MXPA98009659A - Interleucina-1j humana and antagonists of the mi - Google Patents

Abstract

Nucleic acids encoding IL-1y and human and purified IL-1y proteins and fragments thereof are provided. Also provided are polyclonal and monoclonal antibodies, anti-IL-1y antibodies and anti-idiotypic antibodies that can be IL-1 agonists or antagonists and human. The methods of use of the compositions for diagnostic and therapeutic utilities are also provided together with the IL-1 and human antagonists and receptors.

Description

INTERLEUCINA-lj HUMAN AND ANTAGONISTS OF THE SAME FIELD OF THE INVENTION The present invention relates to compositions and methods for affecting the human immune system. In particular, nucleic acids, proteins and antibodies and other antagonists that regulate the response and development of the immune system are provided. The diagnostic and therapeutic uses of these materials are also described.

BACKGROUND OF THE INVENTION Recombinant DNA technology refers, in general, to the technique of integrating genetic information from a donor source into vectors for further processing, such as through introduction into a host, by means of which genetic information transferred is copied and / or expressed in the new environment. Typically, genetic information exists in the form of complementary DNA (cDNA) from messenger RNA (mRNA) that encodes a desired protein product. The carrier is often a plasmid that has the ability to incorporate the cDNA for later replication in a host and, in some cases, actually control the expression of the cDNA and thereby direct the synthesis of the product encoded in the host.

For some time it has been known that the immune response in mammals is based on a series of complex cellular interactions, known as the "immune network". Recent research has provided new insights into the internal workings of this network. Although it remains clear that much of the response, in fact, revolves around the network-like interactions of lymphocytes, macrophages, granulocytes and other cells, immunologists now generally hold the view that soluble proteins, known as lymphokines, Cytokines or monocytes, play an important role in the control of these cellular interactions. In this way, there is considerable interest in the isolation, characterization and mechanisms of action of cell modulation factors, and an understanding of what will lead to significant advances in the diagnosis and therapy of various medical abnormalities, for example, disorders of the immune system. Apparently, lymphokines mediate cellular activities in various ways. These have been shown to support the proliferation, growth and differentiation of pluripotent cells of the haematopoietic stem in a large number of progenitors comprising various cell lineages that make up a complex immune system. The proper and balanced interactions between the cellular components are necessary for a healthy immune response. Different cell lineages often respond in different ways when lymphokines are administered together with other agents. The cell lines especially important for the immune response include two classes of lymphocytes: B cells, which can produce and secrete immunoglobulins (proteins with the ability to recognize and bind to foreign matter to effect their elimination), and T cells from different subseries that secrete lymphokines and induce or suppress B cells and other cells (including other T cells) that make up the immune network. These lymphocytes interact with many other cell types. Another important cell lineage is that of mast cells (which have not been positively identified in all mammalian species), which is a connective tissue cell that contains granules located close to the capillaries throughout the body. These cells are found in especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts. Mast cells play a central role in allergy-related disorders, particularly anaphylaxis as follows: when selected antigens reticulate a class of immunoglobulins bound to receptors on the mast cell surface, the mast cell degrades and releases mediators, for example, histamine, serotonin, heparin and prostaglandins, which causes allergic reactions, for example, anaphylaxis. Research to better understand and treat the various immune disorders has been interrupted by the general inability to keep the cells of the immune system in vi tro. Immunologists have discovered that the culture of these cells can be achieved by the use of T cells and other cellular supernatants, which contain different growth factors, including many of the lymphokines. Okamura et al. (1995) Nature 378: 88-91 describes a novel cytokine that induces certain T cells to produce interferon gamma (IFN-α), the cytokine being called IGIF. The factor has been identified in mouse Kupffer cells and activated macrophages. So far no human equivalent has been described. From the foregoing, it is evident that the discovery and development of new lymphokines could contribute to new therapies for a wide range of degenerative or abnormal states that directly or indirectly include the immune system and / or hematopoietic cells. In particular, the discovery and development of lymphokines that improve or potentiate the beneficial activities of known lymphokines would be highly beneficial. The present invention provides novel compositions of interleukin and related compounds, and methods for their use.

SUMMARY OF THE INVENTION The present invention relates to interleukin-I? (IL-1?) Of primate, for example, of human, and its biological activities. It includes nucleic acids that encode the polypeptides themselves and the methods for their production and use. The nucleic acids of the invention are characterized, in part, by their homology to the cloned complementary DNA sequence (cDNA) contained herein, and / or by functional assays for the activity of IL-1? applied to polypeptides, which are usually encoded by these nucleic acids. Methods for modulation and intervention in the control of an immune response are provided. The invention is based, in part, on the discovery and cloning of human cDNAs that are capable of expressing proteins having IL-1 activity. Equivalent vectors can be constructed using the techniques of the polymerase chain reaction (PCR) and the sequences of the inserts. The invention provides inter alia an IL-1? of substantially pure primate, a fusion protein comprising the sequence of IL-1? of primate and antibodies specific for binding to an IL-1? of primate, and a nucleic acid that encodes an IL-1? human or fusion protein thereof. In the environment of the IL-l? substantially pure, the IL-1? may contain a mature sequence contained within the amino acid sequence defined by SEQ ID NO 2. This last sequence contains a signal sequence that is not a common signal sequence but, as a prosequence analogue for the predominance of the IL-lb that is unfolded by a convertase-like enzyme, it probably runs from position 1 (met) to approximately 36 (asp) of the amino acids. See Dinarello (1994) FASEB J. 1314-1325. The mature protein should start at approximately 37 (tyr). Otherwise, the IL-l? can it present a pattern of post-translational modification other than IL-1? natural In another embodiment, the composition will comprise IL-1? of primate and a pharmaceutically acceptable carrier. In general, the IL-l? can induce the production of IFN-? by a T cell or NK cell, alone or in combination with IL-12 or IL-2. The fusion proteins of the invention comprise IL-1? or a substantial fragment thereof covalently attached to a binding partner. In one embodiment of the fusion protein, the protein may contain a sequence of SEQ ID NO: 2 and / or sequence of another cytokine or chemokine. This protein may comprise a modification in the sequence corresponding to an amino acid residue of SEQ ID NO 2 at positions 88 (tyr) to 96 (met); or may exhibit IL-1α agonist activity, and may comprise a sequence substitution corresponding to the amino acid residue of SEQ ID NO: 2 at positions 37 (tyr) to 82 (ile) or 102 (val) to 191 (asn) ). In another embodiment of the fusion protein, the pharmacokinetic half-life of IL-1? or fragment thereof is increased by conjugation with another polypeptide, for example, to part of an immunoglobulin (Ig) chain, preferably a constant region (Fc), or to a molecule (s) of polyethylene glycol (PEG), in Sometimes called pegylation. These fusion proteins can be mentioned as IL-1β-Ig and PEG-IL-1, respectively. Methods for linking polyethylene glycol (PEG) groups and Ig chains, parts thereof or other polypeptides to proteins are well known in the art. See, for example, the publication of international application No. WO 96/18412, which describes the fusion of polypeptides derived from immunoglobulin chains to a variety of cytokines to increase the circulating half-life of cytokines. Methods for conjugating PEG to proteins have been described, for example, by David et al. (U.S. Patent No. 4,179,337), Nakagawa et al., (U.S. Patent No. 4,791,192), and Nitecki et al.

(U.S. Patent No. 4,902,502). In certain antibody modalities, IL-1? it's a human protein; the antibody develops against a sequence of peptides of SEQ ID NO: 2; the antibody develops for an IL-1? of purified primate, the antibody is a monoclonal antibody; or the antibody is labeled. In different nucleic acid modalities, IL-1? it's human; the nucleic acid (e.g., SEQ ID NO 1) encodes a peptide sequence of SEQ ID NO 2; the nucleic acid is an expression vector; or the nucleic acid comprises a nucleotide of deoxyribonucleic acid. The present invention also comprises a kit containing: an IL-1? of substantially pure primate, or fragment thereof; an antibody that binds specifically to IL-1? of primate; or a nucleic acid encoding an IL-1? human or peptide. Some of these kits will be able to provide qualitative or quantitative analysis. Another embodiment of the invention includes a method of modulating the physiology or development of a cell, which consists of contacting the cell or an immune system containing the cell, with an agonist or antagonist of an IL-1? of primate. This includes methods where it is contacted in combination with IL-2 and / or IL-12. Otherwise, the invention provides a method where the contact is with an antagonist, for example, an antibody against an IL-1? of primate, and can be combined with antagonists for IL-2 and / or IL-12. Frequently, modulation is the regulation of IFN-α production; and does it include contacting an IL-1 agonist? human In other modalities, modulation is the regulation of: an infectious disease; a vaccine response; an allergic reaction; a response mediated by T-helper; or a cancer.

DESCRIPTION OF THE INVENTION All references mentioned herein are incorporated by reference in their entireties. The present invention provides the amino acid sequence and the DNA sequence of the human interleukin molecule having particular defined properties, both structural and biological, referred to herein as interleukin-1? human (IL-l?). A cDNA encoding this molecule was obtained from a cDNA library of activated human monocytes, designated Ml. Some of the normal methods are described or are also referred to, for example, Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al. (1989) Molecular Cloning: Laboratory Manual, (2nd ed.) Vols. • 1-3, CSH Press, NY; Ausubel, et al. Biology, Greene Publishing Associates, Brooklyn, NY; o Ausubel, et al. (1987 5 and periodic supplements) Current Protocols in Molecular Biology, Greene / Wiley, New York; all of which are incorporated herein by reference. The isolation of the human gene solved the uncertainties that make its isolation far from being certain. These uncertainties include: (1) if there is a human counterpart of the mouse protein; (2) the level of similarity of the coding nucleic acid sequences; (3) where the homology of the sequence exists, useful in a PCR method; (4) the level of expression, providing for This means a useful source for the isolation of a portion of the natural cDNA gene; (5) biological activity between species, useful, for example, in a context of • cloning of expression using mouse cells; and (6) immunological cross-reaction of antibodies, important in a method based on antibody binding. Many other problems exist in the successful obtaining of an isolation between species. A complete nucleotide sequence (SEQ ID NO: 1) and corresponding to the amino acid sequence (SEQ ID NO: 2) of IL-l? which encodes the segment is provided in the sequence listing. The human isolate shows homology with the mouse counterpart, approximately 71% identity at the nucleotide level; and approximately 65% at the amino acid level. As used herein, the term IL-1? it should be used to describe a protein comprising a protein or segment of peptide having a mature amino acid sequence as shown in SEQ ID NO: 2, or a substantial fragment thereof. The invention also includes an agonist or antagonist mutein. Typically, these agonists exhibit less than approximately 10% sequence differences, and thus, they will often have between one and eleven substitutions. It also comprises allelic and other variants, for example, natural polymorphs, of the described protein. Typically, it will bind to its corresponding biological receptor with high affinity, for example, at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably better than about 3 nM. The term should also be used herein to mention the related natural forms, eg, alleles, polymorphic variants and metabolic variants of the human protein. This invention also encompasses proteins and peptides which have substantial homology to the amino acid sequence with the amino acid sequence in SEQ ID NO: 2, but which exclude any protein or peptide having substantially the same or less amino acid sequence homology than the one corresponding to the IGIF protein found in the mouse. This will include sequence variants with relatively few substitutions, for example, preferably less than about 3-5. A "fragment," or "segment" of a substantial polypeptide, is an extension of amino acid residues of at least about 8 amino acids, in general at least 10 amino acids, more generally at least 12 amino acids, often at least 14 amino acids, most often at least 16 amino acids, usually at least 18 amino acids, most commonly at least 20 amino acids, usually at least 22 amino acids, more regularly at least 24 amino acids, preferably at least 26 amino acids, at least 26 amino acids, more preferably at least 28 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids. The sequences of segments of different proteins can be compared among themselves on extensions of suitable length. The homology of the sequence of amino acids or identity of the sequence, is determined by optimizing the matches of residues, if necessary, introducing spaces as required. See, for example, Nedleham, et al., (1970) J. Biol. 48: 443-453; Sankoff, et al., (1983) chapter one in Time Warps, String Edits, and Macromolecules: The Theory and Practice of Sequence Comparison. Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA; and University of Wisconsin Genetics Compueter Group (GCG), Madison, Wl; each of which is incorporated herein by reference. This changes when conservative substitutions are considered as coincidences. Conservative substitutions usually include substitutions within the following groups: glycine, alanine,; valine, isoleucine, leucine; aspartic acid, glutaric acid; asparagine, glutamine; serine, threonine, lysine, arginine; and phenylalanine, tyrosine. It is proposed that homologous amino acid sequences include natural and inter-species allelic variations in the cytokine sequence. Common homologous proteins or peptides will have from 50-100% homology (if spaces can be introduced), up to 60-100% homology (if conservative substitutions are included) with a segment of the amino acid sequence of SEQ ID NO: 2 The measures of homology will be at least about 70%, in general at least 76%, more generally at least 81%, often at least 85%, most often at least 88%, commonly at least 90%, most commonly at least 92%, usually at least 94%, more regularly at least 95%, preferably at least 96% and most preferably at least 97%, and in the particularly preferred modalities at least 98% or more. The degree of homology will vary with the length of the segments being compared. Homologous proteins or peptides, such as allelic variants, will share most of the biological activities with the modality described in SEQ ID NO: 2. Preferably, a related polypeptide will contain a plurality of these matching fragments, for example, at least 2, preferably 3, 4, 5 or more of the specific or classified lengths. When used herein, the term "biological activity" is used to describe, without limitation, synergistic induction by splenocytes of IFN-α. in combination with IL-12 or IL-2, with or without anti-type I or anti-type II IL-1 receptor antibodies, or more structural properties such as receptor binding and cross-reactivity with antibodies developed against the same variant or a polymorphic variant of IL-1? described human The terms ligand, agonist, antagonist and analog include molecules that modulate the cellular response characteristic for IL-1 proteins. or similar to IL-1 ?, as well as molecules that exhibit most of the standard structural binding competence characteristics of ligand-receptor interactions, for example, where the receptor is a receptor or a natural antibody. The cellular responses are probably mediated through the binding of IL-1? to cell receptors other than IL-1 type I or type II receptors. Likewise, a ligand is a molecule that serves as a natural ligand to which the receptor, or an analog thereof, binds, or a molecule that is a functional analogue of the natural ligand. The functional analog may be a ligand with structural modifications, or it may be a completely unrelated molecule having a molecular form that interacts with the appropriate ligand binding determinants. Ligands can serve as agonists or antagonists, see, for example, Goodman, et al. (Eds) (1990) Goodman & Gilman's The Pharmacological Bases of Therapeutics, Pergamon Press, New York. The rational design of drugs can also be based on structural studies of the molecular forms of a receptor or antibody and other effectors or ligands. The effectors may be other proteins that mediate other functions in response to ligand binding, or other proteins that normally interact with the receptor.

A means of determining which sites interact with other specific proteins is a determination of the physical structure, for example, X-ray crystallography or two-dimensional NMR techniques. These will provide a guide to know the amino acid residues that form the molecular contact regions. For a detailed description that the structural determination of proteins see, for example, Blundell and Johnson (1976) Protein Cristallography, Academic Press, New York. The IL-1 protein? human has different biological activities. The IL-l? human is homologous to the mouse IGIF protein, but has structural differences. For example, the coding sequence of the IL-1 gene? human has only about 71% homology to the sequence encoding the mouse IGIF nucleotide. At the amino acid level, there is approximately 64% identity. That level of similarity suggests that the new protein IL-1? it is related to IL-la and IL-lß. The mouse IGIF molecule has rather minimally defined biological activities. In particular, it has the ability to stimulate the production of IFN-? which increases the NK activity in splenic cells. See, Okamura et al. (1995) Nature 378: 88-91. The activities of mouse IL-1 and IL-1β and IGIF have been compared for their ability to induce IFN-α alone or in combination with IL-2 or IL-12 in SCID splenocytes and purified NK cells. See, Hunter et al. (1995) J. Immunol. 155: 4347-4354; and Bancroft, et al. (1991) Im unol. Revs. 124: 5-xxx. It was found that IGIF is much more potent in the stimulation of IFN-? than IL-la or IL-lß. In NK cells activated with IL-2, the production of IFN-1? is blocked by the addition of anti-IL-lß antibodies. See Hunter et al. (nineteen ninety five) . However, mouse IGIF can overcome this blockade and induce IFN-α. This is the only known cytokine that is capable of doing this. Furthermore, in vivo administration of the mouse IGIF to mice infected with T. cruzi significantly decreases parasitaemia. The present disclosure also presents new activities that have been discovered using the mouse IGIF molecule. Applicants have confirmed that the mouse IGIF molecule produced by recombinant means similar to the IL-1 protein? human characterized in the present, presents the biological activity of inducing T cells to produce IF-l ?. See the essays described, for example, in Wall Malefyt, et al., In Vries and de Walal Malefyt (eds. 1995) "Interleukin-10" Landes Co., Austin, TX. It also moderately stimulates IFN-? per NK cell. But, as with the human molecules described above, there is a substantial synergy with IL-12.

Biological activity between species has not yet been detected with these assays, for example, the biological activity of mouse IGIF on human cells has not yet been shown, nor has the activity of IL-1 been demonstrated. human in mouse cells. This suggests that the receptors, which are expected to include multiple different polypeptide chains, exhibit species specificity for their corresponding ligands. The ligands IL-la and IL-lβ present signals through heterodimeric receptors. This invention further contemplates the use of isolated nucleic acid or fragments, for example, which encode this protein or a closely related protein or fragments thereof, to modify a corresponding biologically active polypeptide. In addition, this invention covers isolated or recombinant DNA encoding a biologically active protein or polypeptide having the characteristic activity of IF-1 ?. Usually, the nucleic acid is capable of hybridizing, under suitable conditions, with a segment of the nucleic acid sequence of SEQ ID NO: 1. This biologically active protein or polypeptide can be a full-length protein or fragment, and the common will have a segment of the amino acid sequence highly homogeneous to that shown in SEQ ID NO: 2., this invention covers the use of isolated or recombinant nucleic acid, or fragments thereof, which encodes the proteins having the fragments that are homologous to the IF-1 protein? described. The isolated nucleic acids can have the respective regulatory sequences on the 5 'and 3' flanks, for example, promoters, enhancers, poly-A addition signals, and others of the natural gene. An "isolated" nucleic acid is defined herein for a nucleic acid, for example, an RNA, DNA or a mixed polymer, which is substantially pure, for example, separated from other components that naturally accompany a native sequence, such as it can be ribosomes, polymerases and genomic flanking sequences of the species of origin. The term comprises a nucleic acid sequence that has been removed from its natural environment, and includes recombinant or cloned DNA isolates, which are hereby distinguishable from compositions found in the natural state and chemically synthesized analogs or biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule, complete or substantially pure. A nucleic acid isolated will usually be a homogeneous composition of molecules, but in some embodiments it will contain heterogeneity, preferably less. This heterogeneity is usually found at the ends of the polymer or non-crucial portions for a desired biological function or activity. A "recombinant" nucleic acid is defined herein by its production method or structure. With respect to its production method, for example, a product prepared by a process, the process is to use recombinant nucleic acid techniques, for example, including human intervention in the nucleotide sequence, this intervention usually includes the manipulation in vi tro, although under certain circumstances may include more traditional animal reproduction techniques. Otherwise, it can be a nucleic acid prepared by generating a sequence comprising the fusion of two fragments that naturally are not contiguous with each other, but it means to exclude the products of nature, for example, natural mutants as they are in their natural state. Thus, for example, products prepared by transforming cells with any non-natural vector, such as nucleic acids containing sequence derived using any of the synthetic oligonucleotide processes, are included. This process is often performed to replace a codon with a redundant codon that encodes the same amino acid or a conservative amino acid, while it usually introduces or removes a restriction enzyme sequence recognition site. Otherwise, the process is performed to join together the nucleic acid segments of desired functions to generate a single genetic entity that contains a desired combination of functions not found in the commonly available natural forms, eg, coding for a fusion protein. . Recognition sites for restriction enzymes are usually the target of these artificial manipulations, but other site-specific targets, for example, promoters, DNA replication sites, regulatory sequences, control sequences or other useful features can be incorporated by design. A similar concept is proposed for a recombinant polypeptide, for example, fusion. This will include a dimeric repetition. Specifically, synthetic nucleic acids are included which, by redundancy of the genetic code, encode polypeptides similar to fragments of IL-1 ?, and fusions of sequences of different molecules of interleukin or related molecules, for example, growth factors. A "fragment" in a nucleic acid context is defined herein as a contiguous segment of at least about 17 nucleotides, generally at least 21 nucleotides, more generally at least 25 nucleotides, regularly at least 30 nucleotides, more regularly at least 35 nucleotides, often at least 39 nucleotides, more frequently at least 45 nucleotides, usually at least 50 nucleotides, more commonly at least 55 nucleotides, usually at least 60 nucleotides, more regularly at least 66 nucleotides, preferably at least 72 nucleotides, of higher reference at least 79 nucleotides, and in the particularly preferred embodiments will be at least 85 or more nucleotides. In general, the fragments of different genetic sequences can be compared among themselves on extensions of adequate length. A nucleic acid encoding IL-1? it will be particularly useful for identifying genes, mRNAs and cDNA species that code for the closely related protein or proteins, as well as DNAs that code for polymorphic, allelic or other genetic variants, for example, from different individuals. Preferred probes for these scans are those regions of interleukin that are conserved between different polymorphic variants or that contain nucleotides that lack specificity, and preferably will be full length or nearly complete. In other substitutions, specific sequences of polymorphic variants will be more useful. This invention also covers recombinant nucleic acid molecules and fragments having a nucleic acid sequence identical or highly homologous to that of isolated DNA as set forth herein. In particular, the sequences will often be operably linked to the DNA segments that control the transcription, translation and replication of the DNA. These additional segments usually aid in the expression of the desired nucleic acid segment. The homologous nucleic acid sequences, when compared to each other or to the sequences of SEQ ID NO: 1, show significant similarity. Standards for homology in nucleic acids are measures of homology generally used in the art by comparison of sequences or based on the conditions of hybridization. The comparative hybridization conditions are described in greater detail below. Substantial homology in the context of comparing nucleic acid sequences means that the segments, or their complementary strands, when compared, are identical when aligned optimally, with insertions or deletions of suitable nucleotides, at least approximately 60% of nucleotides, generally at least 66%, commonly at least 71%, often at least 76%, most often at least 80%, usually at least 84%, most commonly at least 88%, usually when less 91%, more usually at least 93%, preferably at least 95%, more preferably at least approximately 96% to 98% or more, and in particular modalities, as much as approximately 99% or more of the nucleotides. Otherwise, there is substantial homology when the segments will hybridize under selective hybridization conditions, to a strand or its complement, usually using a sequence derived from SEQ ID NO: 1. common, selective hybridization will occur when there is • at least about 55% of the homology over an extension of at least about 14 nucleotides, more commonly at least about 65%, preferably at least about 75%, and of greater preferably at least about 90%. See, Kanehisa (1984) Nuc. Acids Res. 12: 203-213, which is incorporated herein by reference. • The length of the comparison of homology, as described, may be on longer extensions, and on Certain embodiments will be on an extension of at least about 17 nucleotides, generally at least about 20 nucleotides, usually at least about 24 nucleotides, regularly at least about 28 nucleotides, usually at least about 32 nucleotides, more usually at least about 40 nucleotides, preferably at least about 50 nucleotides, and most preferably at least about 75 to 100 or more nucleotides. Preferably a related nucleic acid will contain a plurality of these corresponding fragments, for example at least 2, preferably 3, 4, 5 or more of specific or classified lengths. Severe conditions, in relation to homology in the context of hybridization, will be severe combined conditions of salt, temperature, organic solvents and other parameters usually controlled in the hybridization reactions. Severe temperature conditions will usually include temperatures in excess of about 30 ° C, more commonly in excess of about 37 ° C, usually in excess of about 45 ° C, more regularly in excess of about 55 ° C, preference in excess of about 65 ° C, and more preferably in excess of about 70 ° C. Severe salt conditions will usually be less than about 500 mM, usually less than about 400 mM, more regularly less than about 300 mM, usually less than about 200 mM, preferably less than about 100 mM and more preferably less than about 80 mM, still less than less than about 20 mM. However, the combination of the parameters is much more important than the measurement of any individual parameter. See, for example, Wetmur and Davidson (1968) J. Mol. Biol. 31: 349-370. The isolated 7β DN can be easily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions and nucleotide extension inversions. These modifications result in novel DNA sequences encoding this protein, its derivatives or proteins having IL-1 activity. These modified sequences can be used to produce mutant proteins (muteins) or to improve the expression of variant species. Improved expression can include gene amplification, increased transcription, increased translation and other mechanisms. These derivatives of IL-l? Mutants include predetermined or site-specific mutations of the protein or its fragments, which include silent mutations using degeneracy of genetic code. "The IL-1" mutant "as defined herein comprises a polypeptide that otherwise falls within the definition of homology of IL-1? as stated before, but which has an amino acid sequence that differs from IL-1? human as it is in nature, either by means of suppression, substitution or insertion. In particular, "IL-1 site-specific mutant" comprises a protein having substantial homology to the sequence of SEQ ID NO: 2, and usually shares most of the biological activities in the manner described herein. Although site-specific mutation sites are predetermined, mutants do not need to be site-specific. The mutagenesis of IL-1? can be achieved by making insertions or deletions of amino acids in the gene, coupled with the expression. Substitutions, deletions, insertions or any combination can be generated to arrive at a final construction. Inserts include amino- or carboxy terminal fusions. Random mutagenesis can be performed on an objective codon and mutants of IL-1? expressed human beings can then be explored for the desired activity. Methods for performing substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, for example by mutagenesis of the M13 primer. See also Sambrook, et al. (1989) and Ausubel, et al. (1987 and periodic supplements). Mutations in DNA normally should not place coding sequences outside of reading frames and preferably will not create complementary regions that can hybridize to produce secondary mRNA structure such as loops or hairpins. The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22: 1859-1862 will produce suitable synthetic DNA fragments. A double-stranded fragment will often be obtained by synthesis of the complementary and tempered strand of the strand together under suitable conditions or by addition of the complementary strand using DNA polymerase with a suitable primer sequence. The polymerase chain reaction techniques (PCR) can often be applied in mutagenesis. Otherwise, mutagenesis primers are commonly methods used to generate defined mutations at predetermined sites. See, for example Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA. As already described, the present invention comprises human IL-1 and l whose sequence is defined in SEQ ID NO: 2 and is described in the foregoing. Allelic variants and others are also contemplated. The present invention also provides recombinant proteins, for example, heterologous fusion proteins using segments from this human protein. A heterologous fusion protein is a fusion of proteins or segments that are natural or not naturally fused in the same way. In this way, the fusion product of a growth factor with an interleukin is a continuous protein molecule having sequences fused to a common peptide bond, usually prepared as a single translation product and having properties from each peptide originally. A similar concept applies to heterologous nucleic acid sequences. In addition, it is possible to prepare new constructs from the combination of similar functional or structural domains from other related proteins, for example, growth factors or other cytokines. For example, binding to the receptor or other segments can be "exchanged" between new polypeptides or different fusion fragments. See, for example Cunningham, et al. (1989) Science 243: 1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263: 15985-15992, each of which is incorporated herein by reference. In this way, new chimeric polypeptides that present new combinations or specificities will result from the functional binding of receptor binding specificities. For example, the receptor binding domains of other related ligand molecules can be added or substituted by other domains of these related proteins or proteins. The resulting protein will often have mixed function and properties. For example, a fusion protein can include a domain that targets a target that can serve to sequester the fusion protein to a specific organ, for example, portions of a ligand that is specifically bound by spleen cells and serves to accumulate in the spleen. The fusion partners and candidate sequences can be selected from the different sequence databases, for example GenBank, c / o IntelliGenetics, Mountain View, CA; and BCG, University of Wisconsin Biotechnology Computing Group, Madison, Wl. The present invention particularly provides muteins that act as agonists or antagonists of IL-1 ?. The structural alignment of IL-1? human and mouse with other members of the IL-1 family show characteristics / conserved residues, particularly 12 ß strands folded into double β-3 leaves. See Bazan et al., (1996) Nature 379: 591. The alignment with the sequence of the IL-1? human (using the initiation residue met of the signal peptide) indicates that the strands ßl (leu 41 to val 47), ß2 (val 55 to asp 59), ß3 (pro 64 to asp 68), ß4 (phe 83 to tyr 88 ), ß5 (met 96 to val 102), ß6 (be "l08 to glu 113), ß7 (lys 115 to lys 120), ß8 (phe 137 to pro 143), ß9 (asn 147 to be 153) ßlO (phe 160 to glu 164), ßll (phe 170 to lys 175), and.β2 (phe 187 to asn 191) correspond to similar sequences in the mouse IGIF, see also, Lodi, et al., (1994) Science 263: 1762-1766, Sayle and Milner-White (1995) TIBS 20: 374-376, and Gronenberg et al (1991) Protein Engineering 4: 263-269.The ligands IL-la and IL-lß bind to a receptor IL-1 type I as the primary receptor and this complex then forms a high affinity receptor complex with the type III IL-1 receptor.The mouse IL-1α does not bind to the known mouse IL-1 receptor I, II (decoy receiver), or III. In addition, the biological activity of mouse IGIF can not be blocked with anti-type I, II or III antibodies. This suggests that the related mouse IGIF binds to receptors related to IL-1 receptors already isolated, but not yet identified as receptors for IGIF. However, the resolved structures for IL-lβ, the natural IL-1 receptor antagonist (IL-IRa), and a co-structure of the IL-lRa / IL-1 receptor type I, suggest how to prepare a mouse IGIF antagonist or of IL-l? human The loop between the ß4 strand and the ß5 strand is the primary binding segment for other ligands from IL-1 to the type III receptor. In the ligands IL-la and IL-lβ, this loop covers 8 residues, whereas in IL-IRa, this loop. it is "trimmed" (only two residues remain). Therefore, IL-IRa binds normally to the type I receptor, but can not interact with the type III receptor. This makes IL-IRa an effective IL-1 antagonist. This suggests that modifications to the loop between ß4 and ß5 strands will result in variants with predictable biological activities. For example, the substitution of KDSEVRGL (the residues between the ß4 and ß5 strand, with the first residue in the ß5 strand) in the mouse IGIF (or the corresponding KDSQPRGM of human IL-1? For EPH) with EPH (corresponding to residues of IL-IRa) must generate an antagonist for IGIF. Otherwise, the replacement of KDSEVR from mouse IGIF with QGEESND (residues of IL-lβ between the ß4 and ß5 strand must allow interaction with the type III receptor (in human IL-1, replaces KDSQPR with QGEESND). IL-IRa from mouse, it was demonstrated that the substitution of mouse IL-IRa residues with these mouse IL-lβ residues introduced IL-1 activity into the IL-IRa (the IL-IRa can now bind to the receptor type III) This will establish whether the type III receptor can be used by mouse IGIF In addition, a construct with KDSEVR substitutes of the mouse IFIF with the flag epitope DYKDDDDK will likely bind to the primary receptor of the IGIF. "Human replacement would be KDSQPR with DYKDDDDK.This mutant protein can act as an antagonist (unable to bind to the secondary receptor mlL-l?). This labeled molecule would be useful for cloning the primary mouse IGIF receptor, which would not be identified as such. similarities in the ligand sequence of IL-1? human, for example, in the corresponding region between residues 88-96, would provide similar interactions with the receptor. Substitutions with mouse sequences or human sequences are indicated. In contrast, conservative substitutions away from the receptor binding interaction versions probably retain most of the biological activities. "Derivatives" of the IL-l? human include mutants of amino acid sequences, glycosylation variants, metabolic derivatives and conjugates covalent or aggregative with other chemical moieties. Covalent derivatives can be prepared by linking the functionalities with the groups found in the amino acid side chains of IL-1? or in the terminal N or C, for example, by means of which they are well known in the art. These derivatives may include, without limitation, aliphatic esters or amides of terminal carboxyls, or residues containing carboxyl side chains, o-acyl derivatives of residues containing hydroxyl groups, and N-acyl derivatives of amino-terminal amino acids or residues containing groups. amino, for example, lysine or arginine. The acyl groups are selected from the group of alkyl portions including normal alkyl of C3 to C18, thereby forming alkanoyl aroyl species. In particular, modifications by glycosylation are included, for example, those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in other processing steps. Particularly preferred means for carrying out these are by exposing the polypeptide to the glycosylation enzymes from the cells that normally provide this processing, for example, the enzymes of mammalian glycosylation. Deglucosylation enzymes are also contemplated. Also included are versions of the same sequence of primary amino acids that have other minor modifications, including phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine or phosphothreonine. An important group of derivatives are the covalent conjugates of the BAS-1 antigens or fragments thereof with other proteins or polypeptides. These derivatives can be synthesized in recombinant cultures, such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in the cross-linking of proteins by reactive side groups. The preferred derivatization sites with the crosslinking agents are in free amino groups, carbohydrate moieties and the cysteine residues. The fusion polypeptides between interleukin and other homologous or heterologous proteins are also provided. The homologous polypeptides may be fusions between different growth factors, resulting, for example, in a hybrid protein having ligand specificity of multiple different receptors, or a ligand that may have enhanced or weakened specificity of binding to its receptor. In the same way, it is possible to construct heterologous fusions that present a combination of properties or activities of the derived proteins. Common examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a receptor, e.g., a ligand-binding segment, so that the presence or location of a desired ligand can be easily determined. See, for example, Dull, et al. U.S. Patent No. 4,859,609. Other gene fusion partners include glutathione-S-transferase (GST), bacterial β-galactosidase, trpE, β-lactamase protein A, alpha amylase, alcohol dehydrogenase and yeast alpha-factor coupling. See, for example, Godowski et al. (1988) Science 241: 812-816. The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra Letts. 22: 1859-1862, will produce suitable synthetic DNA fragments. Frequently a double-stranded fragment will be obtained by synthesizing the complementary strand and quenching the strand together under suitable conditions or by adding the complementary strand using DNA polymerase with a suitable primer sequence. These polypeptides may also have amino acid residues that have been chemically modified by phosphorylation, sulfonation, biotinylation or by the addition or removal of other portions, specifically those having molecular forms similar to phosphate groups. In some embodiments, the modifications will be useful reagent markers, or serve as targets for purification, e.g., affinity ligands. Fusion proteins will usually be prepared by the recombinant nucleic acid methods or by the synthetic polypeptide methods. Techniques for the manipulation and expression of nucleic acids are described, in general, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory manual, (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, and Ausubel, et al. (eds) (1987 and periodic supplements) Current Protocols in Molecular Biology, Greene / Wiley, New York, which are incorporated herein by reference. Techniques for the synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc., 85: 2149-2156; Merrifield (1986) Science, 232: 341-347; and Atherton, et al. (1989) Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford; each of which is incorporated herein by reference. See also Dawson, et al. (1994) Science 266-776-779 for methods for preparing longer polypeptides. This invention also contemplates the use of derivatives of the IL-1 antigens. different human of the variations in the amino acid sequence or glycosylation. These derivatives may include covalent or aggregative association with chemical portions. These derivatives are usually found among the classes: (1) salts, (2) covalent modifications in side chain and terminal residue, and (3) adsorption complexes, for example, with cell membranes. These covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays or in purification methods, such as affinity purification of a receptor or other binding molecule, e.g., an antibody. For example, the ligand of IL-1? can be immobilized by covalent attachment to a solid support such as Sepharose, activated with cyanogen bromide, by methods that are well known in the art, or can be adsorbed onto polyolefin surfaces with or without glutaraldehyde crosslinking, for in the assay or purification of the IL-1 receptor, antibodies or other similar molecules. The IL-l? it may also be labeled with a detectable group, for example, radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent portion for use in diagnostic assays. The IL-l? Human of this invention can be used as an immunogen for the production of antiserum or specific antibodies, for example, capable of distinguishing between mouse IGIF and IL-1? human for interleukin or any fragment of it. The purified interleukin can be used to screen for monoclonal antibodies or antigen binding fragments prepared by immunization with different forms of impure preparations containing the protein. In particular, the term "antibodies" also comprises fragments of natural antibodies that bind to the antigen. The purified interleukin can also be used as a reagent to detect any antibody generated in response to the presence of high expression levels or immunological disorders that cause the production of antibodies to the endogenous cytokine. In addition, fragments of IL-l? they can also serve as immunogens to produce the antibodies of the present invention, as described below. For example, this invention contemplates antibodies that have binding affinity to or may be developed against amino acid sequences shown in SEQ ID NO: 2, fragments thereof or homologous peptides. In particular, this invention contemplates antibodies that have binding affinity to, or are developed against, specific fragments that have been predicted, or are actually exposed to the outer protein surface of the native cytokine. Blocking the physiological response to these interleukins may result in inhibition of ligand binding to the receptor, probably by competitive inhibition. Thus, in vitro assays of the present invention will often utilize antibodies or segments of these antibodies that bind to the ligand, or fragments that bind to substrates in the solid phase. These assays will also allow the diagnostic determination of the effects of mutations and modifications of the binding region, or mutations and modifications of the ligand, for example, ligand analogues. This invention also contemplates the use of competitive drug screening assays, for example, where neutralizing antibodies to interleukin or fragments compete with a test compound for binding to a receptor or antibody. In this way antibodies or neutralizing fragments can be used to detect the presence of any polypeptide that shares one or more binding sites with a receptor and can also • be used to occupy binding sites on a receptor that can otherwise bind to an interleukin. The DNA encoding the protein or fragments thereof can be obtained by chemical synthesis, by scanning cDNA libraries or by scanning genomic libraries prepared from a wide range of cell lines or tissue samples. The natural sequences can be isolated using the normal methods and the sequences that are • provided herein in SEQ ID NO: 1. This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length human interleukin or fragments that can times, for example, be used to generate polyclonal or monoclonal antibodies; for studies of union; by the construction and expression of modified agonist / antagonist molecules; and for structure / function studies. Each variant or its fragments can be expressed in cells hosts that are transformed or transfected with suitable expression vectors. These molecules can be substantially free of proteins or cellular contaminants, different from those from the recombinant host, and are therefore particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and / or diluent. The human protein or portions of it, can be expressed • as fusions with other proteins. Expression vectors are commonly self-replicating DNA or RNA constructs containing the desired receptor gene or its fragments, usually operably linked with suitable genetic control elements that are recognized in a suitable host cell. These control elements are capable of effecting expression inside a suitable host. The specific type of control elements necessary to effect expression will depend on the eventual host cell that is used. In general, the elements of genetic control can include a prokaryotic promoter system or a control system of Eukaryotic promoter expression and will usually include a transcription promoter, an optional operator to control the initiation of transcription, enhancers of the • transcription to raise the level of mRNA expression, a sequence that encodes a ribosome binding site, and sequences that complete transcription and translation. Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell. The vectors of this invention include those that contain AD? encoding a protein, as described, or a fragment thereof encoding a biologically active equivalent polypeptide. The DNA can be under the control of a viral promoter and can encode a selection marker. This invention further contemplates the use of these expression vectors that are capable of expressing eukaryotic cDNA encoding this protein in a prokaryotic or eukaryotic host, where the vector is compatible with the host, and where the eukaryotic cDNA encoding the receptor is inserted into the vector, so that the growth of the host containing the vector expresses the cDNA in question. Usually, expression vectors are designed for stable replication in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell, for example, it is possible to effect transient expression of the interleukin protein or its fragments in different hosts using vectors that do not contain an origin of replication that is recognized by the host cell . It is also possible to use vectors that cause the integration of the human protein or its fragments into the host DNA by recombination. The vectors, as used herein, comprise plasmids, viruses, bateriophages, integrable DNA fragments and other vehicles that allow the integration of DNA fragments into the host's genome. Expression vectors are specialized vectors that contain the elements of genetic control that effect the expression of operably linked genes. Plasmids are the most commonly used vector vector form but all other forms of vectors serving in an equivalent function and which are, or become, known in the art are suitable for use in the present see, for example, Pouweis , et al. (1985 and supplements) Cloning Vectors: A Laboratory Manual, elsevery, N. Y., and Rodriguez, et al., (1988) eds., Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston. 1988, which are incorporated herein by reference. Transformed cells are cells, preferably mammalian, that have been transformed or transfected with receptor vectors constructed using recombinant DNA techniques. The transformed host cells usually express the desired protein or its fragments, but for the purposes of cloning, amplification and manipulation of its DNA, it does not need to express the protein in question. This invention also contemplates the cultivation of transformed cells in a nutrient medium, thus allowing interleukin to accumulate in the culture. The protein can be recovered from the culture or from the culture medium.

For the purpose of this invention, the nucleic sequences are operably linked when they are functionally related to each other. For example, DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in the secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; A ribosome binding site is operably linked to a coding sequence if it is placed to allow translation. Usually, operably linked means contiguous and in the reading frame, however, certain genetic elements such as repressor genes are not contiguously linked if not yet linked to the operator sequences which in turn control the expression. Suitable host cells include prokaryotic, lower eukaryotic and higher eukaryotic cells. Prokaryotic cells include gram negative and gram positive organisms, for example, E. coli and B. s? Btilis. Lower eukaryotes include yeasts, for example, S. cerevisiae and Pichia, and species of the genus Dictyostelium. Higher eukaryotes include cell lines of established tissue cultures from animal cells, both of non-mammalian origin, e.g., insect and bird cells, and of mammalian origin, e.g., of humans, primates and rodents. The prokaryotic host-vector systems include a wide range of vectors for very different species. As used herein, E. Coli and its vectors will be used generically to include equivalent vectors that are used in other prokaryotes. A representative vector for amplifying DNA is pBR322 or some of its derivatives. Vectors that can be used to express the receptor or its fragments include, but are not limited to, vectors such as those containing the lac promoter (pUC series); the trp promoter (pBR322-trp); the Ipp promoter (the pIN series); the lambda-Pp or pR promoters (pOTS); or hybrid promoters, such as ptac (pDR540). See Brosius, et al. (1988) "Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters" in Vectors: A Survey of Molecular Cloning Vectors and Their Uses, (eds. Rodríguez and Dendhardt), Buttersworth, Boston, chapter 10, pp. 205-236, which is incorporated herein by reference. Lower eukaryotes, for example, yeasts and Dictyosteli um, can be transformed with vectors containing the sequence of IL-1 ?. For the purposes of this invention, the most common lower eukaryotic host is the baking yeast, Saccharomyces cerevisiae. This will be used to generically represent lower eukaryotes, although a number of other strains and species are also available. Yeast vectors usually consist of an origin of replication (at least of the type of integration), a selection gene, a promoter, DNA encoding the receptor or its fragments and sequences for translation termination, polyadenylation and termination of transcription. Suitable expression vectors for yeast include: constitutive promoters such as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or inducible promoters such as the alcohol dehydrogenase 2 promoter or metallothionine promoter. Suitable vectors include derivatives of the following types: self-replicating copy number (such as the YRp series), high self-replicating copy number (such as the YEp series); integrating types (such as the YIp series) or mini-chromosomes (such as the YCp series). Higher eukaryotic tissue culture cells are usually the preferred host cells for the expression of the functionally active interleukin protein. At first, any line of cells of higher eukaryotic tissue culture cells is feasible, for example, systems of expression of the insect baculovirus, whether of invertebrate or vertebrate origin. However, mammalian cells are preferred. The transformation or transfection and propagation of these cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, newborn rat kidney cell lines (BRK), insect cell lines, bird cell lines and monkey cell lines (COS). Expression vectors for these cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA cleavage sites (if genomic AD is used), a polyadenylation site, and a Transcription termination site. These vectors also, usually, contain a selection gene or amplification gene. Suitable expression vectors can be plasmids, viruses or carrier retroviruses or promoters from, for example, sources such as adenovirus, SV40, parvovirus, vaccinia virus or cytomegalovirus. Representative examples of suitable expression vectors include pCD? Al; pCD, see Okayama et al. (1985) Mol. Cell Biol. 5: 1136-1142; pMlneo PolyA, see Thomas, et al. (1987) Cell 51: 503-512; and a baculovirus vector such as pAC 373 or pAC 610. For secreted proteins, an open reading frame usually encodes a polypeptide consisting of a mature or secreted product covalently linked in its? terminal to a signal peptide. The signal peptide is split before the secretion of the mature or active polypeptide. The cleavage site can be predicted with a high degree of accuracy or from the empirical rules, for example, von-Heijne (1986) Nucleic Acids Research 14: 4683-4690, and the precise composition of the signal peptide amino acids does not seem be important for its function, for example, Randarll, et al (1989) Science 243: 1156-1159; Kaiser et al. (1987) Science 235: 312-317. It will often be desirable to express these polypeptides in a system that provides a specific or defined glycosylation pattern. In this case, the normal pattern will be that provided in a natural way by the expression system. However, the pattern will be modifiable by exposing the polypeptide, eg, a non-glycosylated form, to the appropriate glycosylating proteins introduced in a heterologous expression system. For example, the interleukin gene can be co-transformed with one or more genes encoding mammalian or other glycosylation enzymes. Using this approach, certain patterns of mammalian glycosylation can be achieved in prokaryotic cells or other cells. The source of IL-l? can a human being be a eukaryotic or prokaryotic host expressing huIL-l? Recombinant DNA, as already described. The source can also be a cell line such as mouse Swiss 3T3 fibroblasts, but other mammalian cell lines in this invention are also contemplated, with the cell line being preferred by human species. Now that the full sequence is known, the IL-1? human, fragments or derivatives thereof, can be prepared by conventional processes for the synthesis of the peptides. These include processes as described in Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984), The Practice of Peptide Synthesis, Springer-Verlag, New York; and Bodansky (1984) The Principies of Peptide Synthesis, Springer-Verlag, New York; all of which are incorporated herein by reference. For example, an azide process, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p-nitrophenyl ester, N-hydroxysuccinimide ester or cyanomethyl ester), a process carbodiimidazole, an oxidative-reductive process or a dicyclohexylcarbodiimide (DCCD) / additive process. The synthesis in solid phase and in phase in solution both are applicable for the aforementioned processes. The IL-1 protein, fragments or derivatives are suitably prepared according to the above processes, as commonly used in the synthesis of peptides, generally by a stepwise process comprising condensing an amino acid to the terminal amino acid, one per one in sequence, or copulating fragments of peptides to the terminal amino acid. Amino groups that are not used in the coupling reaction must usually be protected to prevent coupling in an incorrect location. If solid-phase synthesis is adopted, the C-terminal amino acid is bound to an insoluble or support carrier by its carboxyl group. The insoluble carrier is not particularly limited, as long as it has a binding capacity to a reactive carboxyl group. Examples of these insoluble carriers include halomethyl resins, such as chloromethyl resin or bromomethyl resin, hydroxymethyl resins, phenol resins, ter-alkyloxycarbonyl hydrazide-like resins. An amino acid protected with an amino group is joined in sequence by condensation of its activated carboxyl group and the reactive amino group of the peptide or chain previously formed, to synthesize the peptide, step by step. After synthesizing the entire sequence, the peptide is separated from the insoluble carrier to produce the peptide. This solid phase method is generally described by Merrifield et al. (1963) in J. Am. Chem. Soc. 85: 2149-2156, which is incorporated herein by reference. The prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of separation of peptides, for example, by extraction, precipitation, electrophoresis, different forms of chromatography, and the like. The interleukin of this invention can be obtained in different degrees of purity depending on its intended use. The purification can be carried out with the use of the protein purification techniques described herein, see below or by the use of antibodies, herein described in immunoabsorbent affinity chromatography methods. This immunoabsorbent affinity chromatography is carried out by first binding the antibodies to a solid support and then contacting the bound antibodies with solubilized lysates of suitable cells, lysates of other cells expressing interleukin or lysates or supernatants of cells producing the protein as a result of DNA techniques, see below. In general, the purified protein will be at least about 40% pure, usually at least about 50% pure, usually at least about 60% pure, usually at least about 70% pure, most commonly at least about 80% pure. Pure%, at least approximately 90% pure and most preferred at least approximately 95% pure, and in particular embodiments 97% -99% or more. The purity will usually be based on weight, but it can also be on a molar basis. Different tests will be applied as appropriate. Antibodies can be developed for the different IL-1 proteins? human and fragments thereof, both in the native forms that occur in nature and in their recombinant forms, the difference being that the antibodies for the active ligand will most likely recognize the epitopes that are only present in the native conformations. Anti-idiotypic antibodies are also contemplated, which will be useful as agonists or antagonists of a natural receptor or an antibody. The antibodies, which include the binding fragments and the individual chain versions, against predetermined fragments of the protein can be developed by immunization of animals with conjugates of the fragments with immunogenic proteins. The monoclonal antibodies are prepared from cells that secrete the desired antibody. These antibodies can be screened for binding to normal or defective protein, or they can be screened for agonist or antagonist activity. These monoclonal antibodies will usually bind with at least one KD of about 1 mM, more commonly at least about 300 μM, usually at least about 100 μM, more commonly at least about 30 μM, preferably at least about 10 μM. μM, and more preferably at least about 3 μM or better. Antibodies, including fragments of the antibody that bind to the antigen, of this invention may have significant diagnostic or therapeutic value. These can be potent antagonists that bind to interleukin and inhibit receptor binding or inhibit the ability of IL-1? human to present a biological response. These can also be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides to bind to producer cells, or cells located for the source of interleukin, in addition, these antibodies can be conjugated with drugs or other therapeutic agents, either directly or indirectly through a linker. As used herein, the term "antibody fragment that binds to the antigen" includes, for example, Fab, Fc, F (ab) 2, Fv, and scFv fragments, which are used with their standard immunological meanings. See, for example Klein, Immunology (John Wiley, New York, 1982); Pharman, chapter 14 in Weir, ed. Imm a or chemistry, 4a. edition. (Blackewll Scientific Publishers, Oxford, 1986). These fragments can be prepared from intact antibodies by chemical cleavage by using the recombinant DNA methodology. See, for example, U.S. Patent No. 4,642,334, which describes the production of recombinant Fv fragments and WO 93/11236 for csFv. The antibodies of this invention may also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can bind to interleukin without inhibiting receptor binding. As neutralizing antibodies, these may be useful in competitive binding assays. These will also be useful for detecting or quantifying IL-l ?. The BAS-1 fragments may be linked to other materials, particularly polypeptides, such as polypeptides fused or covalently linked to be used as immunogens. The IL-l? human, its fragments can be fused or covalently bound to a variety of immunogens, such as hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serological Reactions, Dover Publications, New York, and Williams et al., (1967) Methods in Immunology and Immunochemistry, vol.l, Academic Press, New York; each of which is incorporated herein by reference, for descriptions of methods of preparation of polyclonal antiserum. A common method includes the hyperimmunization of an animal with an antigen. The blood of the animal is then collected shortly after the 5 repeated immunizations and the gamma globulin is isolated. In some cases, it is desirable to prepare monoclonal antibodies from different mammalian hosts, such as mice, rodents, primates, humans, etc. The description of the techniques for the preparation of these monoclonal antibodies can be found in, for example, Stites, et al. (eds.), Basic and Clinical Immunology (4th edition), Lange Medical Publications, Los Altos, CA, and references mentioned therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press; Goding (1986) Monoclonal Antibodies: Principies and Practice (2nd edition) Academic Press, New York; and particularly in Kohier and Milstein (1975) in Nature, 256: 495-497, which describes a method of preparation of monoclonal antibodies. Each of these references is incorporated herein as a reference. In summary, this method includes injecting an animal with an immunogen. The animal is then sacrificed and the cells are taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproduction in vi tro. The population of the hybridomas is then screened to isolate individual clones, each of which secretes a single species of antibody to the immunogen. In this manner, the individual antibody species obtained are the products of single B cells immortalized and cloned from the immune animal generated in response to the specific site recognized on the immunogenic substance. Other suitable techniques include in vitro exposure of the lymphocytes to the antigenic polypeptides or otherwise to the selection of antibody libraries on phages or the like. See Huse, et al., (1989) "Generation of a Large Cobminatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. The polypeptides and antibodies of the present invention can be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by binding, either covalently or non-covalently, to a substance that provides a detectable signal. A wide variety of brands and conjugation techniques are known and widely reported in the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescence producing moieties, chemiluminescent portions, magnetic particles and the like. The patents, which show the use of these trademarks, include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Likewise, recombinant or chimeric immunoglobulins 5 can be produced, see Cabilly, US Patent No. 4,816,567. The antibodies of this invention can be used for affinity chromatography in the isolation of IL-1 ?. The columns can be prepared where the antibodies are bound to a solid support, for example particles such as agarose, Sephadex, or the like, where the cell lysate can pass through the column, the column can be washed, followed by concentrations of a moderate denaturant, whereby the purified protein will be released. The antibodies can also be used to screen expression libraries for specific expression products. Usually the antibodies used in this procedure will be labeled with a portion that allows easy detection of the presence of the antigen by binding to the antibody. The antibodies developed against each IL-1? human will also be used to develop anti-idiotypic antibodies. These will be useful in the detection or diagnosis of the different immunological states related to the expression of the protein or cells that express receptors for the protein. These will also be useful as agonists or antagonists of interleukin, which may be competitive inhibitors or substitutes for ligands that occur in the natural state. The natural and recombinant forms of IL-1 molecules? For example, human methods of this invention are particularly useful in kits and assay methods, these methods would also be applied to screening for binding activity, for example, receptors for these proteins. Various methods of automated testing have been developed in recent years to allow the exploration of tens of hundreds of compounds per year. See, for example, the BIOMEK automated work station, Beckman Instruments Palo Alto, California and Fodor et al. (1991) Science 241: 767-773, which are incorporated herein by reference. The last reference describes in the middle to test the binding by a plurality of defined polymers synthesized on a solid substrate. The development of suitable assays for screening a homologous agonist / antagonist receptor or proteins can be greatly facilitated by the availability of large amounts of soluble IL-1, purified in an active state, such as that provided in this invention. The IL-l? purified can be coated directly on plates for use in the aforementioned receptor screening techniques. However, non-neutralizing antibodies to these proteins can be used as capture antibodies to immobilize the respective interleukin on the solid phase, useful, for example, in diagnostic uses. This invention also contemplates the use of IL-1? fragments thereof, peptides and their fusion products in a variety of diagnostic kits and methods for detecting the presence of the protein or its receptor. In an alternative or additional way, antibodies against the molecules can be incorporated into the kits and methods. Usually, the kit will have a compartment that contains the peptide of IL-1? defined or gene segment or a reagent that recognizes one or the other. Typically, recognition reagents, in the case of the peptide, would be a receptor or antibody or in the case of a gene segment, it would usually be a probe for hybridization. A preferred kit for determining the concentration of a sample, for example IL-1 ?, will usually comprise a labeled compound, eg, receptor or antibody, having known binding affinity for IL-1 ?, a source of IL- l? (natural or recombinant) as a control. positive, and a means to separate bound labeled compound from free, for example, a solid phase to immobilize IL-1? in the test sample. Normally, compartments containing reagents, and instructions, will be provided.

Antibodies, including antigen-binding fragments, specific for IL-1? human or a fragment of the peptide, or fragments of the receptor are useful in diagnostic applications, to detect the presence of elevated levels of IL-1? and / or its fragments. Diagnostic assays can be homogeneous (without a separation step between the free reagent and the antibody-antigen complex) or heterogeneous (without a separation step). There are different commercial trials, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme multiplied immunoassay technique (EMIT), substrate-labeled fluorescence immunoassay (SLFIA), and the like. For example, unlabeled antibodies can be employed using a second antibody that is labeled and that recognizes the antibody for IL-1? or for a specific fragment of it. These trials have also been widely described in the literature. See, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH and Coligan (ed.) (1991) and periodic supplements, Current Protocols in Iology, Greene / Wiley, New York. Anti-idiotypic antibodies may have similar use to serve as agonists or antagonists of IL-1 ?.

These should be useful as therapeutic reagents under appropriate circumstances. Reagents for diagnostic tests are often supplied in kits to optimize the sensitivity of the assay. For the present invention, depending on the nature of the assay, the protocol and the label, the labeled or non-labeled antibody, or the labeled receptor are provided. This usually, together with other additives, such as buffer solutions, stabilizers, materials necessary for the production of the signal, as substrates for enzymes and the like. Preferably the kit will also contain instructions for proper use and disposal of the contents after use. Typically, the kit has compartments for each useful reagent and will contain instructions for proper use and to discard reagents. It is desirable that the reagents be provided as a dry lyophilized powder, where the reagents can be reconstituted in an aqueous medium providing adequate concentrations to perform the assay. Any of the aforementioned constituents of the diagnostic assay can be used without modification or can be modified in different ways. For example, labeling by covalent or non-covalent binding can be performed to a portion that directly or indirectly provides a detectable signal. In any of these assays, a test compound, IL-1? or the antibodies thereto can be labeled directly or indirectly. The possibilities for direct labeling include groups of brands: radiolabels such as can be 125 I, enzymes (US Patent No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (US Patent No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift or polarization of fluorescence. The above patents are incorporated herein by reference. The possibilities for indirect labeling include biotinylation of a constituent followed by avidin binding coupled to one of the above label groups. There are also various methods of separating bound ligand from free, or otherwise, test compound bound from free. The IL-l? it can be immobilized in various matrices, followed by washing. Suitable matrices include plastics, such as a plate for ELISA, filters and beads. Methods for immobilizing the receptor to a matrix include, without limitation, direct adhesion to the plastic, the use of a capture antibody, chemical coupling and biotin-avidin. The last step in this method includes the precipitation of the antibody / antigen complex by any of the different methods that includes those that use, for example, an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate. Other suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle et al. (1984) Clin. Chem. 30 (9): 1457-1461, and separation of the magnetic particle from the double antibody as described in U.S. Patent No. 4,459,678, each of which is incorporated herein by reference. The methods for linking the protein or fragments to the different brands have been widely reported in the literature and do not require a more detailed description in the present. Many of the techniques include the use of activated carboxyl groups by the use of carbodiimide or active esters to form peptide bonds, the formation of thioethers by reaction of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin such as maleimide, for the union, or similar. The fusion proteins will also find use in these applications. Another diagnostic aspect of this invention includes the use of oligonucleotide or polynucleotide sequences taken from the sequence of an IL-1 ?. These sequences can be used as probes to detect levels of IL-1? in patients suspected of having an immune disorder. The preparation of the nucleotide sequences of RNA and DNA, the labeling of the sequences and the preferred size of the sequences has received extensive description and analysis in the literature. Normally, an oligonucleotide probe should have at least about 14 nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes can be up to several kilobases. It is possible to use various brands, most commonly radionuclides, particularly 32P. However, it is also possible to employ other techniques, such as the use of biotin-modified nucleotides for introduction into a polynucleotide. Biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescent substances, enzymes or the like. Otherwise, it is possible to use antibodies that can recognize specific duplexes, including DNA duplexes, RNA duplexes, hybrid DNA-RNA duplexes or DNA-protein duplexes. The antibodies in turn can be labeled and the assay can be carried out where the duplex is bound to a surface, so that, with the formation of the duplex on the surface, it may be possible to detect the presence of the antibody bound to the duplex. The use of probes for novel anti-sense RNA can be carried out in any of the conventional techniques, such as nucleic acid hybridization, plus and minus scanning, recombination probing, hybrid translation released (HRT), and hybrid translation. suspended (HART). This also includes amplification techniques such as the polymerase chain reaction (PCR). Diagnostic kits that also test the qualitative or quantitative presence of other markers are also contemplated. The diagnosis or prognosis may depend on the combination of the multiple indicators that are used as markers. This way the kits can try combinations of markers. See, for example Viallet, et al., (1989) Progress in Growth Factor Res., 1: 89-97. This invention also provides reagents with significant therapeutic value. The IL-l? human (natural or recombinant), fragments thereof, mutein agonists and antagonists and antibodies, together with the compounds identified with binding affinity to interleukin or its receptor or antibodies, should be useful in the treatment of states that exhibit expression abnormal of interleukin. This abnormality will usually be manifested by immunological disorders. Furthermore this invention should provide therapeutic value in different diseases or disorders associated with abnormal expression or abnormal activation of the interleukin response. It has been suggested that mouse IGIF is involved in tumors, allergies and infectious diseases, for example, pulmonary tuberculosis, leprosy, fulminant hepatitis and viral infections, such as HIV: In addition, the profile of dendritic cell expression shows IL-1? human, mainly expressed in activated dendritic cells. Activated dendritic cells are also an important producer of IL-12, and it is considered that this IL-12 produced by dendritic cells plays an important role in the direction of a Thl-type response. The combination of IL-l? and IL-12 must be extremely potent in the induction of IFN- ?. It is possible that the combination of these two pro-inflammatory cytokines under certain circumstances may give rise to a septic shock. An antagonist of IL-1 ?, mutein or antibody, would be very useful in this situation. It is possible that a natural IL-1 antagonist? is produced by NK cells or activated T cells to counteract the effects of IL-l ?. The recombinant IL-1, agonists or antagonists of mutein or antibodies of IL-1? they can be purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active ingredients, for example, in conventional pharmaceutically acceptable carriers or diluents, together with physiologically harmless stabilizers and excipients. These combinations can be filtered to be sterile and placed in dosage forms as by lyophilization in small bottles and stored in stabilized aqueous preparations. This invention also contemplates the use of antibodies or binding fragments thereof which do not bind to the complement. The scan of the receiver using IL-l? or fragments thereof can be made to identify molecules that have interleukin binding affinity. Subsequent biological assays can then be used to determine if a receptor can provide competitive binding, which can block intrinsic stimulatory activity. Fragments of the receptor can be used as a blocker or antagonist since it blocks the activity of IL-1 ?. In the same way, a compound that has intrinsic stimulating activity can activate the receptor and in this way is an agonist since it stimulates the activity of IL-1 ?. This invention also contemplates the therapeutic use of antibodies for 'IL-1? and other molecules as antagonists. The amounts of reagents required for effective therapy will depend on very different factors, including the means of administration, the target site, the physiological state of the patient and other medications administered. In this way, dosages for the treatment will be titrated to optimize safety and efficacy. In general, the dosages used in vi tro can provide a useful guide in the amounts useful for the in situ administration of these reagents. Animal testing of effective doses for the treatment of specific disorders will provide greater predictive indications of dosing in humans. Different considerations are described, for example, in Gilman, et al. (Eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8a. Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17a. ed. (1990). Mack Publishing Co. , Easton, Penn. Methods for administration are described in these references and then, for example, for oral, intravenous, intraperitoneal or intramuscular administration, transdermal diffusion and others. The pharmaceutically acceptable carriers will include water, saline, buffer solutions and other compounds that are described, for example, in the Merck Index, Merck & Co., Rahway, New Jersey. Due to the binding with probably high affinity between an IL-1? and its receptors, it is initially expected that the low doses of these reagents will be effective. Thus, dosing ranges will usually be expected in amounts below concentrations of 1 mM, usually concentrations less than about 10 uM, usually less than about 100 nM, preferably less than 10 pM ( picomolar), and more preferably less than about 1 fM (fentomolar), with a suitable carrier. Slow release formulations, or slow release devices will often be used for continuous administration. The IL-l? human, fragments, antagonists and agonists can be administered directly to the host that is going to be treated, or depending on the size of the compounds, it may be desirable to conjugate them to the carrier proteins, such as ovalbumin or serum albumin, before their administration. The therapeutic formulations can be administered in any formulation for conventional dosing. Although it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. The formulations will usually contain at least one active ingredient, as already defined, together with one or more acceptable carriers therefor. Each carrier must be pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not harmful to the patient. The formulations include those suitable for oral, rectal, nasal or parenteral administration (including subcutaneous, intramuscular, intravenous and intradermal). The formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. See, for example, Gilman, et al. (Eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8a. Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17a. ed. (1990). Mack Publishing Co., Easton, Penn .; Avis, et al (eds) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; Lieberman, et al (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, NY; and Lieberman, et al. (eds.) (1990) Dekker, NY. The therapy of this invention can be combined with, or used in association with other therapeutic agents. The description of the ligand IL-1? in the present it provides the means to identify a receiver as already described. This receptor must bind specifically to IL-1 and with reasonably high affinity. Different constructions are available, which allow the marking of the IL-1? to detect your partner. For example, direct dialing of IL-1 ?, merging it on its markers for secondary marking, for example FLAG or other epitope tags, etc., will allow detection of the receiver. This may be histological, such as an affinity method for biochemical purification, or labeling or selection in an expression cloning method. A two-hybrid selection system can also be applied by making suitable constructions with the available sequences of IL-1 ?. See, for example Fields and Song (1989) Nature 340: 245-246. The broad scope of this invention will be better understood by reference to the following examples, which are not intended to limit the invention to the specific embodiments.

EXAMPLES I. General Methods Some of the standard methods are described or referenced in, for example Maniatis, et al., (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.), Vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene Publishing Associates, Brooklyn, NY; o Ausubel, et al. (1987 and supplements) Current Protocols in Molecular Biology, Greene / Wiley, New York. Methods for protein purification include methods such as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization and others. See, for example, Ausubel, et al., (1987 and periodic supplements); Deutscher 81990) "Guide to Protein Purification" in Methods in Enzymology, vol. 182, and other volumes in this series; the manufacturer's literature on the use of protein purification products, for example Pharmacia Piscataway, N.J., or Bio-Rad, Richmond, CA. The combination with recombinant techniques allows fusion for the appropriate segments, for example, to a FLAG sequence or an equivalent that can be fused through a protease-removable sequence. See, for example Hochuli (1989) Chemische Industrie 12: 69-70; Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic Engineering, Principie and Methods 12: 87-98, Plenum Press, N.Y .; and Crowe, et al. (1992) OIAexpress: The High Level Expression & Protein Purification System QUIAGEN, Inc., Chatsworth, CA, Sequence analysis is performed by computer, for example, using available software programs, including those from GCG (U. Wisconsin), and GenBank sources. Public databases of the sequences were also used, for example from GenBank and others. The various techniques applicable for IL-4 and IL-10 can be applied to IL-1 ?, as described, for example, in U.S. Patent No. 5,017,691 (IL-4), U.S. S.N. 07 / 453,951 (IL-10), and U.S. S.N. 08 / 110,683) (IL-10 receptor).

II. Amplification of the fragment of IL-1? human by PCR. Two primers are selected (see SEQ ID NO: 1). RT-PCR is used on a suitable mRNA sample, selected for the presence of the messenger to produce cDNA, for example, a sample of activated human monocyte cells.

III. Distribution of IL-l? in tissue. The message for the gene that codes for IL-1? has been detected in dendritic cells. Expression is elevated in 5-day dendritic cells, dendritic cells activated with LPS, dendritic cells treated with GM-CSF and IL-4 and in a mixture of dendritic cells. Elevated expression has not been detected in B cells or in NK cells. Low levels have been detected in some cDNA libraries of monocytic cells. Expression is usually not detected in fetal tissue, although low levels have been observed in spleen, lung and fetal small intestine. Low levels are also detected in fetal gallbladder and adult amygdala.

IV. Production of mouse IGIF protein A fusion construct of GST was designed for expression in E. coli. The protein was expressed and purified using normal procedures. Similar methods are applicable to IL-1? human A pGex plasmid of mouse IGIF was constructed and transformed into E. coli. Freshly transformed cells were developed in LB medium with a content of 50 μg / ml of ampicillin and induced with IPTG (Sigma, San Luis, MO). After induction overnight, the bacteria were harvested and the packages containing IGIF were isolated. The packages were homogenized in TE buffer (50 mM Tirs-base pH 8.0, 10 mM EDTA and 2 mM pephabloc) in 2 liters. This material was passed three times through a microfluidizer (Microfluidics, Newton, MA). The fluidized supernatant was centrifuged on a Sorvall Gs-3 rotor for one hour at 13,000 rpm. The resulting supernatant with an IGIF content was filtered and passed over a glutathione-SEPHAROSE column equilibrated in 50 mM Tris-base pH 8.0. The fractions containing the IGIF-GST fusion protein were combined and split with thrombin (Enzyme Research Laboratories, Inc., South Bend, IN). The split combination was then passed over a Q-SEPHAROSE column equilibrated in 50 mM Tris-base. The fractions containing the IGIF were combined and diluted in cold distilled H20 to reduce conductivity and again passed over a new Q-Sepharose column. The fractions containing IGIF were combined, an aliquot was taken and stored in the freezer at -70 ° C. The comparison of the CD spectrum with the mouse IL-lβ suggests that the protein was doubled correctly. See Hazuda, et al. (1969) J. Biol. Chem. 264: 1689-1693.

V. Biological assays with mouse IGIF The biological assays confirmed the IFN-α-inducing activity. over T cells. IGIF stimulates the production of IFN-? by purified NK cells, and this induction is strongly synergized with IL-12 or IL-2. Similar tests will be carried out with IL-1? human Mouse IGIF does not seem to work efficiently on human cells, for example, through the human receptor. It has been established that mouse IGIF induces IFN-? by splenocytes of SCID mice, as described above.

Splenocytes in this manner express a receptor for mouse IGIF. Induction is also synergized with IL-12 or IL-2. The mouse IGIF, in combination with IL-12 (and TNF-a also induce NK cells activated with IL-2 to produce IFN-α.) This induction, in the case of NK cells activated with IL-2, appears to be IL-1β-dependent conforming treatment with anti-IL-lβ blocks the production of IFN-α. Mouse IGIF can overcome this blockade and induce IFN- ?.

SAW. Preparation of specific antibodies for IL-1? Inbred Balb / c mice were immunized intraperitoneally with recombinant forms of the human protein, for example, IL-1? soluble, purified or transfected, stable NIH-3T3 cells. The animals are reinforced at appropriate time points with protein, with or without additional adjuvant, to further stimulate the production of antibodies. The serum is collected or the hybridomas produced with splenic cells harvested. Otherwise, Balb / c mice are immunized with cells transformed with the gene or fragments thereof, either with endogenous or exogenous cells, or with isolated membranes enriched for the expression of the antigen. The serum is collected at the appropriate time, usually after numerous other administrations. Various gene therapy techniques may be useful, for example, in the production of proteins in situ, to generate an immune response. It is possible to prepare monoclonal antibodies. For example, splenocytes are fused with a suitable fusion partner and the hybridomas are selected in the depletion medium by standard procedures. Hybridoma supernatants are screened for the presence of antibodies that bind IL-1? human, for example, by ELISA or other assay. Antibodies that specifically recognize IL-1? Human but not species variants can also be selected or prepared. In another method, the synthetic peptides or purified protein are present in an immune system to generate monoclonal or polyclonal antibodies. See, for example, Colign (1991) Current Protocols in Immunology, Wiley / Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press. In suitable situations, the binding reagent is labeled as described above, for example, by fluorescence or otherwise, or immobilized to a substrate by panning methods. Nucleic acids can also be introduced into the cell in an animal to produce the antigen, which serves to develop an immune response. See, for example, Wang, et al. (1993) Proc. Nat'l. Acad. Sci., 90: 4156-4160; Barry, et al. (1994) BioTechniques, 16: 616-619; and Xiang, et al (1995) Im unity 2: 129-135.

XI. Production of fusion proteins with IL-1? Various fusion constructs are prepared with the extracellular domain of IL-1 ?. This portion of the gene is fused with an epitope tag, for example, a FLAG tag or a construction of a two-hybrid system. See, for example Fields and Song (1989) Nature 340: 245-246. The epitope tag can be used in an expression cloning procedure with detection with anti-FLAG antibodies to detect a binding partner, eg, the receptor for IL-1 ?. The two-hybrid system can also be used to isolate proteins that bind specifically to IL-1 ?.

VIII. Mapping of IL-l? human by hybridization in si tu Chromosome extensions were prepared. Hybridization in itself was performed on 'chromosome preparations obtained from human lymphocytes stimulated by phytohemagglutinin grown for 72 h. 5-bromodeoxyuridine was added during the last 7 hours of the culture (60 μg / 1 of medium), to ensure formation of good quality chromosomal bands after hybridization. A suitable fragment, for example PCR fragment, amplified with the aid of primers, on the cDNA template of total B cells, was cloned into a suitable vector. The vector was marked by notch translation with ° H. The radiolabeled probe was hybridized to metaphase extensions, as described in Mattei, et al., (1985) Hum. Genet 69: 327-331.

After coating with the emulsion for nuclear screening (KODAK NTB2), the plates or slides were exposed for 18 days at 4 ° C. To avoid any slippage of the silver grains during the band-forming procedure, the chromosome extensions were first stained with buffered Giemsa solution and the metaphase photographed. The formation of the R band was then performed by the fluorochrome-photolysis-Giemsa (FPG) method and the metaphases were re-imaged before analysis.

IX. Structure Activity Ratio Information on the importance of specific waste is determined using normal procedures and analyzes. The standard mutagenesis analysis is carried out, for example, by generating very different variants in determined positions, for example, in the positions previously identified, and by evaluating the biological activities of the variants. This can be done to the extent of determining the positions that modify the activity, or to focus specific positions to determine the residues that can be substituted to retain, block or modulate the biological activity. Otherwise, the analysis of natural variants may indicate the positions that tolerate natural mutations. This can result from the population analysis of the variation between individuals, or through knowledge or species. The samples of the selected individuals are analyzed, for example, by PCR analysis and sequencing. This allows the evaluation of population polymorphisms.

X. Isolation of a receptor for human IL-1 and IL-1? Human can be used as a specific binding reagent to identify its binding partner, taking advantage of its binding specificity much like how an antibody would be used. A binding reagent is labeled as described above, for example, by fluorescence or otherwise, or is immobilized to a substrate by panning methods. The binding composition is used to screen an expression library prepared from a cell line that expresses a binding partner, i.e., a receptor. Standard staining techniques are used to detect or classify the expressed intracellular or surface receptor, or transformed cells that express on the surface are scanned by panning. The exploration of intracellular expression is carried out by different staining or immunofluorescence procedures. See also McMahan, et al. (1191) EMBO J. 10: 2821-2832. For example, on day 0, permanox two-chamber slides are precoated with 1 ml per fibronectin chamber, 10 ng / nl in PBS, for 30 minutes at room temperature. Rinse once with PBS. Then COS cells are plated at 2-3 x 10 ° cells per chamber in 1.5 ml of growth medium. It is incubated overnight at 37 ° C. During day 1, for each sample, 0.5 ml of a solution of 66 μg / ml DEAE-dextran, 66 μM of chloroquine and 4 μg of DNA in DME without serum are prepared. For each series, a positive control is prepared, for example, of cDNA IL-1? Human FLAG in a dilution of 1 and 1/200, and a negative simulation. The cells are rinsed with DME without serum. The DNA solution is added and incubated for 5 hours at 37 ° C. The medium is removed and 0.5 ml of 10% DMSO in DME is added for 2.5 minutes. Remove and rinse once with DME. Add 0.5 ml of growth medium and incubate overnight. On day 2 the medium is changed. On days 3 or 4 the cells are fixed and stained. The cells are rinsed twice with Hank's buffered saline solution (HBSS) and fixed in 4% paraformaldehyde (PFA) / glucose for 5 minutes. Wash 3X with HBSS, the slides can be stored at -80 ° C after all the liquid is removed. For each chamber, 0.5 ml incubations are performed as follows. HBSS / saponin (0.1%) is added with 32 μl / ml of NaN3 μM for 20 minutes. The cells are then washed with HBSS / saponin IX. Add IL-l? human or the IL-1? / antibody complex to the cells and incubate for 30 min. Wash the cells twice with HBSS / saponin. If appropriate, add the first antibody for 30 min. Add the second antibody, for example, anti-mouse antibody Vector in a 1/200 dilution, and incubate for 30 minutes. Prepare the ELISA solution, for example horseradish peroxidase Vector Elite ABC solution and pre-incubate for 30 minutes. Use, for example, one drop of solution A (avidin) and one drop of solution B (biotin) per 2.5 ml of HBSS / saponin. Wash the cells twice with HBSS / saponin. Add the ABC HRP solution and incubate for 30 min. Wash the cells twice with HBSS, second wash for two minutes, which closes the cells. Then add diaminobenzoic acid (DAB) Vector for 5 to 10 minutes. Use two drops of buffer plus four drops of DAB plus two drops of H20c per 5 ml of vitreous distilled water. Remove the camera carefully and rinse the slide in water. Air dry for a few minutes, then add a drop of Crystal Mount and cover the slide. Bake for 5 minutes at 85-90 ° C. Evaluate the positive staining of the deposits, progressively subcloning to isolate the individual genes responsible for the binding. Otherwise, IL-1 reagents? they are used to purify by affinity or classify cells that express a receptor. See, for example, Sambrook et al, or Ausubel et al. Another strategy is to scan for a membrane-bound receptor by panning. The receptor cDNA is constructed as described above. The ligand can be immobilized and used to immobilize expression cells. Immobilization can be achieved by the use of suitable antibodies that recognize, for example, a FLAG sequence of an IL-1? Fusion construct or by the use of antibodies developed against the first antibodies. The cycles of recursive selection and amplification give rise to the enrichment of the appropriate clones and final isolation of the clones expressing the receptor. Phage display libraries can be explored through IL-1? human Appropriate labeling techniques, for example, anti-FLAG antibodies will allow specific labeling of suitable clones. All citations mentioned herein are incorporated by reference into the same degree as if each individual publication or patent application was specifically and individually indicated as incorporated by reference. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is limited by the terms of the appended claims, together with the full scope of the equivalents for which these claims are entitled; and the invention will not be limited by the specific embodiments that have been presented herein by way of example.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Schering Corporation (B) STREET: 2000 Galloping Hill Road (C) CITY: Kenilworth (D) STATE: New Jersey (E) COUNTRY: USA ( F) POSTAL CODE: 07033-0530 (G) TELEPHONE: 908-298-2906 (H) TELEFAX: 908-298-5388 (I) TELEX: (ii) TITLE OF THE INVENTION: Human cytokine (iii) NUMBER OF SEQUENCES: 2 (Iv): LEGIBLE COMPUTATION FORM: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: Macintosh 7.5.3 (D) SOFTWARE: Microsoft Word 5.1a (v) DATA FROM THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (vi) PREVIOUS APPLICATION DATE: (A) APPLICATION NUMBER: US 08 / 651,998 (B) SUBMISSION DATE: May 20, 1996 ( 2) INFORMATION OF SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1380 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) PECULIARITY: ( A) NAME / KEY: CDS (B) LOCATION: 435..1016 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 AAAGAGATAC TCAGAAAGAG GTACAGGTTT TGGAAGGCAC AGAGCCCCÍLA CTTTTACGGA 60 AGAAAAGATT TCATGAAAAT AGTGATATTA CATTAAAAGA AGTACTCGTA TCCTCTGCCA 120 CTTTATTTCG ACTTCCATTG CCCTAGGAAA GAGCCTGTTT GAAGGCGGGC CCAAGGAGTG 180 CCGACAGCAG TCTCCTCCCT CCACCTTCTT CCTCATTCTC TCCCCAGCTT GCTGAGCCCT 240 TTGCTCCCCT GGCGACTGCC TGGACAGTCA GCAAGGAATT GTCTCCCAGT GCATTTTGCC 30C CTCCTGGCTG CCAACTCTGG CTGCTAAAGC GGCTGCCACC TGCTGCAGTC TAC CAGCTT 360 CGGGAAGAAG AAAGGAACCT CAGACCTTCC AGATCGCTTC CTCTCGCAAC AAACTATTTG 420 TCGCCAGAAT AAAG ATG GCT GCT GAft. CCA GTA GAA GAC AAT TGC ATC AAC 470 Met Wing Wing Glu Pro Val Glu Asp Asn Cys lie Asn 1 5 10 TTT GTG GCA ATG AAA TTT ATT GAC AAT ACG CTT TAC TTT ATA GCT GAA 518 P e Val Wing Met Lys Phe lie Asp Asn Thr Leu Tyr Phe He Wing Glu 15 20 25 GAT GAT GAA AAC CTG GAA TCA GAT TAC TTT GGC AAG CTT GAA TCT AAA 566 Asp Asp Glu Asn Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lye 30 35 40 TTA TCA GTC ATA AGA AAT TTG AAT GAC CAA GTT CTC TTC ATT GAC CAA 614 Leu Ser Val He Arg Asn Leu Asn Asp Gln Val Leu Phe He Asp Gln 45 50 55 60 GGA AAT CGG CCT CTA TTT GAA GAT ATG ACT GAT TCT GAC TGT AGA GAT 662 Gly Asn Arg Pro Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp 65 70 75 AAT GCA CCC CGG ACC ATA TTT ATT ATA AGT ATG TAT AAA GAT AGC CAG 710 Asn Ala Pro Arg Thr He Phe He He Ser Met Tyr Lys Asp Ser Gln 80 85 90 CCT AGA GGT ATG GCT GTA ACT ATC TCT GTG AAG TGT GAG AAA ATT TCA 758 Pro Arg Gly Met Wing Val Thr He Ser Val Lye Cys Glu Lys He Ser 95 100 105 ACT CTC TCC TGT GAG AAC AAA ATT ATT TCC TTT AAG GAA ATG AAT CCT 806 Thr Leu Ser Cye Glu Asn Lys He He Ser Phe Lys Glu Met Asn Pro 110 115 120 CCT GAT AAC ATC AAG GAT ACA AAA AGT GAC ATC ATA TTC TTG CAG AGA 854 Pro Asp Asn He Lys Asp Thr Lys Ser Asp He He Phe Phe Gln Arg 125 130 135 140 AGT GTC CCA GGA CAT GAT AAT AAG ATG CAA TTT GAA TCT TCA TCA TAC 902 Ser Val Pro Gly His Asp Asn Lys Met Gln Phe Glu Ser Ser Tyr 145 150 155 GAA GGA TAC TTT CTA GCT TGT GAA AAA GAG AGA GAC CTT TTT AAA CTC 950 Glu Gly Tyr Phe Leu Wing Cys Glu Lys Glu Arg Asp Leu Phe Lys ^ u 160 165 170 ATT TTG AAA AAA GAG GAT GAA TTG GGG GAT AGA TCT ATA ATG TTC ACT 998 He Leu Lys Lys Glu Asp Glu Leu Gly Asp Arg Ser He Met Phe Thr 175 180 185 GTT CAA AAC GAA GAC TAG CTATTAAAAT TTCATGCCGG GCGCAGTGGC 1046 Val Gln Asn Glu Asp 190 TCACGCCTGT AATCCCAGCC CTTTGGGAGG CTGAGGCGGG CAGATCACCA GAGGTCAGGT 1106 GTTCAAGACC AGCCTGACCA ACATGGTGAA ACCTCATCTC TACTAAAAAT ACAAAAAATT 1166 AGCTGAGTGT AGTGACCCAT GCCCTCAATC CCAGCTACTC AAGAGGCTGA GGCAGGAGAA 1226 TCACTTGCAC TCCGGAGGTG GAGGTTGTGG TGAGCCGAGA TTGCACCATT GCGCTCTAGC 1286 CTGGGCAACA ACAGCAAAAC TCCATCTCAA AAAATAAAAT AAATAAATAA ACAAATAAAA 1346 AATTCATAAT GTGAAAAAAA AAAAAAAAAA AAAG 1380 INFORMATION OF SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 194 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Ala Ala Glu Pro Val Glu Asp Asn Cys He Asn Phe Val Ala Met 1 5 10 15 Lys Phe He Asp Asn Thr Leu Tyr Phe He Wing Glu Asp Asp Glu Asn 20 25 30 Leu Glu Being Asp Tyr Phe Gly Lys Leu Glu Being Lys Leu Being Val He 35 40 45 Arg Asn Leu Asn Asp Gln Val Leu Phe He Asp Gln Gly Asn Arg Pro 50 55 60 Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg 65 70 75 80 Thr lie Phe He He Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met 85 90 95 Wing Val Thr He Ser Val Lys Cys Glu Lys He Ser Thr Leu Ser Cys 100 105 110 Glu Asn Lys He He Ser Phe Lys Glu Met Asn Pro Pro Asp Asn He 115 120 125 Lys Asp Thr Lys Ser Asp He He Phe Phe Gln Arg Ser Val Pro Gly 130 135 140 His Asp Asn Lys Met Gln Phe Glu Ser Ser Tyr Glu Gly Tyr Phe 145 150 155 160 Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu He Leu Lys Lys 165 170 175 Glu Asp Glu Leu Gly Asp Arg Ser He Met Met Phe Thr Val Gln Asn Glu 180 185 190 Asp

Claims (16)

1. An IL-1 antagonist ?.

2. The antagonist of claim 1 comprising an antibody against IL-1? human, or a fragment of union of the same.

3. A pharmaceutical composition for inhibiting the biological activity of IL-1? human comprising an antagonist of any of claims 1 or 2 and a pharmaceutically acceptable carrier.

4. A method of treatment of a condition caused by IL-1? human, comprising the administration of an effective amount of an antagonist or pharmaceutical composition of any of claims 1 to 3, to an individual in need of this treatment.

5. The use of an antagonist of any of claims 1 or 2, for the preparation of a medicament for inhibiting the biological activity of IL-1? human

6. The use of an antagonist of any of claims 1 or 2 for the treatment of a condition caused by IL-1? human

7. An IL-1 receptor? isolated human. -

8. A fusion protein comprising IL-1? human conjugated covalently to polyethylene glycol or a polypeptide.

9. The fusion protein of claim 8, wherein a polypeptide conjugated to IL-1? human is derived from an immunoglobulin chain, preferably an Fc fragment, or is another cytokine or chemokine.

10. A pharmaceutical composition for supplying the biological activity of IL-1? human, comprising a fusion protein of any of claims 8 or 9 and a pharmaceutically acceptable carrier.

11. A method for supplying the biological activity of IL-1? human comprising the administration of an effective amount of a fusion protein or pharmaceutical composition of any of claims 8 to 10 to an individual in need of this activity.

12. The use of a fusion protein of any of claims 8 or 9, for the preparation of a medicament for providing the biological activity of IL-1? human

13. The use of a fusion protein of claim 8 or 9 to deliver the biological activity of IL-1? human

14. An isolated nucleic acid or vector encoding a fusion protein of any of claims 8 or 9.

15. A method for producing a fusion protein comprising IL-1? human conjugated covalently to a polypeptide, which consists of culturing a host cell containing the nucleic acid or vector of claim 14 under conditions in which the nucleic acid or vector is expressed.

16. An anti-idiotypic antibody that is an IL-1 agonist or antagonist.