to IL-6. A IL-6 is neutralized by the containing AGS-33 IL-6 is the major of acute-phase protein in but also in cell such as IL-6 to a substantially increased by cells as by of protein and proteins of not In the presence of the fusion proteins is to the Thus, all three fusion proteins inhibit IL-6-induced acute-phase protein synthesis. To the specificity of the inhibitory fusion we the proliferation of Ba/F3 cells stably transfected with gp130 and IL-6Rα or gp130 and in response to IL-6 or 5 IL-11, for 50 or of cell respectively. is a fusion protein of and activity H. Horsten U. Küster A. Y. S. G. G. Heinrich P.C. Müller-Newen G. Biochem. J. 1998; PubMed Scopus Google Scholar). The proliferation of cells with a of IL-6 is by the fusion protein in a a fusion protein of ng/ml, cell proliferation is of cells in response to is not by IL-6, but not were for the fusion proteins not we that the fusion of the ligand-binding domains of gp130 and IL-6Rα to a more potent inhibitor than sgp130 and sIL-6Rα separately expressed in insect cells. The inhibition of STAT3 phosphorylation in cells by IL-6 with was with inhibition by the combination of sgp130 and sIL-6Rα The of IL-6 and is sufficient for of IL-6 STAT3 activation In to this high of the fusion a of of IL-6 and the combination of sIL-6Rα and sgp130 is to inhibitory activity that the fusion proteins are of increased inhibitory activity with the soluble receptor proteins. In this we present a highly potent IL-6 inhibitor based on the ligand-binding domains of the IL-6 receptor subunits IL-6Rα and gp130. of the IL-6 receptor antagonists are IL-6 of binding sites to gp130 II and They IL-6Rα by binding via the site I but not gp130 M. Grötzinger J. Müllberg J. Brakenhoff J.P.J. J. A. Rose-John S. J. Immunol. 1994; Google R. L. A. A. A. C. S. G. EMBO J. 1994; PubMed Scopus Google Scholar). the of IL-6 with IL-6Rα is of low were by the site I of antagonists to α-receptor binding. the have to be applied in a to IL-6 R. L. A. A. A. C. S. G. EMBO J. 1994; PubMed Scopus Google Scholar). due to the the proteins are highly L. D. A. M. K. T. G. R. G. R. 1997; PubMed Scopus Google Scholar). IL-6 or IL-6Rα antibodies have also been used as IL-6 They were tested in for the treatment of rheumatoid arthritis (3Wendling D. Racadot E. Wijdenes J. J. Rheumatol. 1993; 20: 259-262PubMed Google Scholar) or E. B. H. A. J.M. Wijdenes J. Ther. 1995; Google Scholar) but to be of low recently, potent low IL-6 receptor antagonists were for the first M. A. A. S. Y. T. A. T. Y. K. J. Ther. PubMed Scopus Google Scholar, M. A. A. Y. T. T. K. S. Sci. U. S. A. PubMed Scopus Google Scholar). antagonists have to be applied in the to inhibit of IL-6. A new of cytokine antagonists is based on soluble receptor fragments that bind the with high affinity and In the of IL-6, two receptor subunits are for high affinity IL-6Rα and gp130. the complex of IL-6 and sIL-6Rα acts on cells gp130 (10Taga T. Hibi M. Hirata Y. Yamasaki K. Yasukawa K. Matsuda T. Hirano T. Kishimoto T. Cell. 1989; 58: 573-581Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). sIL-6Rα neutralization of IL-6 by sgp130 due to formation of a soluble high affinity ternary complex (12Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. 1998; 161: 6347-6355PubMed Google Scholar). The new IL-6 receptor antagonist presented in this the that fusion of the ligand-binding domains of IL-6Rα and gp130 result in a antagonist that neutralizes IL-6 with affinity and In the present three different linkers were used to the ligand-binding domains of gp130 and IL-6Rα. It that the fusion proteins inhibitory that the of the linker has been and linkers were All three fusion proteins bind IL-6 as by with The fusion protein present in the insect cell is sufficient to antagonize the activity of IL-6 in the short STAT3 phosphorylation assay using transfected Ba/F3 cells the concentrations of the fusion proteins in the insect cell supernatants are in the of this to an inhibitory activity at a and antagonist of In a assay such as induction of acute-phase protein in cells, the activity of IL-6 was by the of inhibitory fusion protein at a In the Ba/F3 proliferation assay with we an for the inhibition of IL-6 Thus, in and therefore also for of the inhibitory activity of the fusion proteins in an of fusion protein IL-6 be their inhibitory activity the specificity of the fusion proteins is an to their for anti-cytokine therapies. IL-6 because also via gp130 but binds to a different α-receptor, In the of fusion proteins that inhibit IL-6 activity on proliferation for at the concentrations used in our of the three fusion proteins is a potent and specific inhibitor of IL-6 The activity of the fused ligand-binding domains of gp130 and IL-6Rα with the soluble receptors sgp130 and sIL-6Rα is the to be to the of our IL-6-induced STAT3 phosphorylation in Ba/F3 cells is by the presence of of fusion To a inhibition an at of sgp130 and sIL-6Rα has to be This result the high inhibitory activity of the fusion is the for this In the a low of IL-6 that is in the of IL-6 concentrations was the soluble receptors were IL-6 first binds to the sIL-6Rα. This is of low and therefore the complex of IL-6 and sIL-6Rα might In the fusion the complex of IL-6 bound to domains D2 and D3 of IL-6Rα can be immediately by the ligand-binding domains of gp130 inhibitor strategy is also to other cytokines that signal via receptor complexes. in a C. X. F. S. J. K. S. R. C. Yancopoulos G.D. N. PubMed Scopus Google Scholar) used a approach to as highly potent inhibitors for and IL-6. In their the of the respective receptor subunits the regions for binding were fused to the part of human This in of the receptor by formation of the In the of the IL-6 this the to the formation of and As a the heterodimer must be the the cells an of 50 was for the neutralization of IL-6 C. X. F. S. J. K. S. R. C. Yancopoulos G.D. N. PubMed Scopus Google Scholar). our cells with the of is into the the IL-6 in the two different proliferation and the of the respective that is more potent than that the more approach presented in our the of the and to inhibitory fusion proteins. that fusion of the ligand-binding domains of soluble receptor proteins to cytokine inhibitors of activity which might be of for the development of new anti-cytokine therapies. Wijdenes for the of the gp130 also Behrmann for of the