JP2001508652A - Mammalian cytokines related to IL10 - Google Patents

Abstract

  (57) [Summary] Provided are purified genes encoding cytokines from mammals, purified proteins, specific antibodies, and related reagents, including nucleic acids encoding the molecules. Methods for using the reagents and diagnostic kits are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION                    Mammalian cytokines related to IL10                                Field of the invention   The present invention relates to the organism and physiology of mammalian cells, e.g., cells of the mammalian immune system. Compositions related to proteins that function in controlling. For details, Ming describes, for example, the activation, development, differentiation, and activation of various cell types, including hematopoietic cells. Purified genes, proteins, antibodies, and proteins useful for modulating function Provide a series of reagents.                                Background of the Invention   Recombinant DNA techniques generally involve, for example, introduction into a host, donor source Technology to incorporate genetic information from a vector into a vector for subsequent processing, This allows the transferred genetic information to be copied and / or expressed in a new environment. Is done. Usually, the genetic information is a message that encodes the desired protein product. Exist in the form of complementary DNA (cDNA) derived from RNA (mRNA). Career Often, the ability to integrate the cDNA for subsequent replication in the host and If so, it actually controls the expression of the cDNA and is thereby encoded in the host. Plasmids capable of directing the direct synthesis of a product.   For quite some time, the mammalian immune response has been linked to a series of It is known to be based on complex cell interactions. Recent research has shown that It provided new insights into the internal workings of the workpiece. Many of the responses are actually lymphocytes Focusing on network-like interactions of macrophages, granulocytes, and other cells It is clear that immunologists are now generally aware of Soluble proteins known as proteins, cytokines, or monokines Holds that it plays an important role in controlling cellular interactions . Therefore, considerable interest in the isolation, characterization, and mechanism of action of cell regulatory factors And its understanding is important in diagnosing and treating many medical abnormalities (eg, immune system disorders). For treatment Leading remarkable progress.   Lymphokines apparently mediate cellular activity in a variety of ways. They are, Pluripotency to vast numbers of precursors, including diverse cell lineages that make up the complex immune system It has been shown to support proliferation, growth, and differentiation of hematopoietic stem cells. Cell components Proper and balanced interactions between them are necessary for a healthy immune response. Rin When hokines are administered with other drugs, different cell lineages are often Respond in various ways.   Cell lineages of particular importance for the immune response include two classes of lymphocytes: the immunoglobulin. Blin (has the ability to recognize foreign substances and bind to them to effect their removal) B cells capable of producing and secreting proteins) and secreting lymphokines B cells and a variety of other cells (other T cells) T cells of various subsets that induce or suppress vesicles. These lymphocytes Interacts with many other cell types.   Another important cell lineage is mast cells (clearly identified in all mammalian species). Not including granules located near capillaries throughout the body. Connected tissue cells. These cells are found in the lung, skin, and gastrointestinal tract and urinary It is found particularly in high concentrations in incubator tubes. Mast cells are an allergy-related disorder, especially Plays a central role in anaphylaxis as follows: selected antigens Crosslinks a Class of Immunoglobulins Bound to Mast Cell Surface Receptors Mast cells may degranulate and allergic reactions (eg, anaphylaxis) Mediators (eg histamine, serotonin, hepari) And prostaglandins).   Research to better understand and treat various immune disorders is based on the cells of the immune system. Is generally hampered by the inability to maintain in vitro. The immunologists believe that culturing these cells can help T cells and other cell supernatants (which This is achieved through the use of various growth factors, including many lymphokines. Have found that it can be done.   Encodes IL-10 (originally named cytokine synthesis inhibitor (CSIF)) The gene was isolated in the 1980s. See, for example, Mosmann et al., US Pat. No. 5,231,012. checking ... Since then, biology and physiology mediated by cytokines A lot was found about. For example, de Vries and de Waal Malefyt (199 Five)Interleukin-10 See Landes Co., Austin, TX.   From the above, for example, the discovery and development of new lymphokines related to IL-10 But extensive degeneration that is directly or indirectly related to the immune system and / or hematopoietic cells It is clear that it can contribute to a new treatment for the condition or abnormal condition. Details Include lymphokines that enhance or enhance the beneficial activity of known lymphokines Discovery and development are very advantageous. The present invention relates to a new interleukin group Provided are compounds and related compounds, and methods for their use.                                Summary of the Invention   The present invention relates to mammalian (eg, rodents, dogs, cats, primates) interleukins -BKW (IL-BKW) and its biological activity. The invention relates to polypeptides Includes nucleic acids that encode themselves and methods of making and using them. Book The nucleic acids of the invention may, in part, be cloned complementary DNAs (cDNAs) contained herein. ) By homology to the sequence and / or to the polypeptide (typically Functional IL-10-like activity applied to Characterized by ISSEY. Methods to regulate or mediate control of the immune response Provided.   The present invention provides, in part, a novel sequence and structural similarity to cellular IL-10. Based on the discovery of new cytokine sequences. In particular, the present invention provides a mature size of about 158 A gene encoding a protein that is a amino acid is provided. Shows significant sequence homology Functional equivalents include other mammalian species (eg, mice and rats), and Available from non-mammalian species.   In one embodiment, the invention relates to soluble IL-BKW antigenic proteins and peptides. Provides substantially pure or recombinant soluble IL-BKW, including tide fragments I do. Preferably, IL-BKW is a full-length natural soluble from mammals, including primates. It can be a protein or present in a sterile composition. Typically, soluble IL -BKW is the sequence of SEQ ID NO: 2 MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWS Lacks QVSG; is the mature polypeptide of SEQ ID NO: 6; or is the nucleus of SEQ ID NO: 5 Coded by acid. Alternatively, soluble IL-BKW is different from natural soluble IL-BKW Full-length secreted protein showing different post-translational modification patterns.   Functionally, soluble IL-BKW typically immunologically antagonizes IL-10 Active activity.   The present invention also includes the sequence of soluble IL-BKW, but comprising the MNFQQRLQSL WTLAR of SEQ ID NO: 2. Is the sequence of PFCPP LLATASQMQM VVLPCLGFTL LLWSQVSG missing; A fusion protein, which is a peptide; or encoded by SEQ ID NO: 5, provide.   The present invention also provides for soluble IL-BKW protein or peptidic A method of purifying the mixture, wherein the method comprises: Contacting the antibody and separating the bound IL-BKW from other substances. Include.   In another embodiment, the present invention relates to an isolated or isolated IL-BKW encoding Provides a recombinant expression vector. Preferably, the vector comprises SEQ ID NO: 2 or Or 6 secretory sequences and may comprise the sequence of SEQ ID NO: 1 or 5.   The present invention is directed to a positive, substantially pure soluble IL-BKW or fragment. A kit for detection including a control is provided. This kit is also soluble Providing a method for detecting in a sample the presence of an IL-BKW protein or antibody; Comprises testing the sample with a kit.   The present invention also provides a method of regulating the physiology of a cell, the method comprising: Contacting with highly pure soluble IL-BKW. In one embodiment, the cell Is a T cell, and the regulation of physiology is inactivation of the T cell; or Exists in the organization.   The present invention also provides a method for producing soluble IL-BKW, comprising the steps of: Is expressed. A vector can be in a cell, tissue, or organ. You.   Finally, the present invention provides for administering an effective dose of substantially pure soluble IL-BKW to an animal. This provides a method of treating an animal having an abnormal immune response.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows an alignment of IL-10 and related cytokines.                             Detailed description of the invention   All references cited herein are in each individual publication or patent application. , To the same extent as if specifically and individually indicated to be incorporated as reference As incorporated herein by reference. I. General remarks   The present invention relates to (for example, the ability to mediate signals between immune cells or other cells). Encodes various mammalian proteins that are cytokines Amino acid and DNA sequences are provided. For example, Paul (1994)Fundamental I mmunology See Raven Press, N.Y. Full length cytokines and flags Or antagonists are involved in the physiological regulation of cells that express the receptor. Useful. Early data show that cytokines have the opposite effect as IL-10 Suggest that it works. IL-BKW is T cell, B cell, natural killer -Stimulates (NK) cells, macrophages, dendritic cells, hematopoietic progenitors, etc. It seems to have any effect of inhibition. Protein can also be found on proteins Against various epitopes (both linear and conformational epitopes) Useful as antigens (eg, immunogens) for raising antibodies.   CDNA encoding IL-BKW was isolated from a human melanoma cell line. This molecule is It was named mda7 and characterized as a novel melanoma differentiation-related gene. Array Numbers 1 and 2. Jiang et al. (1995)Oncogene 11: 2477-86; Genbank accession number U1626 See 1. This document describes some small homology of mda7 to human IL-10 But the association was unknown. Applicants have analyzed this sequence. And We consider that the coding region was incorrectly identified. Facts coincide with mouse gene sequences I do.   The CDS, proposed by Jiang et al., Ranges from approximately 275 to 895, and codes: Devised:However, Applicants believe that the mature form of IL-BKW, beginning at about residue 49, under: Suggest.   In Table 1, various human IL-10 embodiments are compared to human IL-BKW.                                   Table 1   The mouse clone for IL-BKW (mIL-BKW) also contains activated mouse thymocyte c DNA library (A. Zlotnik, DNAX Res. Institute, Palo Alt. o, CA). SEQ ID NOS: 5 and 6. The signal sequence extends approximately from Metl to GIy23. The mature polypeptide begins approximately at Leu24. Conserved D associated with receptor binding -The helix ranges from almost Glu158 to Leu181. As seen in humans There is no corresponding ATG upstream of the leader sequence, which probably indicates that this molecule Indicates that they are connected.   mIL-BKW has a high degree of amino acid sequence identity to its human counterpart. hIL-10, mIL-1 Alignment between 0, hIL-BKW, and mIL-BKW, D-helix is highly conserved Indicates that See Table 2, and SEQ ID NOs: 2, 3, 6, and 7 .   Table 2 compares human and mouse IL-10 and human and mouse IL-BKW. Compare. In IL-10, this helix appears to be particularly important for receptor binding It is. Therefore, IL-BKW and IL-10 share the same receptor subunit I can do it.                                   Table 2   Chromosome mapping using mIL-BKW as a probe indicates that this cytokine Between the regions homologous to human chromosomes 2q and 1q, It is shown that it is mapped to the central region. Interestingly, mIL-BKW , Located immediately adjacent to the gene for mIL-10. At this particular locus The deficiency is associated with an immune disorder, for example, dominant hemilimb disease. For example, Carter (19 54)Mouse News Lett .11:16; Higgins et al. (1992)Genet . Res.60: 53-60; and And Machado et al. (1976)Am . J. Pathol.85: 515-518.   This protein has a predicted hydrophobic interval that includes residues 25-45 of the protein. It has been characterized as an antigen. However, experiments have shown that membrane morphology exists. I could not confirm. Applicants believe that the predicted hydrophobicity interval is actually Signal sequence that is removed from the protein, as named in the first publication, Consider that it seems to start at either residue 47 (Ser) or 49 (Ala).   Applicants believe that this gene is a small soluble cytokine-like tag of about 158 amino acids. Think of it as coding for protein. The presequence is probably at position 28 or 30 Beginning with an M, thus providing an N-terminal signal sequence of about 17-21 amino acids I do. See Table 1 and SEQ ID NOs: 1 and 2. IL-BKW is a short-chain cytokine Shows the characteristic structural motifs of in members. For example, IL-BKW, mouse and Cellular IL-10, EBV virus IL-10, and equine herpesvirus IL-10 from humans (All sequences are available from GenBank). See Table 1.   The structural homology of IL-BKW to the related IL-10 protein is the mechanism by which this molecule is related. Suggests noh. IL-BKW is a small chain cytokine. Early experiments are new Cytokines are immunized by receptors in the class of cytokine receptors Suggests that it seems to mediate function, but that functional IL-10 receptor complex Seems not to share all parts.   IL-BKW agonists or antagonists also include, for example, IL-10 Functional or receptor antagonists that block binding to sceptors Or may mediate the opposite effect. Therefore, IL-BKW, and Are antagonists of abnormal immune disorders (eg, T cell immune deficiency, chronic inflammation, Or tissue rejection).   Naturally occurring antigens can be used to induce biological or physiological responses in target cells. Can mediate the biochemical response of The embodiments characterized herein are human and human. And other mouse species, but other primates or other species counterparts is expected. For other mammalian species (eg, primates, dogs, cats, and rodents) Additional sequences for the protein in place should also be available. Less than checking ... The following description relates to human IL-BKW for illustrative purposes, but other Are also applicable to related embodiments from the species of   Human and mouse IL-BKW proteins are characteristic structural features of short-chain cytokines Is shown. II. Purified IL-BKW   N-terminal sequence (eg, MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWSQV SG) The amino acid sequence of human IL-BKW lacking comprises one embodiment within SEQ ID NO: 2 Shown. The corresponding mIL-BKW amino acid sequence is shown in SEQ ID NO: 6. Ami These amino acid sequences provided from the N-terminus to the carboxy terminus Sequence information in cytokines that makes it possible to distinguish between protein and protein antigens It is important to provide information and to illustrate many variants. further The peptide sequence is used to generate antibodies that recognize such segments. And the nucleotide sequence is an oligonucleotide Allows the preparation of probes, both of which are genes encoding such sequences Strategy for detection or isolation (eg, cloning). Human sequence Possible glycosylation sites are asn31-thr39 and asn51-ser53 It is asn51-ser53 in mice.   As used herein, the term "human soluble IL-BKW" is used in the context of proteins. The amino acid set forth in SEQ ID NO: 2, for example, described as membrane-bound End part (MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWSQVSG) may be missing Includes proteins having amino acid sequences corresponding to soluble polypeptides. this Is the first 45 residues described as the N-terminus by Jiang et al., Or its significant Missing fragments. "Mouse IL-BKW" has the amino acid sequence corresponding to SEQ ID NO: 6. including. Binding components (eg, antibodies) typically have high affinity (eg, low affinity). Both about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more (Preferably better than about 3 nM) and binds to IL-BKW. Homologous proteins are subhuman Found in other mammalian species (eg, other primates or rodents). Non-feeding Species (eg, birds or amphibians) also have structurally or functionally related genetic Should have offspring and proteins.   As used herein, the term "polypeptide" refers to a significant fragment or cell. And at least about 8 amino acids, typically at least about 12 amino acids. Amino acids, typically at least about 16 amino acids, preferably at least about 20 amino acids Acids, and in a particularly preferred embodiment, at least about 30 or more amino acids, such as It includes a stretch of amino acid residues, such as 35, 40, 45, 50. Such a fragment Start and / or end at virtually every position of every combination (Eg, starting at residues 1, 2, 3, etc. and eg, 150, 149, 148, etc.) Ending in). Particularly interesting peptides are the boundaries of structural domains (eg For example, it has ends corresponding to helices A, B, C, and / or D). table See Table 1, Table 2, and FIG. The sequence of IL-BKW is in the region of residues 126 to 137. And specific identities with cellular IL-10, and other regions have a greater degree of I Note that it shows an L-BKW specific sequence.   The term "binding composition" refers to, for example, in an antibody-antigen interaction, A molecule that binds with specificity. This can also be shared or non-shared Specifically associate with IL-BKW (a naturally occurring physiologically relevant protein Compounds, including those in protein interactions), eg, proteins. Minute The child can be a polymer, or a chemical reagent. Functional analogs require structural alterations A molecular form that can be a protein that has or interacts with an appropriate binding determinant It can be a molecule having a shape. Compounds may be agonists of receptor binding interactions. Or may act as an antagonist. For example, Goodman et al. (Eds.) (1990)Goodman &Gilman's: The Pharmacological Bases of Therapeutics (8th edition), Pergamon P See ress.   Substantially pure typically means that the protein is free of other contaminating proteins, nucleic acids, Or means free of other biological material from the original source organism. Purity can be assayed by standard methods, typically by weight, and Usually at least about 40% pure, generally at least about 50% pure, often less At least about 60% pure, typically at least about 80% pure, preferably at least about 90% pure, and in the most preferred embodiment at least about 95% pure. Cap Rear or excipients are often added.   Polypeptide or fragment solubility depends on environment and polypeptide I do. Temperature, electrolyte environment, polypeptide size and molecular characteristics, and solubility Many parameters, including the nature of the vehicle, affect polypeptide solubility. representative Typically, the temperature at which the polypeptide is used ranges from about 4 ° C to about 65 ° C. . Typically, the temperature during use is above about 18 ° C. For diagnostic purposes, the temperature Is usually about room temperature or warmer, but below the denaturation temperature of the components in the assay. is there. For therapeutic purposes, temperature will usually be at body temperature, typically human and mouse. About 37 ° C, but in some circumstances, the temperature may be in situ or in vivo. Can be raised or lowered with a toro.   The size and structure of the polypeptide are generally substantially stable, And usually should not be in a denatured state. The polypeptide can be, for example, To confer affinity with other polypeptides in a quaternary structure, or Or may be associated with a surfactant.   Solvents and electrolytes are of the type used for the preservation of biological activity. It is a physically compatible buffer and is usually close to a physiological aqueous solvent. Normal, The solvent has a neutral pH, typically between about 5 and 10, and preferably about 7.5 . In some cases, one or more surfactants are added, typically a mild Non-denaturing surfactants such as CHS (cholesteryl hemisuccinate) or CHAPS (3- [3-cholamidopropyl) dimethylammonio] -1-propanesulfonate) Or sufficient to avoid significant disruption of the structural or physiological properties of the protein Low concentration. III. Physical variant   The present invention also has substantial amino acid sequence identity with the amino acid sequence of the IL-BKW antigen. And proteins or peptides that do. A variant is a species, polymorphism, or allele Includes gene variants.   Amino acid sequence homology, or sequence identity, if necessary, Determined by optimizing residue matches by introducing gaps You. Needleham et al. (1970)J . Mol. Biol.48: 443-453; Sankoff et al. (1983) Chapter 1Time Warps , String Edits, and Macromolecules: The Theory and Practice o f Sequence Comparison , Addison-Wesley, Reading, MA; and IntelliGenetic s, Mountain View, CA; and University of Wisconsin Genetics Computer Gr See also software packages from oup, Madison, WI. Save as compatible The sequence identity will vary when considering sequential substitutions. Conservative substitutions typically involve the following: Includes substitutions within the following groups: glycine, alanine; valine, isoleucine, leu Syn; Aspartic acid, Glutamic acid; Asparagine, Glutamine; Serine, G Leonin; lysine, arginine; and phenylalanine, tyrosine. Save is It can apply to biological, functional, or structural features. Homologous amino acid sequence Is typically the natural polymorphism or allele in each respective protein sequence. It is intended to include offspring and interspecific alterations. Representative homologous proteins or pep The tide has 25-100% identity to the amino acid sequence of IL-BKW (gaps may be introduced ) To 50-100% identity (if conservative substitutions are involved). Same Sex measures at least about 35%, generally at least about 40%, often less Both about 50%, typically at least about 60%, usually at least about 70%, preferably Is at least 80%, and more preferably at least about 90%.   The isolated IL-BKW DNA contains nucleotide substitutions, nucleotide deletions, It can be easily modified by insertion and inversion of nucleotide stretch. This These modifications may be made to these antigens, their derivatives, or similar physiological, immunological, Novel DNA sequences encoding proteins with antigenic or other functional activity Is generated. These modified sequences can be used to produce mutant antigens or to express Can be used to enhance Enhanced expression, gene amplification, increased transcription , Increased translation, and other mechanisms. "Mutant IL-BKW" Although it falls within the sequence identity definition of IL-BKW as described above, deletion, substitution, or insertion A different amino acid sequence from IL-BKW that is normally found in nature Polypeptides. This is generally the sequence of SEQ ID NO: 2 or 6. Has significant identity to the protein having the sequence, and its sequence Include proteins that share a specific activity, eg, antigenicity or immunogenicity, and Preferred embodiments include most of the full length disclosed sequences. The full length sequence is Although typically preferred, truncated forms are also useful, as well as found in natural sources. La The selected gene or protein is typically most desired. Similar concept but different IL-BKW protein, especially in various warm-blooded animals, such as mammals and birds Applies to proteins found. These descriptions generally refer to all IL-BKW Specific mouse implementations that are meant to include proteins but are specifically described It is not limited to this.   IL-BKW mutagenesis is also performed by making amino acid insertions or deletions. Can be Substitutions, deletions, insertions, or any combination reaches the final construct Can be generated for Insertions include amino or carboxy terminal fusions. La Random mutagenesis can be performed at the target codon, and the expressed mutant is then It can be screened for the desired activity. For DNA with a known sequence Methods for making substitution mutations at certain sites are well known in the art and include, for example, For example, by M13 primer mutagenesis or polymerase chain reaction (PCR) techniques. An example See, for example, Sambrook et al. (1989); Ausubel et al. (1987 and addenda); and Kunkel et al. 7)Methods in Enzymol .See 154: 367-382.   The present invention also relates to recombinant proteins using segments from these proteins. Proteins, eg, heterologous fusion proteins. Heterologous fusion proteins Is a fusion of proteins or segments that do not fuse normally in the same manner. Kind A similar concept applies to heterologous nucleic acid sequences.   In addition, new constructs incorporate similar functional domains from other proteins. Can be created from matching. For example, target binding or other segments Can be "swapped" between new fusion polypeptides or fragments. For example , Cunningham et al. (1989)Science 243: 1330-1336; and O'Dowd et al. (1988)J . Bi ol .Chem. 263: 15985-15992.   Beaucage and Carruthers (1981)Tetra . Letts.22: 1859-1862 The phosphoramidite method yields a suitable synthetic DNA fragment. Double-stranded hula Often synthesize a complementary strand and combine the strands together under appropriate conditions. DNA Polymerase by Neal or with appropriate primer sequences Either by adding the complementary strand using a polymerase (eg, PCR technique). Can be IV. Functional variant   Blocking of the physiological response to IL-BKW is triggered by Can result from competitive inhibition of the binding of the antibody. Preliminary results indicate that IL-BKW is an IL-10 receptor Does not bind similarly to the described subunits. IL-BKW You Gonists are expected to have the opposite effect as IL-BKW.   The in vitro assays of the invention often involve the isolation of isolated proteins, Soluble fragment containing receptor binding segment of protein, or solid phase Use the fragment bound to the substrate. These assays also provide binding seg Mutations and modifications, or cytokine mutations and modifications, e.g., IL-BKW Allows a diagnostic decision of any effect of the log.   The invention also relates to, for example, neutralizing antibodies to cytokines, or receptor binding. If the combined fragment competes with the test compound, a competitive drug screening assay Intended for use.   A “derivative” of an IL-BKW antigen is an amino acid sequence variant from a naturally occurring form, Includes glycosylation variants and covalent or aggregated conjugates with other chemical moieties. The covalent derivative can be, for example, IL-BKW amino acid side chain or N- Or by linking a functional group to a group found at the C-terminus. For example, Lund  blad and Noyes (1988)Chemical Reagents for Protein Modification, 1-2 Vol., CRC Press, Inc., Boca Raton, FL; Hugli (ed.) (1989)Techniques in Prot ein Chemistry , Academic Press, San Diego, CA; and Wong (1991)Chemistr y of Protein Conjugation and Cross Linking , CRC Press, Boca Raton, FL See also.   In particular, glycosylation modifications are included, for example, during their synthesis and processing. , Or in a further processing step, the glycosylation pattern of the polypeptide Created by changing. For example, Elbein (1987)Ann . Rev. Biochem . See 56: 497-534. Phosphorylated amino acid residues, for example, phosphotyrosine, With other minor modifications, including phosphoserine or phosphothreonine Peptide types having the same primary amino acid sequence are also included.   Also provides fusion polypeptides between IL-BKW and other homologous or heterologous proteins Is done. Many cytokine receptors or other surface proteins are -For example, a homodimeric entity, and the repeat construct It may have various advantages, including reduced susceptibility to dissection. A typical example is A segment of a protein of a porter polypeptide (eg, luciferase) Or a fusion with a domain (eg, a receptor binding segment) Thus, the presence or location of the fused ligand can be readily determined. For example, Dull et al. See U.S. Patent No. 4,859,609. Other gene fusion partners include bacterial β- Galactosidase, trpE, protein A, β-lactamase, α-amylase, Like the FLAG sequence of rucol dehydrogenase, yeast alpha mating factor, and His6 sequence Including a detection or purification tag. For example, Godowski et al. (1988)Science 241: 812-81 See 6.   Fusion peptides are typically prepared by recombinant nucleic acid methods or by synthetic polypeptides. Created either by the tide method. Techniques for nucleic acid manipulation and expression Are generally described, for example, in Sambrook et al. (1989).Molecular Cloning: A Laboratory M anual (2nd edition), Volume 1-3, Cold Spring Harbor Laboratory; and Ausubel et al. 993)Current Protocols in Molecular Biology, Greene and Wiley, NY Is done. Techniques for polypeptide synthesis are described, for example, in Merrifield (1963)J . Amer. Chem. Soc. 85: 2149-2156; Merrifield (1986)Science 232: 341-347; Atherton et al. (1989)Solid Phase Peptide Synthesis: A Practical Approach, IR L Press, Oxford; and Grant (1992)Synthetic Peptides: A User's Guide, W .H. Described in Freeman, NY. Refolding method is applicable to synthetic proteins Can be   The invention also relates to IL-BKW proteins other than alterations in amino acid sequence or glycosylation. The use of derivatives of proteins is contemplated. Such a derivative may be a chemical moiety or a tan. It may include covalent or aggregate binding with the protein carrier. Covalent or aggregated derivatives Can be used as an immunogen, as a reagent in an immunoassay, or as a binding partner. For example, in purification methods such as for affinity purification of other antigens. It is for. IL-BKW is applied to solid supports such as cyanogen bromide activated SEPHAROSE. Both By binding, it can be immobilized by methods well known in the art, or For use in assaying or purifying the body or another binding composition. Adsorbed on polyolefin surfaces with or without taraldehyde crosslinking obtain. IL-BKW protein can also be used, for example, for use in diagnostic assays. It can be labeled with a detectable group. Purification of IL-BKW depends on the immobilized antibody or complementary binding. It can be performed by a binding partner, for example, the binding moiety of the receptor.   The solubilized IL-BKW or fragment of the invention can be used to bind binding specific antisera or Can be used as an immunogen for the production of antibodies. Purified antigen is monochrome Can be used to screen for local antibodies or antigen-binding fragments. An antigen-binding fragment of a natural antibody, e.g., Fab, Fab ', F (ab)_TwoIncluding I do. Purified IL-BKW antigen also responds to the presence of elevated levels of cytokines. Can be used as a reagent to detect naturally occurring antibodies, It may be a diagnosis of an abnormal, physiological condition or medical condition. The present invention relates to SEQ ID NO: 1. Or the amino acid sequence encoded by the nucleotide sequence shown in 5 The raised antibodies, or fragments of proteins containing them, are contemplated. In particular The present invention relates to specific domains (eg, helix A, B, C, or D). Antibodies with or raised against binding affinity are contemplated.   The present invention contemplates the isolation of more closely related species variants. Southern and No Southern blot analysis confirms that similar gene entities are present in other mammals I do. Presumably, IL-BKW is a species variant, eg, rodent, lagomorph (lagomo (rph), carnivores, artiodactyls, artiodactyls, and primates.   The present invention also presents both distinct and similar in structure, expression, and function Means for isolating groups of related antigens of interest. Many physiological effects of molecules Further explanation is given by the isolation and characterization of further different species or polymorphic variants thereof. Greatly promoted. In particular, the present invention relates to additional cognate gene expression in various species. Provide a probe useful for identifying a body.   Isolated genes are expressed in cells lacking IL-BKW expression (eg, the corresponding protein Species types or cells lacking and exhibiting negative background activity ) Can be transformed. This is compared to untransformed control cells. Should be able to analyze the function of IL-BKW.   Important structural elements that lead to various physiological functions mediated by these antigens Incision of the element is modern, especially when comparing members of related classes. Is possible using standard techniques of molecular biology. For example, Cunningham et al. (1989 )Science 243: 1339-1336, a homolog scanning mutagenesis technique; And O'Dowd et al. (1988)J . Biol. Chem. 263: 15985-15992; and Lechleiter et al. (19 90)EMBO J .See the approach used at 9: 4381-4390.   Intracellular functions probably involve receptor signaling. But tampa Internalization of proteins can occur under certain circumstances, and the intracellular components interact with cytokines. An interaction between them can occur. Specific seg of interaction between interacting components and IL-BKW Mention may be by mutagenesis or direct biochemical means, such as cross-linking or affinity. Method. Structural analysis by crystallography or other physical methods It is also applicable. Further research into the mechanisms of signal transduction is By the tea method or by genetic means, for example, the complement analysis of the mutant , Including the study of bound components that may be isolable.   Further studies of IL-BKW expression and regulation will be performed. Control combined with antigen Elements may be discriminating physiological, developmental, tissue specific, or other expression patterns Should be shown. A gene region, eg, upstream or downstream of a regulatory element Is interesting.   Structural studies of IL-BKW antigens have led to the design of new antigens, especially agonists or Deriving analogs that exhibit antagonist properties. This produces the desired spectrum of activity To isolate the indicated antigen, it can be combined with the screening methods described above. V. antibody   Antibodies are directed against various epitopes of the IL-BKW protein, including their naturally occurring forms. Species, polymorphisms, or allelic variants, and their recombinant forms, both (Including fragments of). In addition, antibodies can be natural or modified. Can be raised against IL-BKW in either active or inactive form, including You. Anti-idiotype antibodies are also contemplated.   For a given fragment of an antigen, including binding fragments and single-chain forms Antibodies are used to immunize animals with conjugates of immunogenic proteins and fragments. Thus it can be triggered. Monoclonal antibodies are prepared from cells secreting the desired antibody. Made. These antibodies are screened for binding to normal or defective IL-BKW. Agonis, which can be treated or mediated, for example, by receptors Or antagonist activity. Antibodies include, for example, By sterically blocking the binding to the receptor, Can be antagonistic. These monoclonal antibodies usually have at least About 1 mM K_D, More usually at least about 300 μM, typically at least about 100 μM , More typically at least about 30 μM, preferably at least about 10 μM, and more More preferably, the binding is at least about 3 μM or more.   The antibodies of the present invention may also be useful for diagnostic applications. As a capture or non-neutralizing antibody Thus, they are sought for their ability to bind antigen without inhibiting binding to the receptor. Can be cleaned. As neutralizing antibodies, they are useful in competitive binding assays. Can get. They also detect or determine IL-BKW protein or its receptor. Useful for weighing. For example, Chan (ed.) (1987)Immunology: A Practical G uide , Academic Press, Orlando, FLA; Price and Newman (eds.) (1991)Princ iples and Practice of Immunoassay , Stockton Press, N.Y .; and Ngo (eds.) (1988)Nonisotopic ImmunoassaySee, Plenum Press, N.Y. Cross sucking Deposition or other tests may be based on different types of specificity, e.g., unique or shared species specificity. Identify antibodies that display the spectrum.   In addition, the antibodies (including antigen-binding fragments) of the present invention bind to and bind to antigen. Inhibit functional binding, e.g., to a receptor that can elicit a biological response Can be potent antagonists. These are also useful as non-neutralizing antibodies And if the antibody binds to the antigen, e.g., expresses the antigen on its surface Can be coupled to the toxin or radionuclide so that the cells are killed. In addition, these antibodies can be either directly or indirectly by means of a linker. Can bind to drugs or other therapeutic agents, and provide drug targeting I can do it.   Antigen fragments are fused or covalently bound to be used as immunogens. Can be conjugated to other substances, especially polypeptides. Antigen and And fragments thereof can be used with various immunogens, such as keyhole limpet hemonia Fused or shared with nin, bovine serum albumin, tetanus toxoid, etc. Can be combined. For instructions on how to prepare polyclonal antisera, seeMicrobio logy , Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962)Specificity of Serological Reactions , Dover Publications, New York; Will iams et al. (1967)Methods in Immunology and Immunochemistry, Volume 1, Academic Press, New York; and Harlow and Lane (1988)Antibodies: A Laboratory Manual See, CSH Press, NY.   In some cases, various mammals such as mice, rodents, primates, humans, etc. It is desirable to prepare a monoclonal antibody from a host. Such monochrome For a description of techniques for preparing internal antibodies, see, for example, Stites et al.Basic and Clinical Immunology (4th edition), Lange Medical Publications, Los Altos, CA, And references cited therein; Harlow and Lane (1988).Antibodies: A Lab oratory Manual , CSH Press; Goding (1986)Monoclonal Antibodies: Principle s and Practice (Second Edition), Academic Press, New York; and especially K ohler and Milstein (1975)Nature 256: 495-497 contains a monoclonal antibody One method of generating is discussed.   Other suitable techniques include in vitro exposure of lymphocytes to an antigenic polypeptide, or Or for selection of antibody libraries on phage or similar vectors In vitro exposure of lymphocytes. Huse et al. (1989) "Generation of a Lar ge Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambd a "Science 246: 1275-1281; and Ward et al. (1989).Nature See 341: 544-546 thing. The polypeptides and antibodies of the present invention include modified chimeric or humanized antibodies. It can be used with or without modification. Frequently, polypeptides and antibodies Binds a substance that provides a detectable signal, either covalently or non-covalently. By doing so. A wide variety of labels and conjugation techniques are known. And widely reported in both scientific and patent literature. Appropriate signs include Shooting Sex nuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles Including children. Patents teaching the use of such labels include U.S. Pat. No. 7, No. 3,850,752; No. 3,939,350; No. 3,996,345; No. 4,277,437; No. 4,275 No. 149; and No. 4,366,241. Also produces recombinant immunoglobulin No. 4,816,567; Moore et al., US Pat. No. 4,642,334; And Queen et al. (1989)Proc . Nat'I. Acad. Sci. USA 86: 10029-10033 thing.   The antibodies of the present invention may also be used for affinity chromatography in isolating proteins. Can be used for matography. A column is prepared in which the antibody is bound to a solid support. Can be For example, Wilchek et al. (1984)Meth . Enzymol.See 104: 3-55.   Antibodies raised against each IL-BKW also raised anti-idiotype antibodies. Useful for These are various immunological symptoms related to the expression of each antigen. Is useful for detecting or diagnosing. VI. Nucleic acid   The described peptide sequences and related reagents are, for example, IL-B from natural sources. It is useful for detecting, isolating, or identifying a DNA clone encoding KW. Typically, it is useful for isolating genes from mammals, and The order isolates genes from other species, eg, warm-blooded animals (birds and mammals) Applied for. Cross-hybridization is the same species, e.g., polymorphism modification. Enables isolation of IL-BKW from the body, or other species. Many different approaches are Should be available to successfully isolate a suitable nucleic acid clone.   Purified proteins or peptides of interest can be purified using standard methods, as described above. Therefore, it is useful for producing antibodies. Synthetic peptide or purified tamper Proteins are used by the immune system to produce monoclonal or polyclonal antibodies. Can be presented. For example, Coligan (1991)Current Protocols in Immunology W iley / Greene; and Harlow and Lane (1989)Antibodies: A Laboratory Man ual See Cold Spring Harbor Press.   For example, a specific binding composition may be an expression library made from a cell line that expresses IL-BKW. It can be used for screening of libraries. Screening for intracellular expression It can be performed by various staining or immunofluorescence procedures. The binding composition comprises a surface fusion tag. Can be used to affinity purify or sort cells that express the protein You.   Peptide segments are also suitable for screening libraries. It can be used to predict oligonucleotides. Genetic code, Screenini To select appropriate oligonucleotides useful as probes for Can be used. See, for example, SEQ ID NO: 1 or 5. Polymerase chain reaction In combination with the PCR technique, synthetic oligonucleotides can be Useful for selecting the correct clone. Complementary sequences can also Used as primer or antisense strand. For example, connected vectors Or coupled to poly-A complementary PCR techniques, or complementary to other peptides Note that various fragments coupled to specific DNA are particularly useful I want to be.   The invention is particularly directed to native, lacking portions that encode the untranslated 5 'portions of the described sequences. An isolated DN for encoding a corresponding biologically active IL-BKW polypeptide Intended for use with A or fragments. In addition, the present invention relates to biologically active Encodes a protein or polypeptide, and encodes a DNA sequence as described herein. Covers isolated or recombinant DNA that can hybridize under appropriate conditions You. This biologically active protein or polypeptide is an intact antigen Or fragments, and are disclosed, for example, in SEQ ID NOs: 2 or 6. Amino acid sequence, particularly the mature peptide of SEQ ID NO: 6, or the secreted sequence of SEQ ID NO: 2 Mature polypeptide. Further, the present invention relates to isolated or recombinant DNs. A, or the use of a fragment thereof, which covers secreted IL-BKW Encodes a protein with high identity. The isolated DNA contains 5 'and 3' Each regulatory sequence (eg, promoter, enhancer, Re-A addition signal).   An “isolated” nucleic acid is a nucleic acid (eg, RNA, DNA, or mixed polymer) ), Which are native sequences (eg, ribosomes, polymerase, And / or other components naturally associated with the flanking genomic sequence from the original species). Is substantially separated from. The term refers to a nucleus that has been removed from a naturally occurring environment. Acid sequences and contains recombinant or cloned DNA isolates, and chemically synthesized Includes analogs that have been synthesized or biologically synthesized by heterologous systems. Real A qualitatively pure molecule includes the isolated form of the molecule. Generally, nucleic acids are about 50 kb Less, usually less than about 30 kb, typically less than about 10 kb, and preferably less than about 6 kb Vector or fragment.   An isolated nucleic acid is generally a homogeneous composition of molecules, but in some implementations. Embodiments include minor non-uniformities. This non-uniformity typically results in the desired product. Found at polymer ends or moieties that are not important for physical function or activity.   A “recombinant” nucleic acid is defined by either a method of production or its structure. With respect to the method of production (eg, the product made by the process), the process For example, human intervention in nucleotide sequences, typically selection or production. And the use of recombinant nucleic acid technology. Or two naturally non-contiguous Can be a nucleic acid produced by purifying a sequence containing a fusion of fragments But excludes natural products (eg, naturally occurring variants) . Thus, for example, derivation using any synthetic oligonucleotide process The cells are transformed with any non-naturally occurring vector, as well as the nucleic acid containing the sequence to be Products made by the conversion are included. This is often the representative Typically, the same or conserved amino acids are introduced while introducing or removing sequence recognition sites. This is done to replace codons with encoding duplicate codons.   Alternatively, a desired combination of functions not found in commonly available natural forms The nucleic acid segment of the desired function is This is done to connect the two together. Restriction enzyme recognition sites are often Target for simple artificial manipulation, but other site-specific targets (eg, promoter, DN A replication sites, regulatory sequences, control sequences, or other useful features) Can be impregnated. A similar concept applies to recombinant (eg, fusion) polypeptides. Illustrated. In particular, duplication of the genetic code resembles fragments of these antigens. Fusion of similar polypeptides and sequences from a variety of different species or polymer variants Synthetic nucleic acids encoding the compounds are included.   A "fragment" meaningful in the context of nucleic acids, is at least about 17 nucleotides, Generally at least about 22 nucleotides, usually at least about 29 nucleotides More often, at least about 35 nucleotides, typically at least about 41 nucleotides. Otide, usually at least about 47 nucleotides, preferably at least about 55 nuclei A continuous segment of leotide, and in a particularly preferred embodiment bear at least Is about 60 or more nucleotides (eg, 67, 73, 81, 89, 95, etc.).   DNA encoding the IL-BKW protein encodes a related or similar protein. Genes, mRNA and cDNA species to be loaded, as well as homologous proteins from different species. It is particularly useful for identifying the encoding DNA. Primates, rodents, dogs, cats There are probably homologs in other species, including, and birds. Various IL-BKW tongues The proteins should be homogenous and are included in the present invention. However, for antigen Even those with more distant evolutionary relationships are sufficiently homologous Can be readily isolated under appropriate conditions using these sequences. Primate IL-BKW Proteins are of particular interest.   For example, a recombinant clone derived from a genomic sequence, including introns, For transgenic research, including transgenic cells and organisms It is useful for gene therapy. For example, Goodnow (1992) "Transgenic Animals" R oitt (edit)Encyclopedia of Immunology, Academic Press, San Diego, 1502-15 04; Travis (1992)Science 256: 1392-1394; Kuhn et al. (1991)Science 254: 707 -710; Capecchi (1989)Science 244: 1288; Robertson (1987) (eds.)Teratocarci nomas and Embryonic Stem Cells: A Practical Approach , IRL Press, Oxford; And Rosenberg (1992)J . Clinical Oncology See 10: 180-199.   In the context of nucleic acid sequence comparisons, substantial homology, eg, identity, is a measure of the magnitude of a comparison. If the segment or any of its complements are optimally aligned At least about 50% nucleotides, generally at least about 58%, usually At least about 65%, often at least about 71%, typically at least about 77%, Usually at least about 85%, preferably at least about 95-98% or more, and In embodiments, about 99% or more of the high nucleotides may result in a proper nucleotide insertion or deletion. Loss Means the same as Alternatively, substantial homology is that the segment is selected Under typical hybridization conditions, the strand or its complement, typically For example, when hybridizing using the sequence of IL-BKW of SEQ ID NO: 1 or 5, , Exists. Typically, selective hybridization is at least about 30 nucleotides. At least about 55% identity, preferably about 25 nucleotides over the stretch of nucleotides At least about 75%, and most preferably about It occurs when there is at least about 90% identity over 20 nucleotides. Kanehi sa (1984)Nuc . Acids Res.See 12: 203-213. As described, identity ratio The length of the comparison may exceed the longer stretch, and in certain embodiments, may be less. At least about 17 nucleotides, usually at least about 28 nucleotides, typically At least about 40 nucleotides, and preferably at least about 75 to 100 or more nucleotides Over the stretch of pride.   When referring to homology in the context of hybridization, stringent conditions Is the salt, temperature, organic solvent, and other parameters, typically This is a stringent combination condition controlled by the reaction. Strike The temperature of the lingent is usually about 30 ° C. or higher, usually about 37 ° C. or higher, typically about 55 ° C. C. or higher, preferably about 70 C. or higher. Stringent salt conditions are normal Is less than about 1000 mM, usually less than about 400 mM, typically less than about 250 mM, preferably about 15 mM. Less than 0 mM, and also includes about 100, 50, or 20 mM. But the set of parameters Combination is much more important than any single parameter measure. example For example, Wetmur and Davidson (1968)J . Mol. Biol.See 31: 349-370.   IL-BKW from other mammalian species has been cloned and closely related species Can be isolated by cross-species hybridization. Homology is distantly related Species can be relatively low among species of Hybridization is a good idea. Alternatively, antibodies that show little species specificity Preparation of the preparation may be useful for expression cloning approaches. VII. IL-BKW ； Making mimics   DNA encoding IL-BKW or a fragment thereof can be obtained by chemical synthesis or cDNA library. Screening a wide variety of cell lines or tissue samples Can be obtained by screening a genomic library prepared from . For example, Okayama and Berg (1982)Mol . Cell. Biol.2: 161-170 ； Gubler And Hoffman (1983)Gene 25: 263-269; and Glover (eds.) (1984)DNA Cloning: A Practical Approach See, IRL Press, Oxford. Alternatively, as described herein The sequence provided in provides a useful PCR primer or encodes IL-BKW. Allowing the synthesis or other preparation of the appropriate gene to be administered; naturally occurring embodiments including.   This DNA, in turn, produces polyclonal or monoclonal antibodies, for example. For the synthesis of full-length IL-BKW or fragments that can be used to For the construction and expression of modified molecules; and for structural / functional studies Can be expressed in a wide variety of host cells.   As used herein, a vector is a plasmid, virus, bacterio Phage, integratable DNA fragments, and DNA fragments into the host genome Includes other vehicles that allow for the integration of For example, Pouwels et al. (1985 and Addendum)Cloning Vectors: A Laboratory Manual, Elsevier, N.Y .; and Rodrigu ez et al. (1988) (eds.)Vectors: A Survey of Molecular Cloning Vectors and The ir Uses See, Buttersworth, Boston, MA.   For the purposes of the present invention, DNA sequences are those which are functionally related to each other, Operably linked. For example, the DNA for a presequence or secretory leader is Is expressed as a preprotein or directs the polypeptide to the cell membrane Operably linked to the polypeptide if it is involved in Is tied. The promoter should be able to control if it controls the transcription of the polypeptide. Ribosome binding site, which allows translation. Is operably linked to the coding sequence if it is positioned to Normally operational Concatenated means continuous and within the reading frame, but Certain genetic elements, such as lesser genes, are linked so that they are not contiguous. But still binds to the operator sequence and then controls expression. For example, Ro driguez et al., Chapter 10, pp. 205-236; Balbas and Bolivar (1990).Methods in Enzymo lo gy  185: 14-37; and Ausubel et al. (1993)Current Protocols in Molecular Biol ogy See Greene and Wiley, NY.   Representative examples of suitable expression vectors include pCDNA1; pCD (Okayama et al. (1985)Mol . Cell Biol . 5: 1136-1142); pMClneo Poly-A (Thomas et al. (198 7)Cell 51: 503-512); and baki such as pAC 373 or pAC 610 Urovirus vectors. For example, Miller (1988)Ann . Rev. Microb iol . 42: 177-199.   IL-BKW polypep in a system that provides a specific or defined glycosylation pattern It is often desirable to express the tide. For example, Luckow and Summers (1 988)Bio / Technology 6: 47-55; and Kaufman (1990)Meth . Enzymol.185: 487 See -511.   IL-BKW or its fragment is bound to the phosphatidylinos Can be manipulated to be Toll (PI), but phosphatidylinositol-cleaving enzymes Treatment with an element (eg, phosphatidylinositol phospholipase-C) Can be removed from the membrane. This releases the antigen in a biologically active form and Allows purification by standard procedures of protein chemistry. For example, Low (1989)Biochim . Biophys. Acta  988: 427-454; Tse et al. (1985)Science 230: 1003-1008; and Br unner et al. (1991)J . Cell Biol.See 144: 1275-1283.   Since IL-BKW is currently characterized, fragments or their derivatives It can be prepared by conventional processes for synthesizing peptides. These include St ewart and Young (1984)Solid Phase Peptide Syntehsis, Pierce Chemical C o., Rockford, IL; Bodanszky and Bodanszky (1984)The Practice of Peptid e Synthesis , Springer-Verlag, NeW York; Bodanszky (1984)The Principleso f Peptide Synthesis , Springer-Verlag, NeW York; and Villafranca (eds.) (1991)Techniques in Protein Chemistry II, Academic Press, San Diego, C Includes processes as described in a. VIII. use   The present invention provides, for example, an IL-BKW-mediated condition herein otherwise or for diagnosis. Provide reagents for use in diagnostic applications, as described below in the description of the kit .   The present invention also provides reagents that have significant therapeutic potential. IL-BKW (naturally Native or recombinant), fragments thereof, and antibodies thereto are expressed in IL-B Along with compounds identified as having a binding affinity for KW, It should be useful in treating conditions associated with normal physiology or development. In particular, phosphorus Modulation of the physiology of pa-like cells can be controlled by a suitable therapeutic treatment using the compositions provided herein. Is achieved by placing For example, abnormal expression or abnormal signaling by IL-BKW -Related diseases or disorders are promising for agonists or antagonists Target. New cytokines are used in hematopoietic cells (eg, lymphoid Cell) must play a role in the regulation or development of immunological responses (eg, For example, inflammation and / or autoimmune disorders).   In particular, cytokines, in various situations, cytokine synthesis by cells, It should mediate proliferation and the like. Mutein variants of the naturally occurring form of IL-BKW Or antagonists of IL-BKW, such as blocking antibodies, Situations such as inflammatory or autoimmune responses (rheumatoid arthritis, systemic lupus erythema) Todes (SLE), including Hashimoto's autoimmune thyroiditis)), and acute and chronic Selective and potent for blocking inflammatory inflammatory responses (eg, inflammatory bowel disease) A method can be provided. Samter et al. (Ed.)Immunological Diseases Volumes 1 and 2, Littl See also e, Brown and Co. Block recombinant antibodies or by blocking antibodies By the naturally occurring secreted form of IL-BKW, which can be produced in large quantities Cytokine release is made available in the present invention, for example, in transplant rejection situations It should be adjustable depending on the reagent used.   In addition, certain combination compositions may be used, for example, with other modulators of inflammation. Also useful. Such other molecules are steroids, other types of IL-10 (cells Species variants, or viral IL-10, including, for example, EBV or EHV), and All of their respective antagonists may be included.   A variety of abnormal symptoms can produce IL-BKW mRNA by Northern blot analysis. Are known for each of the cell types indicated. Berkow (ed.)The Merck Manual o f Diagnosis and Therapy , Merck & Co., Rahway, N.J .; Thorn et al.Harrison's P rinciples of Internal Medicine McGraw-Hill, N.Y .; and Weatherall et al. Ed.)Oxford Textbook of Medicine, Oxford University Press, Oxford thing. Many other medical conditions and diseases are caused by macrophages or monocytes. And many of these include agonists or agonists provided herein. Responsive to treatment by antagonists. For example, Stites and Terr (ed. 1991)Basic and Clinical Immunology  Appleton and Lange, Norwalk, Connecticut; And Samter et al. (Eds.)Immunological Diseases See Little, Brown and Co. thing. These problems are addressed by prevention or treatment using the compositions provided herein. Should be susceptible.   IL-BKW, antagonists, antibodies, etc. are purified and then ) Can be administered. These reagents are, for example, physiologically harmless stabilizers, excipients Together with a conventional pharmaceutically acceptable carrier or diluent ( For example, in immunogenic adjuvants), additional active or inactive ingredients and therapeutic use Can be combined for These combinations are sterile filtered and administered As by lyophilization in vials or storage in stabilized aqueous preparations May be placed in a dosage form. The present invention also provides antibodies, including forms that are not complementary binding. Or the use of a binding fragment thereof.   Drug screening using IL-BKW or its fragments Binding affinity for IL-BKW function or other, including isolation of combined components Compounds having a related biological effect can be identified. Then the subsequent biological The assay indicates that the compound has an intrinsic stimulatory activity, Determine if you are a blocker or antagonist in blocking Can be used for Similarly, compounds with intrinsic stimulatory activity are signal-mediated Are agonists in that they can activate the tract and thus stimulate the activity of IL-BKW. The present invention further provides a blocking antibody against IL-BKW as an antagonist, And therapeutic use of stimulating antibodies as agonists. This approach is Should be particularly useful with other IL-BKW species variants.   In addition, IL-BKW can be used to induce leukemia or, for example, HTLV or herpes sp. It can play a role in viral transmission by ills. This is squirrel monkey herpes Induced by infection with a virus. Herpes virus is also a cytokine Encodes a homolog of IL-17 (CTLA-8). Thus, a cytokine, or Antagonists may be useful for antitumor therapy. Virus Interactions Site Cain is used for viral infection or growth processes, or oncological processes (eg, , Tumorigenic transformation and proliferative conditions) can be important as cancer or leukemia Which indicates that. For example, Thorn et al.Harrison's Principles of Internal Med icine See McGraw-Hill, N.Y.   In addition, cytokines appear to be expressed in kidney cells, and organ function For example, it can play an important role in ion exchange or blood pressure regulation. Cytokine Can also have important moisture balance functions. Cytokines are found in the kidney It appears to have some expression.   The amount of reagents required for effective treatment depends on the means of administration, the target site, the physiological condition of the patient, And many other factors, including the other drug being administered. Therefore The treatment dose should be titrated to optimize safety and efficacy. Typically, the doses used in vitro will be based on in situ administration of these reagents. Useful amounts can provide useful guidance. An effective dose of animal to treat the particular disorder Studies provide additional predictive indicators of human dose. Various considerations, for example, Gi lman et al. (eds.) (1990)Goodman and Gilman's: The Pharmacological Bases of Ther apeutlcs , Eighth Edition, Pergamon Press; andRemington's Pharmaceutical Scienc es 17th Edition (1990), Mack PubliShing Co., Easton, Penn. For example For oral, intravenous, intraperitoneal or intramuscular administration, transdermal diffusion, etc. Methods for this are discussed here and below. A pharmaceutically acceptable carrier Is, for example,Merck Index, Merck & Co., Rahway, New Jersey , Saline, buffers, and other compounds. Dosage range is appropriate With the carrier, usually less than 1 mM concentration, typically less than about 10 μM, usually about 1 μM Less than 00 mM, preferably less than about 10 pM (picomolar), and most preferably about 1 It is expected to be less than fM (femtomolar). A delayed release formulation, or Delayed release devices are often utilized for continuous or long term administration. For example, Langer (1990)Science See 249: 1527-1533.   IL-BKW, a fragment thereof, and an antibody against this or this fragment , Antagonists, and agonists are administered directly to the host to be treated. Or ovalbumin prior to its administration, depending on the size of the compound obtained or Or to bind them to a carrier protein such as serum albumin Can be done. The therapeutic formulation can be administered in a number of conventional administration formulations. Active ingredient While capable of being administered alone, it is preferred that it be present as a pharmaceutical formulation. New The formulation is typically prepared with one or more of its acceptable carriers, together with At least one active ingredient as defined in Each carrier has other components Both pharmaceutical and physiological in the sense that it is compatible with the minute and does not hurt the patient Should be acceptable at. Formulations include oral, rectal, nasal, topical, Is a formulation suitable for parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration Is included. The formulations may conveniently be presented in unit dosage form and are well-known in the pharmaceutical arts. It can be prepared by any known method. For example, Gilman et al. (Eds.) (1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics , 8th edition, Pergamon Press; andRemington's Pharmaceutical Sciences, 17th edition (1990), Mack Pub lishing Co., Easton, Penn .; Avis et al. (eds.) (1993).Pharmaceutical Dosage Form s: Parenteral Medications , Dekker, New York; Lieberman et al. (Eds.) (1990).Pharm aceutical Dosage Forms: Tablets , Dekker, New York; and Lieberman et al. (Eds. ) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York See also. The treatment of the present invention may be directed to other drugs, such as other types of IL-10 or other drugs. Can be combined with or used in conjunction with the respective antagonists of You.   Both naturally occurring and recombinant forms of the IL-BKW of the present invention Kits and assay methods that can screen compounds for binding activity of Particularly useful. Some methods for automating assays are tens of thousands of compounds It was recently developed to allow for the screening of objects. For example, Fo dor et al. (1991)Science See 251: 767-773. This document is synthesized on a solid substrate Described are means for testing binding affinity with a number of defined polymers. The development of a suitable assay requires large quantities of purified as provided by the present invention. The availability of soluble IL-BKW can be very easy.   Other methods determine key residues in IL-BKW-IL-BKW receptor interactions Can be used to Mutation analysis is important for interactions and / or signaling. Can be performed to determine key specific residues (eg, Somoza et al. (1993)J . E xptl. Med. 178: 549-558). However, the remains in the A and D helices The groups appear to be most important for receptor interaction.   For example, once an antigen is structurally defined, for example, by tertiary structure data, Antagonists can usually be found. Testing for potential interacting analogs Present by developing highly automated assay methods using purified IL-BKW It is possible. In particular, new agonists and antagonists are described herein. Discovered by using the screening techniques described. A range of IL-BKW Compounds found to have tailored binding affinity for the molecule, for example, Compounds that can act as antagonists for species variants of IL-BKW are particularly heavy It is important.   One method of drug screening is stable with recombinant DNA molecules expressing IL-BKW Utilizing eukaryotic or prokaryotic host cells to be transformed. What are other molecules Cells that separate and express IL-BKW can be isolated. Such cells are viable In fixed or immobilized form for standard binding partner binding assays. Can be used. Parce et al. (1989)Science 246: 243-247; and 0wicki et al. (1990)Pr oc . Nat'l Acad. Sci. USA  See also 87: 4007-4011. These documents refer to cells A sensitive method for detecting a response is described.   Another technique for drug screening has appropriate binding affinity for IL-BKW Includes an approach that provides high-throughput screening for selected compounds And Geysen, published September 13, 1984, in European Patent Application 84/03564. be written. First, a number of different small peptide test compounds are deposited on a solid substrate, for example, Synthesized on plastic pins or some other suitable surface. Fodor et al. ( 1991). All pins were then converted to solubilized unpurified IL- React with BKW or solubilized purified IL-BKW and wash. Next step The step involves detecting bound IL-BKW.   Theoretical drug design may also involve molecules of IL-BKW and other effectors or analogs. It can be based on structural studies of shapes. Effectors mediate other functions in response to binding Other proteins that normally interact with IL-BKW, such as It can be a receptor. Which sites interact with other specific proteins One means for determining is physical structure determination, such as X-ray crystallography or 2 Dimensional NMR technology. These have been modeled, for example, against cellular IL-10. Provide guidance on which amino acid residues form a molecular contact region. You. For a detailed description of protein structure determination, see, for example, Blundell and John son (1976)Protein CrystallographySee, Academic Press, New York . IX. kit   The present invention also relates to the presence of various diagnostic kits and additional IL-BKW or binding partners. IL-BKW protein, a fragment thereof, and a peptide in a method for detecting the presence of The use of tides and their fusion products is contemplated. Typically, the kit is Recognized IL-BKW peptide or gene segment or one or the other Compartment containing any of the following reagents (eg, IL-BKW fragments or antibodies) Have a comment.   Kits for determining the binding affinity of a test compound for IL-BKW are typically Is a test compound; a labeled compound (eg, a known binding affinity for IL-BKW) A binding partner or antibody with a); a source of IL-BKW (naturally occurring or Is recombinant); and free labeled modification, such as a solid phase to immobilize the molecule Means for separating the bound compound from the compound. Once the compound is screenini When labeled, a compound with the appropriate binding affinity for the antigen is IL-BKW signal Whether it acts as an agonist or antagonist on the ring pathway To determine, a suitable biological assay, as is well known in the art, Can be evaluated. The availability of recombinant IL-BKW polypeptides is also Provides a well-defined standard for calibrating the Say.   For example, a preferred kit for determining the concentration of IL-BKW in a sample is representative Include a labeled compound having a known binding affinity for the antigen (eg, binding Partner or antibody), source of cytokine (naturally occurring or recombinant) ) And means for separating bound compounds from free labeled compounds (Eg, a solid phase for immobilizing IL-BKW). Containing reagents and instructions A compartment is usually provided.   Antibodies specific to IL-BKW or fragments (including antigen-binding fragments) To detect the presence of elevated levels of IL-BKW and / or fragments thereof This is useful for diagnostic applications. Such diagnostic assays include lysates, live cells, and solid Defined cells, immunofluorescence, cell cultures, body fluids, and It may include the detection of antigens associated with the antigen. Diagnostic assays are homogeneous (free reagent and No separation step between antigen binding partner complex) or heterogeneous (with separation step) Can be Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Enzyme immunoassay (EIA), enzyme multiplied immunoassay technology (EMIT) There are various commercially available assays, such as the Substrate Labeled Fluorescent Immunoassay (SLFIA) Exists. For example, Van Vunakis et al. (1980)Meth Enzymol. 70: 1-525; Harlow And Lane (1980)Antibodies: A Laboratory Manual, CSH Press, NY; and Col igan et al. (eds.) (1993)Current Protocols in Immunology, Greene and Wiley, NY checking ...   Anti-idiotypic antibodies are characteristic of antibodies to IL-BKW in various abnormal conditions. As such, they may have a similar use to diagnose their presence. For example, IL -BKW overproduction is particularly important for proliferative disorders such as cancer or abnormal activation or differentiation. Cellular conditions produce a variety of immunological responses that may be characteristic of abnormal physiological conditions obtain.   Frequently, the reagents for a diagnostic assay are designed to optimize the sensitivity of the assay, Supplied in kit. For the present invention, assays, protocols, and labeling Depending on the nature, either labeled or unlabeled antibodies Alternatively, a binding partner or labeled IL-BKW is provided. This is usually Other substances such as buffers, stabilizers, substances necessary for signal generation such as enzyme substrates, etc. With additives. Preferably, the kit also contains the appropriate use and contents after use Include instructions for disposal. Typically, kits contain only useful reagents. It has a compartment for Desirably, the reagent is suitable for performing the assay. Dried lyophilized, if it can be reconstituted in an aqueous medium providing a sharp concentration of reagent Provided as a powder.   Many of the above components of drug screening and diagnostic assays are unmodified Or can be modified in various ways. For example, the label is detectable Or non-covalent attachment of moieties that provide a direct or indirect signal Can be achieved by: In any of these assays, the binding -Toner, test compound, IL-BKW, or antibody against Can be labeled with any of the targets. Possibilities for direct labeling include: Include:¹²⁵Radiolabels such as I, peroxidase and alkaline phosphata Enzymes (US Pat. No. 3,645,090) and fluorescence intensity, wavelength shift, Or a fluorescent label capable of monitoring changes in fluorescence polarization (US Pat. No. 3,940,475). The potential for indirect targets is the biotinylation of one component, followed by the targets described above. Includes binding to avidin attached to one of the groups.   Also, IL-BKW bound from free IL-BKW or from free test compound There are many ways to separate the combined test compounds. IL-BKW can be used in various matrices It can be fixed and then washed. Suitable matrices, such as ELISA plates Plastics, filters, and beads. For example, Coligan et al. ( (Ed.) (1993)Current Protocols in Immunology, Volume 1, Chapter 2, Greene and Wile See y, NY. Other suitable separation techniques are described in Rattle et al. (1984)Clin . Chem. 30: Fluorescein antibody magnetizable particle method according to 1457-1461, and U.S. Pat. Including but not limited to double antibody magnetic particle separation as described in US Pat.   Methods for linking proteins or fragments thereof to various labels are widely available in the literature. Well-reported and does not require detailed discussion herein. Lots of technology Is either carbodiimide or active ester to form a peptide bond Use of activated carboxyl groups, use of chloroacetyl for binding With activated olefins such as activated halogens or maleimides Formation of thioethers by reaction of groups. The fusion protein also Find use in these applications.   Another diagnostic aspect of the invention relates to oligonucleotides or polynucleotides derived from the sequence of IL-BKW. Includes the use of oligonucleotide sequences. These sequences may be in an abnormal state (eg, IL-BKW messages in samples from patients suspected of having inflammation or autoimmunity) Can be used as a probe to detect the level of. Cytokines are active May be markers or mediators for And when in a prophylactic manner, for example, further treatment is required before significant progress It can be useful to determine the number of activated cells to determine what to obtain. RNA Preparation of both DNA and DNA nucleotide sequences, labeling of sequences, and sequence preferred Size is well explained and discussed in the literature. For example, Langer-Safer et al. ( 1982)Proc . Nat'l. Acad. Sci.79: 4381-4385; Caskey (1987)Science 236: 96 2-967; and Wilchek et al. (1988)Anal . Biochem.See 171: 1-32.   Diagnostic kits that also test for qualitative or quantitative expression of other molecules are also contemplated. You. Diagnosis or prognosis depends on the combination of several indicators used as markers. Can exist. Thus, the kit may test for a combination of markers. For example, Viallet et al. (1989)Progress in Growth FactorRes .See 1: 89-97 When. Other kits can be used to evaluate other cell subsets. X. Isolating the IL-BKW receptor   Once the ligand for the specific ligand-receptor interaction is isolated, There are methods for isolating Gearing et al. (1989)EMBO J .See 8: 3667-3676 Teru. For example, IL-BKW cytokines without interrupting their binding to the receptor The means for labeling can be determined. For example, the affinity label can Can be fused to either the N-terminus or the carboxyl terminus, but based on IL-10 , The amino terminus seems to work better. Such a tag may be a FLAG epitope It can be a tag or, for example, an Ig or Fc domain. Expression libraries For example, cell sorting, or detecting subpopulations expressing such binding components. Other screens to screen for specific binding of cytokines Can be adjusted. For example, Ho et al. (1993)Proc . Nat'l. Acad. Sci. USA 90: 11267 -11271; and Liu et al. (1994)J . Immunol.See 152: 1821-29. Or , A panning method may be used. Seed and Aruffo (1987)Proc . Nat ' l. Acad. Sci. USA  84: 3365-3369.   Applying protein cross-linking technology with label, IL-BKW cytokine binding partner Can be isolated. This includes, for example, cytokines in a ligand-receptor-like manner Enables the identification of proteins that interact specifically with.   Early experiments determine whether known IL-10R is involved in the response to IL-BKW Done in order to. Functional IL-10 receptor complex, IL-BKW receptor complex (Either specific receptor subunit or co-receptor subunit It is entirely possible that many or all of the components can be shared.   Many modifications and variations of this invention will be apparent to those skilled in the art, It can be done without departing from God and scope. Certain fruits described herein The embodiments are provided for illustrative purposes only, and the present invention is intended to cover such claims. Along with the full scope equivalent to that given, by the terms of the appended claims. Only should be limited.                                  Example General method   Some of the standard methods are described, for example, in Maniatis et al. (1982)Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor Pre ss; Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual(2nd edition), 1st -3, CSH Press, NY; Ausubel et al.Biology, Greene Publishing Associates, B rooklyn, NY; or Ausubel et al. (1987 and addenda)Current Protocols in Molec ular biology , Greene and Wiley, New York; Innls et al. (Eds.) (1990).PCR Protoco ls: A Gulde to Methods and Applications , Academic Press, N.Y. Referenced. Methods for protein purification include ammonium sulfate precipitation, Includes methods such as chromatography, electrophoresis, centrifugation, crystallization, etc. It is. See, for example, Ausubel et al. (1987 and periodic addendum); Deutscher (1990) "Guide t o Protein Purification "Methods in EnzymologyVolume 182 and this series Other volumes; and manufacturer's literature in the use of protein purification products, such as See, for example, Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA. . Combinations with recombinant techniques can be used to, for example, FLAG sequences or Allows fusion to an equivalent that can be fused via a protease-removable sequence . For example, Hochuli (1989)Chemische Industrie 12: 69-70; Hochuli (1990) “P urification of Recombinant Proteins with Metal Chelate Absorbent '' Setlow (Edit)Genetic Engineering , Principle and Methods 12: 87-98, Plenum Press , N.Y .; and Crowe et al. (1992)QIAexpress: The High Level Expression & Prot e in Purification System  See QUIAGEN, Inc., Chatsworth, CA. cell Culture techniques are described in Doyle et al. (Eds.) (1994)Cell and Tissue Culture: Laboratory Proced ures , John Wiley and Sons, NY.   Standard immunological techniques are described, for example, in Hertzenberg et al. (Ed. 1996)Weir's Handbook of Experimental Immunology Volume 1-4, Blackwell Science; Coligan (1991)Cu rrent Protocols in Immunology  Wiley / Greene, NY; andMethods in Enzymol ogy Volumes 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163 be written.   FACS analysis was performed by Melamed et al. (1990).Flow Cytometry and Sorting Wiley-Liss, In c., New York, NY; Shapiro (1988)Practical Flow Cytometry Liss, New York , NY; and Robinson et al. (1993).Handbook of Flow Cytometry Methods WIley-L iss, New York, NY. Fluorescent labeling of appropriate reagents can be performed using standard methods. I went. Example 1: Cloning of human and mouse IL-BKW   PBMC, for example, Coligan et al.Current Protocols in Immunology Greene / Wiley Prepared from healthy human blood donors by conventional Ficoll gradient as described in. Cells, preferably monocytes, from this preparation are stimulated, for example, with PHA. these Using RNA from activated monocytes, cDNA based on sequence information for mda7 Is isolated.   PCR products are cloned using the TA Cloning Kit (Invitrogen). You. The resulting cDNA plasmid is converted to an automated sequencer (Applied Biosystems) Sequence from both ends with.   Clones for mIL-BKW were cloned into NK1.1 +, CD4 + activated mouse thymocyte cDNA Bully (A. Zlotnik, DNAX Research Institute, Palo Alto, CA) Was. Sequencing of this clone showed high levels of hIL-BKW and mIL-10 and hIL-10. Shows amino acid sequence identity. In particular, a high degree of identity between the four cytokines , Found in the D helix domain involved in receptor binding. Example 2: Mammalian IL-BKW cell expression   Study distribution on similar cell types due to sequence similarity to human IL-10 I do. Probes specific for the cDNA encoding primate IL-BKW, for example, random Label by priming.   IL-BKW / ak155 is strongly transcribed in various T cell tissues and clones.   Southern analysis: DNA (5 μg) from the primary amplified cDNA library was Cleavage with restriction enzyme to release insert, run on 1% agarose gel and Transferred to membrane (Schleicher and Schuell, Keene, NH).   Use multi-tissue Northern blots to determine the size and tissue distribution of hIL-BKW mRNA. Were determined. Many mRNA transcripts varying in size from 1.0 kb to 4.4 kb, mostly Of the tissues. The stringency of hybridization and Based on the wash conditions of the blot, these transcripts were alternatively spliced. Most likely to show mRNA, not a distinct family member. Cancer cell line A similar northern blot using (Clontech) was 2.2 colorectal carcinoma SW480. Strongly hybridizes to the kb mRNA transcript, but this transcript is It is clear that there is no deviation.   HI in monocytes / macrophages, dendritic cells, T cells, B cells, and NK cells The expression profile of L-BKW was analyzed by cDNA library Southern blot hybridization. Analyzed by the The hIL-BKW probe was used after digestion with NotI and SalI. Hybridize to μg of each cDNA library and pSport vector (Gibco-BRL) Released the cDNA insert. Among T cells, B cells, and NK cells, hIL-BKW Expression is restricted by the activated Th0 clone (Mot72) and the activated Th1 clone (HY06). Limited. Much higher levels of hIL-BKW expression were found in LPS, IFN-γ, and anti-IL-10 Found on elutriated monocytes activated by antibodies. In contrast, the present inventors Libraries made from these same monocytes treated with IL-10 instead of anti-IL-10 , A clear decrease in the level of IL-BKW expression was observed, This suggested that the expression of IL-BKW was strongly regulated by IL-10.   Samples for mRNA isolation for analysis of mIL-BKW include: resting mouse line Fibroblast cell line (C200); Braf: ER (Braf fusion to estrogen receptor) Transfected cells, control (C201); TH1-polarized T cells (Mel 14 bright, CD4 + cells from spleen, polarized for 7 days with IFN-γ and anti-IL-4; T20 0); TH2 polarized T cells (Mel14 bright, polarized for 7 days with IL-4 and anti-IFN-γ) CD4 + cells from spleen; T201); highly TH1-polarized T cells (Openshaw et al. (1995)J . Exp. Med.182: 1357-1367; pooled for 2, 6, 16 hours T cells activated by anti-CD3; T202); highly TH2-polarized T cells (Openshaw et al. (1995)J . Exp. Med. 182: 1357-1367; with anti-CD3 pooled for 2, 6, 16 hours Activated; T203); CD44-CD25 + pre-T cells sorted from the thymus (T2 04); Tm1 T cell clone D1.1 (T2 05); TH1 T cell clone D1.1 (T206) stimulated with 10 μg / ml ConA for 15 hours; antigen TH2 T cell clone CDC35 (T207) resting for 3 weeks after last stimulation with Time 10 μg / ml ConA stimulated, TH2 T cell clone CDC35 (T208); resting, spleen Mel14 + naive T cells from the gut (T209); IF pooled for 6, 12, 24 hours Mel14 + T cells (T210) polarized to Th1 with N-γ / IL-12 / anti-IL-4; 6, 13, 24 h Mel14 + T cells (T211) polarized to Th2 with IL-4 / anti-IFN-γ pooled between cells; Extremely mature B cell leukemia cell line A20 (B200); unstimulated B cell line CH12 (B201); spleen Unstimulated large B cells (B202); B cells from LPS-activated total spleen (B203) Resting, metrizamide-rich dendritic cells from spleen (D200); Dendritic cells from bone marrow (D201); monocyte cell line RAW 264.7 (M2 00); Bone marrow macrophages (M201) derived from GM and M-CSF; resting macro Phage cell line J774 (M202); pooled macro at 0.5, 1, 3, 6, 12 hours Phage cell line J774 + LPS + anti-IL-10 (M203); pooled 0.5, Macrophage cell line J774 + LPS + IL-10 (M204) at 1, 3, 5, 12 hours; Aerosolch lime and pooled for aerosol OVA challenge 7, 14, 23 hours Arranged mouse lung tissue (Garlisi et al. (1995)Clinical Immunology and Immun opathology  75: 75-83; X206); Nippostrongulus infected lung tissue ( Coffman et al. (1989)Science 245: 308-310; X200); total adult lung, normal (O200); total lung, rag-1 (Schwarz et al. (1993)Immunodeficiency See 4: 249-252 O205); IL-10 K.O. Spleen (Kuhn et al. (1991)Cell 75: 263-274 X201); total adult spleen, normal (O201); total spleen, rag-1 (O207); IL-10 K.O. Pair group (O202); Total Pair group, normal (O210); IL-10 K.O. Mesenteric lymph Node (X203); mesenteric lymph node, normal (O211); IL-10 K.O. Colon (X203); total Intestine, normal (O212); NOD mouse pancreas (Makino et al. (1980)Jikken Dobutsu 29: 1-13 X205); total thymus, rag-1 (O208); total kidney, rag-1 (O209); total heart Gut, rag-1 (O202); total brain, rag-1 (O203); total testis, rag-1 (O204); total liver, r ag-1 (O206); rat normal joint tissue (O300); and rat arthritis joint tissue (X3 00).   Expression by cDNA Southern analysis indicates that TH2-polarized T cells (Mel14 bright, spleen Their CD4 + cells, polarized for 7 days with IL-4 and anti-IFN-γ; very high in T201) Highly TH2-polarized T cells (Openshaw et al. (1995)J . Exp. Med.182: 1357-136 See 7; activated with pooled anti-CD3 for 2, 6, 16 hours; high in T203) And Tl-polarized T cells (Mel14 bright, CD4 + cells from spleen, IFN- Polarized with gamma and anti-IL-4 for 7 days; T200); and pooled for 6, 13, 24 hours It was remarkable in Mel14 + T cells (T211) polarized to Th2 with IL-4 / anti-IFN-γ. Example 3: Chromosome mapping of mouse and human IL-BKW   The mIL-BKW gene is mapped to the mouse chromosome using procedures well known in the art. I did it. For example, Copeland et al., (1993)Science See 262: 57-66. mIL-10 Maps directly to IL-BKW on chromosome 1.   Isolated human IL-B encoding IL-BKW to map human counterparts Use KW cDNA. Chromosome mapping is a standard technique, as described above. An example For example, mouse somatic cell hybridization with BIOS Laboratories (New Haven, CT) and PCR See the method for using the touch panel. Map human gene to human chromosome 1 And map from the mouse synteny information to a presumably 1q32 region. Is performed. Example 4: Purification of IL-BKW protein   Large numbers of transfected cell lines can be used at high levels compared to other cells. Screen for cells expressing tokine. Screen various cell lines And select for favorable properties in handling. Natural IL-B KW can be obtained from natural sources or transformed cells using an appropriate expression vector. Can be isolated by expression from the vesicles. Purification of the expressed protein can be accomplished using standard techniques. Or efficient purification from cell lysates or supernatants with high efficiency Can be combined with manipulated means for FLAG or His₆This segment Can be used for such purification features. Alternatively, affinity chromatography Can be used with specific antibodies. See below.   Produce proteins in E. coli, insect cells, or mammalian expression systems . Mouse fusion protein COP5 cells as an IgG fusion with an IGase cleavage site Production resulted in an N-terminally blocked, secreted, product. Micro distribution Sequencing analysis showed that the actual N-terminus was QEF ... Class having human sequence Production of similar substances provides the N-terminus of AQG ...   Recombinant human IL-BKW produced from standard constructs in mammalian cells is soluble in medium It appears to be found in small quantities in form. Protein is cell surface proteoglyca There is evidence that they bind tightly to the protein and are difficult to dissolve. Hepa Phosphorus treatment causes some release of the protein. Surface proteogli Proteins can be produced using can-deficient cells. Example 5: Isolation of homologous IL-BKW gene   IL-BKW cDNA can be a library from a desired source, such as a primate cell cDNA. Used as a hybridization probe to screen libraries Can be used. Many different species are required for easy hybridization. Both for stringency and for presence using probes. Can be screened. Using appropriate hybridization conditions, Alternatively, select for clones that show hybridization specificity.   By hybridization using degenerate probes based on peptide sequences Screening also allows for the isolation of suitable clones. Alternatively, PCR Use of the appropriate primers for cleaning will enrich the appropriate nucleic acid clones Is generated.   Similar methods are used to isolate either species, polymorphisms, or allelic variants Applicable to Species variants can be cloned from one species as full-length isolates or probes. Based on fragment isolation, using species cross-hybridization techniques Let go.   Alternatively, using antibodies raised against human IL-BKW, for example, Screening for cells expressing cross-reactive protein from cDNA library To run. Purified protein or a given peptide can be standard, as described above. Useful for producing antibodies by the method. Synthetic peptide or purified tan Presents proteins to the immune system to produce monoclonal or polyclonal antibodies You. For example, Coligan (1991)Current Protocols in Immunology Wiley / Greene And Harlow and Lane (1989)Antibodies: A Laboratory Manual Cold Spri See ng Harbor Press. The resulting antibodies are screened as described. Used for cleaning, purification, or diagnostics. Example 6: Preparation of antibody specific for IL-BKW   The synthetic peptide or purified protein is presented to the immune system and Or polyclonal antibodies. For example, Coligan (1991)Current Pro tocols in Immunology  Wiley / Greene; and Harlow and Lane (1989)Antibod ies: A Laboratory Manual  See Cold Spring Harbor Press. Polyclaw Null serum or hybridomas can be prepared. In appropriate circumstances, the binding reagent will As described above, for example, they may be labeled with fluorescent or other Or immobilized on a substrate.   Many monoclonal antibodies were obtained. Of the purified, half are IgG And the other half was IgM. Most of these are good Western blocks Reagent. Some of the recombinant hIL-BKWs from other strains are expressed on the cell surface and Others were appropriate for immunohistochemistry. "Positive" seen in the tonsil section by the latter The "live" cells appear to be macrophage-like. Example 7: IL-10 regulated expression of IL-BKW   To study the regulatory effect of IL-10 directly by Northern blot analysis, RNA was treated with either IL-10 or anti-IL-10 antibody, elutriated L Prepared from PS and IFN-γ activated monocytes. Just Southern of the cDNA library Strong induction of IL-BKW mRNA was treated with anti-IL-10 as assessed by blot. Treatment of these cells under the same conditions in the presence of IL-10, as observed on activated monocytes Prevented synthesis of the IL-BKW message almost completely. Example 8: Evaluation of breadth of biological function   The biological activity of IL-BKW is based on sequence and structural homology between IL-BKW and IL-10. And tested. First, assays that demonstrated the biological activity of IL-10 were tested. These are found in human peripheral blood mononuclear cells, human monocytes, and human T cell clones. Including assays. A. Effect on cell surface molecule expression on human monocytes.   Monocytes were purified by negative selection from peripheral blood mononuclear cells of normal healthy donors. Was. Simply put, 3 × 10⁸The mononuclear cells of the ficoll band were treated with 200 μl of αCD2 (Leu-5A ), 200 μl αCD3 (Leu-4), 100 μl αCD8 (Leu 2a), 100 μl αCD19 (Leu -12), 100 μl αCD20 (Leu-16), 100 μl αCD56 (Leu-19), 100 μl αCD6 7 (IOM 67) (Immunotech, Westbrook, ME) and anti-glycophorin antibody (10F7 Cocktail of monoclonal antibody consisting of MN, ATCC, Rockville, MD (Becton-Di) ckenson; Mountain View, CA). Antibody bound The cells are washed and then magnetic beads (Dynal, Oslo, Norwa) at a bead to cell ratio of 20: 1. Incubated with sheep anti-mouse IgG conjugated to y). Antibody-bound cells Separated from monocytes by application of a magnetic field. Then, human monocytes were replaced with polypropylene 96 In a well plate (Costar), IL-BKW (substance expressing baculovirus at 1/100 dilution) Or 1 μg / ml substance expressing mammalian cop 5) or absence of IL-10 (200 U / ml) Medium containing 1% human AB positive in the presence or absence (Gemini Bioproductsw, Ca labasas, CA) for 40 hours. In addition, the same culture was analyzed for the presence of IFN-γ Established below (100 U / ml).   Analysis of cell surface molecule expression was performed by direct immunofluorescence. In short, 2 × 10^FiveOf purified human monocytes in phosphate buffered saline containing 1% human serum Incubated for 20 minutes on ice in (PBS). Pellet cells at 200 xg Was. Cells were resuspended in 20 ml of PE or FITC labeled mAb. Another 20 minutes on ice After incubation between the cells, the cells are washed with PBS containing 1% human serum and then Washed twice with BS alone. Fix cells in PBS containing 1% paraformaldehyde And the FACScan flow cytometer (Becton Dickenson; Mountain View, C Analyzed in A) and the results are expressed in Table 3 as mean fluorescence intensity. Use the following Ab Used: CD11b (anti-mac1), CD11c (gp150 / 95), CD14 (Becton-Dickinson) Leu-M3), CD54 (Leu 54), CD80 (anti-BB1 / B7), HLA-DR (L243), and CD86 (FUN1) (Pharmingen), CD64 (32.2) (Medarex), CD40 (mAb89) (Schering -Plough France).                                   Table 3               Effect of IL-BKW on cell surface phenotype of human monocytes   IL-BKW enhanced the expression of CD54 (ICAM-1), CD80, and CD86, whereas HLA-DR Expression was reduced (see Table 3). IL-BKW, CD40, CD54 by IFN-γ, CD80 and CD86 upregulation was further enhanced. In the presence of IFN-γ Thus, IL-BKW enhanced the expression of HLA-DR. Changes in the effect of IL-BKW alone were measured for CD11b, C Observed for D11c and CD14 expression, when combined with IFN-γ, IFN-γ alone and Cell surface expression was reduced in comparison. In both the presence and absence of IFN-γ, IL -10 down regulates CD54, CD80, CD86, and HLA-DR. The results show that IL-20 differs in expression of cell surface molecules on monocytes compared to IL-10 It has an effect. B. Effect of IL-BKW on cytokine production by human monocytes.   Human monocytes were isolated as described and IL-BKW (1/100 diluted baculo Yssel's containing 1% human AB serum in the absence or presence of irs expressed substance) The cells were cultured in a medium (Gemini Bioproducts, Calabasas, CA). In addition, monocytes are 24 hours with a predetermined amount of LPS (E. coli 0127: B8 Difco) in the absence or presence of L-BKW And stimulate cytokines (IL-1b, IL-6, TNFα, GM-CSF, and And IL-10) were determined by ELISA. Cytoplasm for cytokines For staining, monocytes were treated with IL-BKW (substance expressing baculovirus at 1/100 dilution or Are 1 mg / ml mammalian cop 5 expressed substances) and / or 5 mg / ml LPS (E. coli 01 27: B8 Difco) and 10 mg / ml Brefeldin A (Epicentre technologies Madison W Cultured in Yssel's medium for 12 hours in the absence or presence of I) (1,000,000 / ml) ). The cells are washed with PBS and placed in a 2% formaldehyde / PBS solution at room temperature for 20 minutes. Incubated for minutes. Subsequently, cells are washed and resuspended in permeabilization buffer (PB S / BSA (0.5%) / 0.5% saponin (Sigma) in azide (1 mM)) and 20 minutes at room temperature Incubated. Cells (2 × 10^Five) And centrifuge in permeabilization buffer : Resuspend in 20 ml of directly conjugated anti-cytokine mAb diluted: 10 for 20 minutes at room temperature did. The following antibodies were used: IL-1α PE (364-3B3-14); IL-6-PE (MQ2-13A5); TN Fα-PE (MAb11); GM-CSF-PE (BVD2-21C11); and IL-12-PE (C11.5.14) (Pharmi ngen San Diego, CA). Subsequently, cells are washed twice with permeabilization buffer and PB Wash once with S / BSA / azide and use a FACScan flow cytometer (Becton Dick enson; Mountain View, CA). The results are shown in Table 4 as% positive cells and And the mean fluorescence intensity of the positive group.                                   Table 4            Effect of IL-BKW on cytokine production by human monocytes                       (% Intracellular staining / average fluorescence intensity)   IL-BKW is expressed by human monocytes on IL-6, TNFα, IL-10, IL-1b, and GM-CSF. Expression was induced (Table 5). LPS is also responsible for these cytokines in a dose-dependent manner. Expression was induced. The addition of IL-BKW in combination with LPS is either LPS or IL-BKW. Enhanced production of IL-6, TNFα, IL-10, IL-1b, and GM-CSF compared to Gave birth.                                   Table 5            Effect of IL-BKW on cytokine production by human monocytes   Similar results were obtained when cytokine production was assessed by cytoplasmic staining . IL-BKW shows the percentage of positive cells as well as the fluorescence intensity (this (Which reflects the amount of cytokines produced) Induced the expression of IL-1, IL-6, and TNFα by human monocytes. LPS is IL-1, IL -6, and TNFα expression; and the addition of IL-BKW to these cultures This production was further increased. This enhanced production is transferred to baculovirus and Observation was made of material expressed in both dairy animals. See Table 4. C. Effect of IL-BKW on cytokine production by human peripheral blood mononuclear cells (PBMC) .   Total PBMC were collected by centrifugation as described (Boyum et al.) With ficoll-hypaque. It was isolated from the buffy coat of a normal healthy donor and IL-BKW (1/100 dilution buffer). 1% human AB serum in the absence or presence of urovirus-expressed substances) The cells were cultured in Yssel's medium (Gemini Bioproducts, Calabasas, CA). Culture cells 2 × 10 in the ground⁶Incubated with cells / ml, or with PHA (100ng / ml) or Was activated by IL-2 (100 U / ml) (R & D Systems). In addition, PBMC, IL- IL-10 alone (100% diluted baculovirus expressed in combination with BKW) U / ml) or IL-10. Cytokine secretion, cytokine-specific ELISA Was determined on supernatants collected at 72 hours.   IL-BKW induced PBMC expression of IL-6, TNFα, IL-10, and INF-γ ( See Table 6). Furthermore, when PBMA is activated by PHA or IL-2, , IL-BKW enhanced the production of IL-6, TNFα, IL-10, and INF-γ. IL-10 is I L-BKW, PHA, IL-2, or a combination of IL-BKW with either PHA or IL-2 Inhibited the production of IL-6, TNFα, and IFN-γ after activation of PBMCs.                                   Table 6              Effect of IL-BKW on cytokine production by PBMCD. Effect of IL-BKW on proliferation of human peripheral blood mononuclear cells (PBMC).   Total PBMC were collected by centrifugation as described (Boyum et al.) With ficoll-hypaque. It was isolated from the buffy coat of a normal healthy donor. PBMCs were prepared using a predetermined amount of IL-BKW (mammalian cop 5 96-well plate (Falcon, Becton-D) in the absence or presence of expressed substances) ickinson, NJ) in 200 ml of Yssel's medium containing 1% human AB serum (Gemini Bioprod ucts, Calabasas, CA). Cells were cultured in medium alone or 100 U / ml IL The cells were cultured for 120 hours in combination with -2 (R & D Systems). 3H-thymidine (0.1mCi) Was added during the last 6 hours of culture, and 3H-thymidine incorporation was Determined by Tilation Count. The result is the average cpm of triplicate cultures. To represent.   IL-BKW dose-dependently induces low levels of PBMC proliferation. See Table 7 . Moreover, IL-BKW also enhances the proliferative response of PBMC to IL-2 in a dose-dependent manner You.                                   Table 7                    Effect of IL-BKW on proliferation by PBMC  Natural, recombinant, and fusion proteins include, for example, T cells, B cells, NK cells, Many other biological updates, such as macrophages, dendritic cells, and hematopoietic precursors Tested for agonist and antagonist activity in sire system. IL-10 structural phase Assays related to IL-10 activity were analyzed for correlation.   IL-BKW was transfected into cells and cells expressing the IL-10 receptor. Evaluate for agonist or antagonist activity on trolls. example For example, Ho et al. (1993)Proc . Nat'l Acad. Sci. USA 90, 11267-11271; Ho et al. (1995)Mol . Cell. Biol. 15: 5043-5053; and Liu et al. (1994)J . Immunol.152: 1821-1 See 829.   Some, based on structural homology to IL-10, convert IL-BKW to macrophages / dendritic cells. T cells in response to antigen or allostimulation, effects on cell activation and antigen presentation assays Assess cytokine production and proliferation. For example, de Waal Malefyt et al. (1 991)J . Exp. Med.174: 1209-1220; de Waal Malefyt et al. (1991)J . Exp. Med. 1 74: 915-924; Fiorentino et al. (1991)J . Immunol.147, 3815-3822; Fiorentino et al. (1991)J . Immunol.146: 3444-3451; and Groux et al. (1996)J . Exp. Med.184 : 19-29.   IL-BKW is also evaluated for its effect on NK cell stimulation. Assays Hsu et al. (1992)Internat . Immunol.4: 563-569; and Schwarz et al. (1994)J . Immunother. 16: 95-104.   B cell proliferation and differentiation effects are described, for example, by Defrance et al. (1992)J . Exp. Med.175 : 671-682; Rousset et al. (1992)Proc . Nat'l Acad. Sci. USA 89: 1890-1893 For example, by methodologies including IgG2 and IgA2 switch factor assays ,analyse. Unlike the COS7 supernatant, the NIH3T3 and COP supernatants were Note that it does not obviously interfere with b.   All references cited herein are each individual publications or patent publications. In particular, the request should be incorporated by reference in its entirety for all purposes. And to the same extent as if individually indicated, is incorporated herein by reference.   Many modifications and variations of this invention will be apparent to those skilled in the art and And without departing from the scope. Certain embodiments described herein are examples And the present invention is not deemed to be given the scope of such claims. Should be limited only by the appended claims, along with the full scope of equivalents to You.

[Procedural amendment] [Submission date] August 6, 1999 (1999.8.6) [Content of amendment] Claims 1. Substantially pure or recombinant soluble IL-BKW protein. 2. An antigenic protein or peptide fragment of IL-BKW according to claim 1. 3. 3. The IL-BKW of claim 2, which is a full-length natural soluble protein from a mammal, including a primate or a mouse. 4. 3. The IL-BKW according to claim 2, wherein a) is a soluble IL-BKW lacking the sequence MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWSQVSG of SEQ ID NO: 2; b) is a mature polypeptide of SEQ ID NO: 6; or c. ) IL-BKW encoded by the nucleic acid of SEQ ID NO: 5. 5. 3. The IL-BKW of claim 2, which is a full-length secreted protein that exhibits a different post-translational modification pattern from native IL-BKW. 6. 3. The IL-BKW according to claim 2, which exhibits an antagonist immunological activity of IL-10. 7. 3. A fusion protein comprising the sequence of the protein or peptide according to claim 2, wherein the protein or peptide lacks: a) the sequence of MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWSQVSG of SEQ ID NO: 2; A fusion protein, which is a peptide; or c) is encoded by the nucleic acid of SEQ ID NO: 5. 8. A sterile composition comprising the protein or peptide according to claim 2. 9. 7. A method for purifying an IL-BKW protein or peptide according to claim 6 from other substances in a mixture, the method comprising: contacting the mixture with an antibody against the protein; Separating. 10. An isolated or recombinant expression vector encoding the soluble IL-BKW of claim 1. 11. 11. The vector of claim 10, wherein the nucleic acid encodes a secreted sequence of SEQ ID NO: 2 or 6. 12. The vector according to claim 10, comprising the sequence of SEQ ID NO: 1 or 5. 13. A detection kit comprising a positive control which is the substantially pure soluble IL-BKW or fragment according to claim 1. 14． A method for detecting in a sample for the presence of an IL-BKW nucleic acid, protein, or antibody, comprising testing the sample with the kit of claim 11. 15. A method of regulating the physiology of a cell, comprising contacting the cell with the substantially pure soluble IL-BKW of claim 1. 16. 16. The method of claim 15, wherein the cell is a T cell, and wherein the regulation of physiology is inactivation of the T cell. 17． 16. The method of claim 15, wherein said cells are in a tissue. 18. A method for producing soluble IL-BKW, comprising a step of expressing the vector according to claim 10. 19. A cell, tissue or organ comprising the vector of claim 10. 20. A composition for treating a mammal having an abnormal immune response, comprising an effective dose of substantially pure soluble IL-BKW.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 G01N 33/53 P C12Q 1/68 C12N 5/00 B G01N 33/53 A61K 37/02 ( 81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ) , BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AU, AZ, BA, BB, BG, BR, BY, CA, CN, CZ, EE, GE, G , HU, ID, IL, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LV, MD, MG, MK, MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UZ, VN, YU (72) Inventor Dewar Malephite, Lane USA 94040, Sunnyvale, Muender Avenue 891

Claims (1)

[Claims]   1. Substantially pure or recombinant soluble IL-BKW protein.   2. An antigenic protein or peptide fragment of IL-BKW according to claim 1. .   3. Full-length natural soluble proteins from mammals, including primates or mice The IL-BKW according to claim 2, which is   4. The IL-BKW according to claim 2, wherein   a) Sequence of SEQ ID NO: 2 MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWSQVSG Is soluble IL-BKW lacking;   b) is the mature polypeptide of SEQ ID NO: 6; or   c) encoded by the nucleic acid of SEQ ID NO: 5, IL-BKW.   5. Full-length secreted protein with a different post-translational modification pattern than native IL-BKW The IL-BKW according to claim 2, which is a protein.   6. 3. The IL-BKW according to claim 2, which exhibits an antagonist immunological activity of IL-10.   7. A fusion protein comprising the protein or peptide sequence according to claim 2. Wherein the protein or peptide is   a) arrangement of MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWSQVSG of SEQ ID NO: 2 Missing columns;   b) is the mature polypeptide of SEQ ID NO: 6; or   c) encoded by the nucleic acid of SEQ ID NO: 5, Fusion protein.   8. A sterile composition comprising the protein or peptide according to claim 2.   9. 7. The IL-BKW protein or peptide of claim 6 from other substances in the mixture. A method for purifying a peptide, comprising contacting the mixture with an antibody against the protein. And separating the bound IL-BKW from other substances.   10. An isolated or recombinant encoding the soluble IL-BKW of claim 1. Body expression vector.   11. The nucleic acid encodes a secreted sequence of SEQ ID NO: 2 or 6. The vector according to claim 10.   12. The vector according to claim 10, comprising the sequence of SEQ ID NO: 1 or 5.   13. A substantially pure soluble IL-BKW or fragment according to claim 1. A detection kit including a positive control.   14． Detect in a sample for the presence of IL-BKW nucleic acids, proteins, or antibodies Testing the sample with the kit of claim 11. how to.   15. A method for regulating the physiology of a cell, the method comprising: Contacting with highly pure soluble IL-BKW.   16. The cell is a T cell and the regulation of physiology is inactivation of the T cell The method of claim 15, wherein   17． 16. The method of claim 15, wherein said cells are in a tissue.   18. A soluble IL-BKW comprising a step of expressing the vector according to claim 10. How to manufacture.   19. A cell, tissue or organ comprising the vector of claim 10.   20. Mammals with an abnormal immune response are treated with substantially pure soluble IL-BKW. A method of treating by administering an effective dose to the mammal.