INTERLEUKIN-6 RECEPTOR ANTAGONISTS

Abstract: This invention provides a class of interleukin-6 (IL-6) muteins which act as IL-6 receptor antagonists, thereby inhibiting the normal function of naturally-occuring IL-6. These IL-6 receptor antagonists are preferably IL-6 molecules containing one or more mutations in the Site II region comprising amino acids 145-163. This invention also provides pharmaceutical compositions comprising IL-6 receptor antagonists with a pharmaceutically acceptable carrier. This invention further provides methods for treating IL-6 related diseases such as sepsis and multiple myeloma, the methods comprising administering to a patient an IL-6 receptor antagonist.

Description: INTERLEUKIN-6 RECEPTOR ANTAGONISTS Field of the Invention This invention is in the field of immunology and control of host defense mechanisms. More specifically, this invention relates to the discovery of a class of interleukin-6 muteins which interfere with the interaction between interleukin-6 and its two receptor proteins. This invention also relates to the use of such muteins to control and prevent interleukin-6 related diseases. Background of the Invention Interleukin-6 (IL-6) is a multi-functional cytokine playing a central role in host defense mechanisms. Heinrich et al., Biochem . J. , 265 , 621 (1990) ; Van Snick, J. Annu . Rev . Immunol . , 8, 253 (1990); and Hirano et al., Immunol . Today, 11, 443 (1990) . However, in a variety of human inflammatory, autoimmune, and neoplastic diseases, abnormal IL-6 production is observed and has been suggested to play a role in the pathogenesis of those diseases. Hirano et al., supra ; Sehgal, Proc . Soc. Exp. Biol . Med . , 195, 183 (1990); Grau, Eur . Cytokine Net, 1 , 203 (1990); Bauer et al., Ann . Hematol . , 62, 203 (1991); Campbell et al., J . Clin . Invest . , 7, 739, (1991) ; and Roodman et al., J. Clin . Invest . , 89, 46 (1992). Inhibitors of IL-6 bioactivity might thus be useful to study its role in disease and could have broad therapeutic applications. IL-6 overproduction is involved in sepsis (Starnes, Jr. et al., J . Immunol . , 145, 4185 (1990)) , and is also implicated in multiple myeloma disease, or plasma cell leukemia (Klein et al., Blood, 78, 1198 (1991)). Other diseases include bone resorption (osteoporosis) (Roodman et al., J . Clin . Invest . , 89, 46 (1992); Jilka et al., Science, 257, 88-91 (1992)), cachexia (Strassman et al., J . Clin . Invest . , 89, 1681 (1992)), psoriasis, mesangial proliferative glomerulonephritis, renal cell carcinoma, Kaposi's sarcoma, rheumatoid arthritis, hypergammaglobulinemia (Grau et al., J. Exp. Med . 172, 1505 (1990)), Castleman's disease, IgM gammopathy, cardiac myxoma and autoimmune insulin-dependent diabetes (Campbell et al., J . Clin . Invest . , 87, 739 (1991)). IL-6 functions through interaction with at least two specific receptors on the surface of target cells. Taga et al., J. Exp . Med . , 166, 967 (1987); and Coulie et al., Eur . J. Immunol . , 17, 1435 (1987) . The cDNAs for these two receptor chains have been cloned, and they code for two transmembrane glycoproteins: the 80 kDa IL-6 receptor ("IL-6R") and a 130 kDa glycoprotein called "gpl30". Yamasaki et al., Science, 241, 825 (1988); and Hibi et al., Cell, 63, 1149 (1990) . IL-6 interacts with these glycoproteins following a unique mechanism. First, IL-6R binds to IL-6 with low affinity (Kd = about 1 nM) without triggering a signal. Taga et al., Cell , 58, 573 (1989) . The IL-6/IL-6R complex subsequently associates with gpl30, which transduces the signal. Hibi et al., supra ; and Taga et al., supra . Gpl30 itself has no affinity for IL-6 in solution, but stabilizes the IL-6/IL-6R complex on the membrane, resulting in high affinity binding of IL-6 (Kd = about 10 pM) . Hibi et al., supra . It was recently found that gpl30 is also a low affinity receptor for oncostatin M and an affinity converter for the LIF receptor (Gearing et al., Science, 255, 1434 (1992)). Mature human (h) IL-6 is a 185 amino acid polypeptide containing two disulfide bonds (Cys 45 to c Y s 5 i and C Y S 4 to Y S 84 - Clogston et al., Arch. Biochem . Biophys . , 272, 144 (1989). The first 28 residues can be deleted without affecting bioactivity. Brakenhoff et al., J . Immunol . , 143, 1175 (1989) . Bioactivity of hIL-6 appears to be conformation dependent. Large internal deletions disrupt the overall structure of the molecule and completely abolish activity. Snouwaert et al., J. Immunol . , 146, 585 (1991); and Fontaine et al. , Gene, 104, 227 (1991) . Maintenance of the second (but not the first) disulfide bond is critical, especially in bioassays involving human cell lines. Snouwaert et al., J " . Biol . Chem . , 266, 23097 (1991). Regions critical to activity comprise residues Ile 30 to Asp 35 (see Brakenhoff et al., supra ; Fontaine et al., supra ; and Arcone et al., FEBS Letters, 288, 197 (1991)) , Ala 154 to Thr 164 (see Ida et al. , Biochem . Biophys . Res . Commun . , 165, 728 (1991); and Nishimura et al., FEBS Letters, 281, 167 (1991)) and Arg 183 to Met 185 (see Krϋttgen et al., FEBS Letters, 262, 323 (1990); Brakenhoff et al., J . Immunol . , 145, 561 (1990); and Krϋttgen et al., FEBS Letters, 273, 95 (1990)). Substitution analysis of individual residues have implicated Leu 159 , Met 162 and Leu 165 to be important both for activity and binding to IL-6R (see Nishimura et al., supra . A positive charge and α-helical C-terminal structure were found to be essential for activity. Lϋtticken et al., FEBS Letters, 282 , 265, (1991) . One method for neutralization of IL-6 activity is the use of antibodies to IL-6. Neutralizing monoclonal antibodies (MAbs) to IL-6 can be divided in two groups, based on the recognition of two distinct epitopes on the IL-6 molecule, designated Site I and Site II. Site I is a conformational epitope composed of both amino terminal and carboxy terminal portions of the IL-6 molecule: the amino terminal portion includes amino acids Ile 30 -Asp 35 ; while the carboxy terminal portion includes critical amino acids Arg 183 -Met 185 . Site II includes critical amino acids Ala 154 -Thr 163 . Brakenhoff et al., supra , (1990). Another way to neutralize IL-6 activity is to inhibit the ligand-receptor interactions with specific receptor-antagonists. The feasibility of this general type of approach was recently demonstrated with a naturally occurring receptor antagonist for interleukin-1. Hannum et al., Nature, 343, 336-340 (1990) . However, no natural receptor-antagonist has been identified for IL-6 so far. Nor has any hIL-6 variant with antagonistic properties been discovered. This invention uses the information gleaned from the Site I and Site II work with MAbs to construct hIL-6 variants that act as IL-6 receptor antagonists. Summary of the Invention This invention relates to the discovery of a role for site I in IL-6 binding to IL-6R and for site II in IL-6/gpl30 interaction. According to the IL-6 receptor model, IL-6 variants that bind normally to IL-6R, and whose subsequently formed IL- 6 variant/IL-6R complex fails to interact with gpl30, will function as receptor-antagonists. The inventors herein have analyzed substitution mutants in the Site II region for residual bioactivity in various IL-6 bioassays. One of the isolated mutants showed a 1,000 to 10, 000-fold reduction in specific activity in assays with human cells, and could specifically antagonize the activity of wild-type recombinant (r) hIL-6 in two of three human assays. It is accordingly a primary object of the present invention to provide a newly identified class of molecules that are antagonists of IL-6 as newly discovered agents. It is a further object of this invention to provide oligonucleotide sequences encoding IL-6 receptor antagonists. It is another object of this invention to provide pharmaceutical compositions for the treatment of IL-6 related diseases, especially sepsis and multiple myeloma. Accordingly, in one aspect of this invention, a group of IL-6 receptor antagonist molecules are provided. In a preferred embodiment, the IL-6 receptor antagonists are IL-6 molecules containing one or more site II mutations. In another aspect of this invention, pharmaceutical compositions are provided comprising an IL-6 receptor antagonist and a pharmaceutically acceptable carrier. In a further aspect of this invention, a method for treating IL-6 related diseases is provided, said method comprising administering to a patient in need of such treatment a pharmaceutical composition containing an amount of an IL-6 receptor antagonist effective for treating sepsis and a pharmaceutically acceptable carrier. In preferred embodiments, the IL-6 related disease is sepsis or multiple myeloma. Brief Description of the Drawings Figure 1 shows a Coo assie blue-stained SDS-polyacrylamide gel of preparations of various IL-6 mutants. The lanes contain: (1) rhIL-6 HGF7; (2) rhIL-6 T 163 P; (3) rhIL-6 Q 16 o E ' τ i 63 p - Tne a row denotes the migration position of mature rhIL-6. Figure 2 shows the dose response curve of wild type IL-6 and two IL-6 mutants in various assays with human cell lines. (A) shows the amount of IgGl synthesis by CESS cells; (B) shows the amount of Cl esterase inhibitor production by HepG2 cells; and (C) shows the amount of tritiated thymidine incorporated by human myeloma cell line XG-1. Figure 3 shows the inhibition of recombinant human IL-6 Qιeo E ' T 163 P on w; ld tYP e IL-6 activity in (A) the CESS assay; and (B) the HepG2 assay. Figure 4 shows the amount of Cl esterase inhibitor production by HepG2 cells in the presence of wild type IL-6 with and without IL-6 Q 160 E/ T 163 P. Figure 5 shows the amount of Cl esterase inhibitor production by HepG2 cells in the presence of media, wild type IL-6 (5 ng/ml) or gamma interferon (1 ng/ml) with and without IL-6 Q^ Q E , T 163 P - Figure 6 compares the inhibition of IL-6 binding to IL-6 receptor-bearing cells (NIH-3T3 fibroblasts transfected with an expression vector encoding IL-6R) with rhIL-6 HGF7 and IL-6 Q 160 E, Tι 63 P- Detailed Description of the Invention The invention described herein draws on previously published work and pending patent applications. By way of example, such work consists of scientific papers, patents or pending patent applications. All of these publications and applications, cited previously or below are hereby incorporated by reference. Although any similar or equivalent methods and materials may be employed in the practice or testing of the present invention, the preferred methods and materials are now described. Definitions: The term "interleukin 6" or "IL-6", as used herein refers to IL-6 and to fragments, deletions, additions, substitutions, mutations and modifications thereof which retain the biological characteristics of the natural IL-6. Unless otherwise specified, the term refers to human IL-6. The term "IL-6 related diseases" as used herein, refers to diseases associated with IL-6 overproduction including sepsis, multiple myeloma disease (plasma cell leukemia) , bone resorption (osteoporosis) , cachexia, psoriasis, mesangial proliferative glo erulonephritis, renal cell carcinoma, Kaposi's sarcoma, rheumatoid arthritis, hypergammaglobulinemia, Castleman's disease, IgM gam opathy, cardiac myxoma and autoimmune diabetes. The term "IL-6 receptor antagonist", as used herein refers to molecules that interfere with the normal functioning of IL-6, as determined by specific inhibition of the wild type IL-6 molecule in in vitro bioassays, such as: (1) inhibition of IL-6 induction of production of acute phase proteins; (2) inhibition of IL-6 induction of myeloma or plasmacytoma growth; and (3) inhibition of IL-6 induction of immunoglobulin synthesis by human B cells. When the particular IL-6 receptor antagonist is a polypeptide of determined sequence, this invention also contemplates the term to include fragments, deletions, additions, substitutions, mutations and modifications thereof which retain the biological characteristics of the determined polypeptide. A "mutation" in a protein alters its primary structure due to changes in the nucleotide sequence of the DNA which encodes it. These mutations include allelic variants. A "modified" protein differs from the unmodified protein as a result of post-translational events which change the glycosylation or lipidation pattern, or the primary, secondary, or tertiary structure of the protein. Changes in the primary structure of a protein can also result from deletions, additions, or substitutions. A "deletion" is defined as a polypeptide in which one or more internal amino acid residues are absent. An "addition" is defined as a polypeptide which has one or more additional internal amino acid residues as compared to the wild type. A "substitution" results from the replacement of one or more amino acid residues by other residues. A protein "fragment" is a polypeptide consisting of a primary amino acid sequence which is identical to a portion of the primary sequence of the protein to which the polypeptide is related. Preferred "substitutions" are those which are conservative, i.e., wherein a residue is replaced by another of the same general type. As is well understood, naturally-occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar. Furthermore, three of the encoded amino acids are aromatic. It is generally preferred that encoded peptides differing from the determined IL-6 receptor antagonist contain substituted codons for amino acids which are from the same group as that of the amino acid replaced. Thus, in general, the basic amino acids Lys, Arg, and His are interchangeable; the acidic amino acids Asp and Glu are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gin, and Asn are interchangeable; the nonpolar aliphatic amino acids Gly, Ala, Val, lie, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, lie and Leu are more closely related) , and the aromatic amino acids Phe, Trp and Tyr are interchangeable. It should further be noted that if IL-6 receptor antagonist polypeptides are made synthetically, substitutions by amino acids which are not naturally encoded by DNA may also be made. For example, alternative residues include the omega amino acids of the formula NH 2 (CH 2 ) n COOH wherein n is 2-6. These are neutral, nonpolar amino acids, as are sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr or Phe; citrulline and methionine sulfoxide are neutral polar, cyclohexylalanine is neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties. The "biological characteristics" of a protein refers to the structural or biochemical function of the protein in the biological process of the organism in which it participates. Examples of biological characteristics of IL-6 receptor antagonists include: (l) inhibition of IgG 1 synthesis by CESS cells induced by wild type IL-6; (2) induction of Cl esterase inhibitor synthesis by HepG2 cells induced by wild type IL-6; (3) ability to bind to the IL-6 receptor without activity on IL-6-responsive cells; (4) competition with wild type IL-6 for binding to the IL-6 receptor; and (5) inhibition of biological activity of wild type IL-6 on target cells. As used herein, "Site I" refers to a conformational epitope on the IL-6 molecule recognized by the monoclonal antibody MAb CLB.IL-6/8 (see Brakenhoff et al., J . Immunol . , 145, 561 (1990)). The epitope includes sites at both the amino terminal and carboxy terminal portions of the IL-6 molecule: the amino terminal portion includes amino acids Ile 30 -Asp 35 ; while the carboxy terminal portion includes critical amino acids Arg 183 -Met 185 . "Site II" includes critical amino acids Ala 154 -Thr 163 , and other regions corresponding to the conformational epitope on the IL-6 molecule recognized by the monoclonal antibody MAb CLB.IL-6/16 (see Brakenhoff et al., J. Immunol . , 145, 561 (1990)). Preparation of IL-6 Receptor Antagonists IL-6 receptor antagonists may be produced synthetically by the method of Merrifield et al. IL-6 receptor antagonists may be produced recombinantly as shown in U.S. Patent No. 4,966,852. For example, the cDNA for the protein can be incorporated into a plasmid for expression in prokaryotes or eukaryotes. U.S. Patent No. 4,847,201, which is hereby incorporated by reference in its entirety, provides details for transforming microorganisms with specific DNA sequences and expressing them. There are many other references known to those of ordinary skill in the art which provide details on expression of proteins using microorganisms. Many of those are cited in U.S. Patent No. 4,847,201, such as Sambrook et al., Molecular Cloning, Cold Spring Harbor Press (2d ed.. 1989) . The following is an overview about transforming and expressing IL-6 receptor antagonists in microorganisms. IL-6 receptor antagonists DNA sequences may be incorporated into plasmids, such as pUNC13 or pBR3822, which are commercially available from companies such as Boehringer-Mannhei . Once the IL-6 receptor antagonist DNA is inserted into a vector, it can be cloned into a suitable host. The DNA can be amplified by techniques such as those shown in U.S. Patent No. 4,683,202 to Mullis and U.S. Patent No. 4,683,195 to Mullis et al. After the expression vector is transformed into a host such as E . coli the bacteria may be fermented and the protein expressed. Bacteria are preferred prokaryotic microorganisms and E . coli is especially preferred. A preferred microorganism useful in the present invention is E . coli K-12, strain MM294 deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852, United States of America (hereinafter referred to as "ATCC") , on February 14, 1984, under the provisions of the Budapest Treaty, Accession Number 39607. Alternatively, IL-6 receptor antagonists may be introduced into mammalian cells. These mammalian cells may include CHO, COS, C127, Hep G2, SK Hep, baculovirus, and infected insect cells (see also U.S. Patent No. 4,847,201, referred to above) . See also Pedersen et al., J . Biol . Chem . , 265, 16786- 16793 (1990) . Some specific details about the production of a recombinant protein typically involve the following: Suitable Hosts, Control Systems and Methods First, a DNA encoding the mature protein (used here to include all muteins) ; the preprotein; or a fusion of the IL-6 receptor antagonist protein to an additional sequence which does not destroy its activity or to additional sequence cleaved under controlled conditions (such as treatment with peptidase) to give an active protein, is obtained. If the sequence is uninterrupted by introns it is suitable for expression in any host. If there are introns, expression is obtainable in mammalian or other eucaryotic systems capable of processing them. This sequence should be in excisable and recoverable form. The excised or recovered coding sequence is then placed in operable linkage with suitable control sequences in a replicable expression vector. The vector is used to transform a suitable host and the transformed host cultured under favorable conditions to effect the production of the recombinant IL-6 receptor antagonists. Genomic or cDNA fragments are obtained and used directly in appropriate hosts. The constructions for expression vectors operable in a variety of hosts are made using appropriate replications and control sequences, as set forth below. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene. Generally, procaryotic, yeast, or mammalian cells are presently useful as hosts. Host systems which are capable of proper post-translational processing are preferred. Accordingly, although procaryotic hosts are in general the most efficient and convenient for the production of recombinant proteins, eucaryotic cells, and, in particular, mammalian cells are preferred for their processing capacity, for example, the ability to form the proper glycosylation patterns. In addition, there is more assurance that the native signal sequence will be recognized by the mammalian host cell, thus making secretion possible, and purification thereby easier. Control Seguences and Corresponding Hosts Procaryotes most frequently are represented by various strains of E . coli . However, other microbial strains may also be used, such as bacilli, for example Bacillus subtilis, various species of Pseudomonas, or other bacterial strains. In such procaryotic systems, plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used. For example, E . coli is typically transformed using derivatives of pBR322, a plasmid derived from an E . coli species by Bolivar et al., Gene, 2, 95 (1977) . pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides additional markers which can be either retained or destroyed in constructing the desired vector. Commonly used procaryotic control sequences are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, which include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature, 198, 1056 (1977)); the tryptophan (trp) promoter system (Goeddel, et al., Nucleic Acids Res . , 8, 4057 (1980)); the T7 promoter (Studier et al., Meth . Enzymol . , 185, 60 (1990)); and the λ derived P L promoter and N-gene ribosome binding site (Shimatake et al., Nature, 292, 128 (1981)), which has been made useful as a portable control cassette, as set forth in U.S. Patent No. 4,711,845, issued December 8, 1987. However, any available promoter system compatible with procaryotes can be used. In addition to bacteria, eucaryotic microbes, such as yeast, may also be used as hosts. Laboratory strains of Saccharomyces cerevisiae , Baker's yeast, are most used although a number of other strains are commonly available. Examples of plasmid vectors suitable for yeast expression are shown in Broach, Meth . Enz . , 101, 307 (1983); Stinchcomb et al., Nature, 282, 39 (1979); and Tschempe et al., Gene, 10, 157 (1980) and Clarke et al., Meth . Enz . , 101, 300 (1983). Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess et al., J . Adv . Enzyme Reg . , 7, 149 (1968); Holland et al., Biochemistry, 17, 4900 (1978)). Additional promoters known in the art include the promoter for 3- phosphoglycerate kinase (Hitzeman et al., J . Biol . Chem . , 255, 2073 (1980)), and those for other glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucoεe isomerase, and glucokinase. Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization (Holland, supra) . It is also believed that terminator sequences are desirable at the 3' end of the coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes. Many of the vectors illustrated contain control sequences derived from the enolase gene containing plasmid peno46 (Holland et al., J . Biol . Chem . , 256, 1385 (1981)) or the LEU2 gene obtained from YEpl3 (Broach et al., Gene, 8, 121 (1978)), however, any vector containing a yeast compatible promoter, origin of replication and other control sequences is suitable. It is also, of course, possible to express genes encoding polypeptides in eucaryotic host cell cultures derived from ulticellular organisms. See, for example, Tissue Culture , 1973, Cruz and Patterson, eds., Academic Press. Useful host cell lines include murine myelomas N51; VERO, HeLa cells, Chinese hamster ovary (CHO) cells, COS, C127, Hep G2, SK Hep, baculovirus, and infected insect cells. Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and later promoters from Simian Virus 40 (SV40) (Fiers et al., Nature, 273 , 113 (1978)), or other viral promoters such as those derived from polyoma, Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses, or im unoglobulin promoters and heat shock promoters. General aspects of mammalian cell host system transformations have been described by Axel, U.S. Patent No. 4,399,216, issued August 16, 1983. It now appears also that "enhancer" regions are important in optimizing expression; these are, generally, sequences found upstream of the promoter region. Origins of replication may be obtained, if needed, from viral sources. However, integration into the chromosome is a common mechanism for DNA replication in eucaryotes. Plant cells are also now available as hosts, and control sequences compatible with plant cells such as the nopaline εynthase promoter and polyadenylation signal sequences (Depicker et al., J . Mol . Appl . Gen . , 1, 561 (1982)) are available. Methods and vectors for transformation of plant cells have been disclosed in PCT Publication No. WO 85/04899, published November 7, 1985. Host strains useful in cloning and expression herein are as follows: For cloning and sequencing, and for expression of construction under control of most bacterial promoters, E . coli strain MM294 obtained from E . coli Genetic Stock Center GCSC #6135. For expression under control of the P L N RBS promoter, E . coli strain K12 MC1000 lambda lysogen, N 7 N 53 cI857 SusP80, a strain deposited with the ATCC on December 2, 1983 under the provisions of the Budapest Treaty, Accession Number 39531, may be used. E . coli DG116, which was deposited with the ATCC on April 7, 1987, under the provisions of the Budapest Treaty, Accession No. 53606, may also be used. For M13 phage recombinants, E . coli strains susceptible to phage infection, such as E. coli K12 strain DG98, can be employed. The DG98 strain has been deposited with the ATCC on July 13, 1984, under the provisions of the Budapest Treaty, Accession No. 39768. Mammalian expression can be accomplished in COS-A2 cells, COS-7, CV-1, murine myelomas N51, VERO, HeLa cells, Chinese hamster ovary (CHO) cells, COS, C127, Hep G2, SK Hep, baculovirus, and infected insect cells. Insect cell-based expression can be in Spodoptera frugiperda . Transformations Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described by Cohen, Proc . Nat f l . Acad . Sci . (USA) , 69, 2110 (1972), is used for procaryotes or other cells which contain substantial cell wall barriers. Infection with Agrobacterium tumefaciens (Shaw et al., Gene, 23, 315 (1983)) is used for certain plant cells. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham et al., Virology, 52, 546 (1987) is preferred. Transformations into yeast are carried out according to the method of Van Solingen et al., J . Bact . , 130 , 946 (1977) and Hsiao et al., Proc . Nat 'l . Acad . Sci . (USA) , 76, 3829 (1979) . Vector Construction Construction of suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired. Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In general, about 1 μg of plasmid or DNA sequence is cleaved by 1 unit of enzyme in about 20 μl of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate. Incubation times of about 1 hour to 2 hours at about 37°C are workable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods of Enzymology, 65, 499-560 (1980) . Synthetic oligonucleotides may be prepared by the triester method of Matteucci et al., J . Am . Chem . Soc , 103 , 3185-3191 (1981) , or using automated synthesis methods. Kinasing of single strands prior to annealing or for labelling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl 2 , 5 mM DTT, 1-2 mM ATP. If kinasing is for labelling of probe, the ATP will contain high specific activity γ- 32 P. Ligations are performed in 15-30 μl volumes under the following standard conditions and temperatures: 20 M Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 μg/ml bovine serum albumin (BSA) , 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end" ligation) or 1 M ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end" ligation) . Intermolecular "sticky end" ligations are usually performed at 33-100 μg/ml total DNA concentrations (5-100 nM total end concentration) . Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at 1 μM total ends concentration. In the vector construction employing "vector fragments", the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) in order to remove the 5' phosphate and prevent religation of the vector. BAP digestions are conducted at pH 8 in approximately 150 mM Tris, in the presence of Na 2+ and Mg 2+ using about 1 unit of BAP per μg of vector at 60°C for about 1 hour. In order to recover the nucleic acid fragments, the preparation is extracted with phenol/chloroform and ethanol precipitated. Alternatively, religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments. Modification of DNA Sequences For portions of vectors derived from cDNA or genomic DNA which require sequence modifications, site specific primer directed mutagenesis is used. This technique is now standard in the art, and is conducted using a primer synthetic oligonucleotide complementary to a single stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage. Theoretically, 50% of the new plaques will contain the phage having, as a single strand, the mutated form: 50% will have the original sequence. The plaques are hybridized with kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered. Verification of Construction Correct ligations for plasmid construction could be confirmed by first transforming E . coli strain MM294, or other suitable host, with the ligation mixture. Successful transformants are selected by ampicillin, tetracycline or other antibiotic resistance or using other markers depending on the mode of plasmid construction, as is understood in the art. Plasmids from the transformants are then prepared according to the method of Clewell et al., Proc . Nat 'l . Acad . Sci . (USA) , 62, 1159 (1969), optionally following chloramphenicol amplification (Clewell J . Bacteriol, 110, 667 (1972)). The isolated DNA is analyzed by restriction and/or sequenced by the dideoxy method of Sanger et al., Proc . Nat 'l . Acad . Sci . (USA) , 74, 5463 (1977) as further described by Messing et al. , Nucleic Acids Res . , 9, 309 (1981), or by the method of Maxam et al., Methods in Enzymology, 65, 499 (1980) . Purification of IL-6 Receptor Antagonists IL-6 receptor antagonists may be produced in bacteria, such as E. coli , and subsequently purified. Generally, the procedures shown in U.S. Patent Nos. 4,511,502; 4,620,948; 4,929,700; 4,530,787; 4,569,790; 4,572,798; and 4,748,234 can be employed. These patents are hereby incorporated by reference in their entireties. Typically, the heterologous protein (i.e., IL-6 receptor antagonist) is produced in a refractile body within the bacteria. To recover and purify the protein, the cells are lysed and the refractile bodies are centrifuged to separate them from the cellular debris (see U.S. Patent No. 4,748,234 for lowering the ionic strength of the medium to simplify the purification) . Thereafter, the refractile bodies containing the IL-6 receptor antagonist are denatured, at least once (typically in a non-reducing environment) , and the protein is oxidized and refolded in an appropriate buffer solution for an appropriate length of time. IL-6 receptor antagonists may be purified from the buffer solution by various chromatographic methods, such as those mentioned above for the mammalian cell derived IL-6 receptor antagonists. Preferably, IL-6 receptor antagonists are purified by affinity chromatography using anti-IL-6 monoclonal antibodies. Additionally, the methods shown in U.S. Patent No. 4,929,700 may be employed. Formation and Administration IL-6 receptor antagonists are administered at a concentration that is therapeutically effective to treat and prevent IL-6 related diseases, including sepsis and multiple myeloma. To accomplish this goal, IL-6 receptor antagonists are preferably administered intravenously. Methods to accomplish this administration are known to those of ordinary skill in the art. Before administration to patients, formulants may be added to IL-6 receptor antagonists. A liquid formulation is preferred. For example, these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Preferably carbohydrates include sugar or sugar alcohols such as mono, di, or polysaccharideε, or water soluble glucans. The saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof. Sucrose is most preferred. "Sugar alcohol" is defined as a C 4 to C 8 hydrocarbon having an -OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is most preferred. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. Preferably,the sugar or sugar alcohol concentration is between 1.0 w/v% and 7.0 w/v%, more preferable between 2.0 and 6.0 w/v%. Preferably amino acids include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added. Preferred polymers include polyvmylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. It is also preferred to use a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution. Most any physiological buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred. Most preferred is a citrate buffer. Preferably, the concentration is from 0.01 to 0.3 molar. Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268,110. Additionally, IL-6 receptor antagonists can be chemically modified by covalent conjugation to a polymer to increase its circulating half-life, for example. Preferred polymers, and methods to attach them to peptideε, are shown in U.S. Patent Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546 which are all hereby incorporated by reference in their entireties. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG) . PEG is soluble in water at room temperature and has the general formula: R(0-CH 2 -CH 2 ) n O-R where R can be hydrogen, or a protective group such as an alkyl or alkanol group. Preferably, the protective group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG has a preferred average molecular weight between 1000 and 40,000, more preferably between 2000 and 20,000, most preferably between 3,000 and 12,000. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with a free amino group on the inhibitor. However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/IL-6 receptor antagonist of the present invention. Water soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG) , etc. POG is preferred. One reason is because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body. The POG haε a preferred molecular weight in the εame range as PEG. The structure for POG is shown in Knauf et al., J . Bio . Chem . , 263 , 15064-15070 (1988) and a discusεion of POG/IL-2 conjugates is found in U.S. Patent No. 4,766,106, both of which are hereby incorporated by reference in their entireties. After the liquid pharmaceutical composition is prepared, it is preferably lyophilized to prevent degradation and to preserve sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art. Administration to Affected Individuals As stated above, IL-6 receptor antagonists are useful to treat human patients with IL-6 related diseases, including sepsis and multiple myeloma. Generally, sepsis patients are characterized by high fever (>38.5°C) or hypothermia (<35.5°C), low blood pressure, tachypnea (> than 20 breaths/minute) , tachycardia (> than 100 beats/minute) , leukocytosis (> 15,000 cells/mm 3 ) and thrombocytopenia (< than 100,000 platelets/mm 3 ) in association with bacteremia. IL-6 receptor antagonists are to be administered as soon as a patient is suspected of being septic; presenting themselves with a > 20% drop in fibrinogen or appearance of fibrin split products, a rise in the patient's temperature and the diagnosis of leukopenia and hypotension associated with sepsis. As also stated above, the preferred route is by intravenous administration. Generally, IL-6 receptor antagonists are given at a dose between 1 μg/kg and 20 mg/kg, more preferably between 20 μg/kg and 10 mg/kg, most preferably between 1 and 7 mg/kg. Preferably, it is given as a bolus dose, to increase circulating levels by 10-20 fold and for 4-6 hours after the bolus dose. Continuous infusion may also be used after the bolus dose. If so, IL-6 receptor antagonists may be infused at a dose between 5 and 20 μg/kg/minute, more preferably between 7 and 15 μg/kg/minute. When used to treat sepsis, IL-6 receptor antagonists may be given in combination with other agents which would be effective to treat sepsis. For example, the following may be administered in combination with IL-6 receptor antagonists: antibiotics that can treat the underlying bacterial infection; monoclonal antibodies that are directed against bacterial cell wall components; receptors that can complex with cytokineε that are involved in the sepsis pathway; antibodies to cell adhesion molecules such as LFA-1; and generally any agent or protein that can interact with cytokines or complement proteins in the sepsis pathway to reduce their effects and to attenuate sepsis or septic shock. IL-6 receptor antagonists may also be administered in conjunction with other similar odulatory cytokineε including LIF, oncostatin M, CNTF and IL-11. Antibiotics that are useful in the present invention include those in the general category of: beta-lactam rings (penicillin) , amino sugars in glycosidic linkage (amino glycosides) , macrocyclic lactone rings (macrolides) , polycyclic derivatives of napthacenecarboxamide (tetracyclines) , nitrobenzene derivatives of dichloroacetic acid, peptides (bacitracin, gramicidin, and polymyxin) , large rings with a conjugated double bond system (polyenes) , εulfa drugε derived from εulfanilamide (sulfonamides) , 5-nitro-2-furanyl groups (nitrofurans) , quinolone carboxylic acids (nalidixic acid) , and many others. Other antibiotics and more versions of the above specific antibiotics may be found in Encyclopedia of Chemical Technology, 3rd Edition, Kirk-Othy er (ed.), Vol. 2, pages 782-1036 (1978) and Vol. 3, pages 1-78, Zinsser, MicroBiology, 17th Ed., Joklik et al. (Eds.) 235-277 (1980), or Dorland's Illustrated Medical Dictionary, 27th Ed., W.B. Saunders Company (1988). Monoclonal antibodieε that may be administered along with IL-6 receptor antagonists include those found in PCT WO 88/03211, to Larrick et al., entitled Gram-Negative Bacterial Endotoxin Blocking Monoclonal Antibodies, and U.S. Serial No. 07/876,854, filed April 30, 1992, to Larrick et al. Both applications disclose specific monoclonal antibodies that are useful to treat sepsis and which bind to various antigens on the E . coli bacterial cell wall. A specifically preferred monoclonal antibody is that which is produced by hybridoma deposited with the ATCC on May 19, 1987, under the provisions of the Budapest Treaty, Acceεεion No. HB9431. Other agents which may be combined with IL-6 receptor antagonists include monoclonal antibodies directed to cytokines involved in the sepsiε pathway, such as those monoclonal antibodies directed to IL-6 or M-CSF, see U.S. Serial No. 07/451,218, filed December 15, 1989 to Creasey et al. and monoclonal antibodies directed to TNF, see Cerami et al., U. S. Patent No. 4,603,106. Inhibitors of protein that cleave the mature TNF prohormone from the cell in which it was produced, see U.S. Serial No. 07/395,253, filed August 16, 1989, to Kriegler et al. Antagonists of IL-1, such aε εhown in U.S. Serial No. 07/517,276, filed May 1, 1990 to Haskill et al. Inhibitors of IL-6 cytokine expression such as inhibin, εuch as shown in U.S. Serial No. 07/494,624, filed March 16, 1992, to Warren et al., and receptor based inhibitors of various cytokines such as IL-1. Antibodies to complement may also be employed. Generally, IL-6 receptor antagonists may be useful for those diseases that occur due to the up-regulation of tissue factor brought on by TNF, IL-1 or other cytokines. The present invention will now be illustrated by reference to the following examples which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be construed as restricting the invention in any way. Examples Materials and Methodε Antibodies and Cytokines: The production and purification of an IL-6 specific MAb has been deεcribed before in detail (εee Brakenhoff et al. (1990)). The wild-type rIL-6 preparation used throughout these experiments as a standard is purified from E . coli carrying the HGF7 plasmid (see Brakenhoff et al., J. Immunol . , 139, 4116 (1987)). HGF7 encodes an IL-6 fusion protein consisting of a 9 amino acid jβ-galactosidaεe derived leader followed by 4 glycines, an aspartic acid residue, and Arg 17 -Met 185 of mature IL-6. Purification of HGF7 has been described before in Brakenhoff et al. (1990) . Specific activity of purified rhIL-6 HGF7 as determined in the B9 assay is approximately 10 9 U/mg. Recombinant IFN- was obtained from Genentech (San Francisco, California) . Bacterial Strains and Vectors: Construction of the expression vector pUK-IL-6 is described in Brakenhoff et al., 1989. Expression of vectors occurred in an E . coli DH5α (GIBCO BRL) host. Example 1 Preparation of IL-6 Variants A. Preparation of an IL-6 Variant Library Random mutagenesiε of the IL-6 region around Trp 158 was performed to identify residues which might be important to the biological activity of IL-6. The vector pUK-IL-6 was used for construction of the library of rhIL-6 mutants with random substitutions in residues Gln 153 -Thr 163 . Restriction fragments with the desired substitutions suitable for subcloning in pUK-IL-6, were obtained in two steps by overlap extension PCR (see Ho et al., Gene, 77, 51 (1989)) . In the first PCR reactionε, pUK-IL-6 was used as template. Fragment 1, running from the unique Xbal site in the IL-6 coding region, to amino acid Thr 163 , was generated by combining a 5' primer (A) with (SEQ ID NO: 1) (nucleotides (ntε) 477-498 of IL-6 coding region (see Brakenhoff et al., supra, 1987, and a 3' primer (B) with (SEQ ID NO: 2) (nts 537-570, corresponding to residueε Gln 153 -Thr 163 ) . To obtain randomly distributed substitutions in primer B a similar approach was used as described by Derbyshire et al., Gene, 46, 145 (1986) . Instead of contaminating each nucleotide reservoir with the three other monomers, the fifth channel of the oligo synthesizer (Applied Biosystems type 381A, Warrington, UK) was used during oligo synthesis: during each synthesis step both the channel containing 100 mM of the wild-type nucleotide and the fifth channel containing 1.25 mM of each of the four dNTP's were mixed in 1:1 ratio. With an oligo length of 34 thiε reεults in approximately 36% single, 36% multiple, and 28% no mutations per oligo. Fragment 2, running from Gln 153 to the Banll site, was generated by using a 5' primer C complementary to primer B (SEQ ID NO: 3) (nts 538-571) synthesized in the same manner as primer B. This oligo was combined with a 3' primer D, (SEQ ID NO: 4) (nts 609-629) . PCRs were carried out with Taq polymerase as specified by the manufacturer (Perkin Elmer Cetus) by using 10 ng of template DNA and 100 ng of each primer (annealing 2 minutes at 50°C, extension 2.5 minutes at 65°C, denaturation 1.5 minutes at 95°C; 30 cycles). After the first PCR reactions, fragments 1 and 2 were purified from low melting agarose and approximately 100 ng of each served as template in the second PCR reaction with primers A and D. After phenol/CHCl 3 extraction the second PCR product was digested with Xbal and Banll, gel purified and subcloned in Xbal-Banll digested pUK-IL-6. Following transformation to E . coli DH5α approximately 1,000 colonies were obtained. DNA manipulation procedures were performed aε deεcribed in Brakenhoff et al., 1989 and Brakenhoff et al., 1990. Nucleotide sequences of selected mutants (see below) were obtained with cDNA derived oligonucleotide primers on dsDNA by using the "Sequenase" kit (United Stateε Biochemical Corporation, Cleveland, OH) . B. Preparation and Screening of IL-6 Variantε Variantε were subsequently selected for binding to a site I specific MAb (MAb CLB.IL-6/8) and losε of binding to a εite II εpecific MAb (MAb CLB.IL-6/16) in ELISAs and the nucleotide sequence of plasmids encoding mutants with this phenotype was determined. 400 a picillin reεiεtant colonieε were toothpicked in wells of 96-well flat bottom microtiterplates (NUNC) containing 100 μl LC amp medium (10 g of bactotryptone, 5 g of yeaεt extract, 8 g of NaCl, and 2 ml of Tris base per liter supplemented with 100 μg/ml final concentration of ampicillin) . Following overnight culture at 37°C, bacteria were lysed by addition of lysozyme to 1 mg/ml and further incubation for 30 inuteε at 37°C. One in 10 dilutionε of the crude extracts in phosphate-buffered saline (PBS), 0.02% Tween-20, 0.2% gelatin were directly tested for reactivity in sandwich ELISAs with MAb CLB.IL-6/8 or 16 coated to the plastic and biotinylated polyclonal goat anti-rhIL-6 as detecting antibody. Bound polyclonal anti-rhIL-6 was detected with horεeradiεh peroxidase conjugated streptavidin (Amersham, Amersham UK) . Similarly prepared extracts of E. coli carrying pUK-IL-6 were used as poεitive control. Of the MAb CLB.IL-6/8 poεitive/MAb CLB.IL-6/ 16 negative mutants the nucleotide sequence of the Xbal-Banll fragment was subsequently determined. ELISA procedures and biotinylation of polyclonal antibodies have been described in detail by Brakenhoff et al., 1990, supra and Helle et al., J . Immunol . Methods, 138 , Al (1991) . As shown in Table 1, the MAb CLB.IL-6/16 epitope is disrupted by single substitution of Gln 155 , Asn 156 , Trp 158 , and Thr 163 . The double and triple εubstitution mutants suggest that residueε Ala 154 , Leu 159 , Gln 160 and Met 162 might alεo be important for the MAb CLB.IL-6/16 epitope. Table 1. Bioactivity of rhIL-6 mutants that do not bind to MAb CLB.IL-6/16 TABLE 1 B9 CESS Assay IL-6 Variant Asεay (u/μq) a ( V / UO) a mature rhIL-6 6 x 10' 1 x 10 4 Gln 155 His 5 x 10 c 3 x 10* Asn 156 Lyε 1 x 10 6 6 x 10 4 Trp 158 * (Gln) 5 x 10 5 4 x 10 2 Trp 158 Gly 4 x 10 6 4 x 10 3 Trp 158 Arg 1 x 10 c 5 x 10 3 Thr 163 Pro 2 X 10 6 < 20 Ala 154 Glu, Gln 160 His 5 x 10 5 not detectable Gln 155 His, Gln 157 Pro 6 x 10 c 2 x 10 3 Trp 158 Cyε, Met 162 Ile 6 x 10 < 10 4 Trp 158 Arg, Ser 170 Asn 5 X 10' 2 x 10 3 Gln 160 Glu, Thr 163 Pro 3 X 10 c < 20 Ile 137 Leu, Leu 159 Arg, Met 162 Ile 1 X 10' 5 x 10 3 a Concentration and biological activity in B9 and CESS assay of SDS-extracts of rhIL-6 mutants that bound to MAb CLB.IL-6/8 but not to MAb CLB.IL-6/16 were measured as described above. 1 unit is the amount of variant giving half-maximal stimulation in each assay. Values derived from one of two asεays are shown. C. Bioactivity of IL-6 Mutants The biological activity of crude extracts of various mutant proteins was subsequently measured both in the B9 assay and on IgGl production by CESS cells. The B9 assay measures the murine hybridoma growth factor activity of rhIL-6 and variants as described in Aarden et al., Eur . J . Immunol . , 17, 1411 (1987) and Helle et al., Eur . J . Immunol . , 18, 1535 (1988) . The CESS assay measureε B-cell εti ulatory factor-2 activity of rhIL-6 variantε eεεentially as described by Poupart et al., EMBO J. , 6, 1219 (1987). Briefly, CESS cells (6 x 10 3 cells/200 μl well in 96-well flat-bottom microtiterplates, in IMDM-5% FCS-Trf) were incubated for 4 days with serial dilutions of rhIL-6 or rhIL-6 variant containing samples in triplicate. IL-6 induced IgGl production by the cells waε subsequently meaεured in a εandwich ELISA by using a mouse MAb specific for human IgGl (MH161-1M, Department of Immune Reagents, Central Laboratory of the Netherlands Red Croεε Blood Tranεfuεion Service (CLB) , Amsterdam, The Netherlands) in combination with a horεeradish peroxidase conjugated murine MAb specific for human IgG (MH16-1 ME, CLB) with a human serum aε εtandard (H00-1234, CLB) . ELISA procedures were as described above. To measure the bioactivity of the MAb CLB.IL-6/8 positive/MAb CLB.IL6/16 negative mutantε, overnight cultureε of E . coli DH5α carrying the mutant conεtructε were diluted 1:50 in 250 ml LC amp medium and εubεequently cultured to an OD550 of 1.5. Bacteria were harveεted by centrifugation, resuspended in 5 ml lysis buffer (PBS, 1% Tween-20, 10 mM EDTA, 2 mM PMSF) and lysed by sonication. To solubilize rhIL-6 containing incluεion bodieε, SDS was subsequently added to 1%. After 1 hour incubation at room temperature, SDS-insoluble material waε removed by centrifugation (15 minutes at 13,000 g) . Bioactivity of this SDS εolubilized material was directly measured in the B9 and CESS asεays starting from a 1/1000 dilution. The IL-6 variant concentration of these preparations waε determined by meanε of a competitive inhibition radioimmunoassay (RIA) with IL-6 εpecific MAb CLB.IL-6/7 coupled to Sepharose 4B (Pharmacia LKB) and 125 I-rhIL-6 HGF7, in the presence of 0.1% SDS. Unlabelled rhIL-6 HGF7 served as a standard. MAb CLB.IL-6/7 binds heat and SDS denatured IL-6 and recognizes IL-6 residues Thr 143 -Ala 146 as determined by pepscan analysis (see Fontaine et al., Gene, 104, 227 (1991) and Arcone et al., FEBS Letters, 288, 197 (1991)). As shown in Table 1, all mutants were biologically active in the murine B9 hybrido a proliferation assay. However, although very active in the B9 assay, no activity could be detected for the rhIL-6 Thr 163 Pro (rhIL-6 T 163 P) single-mutant and rhIL-6 Gln 160 Glu, Thr 163 Pro (rhIL-6 Q 160 E, T 163 P) double-mutant preparation on human CESS cells. (The nomenclature X n Y following a protein indicates that amino acid X at residue n has been replaced by amino acid Y, where X and Y are the commonly used three-letter or one-letter abbreviations for the naturally-occurring amino acids.) D - Expression and Purification of Two IL-6 Mutants To confirm the role of the two mutants active in the B9 assay and inactive in the CESS assay described above, the IL-6 cDNA inserts from the vectors pUK-IL-6 T 163 P and pUK-IL-6 Q 160 E, T 163 P were removed with Ncol and BamHI and subcloned in NcoI-BamHI digeεted pET8c. Plaεmid DNA waε prepared from E. coli DH5α carrying the pET8c conεtructε and transformed to E . coli BL21 (DE3). E . coli BL21 (DE3) carrying these expreεεion plaεmidε were subsequently cultured to an OD550 of 0.6 in LC amp medium and expression was induced by addition of 0.5 mM IPTG (Sigma) . After a 3 hour induction period, the bacteria were harvested by centrifugation and the IL-6 variants were purified essentially as described by Arcone et al., Eur . J . Biochem . , 198, 541 (1991) , with some modifications. Briefly, following centrifugation, bacteria were resuspended in 1/20 of the culture volume 10 mM Tris-HCl pH 7.4, 2 mM PMSF and frozen at -20°C. Following thawing bacteria were lysed by sonication. The sonicate was then applied upon a sucrose cushion (40% sucrose, 10 mM Tris-HCl pH 7.4) and centrifuged for 1 hour at 47,000 g. Pelleted inclusion bodies were subεequently waεhed once with PBS, 0.5% Tween-20, 10 mM EDTA, 2 mM PMSF and diεεolved in 6 M guanidine-HCl, 25 mM Triε-HCl pH 7.4 (0.4 g wet weight/liter) . Following two times dialysis against 20 volumes of 25 mM Tris-HCl pH 8.5, aggregates were removed by centrifugation for 1 hour at 11,000 g and the dialysate waε concentrated 30-fold with an A icon YM10 Filtration unit (Amicon Corp., Danverε, MA) . The concentrate was then directly applied on a fast Q Sepharose anion exchange column attached to a FPLC (Pharmacia LKB) . Bound rhIL-6 variants were εubεequently eluted with a linear gradient of NaCl in 25 mM Triε-HCl pH 8.5 and eluted at approximately 100 mM NaCl. The variantε were subsequently sterile filtered and stored at -70°C. Protein concentration waε determined both by meaεuring the optical denεity of the preparations and by the Bradford method (Anal . Biochem . , 72, 248 (1976)) using BSA as a standard. Bradford and OD 280 correlated best when asεuming the OD 280 of a 10 mg/ml εolution of IL-6 is 10. Figure 1 showε a Coomassie blue stained SDS-polyacrylamide gel of the mutant preparations. The variants migrated with approximately the same molecular weight aε mature rhIL-6. After the final purification step, two bands were observed in the rhIL-6 Qi 6θ E ' T 163 P preparation. Following Western blotting, both bands were recognized by an IL-6 specific MAb, suggesting that the lower band is a degradation product of the upper band (data not shown) . E. Bioactivity of Two IL-6 Mutants The bioactivity of these two mutants were tested both in the CESS asεay and in two other available bioassays for IL-6: the HepG2 aεsay and the XG-1 asεay. The HepG2 assay measures the hepatocyte εtimulating activity of rhIL-6 variants through the induction of Cl esterase inhibitor (Cl inh.) production by HepG2 cells aε described by Zuraw et al., J . Immunol . , 265, 12664 (1990) . Following culturing to confluency (5 x 10 5 cells in 0.5 ml wells (Costar) in Iεcove's Modified Dulbecco'ε Medium (IMDM) supplemented with 5% FCS, 5 x 10 "5 M 2-ME, penicillin (100 IU) , streptomycin (100 μg/ml) and human transferrin (20 μg/ml; Behringwerke, Marburg, Germany) (IMDM-5% FCS-Trf) , HepG2 cells were washed twice and stimulated with serial dilutions of rhIL-6 or rhIL-6 mutants for 48 hours in the same medium in duplicate. In some experiments, cells were washed again after 24 hours and the stimulus was repeated for 24 hours. This procedure results in a higher stimulation index. After the incubation period, Cl inh. synthesiε waε εubsequently measured by sandwich RIA with anti-Cl inh. MAb RII coupled to Sepharose 4B and 125 I-labelled sheep polyclonal anti-Cl inh. IgG with normal human plasma as a εtandard as described in Nuijens et al., J. Clin . Invest . , 84, 443 (1989) and Eldering et al., J. Biol . Chem . , 263 , 11776 (1988). The XG-1 assay measures IL-6 activity on human myeloma cell line XG-1 esεentially aε deεcribed in Jourdan et al., J . Immunol . , 147, 4402 (1991) . Briefly, the cellε were washed twice, incubated in IMDM-5% FCS-Trf for 4 hours at 37°C and then washed again. 10 4 cells/well in 200 μl IMDM-5% FCS-Trf in 96-well flat-bottom microtiterplates were subsequently incubated in triplicate with serial dilutions of rhIL-6 or rhIL-6 variants for 3 days. Following this culture period, proliferation of the cells was meaεured by labelling the cells with 7.4 kBq of [ 3 H]Thymidine (74 Gbq/ mol) for 4 hours and counting the radioactivity incorporated in the nuclei. Figure 2a-c showε repreεentative doεe reεponse curves of the mutantε in three different aεεayε with human cell lineε. In Table 2, the specific activities of the mutantε in the human assays is depicted, together with the specific activities in the (murine) B9 aεεay, relative to that of wild-type rhIL-6 HGF7. In the experiment in Figure 2a iε εhown that also the purified rhIL-6 Q 160 E, T 163 P double-mutant did not induce IgGl syntheεiε by the CESS cellε. In εome experimentε however (see e.g. Figure 3a) , a small increase in background IgGl production was observed. As shown in Figure 2b, a weak induction of the acute phase protein Cl esterase inhibitor (Cl inh.) was reproducibly observed at high concentrations of the variant, with a strongly reduced plateau level as compared to wild-type rhIL-6 HGF7. However, although the εpecific activity of the double-mutant in inducing proliferation of the human myeloma cell line XG-1 (see Jourdan et al., supra and Figure 2c) was approximately 1,000-fold reduced as compared to wild-type IL-6, almoεt the same plateau level was reached. On B9 cells the specific activity of rhIL-6 Q 16 o E ' τ i 63 p was only 10-fold reduced (Table 2) . RhIL-6 T 163 P was more active than the double-mutant in all assays, with a reduced plateau in CESS and HepG2 assays. The activity of the rhIL-6 Qi 6 o E , τ i 63 p mutant on XG-1 cells was not due to contamination by wild-type rhIL-6 because it could be inhibited by MAb CLB.IL-6/8, but not by MAb CLB.IL-6/16 (data not εhown) . Table 2. Specific activities (U/μg) of purified rhIL-6 variants in IL-6 bioaεεays TABLE 2 XG-1 IL-6 Variant B9 (x!0~ 5 ) CESS (x!0 ~3 ) HepG2 (x!0~ 4 ) mature rhIL-6 11 ± 3 5.2 ± 0.3 542 ± 12 ± 0.3 209 HGF7 6 ± 2 4.7 ± 1.2 666 ± 4.9 ± 165 2.5 Thr 163 Pro 2.1 ± 0.3 0.003 ± 11 ± 10 0.024 ± 0.003 0.0175 Gln 160 Glu, 0.9 ± 0.4 < 0.001 < 0.1 0.004 ± Thr 163 Pro 0.002 F. IL-6 receptor antagonistic Activity of Two IL-6 Mutants We tested these mutants for ability to antagonize the activity of wild-type rhIL6 HGF7 on the cell lines. In Figure 3a and b is shown that rhIL-6 Qi 6 o E / τ i 63 p completely inhibited the wild-type IL-6 activity on CESS and HepG2 cells. In these experiments, 50% inhibition of IL-6 activity in CESS and HepG2 asεayε waε obεerved with approximately 50 ng/ml and 1 μg/ml of rhIL-6 Q 160 E / T 163 P, respectively, corresponding to 20 and 200-fold the concentration of rhIL-6 HGF7 used to stimulate the cells. 100% inhibition was observed when the double-mutant was used in respectively 1,000 and 3,600-fold exceεε over wild-type. No inhibitory effectε were observed on XG-1 cells. Of rhIL-6 T 163 P no antagonistic activity could be detected (data not shown) . Figure 4 showε that the inhibitory effect of rhIL-6 Q 16 o E ' τ i 63 p on IL_6 activity in the HepG2 aεεay could be reversed by high concentrations of rhIL-6 HGF7, suggesting competitive inhibition of IL-6 receptor binding by rhIL-6 Qιeo E τ i 63 p * A similar result was found with CESS cells (data not shown) . HepG2 cells can syntheεize Cl inh. in response to both IL-6 and IFN-7 via separate mechanisms (see Zuraw et al., supra) . To further demonstrate the specificity of inhibition by the double-mutant, we tested whether rhIL-6 Qi 6 o E » τ i 63 p could inhibit IFN-γ induced Cl inh. εyntheεiε by the HepG2 cells. Aε shown in Figure 5, the Cl esterase inhibitor synthesis induced by 5 ng/ml of rhIL-6 HGF7 was inhibited to background levels, whereas the Cl inh. synthesis induced by 1 ng/ml of IFN-γ was unimpaired. G. IL-6R Binding of Two IL-6 Mutants The fact that the rhIL-6 Q 160 T i 63 P c ° uld still be recognized by site I-specific MAb CLB.IL-6/8 and that it could antagonize wild-type IL-6 activity on CESS and HepG2 cellε εuggested that the 80 kDa binding site was still intact. To teεt thiε hypothesis binding of this variant to NIH-3T3 fibroblasts transfected with an expression vector encoding the 80 kDa IL-6 receptor (see Rose-John et al., J . Biol . Chem . , 266, 3841 (1991)) was compared to that of wild-type rhIL-6 in a competitive inhibition assay. Figure 7 shows that the double-mutant was approximately 4-fold less efficient in inhibiting binding of 125 I-rhIL-6 to the cells, than rhIL-6 HGF7. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A NAME: Cetus Oncology Corporation (B STREET: 4560 Horton Street (C CITY: Emeryville (D STATE: California (E COUNTRY: United States of America (F POSTAL CODE: 94608 (A NAME: Central Labratory of the Netherlands Red Cross Blood Transfusion Service (B STREET: P.O. Box 9190 (C CITY: 1006 AD Amsterdam (E COUNTRY: Netherlands (A NAME: Brakenhoff, Just P.J. (B STREET: Wenslauerstraat 35 hs (C CITY: 1053 AW Amsterdam (E COUNTRY: Netherlands (A NAME: Aarden, Lucien A. (B STREET: Noordmeerweg 10 (C CITY: 1151 CV Broek in Waterland (E COUNTRY: Netherlands (ii) TITLE OF INVENTION: Interleukin-6 Receptor Antagonists (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Marshall, O'Toole, Gerstern, Murray & Borun (B) STREET: 6300 Sears Tower, 233 South Wacker Drive (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: United States of America (F) POSTAL CODE: 60606-6402 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 20-OCT-1993 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 07/959,942 (B) FILING DATE: 20-OCT-1992 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Rin-Laures, Li-Hsien (B) REGISTRATION NUMBER: 33,547 (C) REFERENCE/DOCKET NUMBER: 31389 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 312/474-6300 (B) TELEFAX: 312/474-0448 (C) TELEX: 25-3856 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GAATCTAGAT GCAATAACCA CC 22 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TGTCATGTCC TGCAGCCACT GGTTCTGTGC CTGC 34 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CAGGCACAGA ACCAGTGGCT GCAGGACATG ACAA 34 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CGAAGAGCCC TCAGGCTGGA C 21

Claims: WE CLAIM: 1. An interleukin-6 (IL-6) receptor antagonist. 2. The IL-6 receptor antagonist of claim 1 in isolated and purified form. 3. The IL-6 receptor antagonist of claim 1 wherein the antagonist is an IL-6 molecule containing one or more mutations. 4. The IL-6 receptor antagonist of claim 3 wherein the IL-6 molecule contains one or more Site II mutations. 5. The IL-6 receptor antagonist of claim 4 containing a single Site II mutation. 6. The IL-6 receptor antagonist of claim 5 wherein the single Site II mutation is Thrl63 'Pr 163- 7. The IL-6 receptor antagonist of claim 5 wherein the single Site II mutation is Glen160¯ Glu160. 8. The IL-6 receptor antagonist of claim 4 wherein the antagonist is an IL-6 molecule containing the two Site II mutations Thr163°Prol63 and Gln160Glu160. 9. A pharmaceutical composition comprising an IL-6 receptor antagonist and a pharmaceutically acceptable carrier. 10. The pharmaceutical composition of claim 9 wherein the antagonist is an IL-6 molecule containing one or more mutations. 11. The pharmaceutical composition of claim 10 wherein the IL-6 molecule contains one or more Site II mutations. 12. The pharmaceutical composition of claim 11 wherein the antagonist contains a single Site II mutation. 13. The pharmaceutical composition of claim 12 wherein the single Site II mutation is Thr163Pro163. 14. The pharmaceutical composition of claim 12 wherein the single Site II mutation is Gln160Glu160. 15. The pharmaceutical composition of claim 11 wherein the antagonist is an IL-6 molecule containing the two Site II mutations Thr163-Prol63 and G1n160Glu160. 16. A method for treating an IL-6 related disease comprising administering to a patient in need of such treatment a pharmaceutical composition comprising an amount of an IL-6 receptor antagonist effective for treating the IL-6 related disease and a pharmaceutically acceptable carrier. 17. The method of claim 16 wherein the antagonist is an IL-6 molecule containing one or more mutations. 18. The method of claim 17 wherein the IL-6 molecule contains one or more Site II mutations. 19. The method of claim 18 wherein the antagonist contains a single Site II mutation. 20. The method of claim 19 wherein the single Site II mutation is Thr163-Prol63. 21. The method of claim 19 wherein the single Site II mutation is Gln160-Glul60. 22. The method of claim 18 wherein the antagonist is an IL-6 molecule containing the two Site II mutations Thr163Pro163 and Gln160-GlU160 23. The method of claim 16 wherein the IL-6 related disease is sepsis. 24. The method of claim 16 wherein the IL-6 related disease is multiple myeloma.

Inventors: AARDEN LUCIEN A, BRAKENHOFF JUST PJ, AARDEN, LUCIEN, A., BRAKENHOFF, JUST, PJ.
Applicants: AARDEN LUCIEN A, BRAKENHOFF JUST PJ, CETUS ONCOLOGY CORP, RED CROSS BLOOD TRANSF SERV NL, CENTRAL LABORATORY OF THE NETHERLANDS RED CROSS BL, CETUS ONCOLOGY CORPORATION
Application year: 1993
Main classification: C12N15
IPC codes: A61K38, A61P29, A61P31, A61P35, A61P37, C07K14, C12N1, C12N15, C12P21, C12R1