MXPA99005980A - Mammalian cytokine related to il10 - Google Patents

Abstract

Purified genes encoding cytokine from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding this molecule are provided. Methods of using said reagents and diagnostic kits are also provided.

Description

MAMMALIAN CYTOKINE RELATED TO IL-10 FIELD OF THE INVENTION The present invention pertains to compositions related to proteins that function to control the biology and physiology of mammalian cells, e.g., cells of the mammalian immune system. In particular, it provides purified genes, proteins, antibodies and related reagents useful for, for example, regulating the activation, development, differentiation and function of various cell types, including hematopoietic cells.

BACKGROUND OF THE INVENTION Recombinant DNA technology refers in a general way to the technique of integrating into vectors genetic information from a donor source for subsequent processing, such as through introduction into a host, from where the transferred genetic information is copied and / or is expressed in the new medium. Commonly, genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) which codes for a desired protein product. Frequently, the carrier is a plasmid that has the ability to incorporate cDNA for subsequent replication in a host and, in some cases, to normally control the expression of cDNA and thereby direct the synthesis of the product encoded in the host. It is known, for some time, that the immune response of mammals is based on a series of complex cellular interactions, called "the immune network". Recent research has provided new insights into the internal workings of this network. While it is clear that most of the response revolves, in fact, around the network-like interactions of lymphocytes, macrophages, granulosites and other cells, immunologists now generally hold the view that soluble proteins, known as lymphokines, cytokines or monocins, play a critical role in the control of these cellular interactions. Therefore, there is considerable interest in the isolation, characterization and mechanisms of action of cell modulating factors, whose understanding will lead to significant advances in the diagnosis and therapy of numerous medical abnormalities, for example, system disorders. immune. Apparently lymphokines mediate cellular activities in a variety of ways. It has been shown that they support the proliferation, growth and differentiation of pluripotent hematopoietic stem cells towards vast numbers of progenitors that consist of diverse cell lineages which constitute a complex immune system. The appropriate and balanced interactions between the cellular components are necessary for a healthy immune response.

Frequently, different cell lineages respond in a different way when lymphokines are administered in conjunction with other agents. The lineages of cells especially important for the immune response include two classes of lymphocytes: B cells, which can produce and secrete immunoglobulins (proteins with the ability to recognize and bind with foreign matter to effect their elimination), and T cells of several subsets that secrete lymphokines and induce or suppress B cells and some other cells (including other T cells) constituting the immune network. These lymphocytes interact with many other cell types. Another important cell lineage is that of the barley cell (which has not been positively identified in all mammalian species), which is a connective tissue cell that contains granules that are located close to the capillaries throughout the body. These cells are found in especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts. Mast cells play a central role in allergy-related disorders, particularly anaphylaxis, in the following manner: When the selected antigens are cross-linked with a class of immunoglobulins bound to the receptors on the surface of the barley cell, the barley cell is degranulated and releases mediators, for example, histamine, serotonin, heparin and prostaglandins, which cause allergic reactions, for example anaphylaxis. Research to better understand and treat the various immune disorders has been hampered by the general inability to maintain cells of the immune system in vitro. Immunologists have discovered that the culture of these cells can be achieved through the use of supernatants of T cells and other cells, which contain various growth factors, including many of the lymphokines. The gene coding for IL-10, originally called Cytokine Synthesis Inhibiting Factor (CSIF), was isolated in the 1980s. See for example, Mosmann, et al., Patent E.U.A. No. 5,231, 012. Since then, much has been learned about the biology and physiology mediated by the cytokine. See, for example, de Vries and Waal Malefyt (1995) Interleukin-10 Landes Co., Austin, Texas. From the above, it is evident that the discovery and development of new lymphokines, for example those related to IL-10, could contribute to new therapies for a wide range of degenerative or abnormal conditions that directly or indirectly involve the immune system and / or the hematopoietic cells. In particular, the discovery and development of lymphokines that increase or enhance the beneficial activities of known lymphokines would be highly advantageous. The present invention provides novel compositions of etherleucine and related compounds, and methods for their use.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to mammalian interleukin-BKW (IL-BKW), for example, rodent, canine, feline, primate, and their biological activities. This includes the nucleic acids that code for the polypeptides themselves and the methods for their production and use. The nucleic acids of the invention are characterized, in part, by their homology to the cloned complementary DNA (cDNA) sequences described herein, and / or by functional assays for the IL-10 type activities applied to the polypeptides, which they are typically encoded by these nucleic acids. Methods are provided to modulate or intervene in the control of an immune response. The present invention is based in part on the discovery of a new cytokine sequence that exhibits high sequence and structure similarity to cellular IL-10. In particular, it provides a gene that encodes a protein whose mature size is approximately 158 amino acids. Functional equivalents that exhibit significant sequence homology will be available from other mammals, for example mouse and rat, and non-mammalian species. In one embodiment, the invention provides a recombinant or substantially pure soluble IL-BKW, which includes an antigenic peptide protein or fragment of soluble IL-BKW. Preferably, IL-BKW is a full length natural soluble protein from a mammal, which includes a primate, or it can be in a sterile composition. Typically, soluble IL-BKW lacks the sequence MNFQQRLQSL WTLARPFCPP LLATASQMQM WLPCLGFTL LLWSQVSG of SEQ ID NO: 2; is a mature polypeptide of SEQ ID NO: 6; or is encoded by a nucleic acid of the sequence SEQ ID NO: 5. Alternatively, soluble IL-BKW is a full length secreted protein that exhibits a post-translational modification pattern distinct from natural soluble IL-BKW. Functionally, soluble IL-BKW will typically exhibit an immunologically functional activity antagonist to IL-10. The invention further provides a fusion protein consisting of a sequence of soluble IL-BKW, but lacking the sequence of MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWSQVSG of SEQ ID NO: 2; is a mature polypeptide of SEQ ID NO: 6; or is encoded by SEQ ID NO: 5. The invention further provides a purification method for a protein or peptide of soluble IL-BKW from other materials in a mixture consisting of contacting said mixture with an antibody for said protein, and separate the bound IL-BKW from the other materials. In other embodiments, the invention provides a recombinant or isolated expression vector encoding a soluble IL-BKW. Preferably, the vector encodes a secreted sequence of SEQ ID NO: 2 or 6 and may comprise the sequence of SEQ ID NO: 1 or 5.

The invention provides detection equipment consisting of a positive control which is an IL-BKW or a substantially pure soluble fragment. The equipment also provides a method to detect in a sample the presence of a soluble IL-BKW protein or antibody, which includes testing said sample with the equipment. The invention further provides a method for modulating the physiology of a cell that includes contacting the cell with a substantially pure soluble IL-BKW protein. In certain embodiments, the cell is a T cell and the modulation of physiology is the inactivation of the T cell; or the cell is in a tissue. The invention further provides a method for making a soluble IL-BKW protein comprising expressing a vector. The vector can be in a cell, tissue or organ. Finally, the invention provides a method of treatment for an animal that has an abnormal immune response by administering to the animal an effective dose of a substantially pure soluble IL-BKW protein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the alignment of IL-10 and related cytokines.

DETAILED DESCRIPTION OF THE INVENTION All references cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated for reference.

I. General The present invention provides amino acid sequences and DNA sequences that encode several mammalian proteins that are cytokines, which are for example secreted molecules that can mediate a signal between immune cells and other cells. See for example, Paul (1994) Fundamental Immunoloqy, Raven Press, N.Y. Full-length cytokines, and fragments, or antagonists will be useful in the physiological modulation of cells expressing a receptor. Initial data suggest that the cytokine functions to affect the opposite effects caused by IL-10. It is likely that IL-BKW has a stimulatory or inhibitory effect on T cells, B cells, natural killer cells (NK), macrophages, dendritic cells, hematopoietic progenitors, etc. The proteins will also be useful as antigens, for example, immunogens, to create antibodies for some epitopes, both linear and conformational, on the protein. A cDNA encoding IL-BKW was isolated from a human melanoma cell line. The molecule was named mda7, and it was characterized as a novel gene associated with melanoma differentiation.

SEQ ID NO: 1 and 2. See Jiang, et al. (1995) Oncoaene 11: 2477-86; Genbank access number U16261. That document reported some small homology of mda7 with human IL-10, but the relevance is unknown. Applicants have analyzed the sequence and believe that the coding region has been misidentified, a fact consistent with the mouse gene sequence. The CDS was proposed by Jiang, and others to run approximately from 275. . 895 to code: MNFQQRLQSL WTLARPFCPP LLATASQMQM WLPCLGFTL LLWSQVSGAQ GQEFHFGPCQ VKGWPQKLW EAF AVKDTM QAQDNITSAR LLQQEVLQNV SDAESCYLVH TLLEFYLKTV FKNYHNRTVE VRTLKSFSTL ANNFVLIVSQ LQPSQENEMF SIRDSAHRRF LLFRRAFKQL DVEAALTKAL GEVDILLTWM QKFYKL (SEQ ID NO: 2) However, the applicants propose a mature form of IL-BKW that starts at approximately residue 49, or: AQGQEFHFGP CQVKGWPQK LQFDWLRQ DQG WR WKH RI WKH DQG WKH WKH WKH WKH WKH WKH WKH WKH WKH WKH WKH WKH WKH.

Table 1 compares some modalities of human IL-10 with human IL-BKW.

TABLE 1 L-BKW .MQMWLPCL GFTLLLWSQV SGAQGQEFHF GPCQVK.GW PQKL ... WEA IL-10 MHSSALLCCL .. VLLTGVRA SPGQGTQSEN SCTHFP.GNL PNMLRDLRDA L-XX M VNFILRCG .. L VT SL AIAKHKQSSF TKSCYPRGTL SQAVDALYIK IL-BKW F AVKDTMQA QDNITSAR.L LQQEVLQNVS DAESCY VHT L EFY KTVF IL-10 FSRVKTFFQM KDQLDNL .. LKESLLEDFK GYLGCQALSE MIQFYLEEVM IL-XX AA LKATIP. EDRIKNIR.L LKKKTKKQFM K..NCQFQEQ LLSFFNEDVF IL-BKW KNYHNRTVEV RT KSFSTLA NNFVLIVSQL QPSQENEMFS IRDSAHRRFL IL-10 PQAENQDPDI .. KAHVNSLG ENLKTLRLRL RRCHR ... FL PCENKSKAVE IL-XX GQLQLQG CKKIRFV EDFHTLRQKL SHCIS ... CA SSAREMKSIT IL-BKW LFRRAFKQLD VEAALTKALG EVDILLT MQ KFYK .... IL-10 QVKNAFNKLQ. EKGIYKAMS EFDIFINYIE AYMTMKIRN IL-XX RMKRIFYRIG .NKGIYKAIS ELDILLS IK KLLESSQ .. hIL-BK (SEQ ID NO: 2) IL-10 (SEQ ID NO: 3) hIL-XX (SEQ ID NO: 4) A murine clone for IL-BKW (mlL-BKW) was also isolated from an activated genotope of mouse thymocyte cDNA (A. Zlotnik, DNAX Res. Institute, Palo Alto, CA). SEQ ID NO: 5 and 6. The signal sequence runs from approximately Met to Gly23. The mature polypeptide begins approximately in Leu24. Helix D conserved, involved in receptor binding, runs from approximately Glu158 to Leu181. There is no ATG towards the corresponding 5 'end of the leader sequence as it is found in humans, possibly indicating that this molecule is bound to the membrane. MlL-BKW possesses a high degree of amino acid sequence identity with its human counterpart. An alignment between hIL-10, mlL-10, hIL-BKW, and mlL-BKW reveals that helix D is highly conserved. See Table 2 and SEQ ID NO: 2, 3, 6 and 7. Table 2 compares human and mouse IL-10 and human and mouse IL-BKW. In IL-10, this helix seems particularly important in receptor binding. Therefore, IL-BKW and IL-10 can share the same receptor subunit.

TABLE 2 hillo ..MHSSALLC C VLLTGVR AS..PGQGTQ SENSCTHFPG NLPNMLRDLR mlLlO ..MPGSALLC C LLLTGMR IS..RGQYSR EDNNCTHFPV GQSHMLLELR hILBKW ..MQMWLPC LGFTLLLWSQ VSGAQGQEFH F.GPCQVKGV .... VPQK MiLB W-KW MSWGLQILPC LSLILLL NQ VPGLEGQEFR S.GSCQVTGV. ... VLPEL hI lO DAFSRVKTFF QMKDQLDNL. LLKESLLEDF KGYLGCQALS EMIQFYLEEV miLIO TAFSQVKTFF QTKDQLDNI. LLTDSLMQDF KGYLGCQALS EMIQFYLVEV hILBKW EAF AVKDTM QAQDNITSAR LLQQEVLQNV SDAE? CYLVH TLLEFYLKTV m LBKW EAF TVKNTV QTQDDITSIR LLKPQVLRNV SGAESCYLAH SL KFYLNTV Hillo LRR MPQAENQDPD IK..AHVNSL GENLKTLRLR ... CHRF LPCENKSKAV mlLlO MPQAEKHGP? IK ..? HLNSL GEKLKTL-RMR LRR ... CHRF PCENKSKAV Hilbk FKNYHNRTVE VRTLKSFSTL ANNFVLIVSQ LQPSQENEMF SIRDSAHRRF mlLBK FKNYHSKIAK FKVLRSFSTL ANNFIVIMSQ LQPSKDNSML PISESAHQRF Hillo EQVKNAFNKL Q. EKGIYKAM SEFDIFINYI EAYMTMKIR N mlLlO EQVKSDFNKL Q. DQGVYKAM NEFDIFINCI EAYMMIKMK S ILBKW LLFRRAFKQL DV EAALTKAL GEVDILLTWM QKFYK .. mILBKW LLFRAFKQL DT EVALVKAF GEVDILLTWM QKFYHL ... J D-helix I hIL-10 (SEQ ID NO: 3) mIL-10 (SEQ ID NO: 7) h L-BKW (SEQ ID NO: 2) mlL-BKW ( SEQ ID NO: 6) Chromosome mapping using MLL-BKW as a probe revealed that this cytokine maps to the central region of mouse chromosome 1 between regions of homology to human chromosomes 2q and 1q. Interestingly, mlL-BKW is located adjacent to the mlL-10 gene. Defects in this particular locus have been implicated in immune disorders, for example dominant hemimelia. See, for example, Carter (1954) Mouse News Lett. 11:16; Higgings, and others (1992) Genet. Beef. 60: 53-60; and Machado, et al. (1976) Am. J. Pathol. 85: 515-518. The protein has been characterized as an antigen with a predicted hydrophobic stretch that spans 25-45 residues of the protein. But the experiments failed to confirm that there is a membrane form. Applicants believe that the predicted hydrophobic stretch is normally a signal sequence that is removed from the mature protein, which probably starts either at residue 47 (Ser) or 49 (Ala), as designated in the original publication . Applicants believe that the gene encodes a small soluble cytokine type protein of approximately 158 amino acids. The pre-sequence probably starts either in the M of position 20 or 30, thereby providing an N-terminal signal sequence of approximately 17-21 amino acids. See table 1 and SEQ. ID. NO: 1 and 2. IL-BKW exhibits structural motifs characteristic of a member of short chain cytokines. Compare for example, IL-BKW, IL-10 cellular from mouse and human, IL-10 from viral EBV and IL-10 from equine herpes virus, all sequences are available in GenBank. See table 1 The structural homology of IL-BKW with the related IL-10 proteins suggest a related function of this molecule. IL-BKW is a small chain cytokine. Initial experiments suggest that the new cytokine probably mediates immune functions through a receptor in the class of cytokine receptors, although it seems that it does not share all the parts of a receptor complex for functional IL-10. IL-BKW agonists, or antagonists, can also act as functional or receptor antagonists, for example, that block the binding of IL-10 to its receptor, or mediate the opposite actions. In this manner, IL-BKW, or its antagonists, may be useful in the treatment of abnormal immune disorders for example, immune deficiencies of T cells, chronic inflammation or tissue rejection. Natural antigens are capable of mediating various biochemical responses that lead to biological or physiological responses in target cells. The modalities characterized in this come from human and mouse, but other counterparts of primate or other species are expected to exist in nature. Additional sequences for proteins in other mammalian species, for example, primates, canines, felines and rodents, should also be available. See ahead. The following descriptions are intended for purposes of exemplification to human IL-BKW, but are applicable in a manner similar to the related modalities of other species.

Human and mouse IL-BKW proteins exhibit structural characteristics characteristic of short-chain cytokines.

II. I L-BKW purified. The amino acid sequence of IL-BKW lacking the N-terminal sequence for example, MNFQQRLQSL WTLARPFCPP LLATASQMQM WLPCLGFTL LLWSQVSG, is shown as a modality within SEQ ID NO: 6. The corresponding amino acid sequence for mlL-BKW is shown in SEQ ID NO: 6. These amino acid sequences, provided as amino to carboxy, are important to provide the sequence information in the cytokine allowing distinguishing the antigen from the protein of other proteins and exemplifying numerous variants. In addition, the peptide sequences allow the preparation of peptides to generate antibodies to recognize such segments, and the nucleotide sequences allow the preparation of oligonucleotide probes, both of which are strategies for the detection or isolation, for example, of cloning of genes. that encode such sequences. Potential glycosylation sites in the human sequence are asn31-thr39 and asn51-ser53; and in the mouse asn51-ser53. As used herein, the term "soluble human IL-BKW" should encompass, when used in a protein context, a protein having an amino acid sequence corresponding to a soluble polypeptide shown in SEQ ID NO: 2 for example, lacking the amino terminal portion described as associated with the membrane (MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWSQVSG). Such a polypeptide will lack the first 45 residues described as the N-terminus by Jiang, and others, or significant fragments thereof. "Mouse IL-BKW" should encompass the amino acid sequence corresponding to SEQ ID NO: 6. Binding components eg, antibodies, will typically bind to an IL-BKW with a high affinity, eg, at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and most preferred better than about 3 nM. The homologous proteins will be found in species of mammals other than humans, for example, other primates or rodents. Non-mammalian species must also possess structurally or functionally related genes and proteins, for example, birds or amphibians. The term "polypeptide" as used herein includes a significant fragment or segment, and encompasses a stretch of amino acid residues of at least about 8 amino acids, generally at least about 12 amino acids, typically at least about 16 amino acids , preferably at least about 20 amino acids and, in the particularly preferred embodiments, at least about 30 or more amino acids for example, 34, 40, 45, 50, etc. Such fragments may have ends that start and / or end in virtually all positions, starting at residues 1, 2, 3, etc., and ending at, for example, 150, 149, 148, etc., in all combinations. Particularly interesting peptides have ends corresponding to structural domain boundaries for example, helices A, B, C, and / or D. See Table 1, Table 2, and Figure 1. Note that the IL-BKW sequences exhibit an identity Particularly with cellular IL-10 in the region of residues 126-137, and the other regions exhibit higher specific sequence degrees of IL-BKW. The term "binding composition" refers to molecules that bind with specificity to IL-BKW for example, in an antigen-antibody interaction. It also includes compounds, for example, proteins that specifically associate with IL-BKW, including a physiologically relevant protein-protein interaction, either covalent or non-covalent. The molecule can be a polymer, or a chemical reagent. A functional analog may be a protein with structural modifications, or this may be a molecule having a molecular form that interacts with the appropriate binding determinants. The compounds can serve as agonists or antagonists of a receptor binding interaction, see for example, Goodman, et al. (Eds.) (1990) Goodman &; Gilman's: The Pharmacoloqical Bases of Therapeutics (8th ed.), Pergamon Press. "Substantially pure" typically means that the protein is free of other proteins, nucleic acids or other contaminating biological products, derived from the original source organism. The purity can be evaluated by standard methods, typically by weight, and ordinarily will be at least about 40% pure, generally at least about 50% pure, often at least about 60% pure, typically at least about 80% pure, preferably at least about 90% pure, and in the most preferred modalities, at least about 95% pure. Vehicles or excipients will be added frequently. The solubility of a polypeptide or fragment depends on the environment and the polypeptide. Many parameters affect the solubility of the polypeptide, including the temperature, the electrolyte medium, size and molecular characteristics of the polypeptide, and the nature of the solvent.

Typically, the temperature at which the polypeptide is used varies from about 4 ° C to about 65 ° C. Usually the temperature used is greater than 18 ° C. For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturing temperature of the components in the test. For therapeutic purposes, the temperature will usually be body temperature, typically close to 37 ° C for humans and mice, although under certain situations the temperature may be elevated or decreased in situ or in vitro. The size and structure of the polypeptide should generally be in a substantially stable state, and usually not in the denatured state. The polypeptide may be associated with other polypeptides in a quaternary structure for example, to confer solubility, or associated with lipids or detergents.

The solvent and the electrolytes will usually be a biologically compatible regulatory solution, of a type used for the conservation of biological activities, and will usually approximate the aqueous physiological solvent. Usually the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5. In some instances one or more detergents will be added, typically one that is mild and non-denaturing eg, CHS (cholesteryl hemisuccinate) or CHAPS (3- [3-colamidopropyl) dimethylammonium] -1-propane sulfonate), or in a concentration low enough to avoid significant disruption of the structural or physiological properties of the protein. lll Physical Variants This invention also encompasses proteins or peptides having substantial amino acid sequence identity with the amino acid sequence of the antigen for IL-BKW. Variants include species variants, polymorphic or aielic. The amino acid sequence homology, or sequence identity is determined by optimizing the residue matches, if necessary, by entering spaces as required. See also Needleham, and others. (1970) J. Mol. Biol. 48: 443-453; Sankoff, and others. (1983) Chapter One in Time Warps, String Edits, and Macromolecules: The Theorv and Practice of Sequence Comparison, Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA; and The University of Wisconsin Genetics Computer Group Computation in Genetics of the University of Wisconsin), Madison, Wl. Sequence identity changes when conservative substitutions are considered as pairs. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, aginin; and phenylalanine, tyrosine. Conservation can be applied to biological characteristics, functional characteristics or structural characteristics. Homologous amino acid sequences are typically designed to include natural polymorphic or allelic and interspecies variations in each respective protein sequence. Typical homologous proteins or peptides will have from 25-100% identity (if spaces can be entered), at 50-100% identity (if conservative substitutions are included) with the amino acid sequence of IL-BKW. The identity measurements will be at least about 35%, generally at least about 40%, frequently at least about 50%, typically at least about 60%, usually at least about 70%, preferably at least 80 %, and very preferred at least about 90%. The IL-BKW isolated DNA can be easily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and nucleotide stretch inversions. These modifications result in novel DNA sequences that encode these antigens, its derivatives or proteins having physiological, immunogenic, antigenic or other similar functional activity. These modified sequences can be used to produce mutant antigens or to increase expression. Increased expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. "IL-BKW mutants" encompasses a polypeptide that otherwise falls within the definition of sequence identity of IL-BKW as indicated above, but having an amino acid sequence that differs from that of IL-BKW as normally found in nature, either through deletion, substitution or insertion. This generally includes proteins that have significant identity with a protein having a sequence of SEQ ID NO: 2 or 6 and that shares several biological activities for example antigenic or immunogenic with those sequences and in the preferred embodiments contains most of the described sequences of full length Full length sequences will typically be preferred although truncated versions will also be useful, similarly, proteins or genes found in natural sources are typically the most desired. Similar concepts apply to different IL-BKW proteins, particularly those found in several warm-blooded animals, e.g., mammals and birds. These descriptions generally mean that they encompass all IL-BKW proteins, not being limited to the particular mouse modalities specifically discussed.

The mutagenesis of IL-BKW can also be conducted by inserting or deleting amino acids. The substitutions, deletions, insertions or any combinations can be generated to get to build the final product. The insertions include amino- or carboxy-terminal fusions, random mutagenesis can be conducted in a white codon and the expressed mutants can then be evaluated for the desired activity. Methods for making mutations by substitution at predetermined sites in DNA having a known sequence are well known in the art, for example, by mutagenesis of M13 primer or polymerase chain reaction (PCR) techniques. See for example Sambrook, and others. (1989); Ausubel, and others. (1987 and supplements); and Kunkel, and others. (1987) Methods in Enzvmol. 154: 367-382. The present invention also provides recombinant proteins for example, heterologous fusion proteins using segments derived from these proteins. A heterologous fusion protein is a fusion of proteins or segments that are naturally not fused in the same way. A similar concept applies to heterologous nucleic acid sequences. In addition, new constructions can be made from the combination of similar functional domains from other proteins. For example, binding to the blank or other segments can be "swapped" between different new fusion polypeptides or fragments. See for example, Cunningham, and others. (1989) Science 243: 1330-1336; and O'Dowd, and others. (1988) J. Biol. Chem. 263: 15985-15992. The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22: 1859-1862, will produce appropriate synthetic DNA fragments. A double-stranded fragment will often be obtained either by synthesizing the complementary strand and attaching the strand to join them under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence, eg PCR IV Functional Variants Blocking the physiological response to IL-BKW can result from competitive inhibition of ligand binding to its receptor. Preliminary results suggest that IL-BKW does not bind to it as the described subunit of the receptor for IL-10. It could be expected that an IL-BKW antagonist has the opposite effect to that of IL-BKW. In vitro tests of the present invention will often use isolated protein, soluble fragments consisting of receptor binding segments of these proteins or fragments adhered to solid phase substrates. These tests will also allow the diagnostic determination of the effects of either the mutations and modifications in the binding segment, or the mutations and modifications of cytokine, for example IL-BKW analogues.

This invention further contemplates the use of competitive drug evaluation tests for example, wherein the neutralizing antibodies to the cytokine or to the receptor binding fragments compete with a test compound. The "derivatives" of IL-BKW antigens include amino acid sequence mutants from naturally occurring forms, glycosylation variants and covalent conjugates or conjugates aggregated with other chemical moieties. The covalent derivatives can be prepared by linking functionalities to groups that are found in the amino acid side chains of IL-BKW or at the N- or C-terminal ends for example, by standard methods. See, for example, Lundblad and Noyes (1988) Chemical Reagents for Protein Modification, vols. 1-2, CRC Press, Inc., Boca Raton, FL; Hugli (ed.) (1989) Techniques in Protein Chemistry, Academic Press, San Diego, CA; and Wong (1991) Chemistry of Protein Conjugation and Cross Linkinq, CRC Press, Boca Raton, FL. In particular, alterations by glycosylation are included, for example made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in subsequent processing steps. See for example Elbein (1987) Ann, Rev. Biochem. 56: 497-534. Also covered are versions of the peptides with the same "primary amino acid sequence" which have other minor modifications, including the phosphorylated amino acid residues eg, phosphotyrosine, phosphoserine or phosphothreonine.Fusion polypeptides are also provided between the IL-BKW and other homologous or heterologous proteins Many cytokine receptors or other surface proteins are multimeric, eg, homodimeric entities and a repeat structure may have several advantages, including decreased susceptibility to proteolytic digestion Typical examples are fusions of a reporter polypeptide for example, luciferase, with a segment or domain of a protein, e.g., a receptor binding segment, such that the presence or location of the fused ligand can be readily determined See for example Dull et al., U.S. Patent No. 4,859,609. Other gene fusion partners include ß-gal bacterial actsidase, trpE, protein A, β-lactamase, alpha-amylase, alcohol dehydrogenase, yeast alpha complementary factor, and purification or detection labels such as a FLAG sequence of the His6 sequence. See for example Godowski et al., (1988) Science 241: 812-816. The fusion peptides will typically be made either by recombinant nucleic acid methods or by synthetic methods for polypeptides. Techniques for the manipulation and expression of nucleic acid are described generally for example in Sambrook et al., (1989) Molecular Cloninq: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al., (eds.) (1993) Current Protocols in Molecular Bioloqv, Greene and Wiley, NY. Techniques for polypeptide synthesis are described, for example, in Merrifield (1963) J. Amer. Chem.

Soc. 85: 2149-2156; Merrigield (1986) Science 232: 341-347; Atherton, and others. (1989) Solid Phase Peptide Svnthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992) Synthetic Peptides: A User's Guide, W.H. Freeman, NY. The methods of structural conformation can be applicable to synthetic proteins. This invention also contemplates the use of IL-BKW protein derivatives other than variations in the amino acid sequence or glycosylation. Such derivatives may involve covalent or aggregative association with chemical portions or with protein carriers. The covalent or aggregative derivatives will be useful as immunogens, as reagents in immunoassays, or in such purification methods, as for the affinity purification of binding partners for example, other antigens. An IL-BKW can be immobilized by covalent attachment to a solid support such as a SEPHAROSE activated with cyanogen bromide, by methods that are well known in the art, or adsorbed on polyolefin surfaces, with or without crosslinking with glutaraldehyde, for be used in the testing or purification of antiILL-BKW antibodies or an alternative binding composition. The IL-BKW proteins can also be labeled with a detectable group, for example to be used in diagnostic tests. The purification of IL-BKW can be effected by an immobilized antibody or a complementary binding partner, for example the binding portion of a receptor. An IL-BKW protein or solubilized fragment of this invention can be used as an immunogen for the production of antiserum or antibodies specific for binding. The purified antigen can be used to evaluate monoclonal antibodies or antigen-binding fragments, encompassing antigen-binding fragments of natural antibodies by example Fab, Fab ', F (ab) 2, etc. The purified IL-BKW antigens can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of the cytokine, which can be diagnostic of a disease condition or specific physiological condition or abnormal condition. This invention contemplates antibodies created against amino acid sequences encoded by the nucleotide sequence shown in SEQ ID NO: 1 of 5, or fragments of proteins containing them. In particular, this invention contemplates antibodies that have binding affinity to, or are created against, specific domains for example, helices A, B, C or D. The present invention contemplates the isolation of additional variants of species closely related The Southern blot and Northern blot analyzes will establish which similar genetic entities exist in other mammals. It is likely that IL-BKW are widely distributed in species variants for example, rodents, lagomorphs, carnivores, artiodactyls, perissodactyls and primates.

The invention also provides means for isolating a group of related antigens that show both distinction and similarity in structure, expression and function. The elucidation of many of the physiological effects of the molecules will be greatly accelerated by the isolation and characterization of additional polymorphic variants or of different species thereof. In particular, the present invention provides useful probes for identifying additional homologous genetic entities in different species. The isolated genes will allow the transformation of cells lacking the expression of an IL-BKW, for example, either types or cells of species lacking the corresponding proteins and exhibiting negative background activity. This should allow analysis of the function of IL-BKW compared to non-transformed control cells. It is possible to dissect critical structural elements, which affect the various physiological functions mediated through these antigens, using standard techniques of modern molecular biology, particularly when comparing members of the related class. See, for example, the peer-reviewed mutagenesis technique described in Cunningham, et al. (1989) Science 243: 1339-1336; and the methods used in O'Dowd, and others. (1988) J. Biol. Chem. 263: 15985-15992; and Lechleiter, and others. (1990) EMBO J. 9: 4381-4390. Intracellular functions will probably involve receptor signaling. However, protein internalization may occur under certain circumstances, and the interaction between intracellular components and the cytokine may occur. The specific segments of interaction of IL-BKW with the interacting components can be identified by mutagenesis or direct biochemical means, for example, cross-linking methods or affinity methods. Structural analysis by crystallographic methods or other physical methods will also be applicable. Further investigation of the signal translation mechanism will include the study of associated components which can be isolated by affinity methods or by genetic means for example, mutant complementation analysis. Further study of the expression and control of IL-BKW will be pursued. The controlling elements associated with the antigens must exhibit differential physiological, developmental, tissue-specific or other expression patterns. The genetic regions towards the 5 'end or towards the 3' end for example the control elements are of interest. Structural studies of IL-BKW antigens will lead to the design of new antigens, particularly analogs that exhibit agonist or antagonist properties on the molecule. This can be combined with previously described assessment methods to isolate antigens that exhibit the desired spectrum of activities.

V. Antibodies Antibodies can be created for several epitopes of IL-BKW proteins, including species, polymorphic, or allelic variants and fragments thereof, both in their naturally occurring forms or in their recombinant forms. Additionally, antibodies can be created for IL-BKW proteins either in their active forms or in their inactive forms, including native or denatured versions. Anti-idiotypic antibodies are also contemplated. Antibodies, including binding fragments and single chain versions, against predetermined fragments of the antigens can be created by immunizing animals with conjugates of the fragments with immunogenic proteins. The monoclonal antibodies are prepared from cells that secrete the desired antibody. These antibodies can be evaluated for their binding to normal or defective IL-BKW, or evaluated for their agonist or antagonist activity, for example mediated through a receptor. The antibodies can be agonists or antagonists, for example by steric blocking of receptor binding. These monoclonal antibodies will usually bind at least one KD of about 1 mM, more usually at least about 300 μM, typically at least about 100 μM, more typically at least 30 μM, preferably at least about 10 μM, and very preferred at least about 3 μM or better.

The antibodies of this invention may also be useful in diagnostic applications. As for the capture of non-neutralizing antibodies, these can be evaluated in terms of their ability to bind antigens without inhibiting receptor binding. As neutralizing antibodies, these may be useful in competitive binding tests.

These will also be useful for detecting or quantifying IL-BKW protein or its receptors. See for example, Chan (ed.) (1997) Immunologv: A Practical Guide, Academic Press, Orlando, Florida; Price and Newman (eds.) (1991) Principles and Practice of Immunoassav, Stockton Press, N.Y .; and Ngo (ed.) (1988) Nonisotopic Immunoassav, Plenum Press, N.Y. Cross-absorbance tests or other tests will identify antibodies that exhibit various spectra of specificities, eg, unique or shared species specificities. In addition, the antibodies, including the antigen-binding fragments, of this invention can be potent antagonists that bind to the antigen and inhibit functional binding, for example, to a receptor that can produce a biological response. These may also be useful as non-neutralizing antibodies and may be coupled to toxins or radionuclides so that when the antibody binds to the antigen, a cell expressing it, for example, on its surface, is annihilated. In addition, these antibodies can be conjugated with drugs or other therapeutic agents, either directly or indirectly through a linker, and can affect the purpose of the drug.

The antigen fragments may be linked to other materials, particularly polypeptides, in the form of covalently linked or fused polypeptides which are used as immunogens. An antigen and its fragments can be fused or covalently linked to a variety of immunogens, such as limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbioloqy, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificitv of Seroloqical Reactions, Dover Publications, New York; Williams, and others. (1967) Methods in Immunoloqy and Immunochemistry, vol. 1, Academic Press, New York; and Harlow and Lane (1988) Antibodies: A Laboratorv Manual. CSH Press, NY, for descriptions of polyclonal antisera preparation methods. In some cases, it is desired to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. One can find the description of preparation techniques of such monoclonal antibodies in for example Stites, and others (eds) Basic and Clinical Immunoloqy (4th ed.), Lange Medical Publications, Los Altos, CA, and the references cited therein.; Harlow, and Lane (1988) Antibodies: A Laboratorv Manual, CSH Press; Goding (1986) Monoclonal Antibodies: Principies and Practice (2nd ed.), Academic Press, New York; and particularly in Kohier and Milstein (1975) in Nature 256: 495-497, which discusses a method for the generation of monoclonal antibodies. Other appropriate techniques involve the in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively the selection of antibody libraries on phage or similar vectors. See Huse, and others (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda, "Science 246: 1275-1281; and Ward, and others (1989) Nature 341: 544-546. The polypeptides and antibodies of the present invention can be used with or without modification, including humanized or chimeric antibodies. Frequently, polypeptides and antibodies can be labeled by binding, either covalently or non-covalently, with a substance that provides a detectable signal. A wide variety of brands and conjugation techniques are known and are reported extensively in both patent and scientific literature. Appropriate labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent portions, magnetic particles, and the like. The patents that teach the use of such marks include E.U.A. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. In addition, recombinant immunoglobulins can be produced, see Cabiily, Patent E.U.A. No. 4,816,567; Moore, and others, patent E.U.A. 4,642,334; and Queen, and others. (1989) Proc. Nat'1 Acad. Sci. USA 86: 10029-10033. The antibodies of this invention can also be used for affinity chromatography in protein separation. Columns can be prepared where antibodies are attached to a solid support. See for example, Wilchek et al. (1984) Meth. Enzvmol 104: 3-55.

Antibodies raised against each IL-BKW will also be useful for creating anti-idiotypic antibodies. These will be useful for the detection or diagnosis of various immunological conditions related to the expression of the respective antigens.

SAW. Nucleic acids The peptide sequences described and the related reagents are useful for detecting, isolating or identifying a DNA clone encoding IL-BKW, for example, from a natural source. Typically, this will be useful for isolating a gene from a mammal, and similar procedures will be applied to separate genes from other species, for example, warm-blooded animals, such as birds and mammals. The cross-hybridization will allow the separation of IL-BKW from the same species, for example polymorphic variants or from other species. A number of different methods should be available to successfully isolate an appropriate nucleic acid clone. The purified protein or the defined peptides are useful for generating antibodies by standard methods, such as those described above. The synthetic peptides or the purified protein can be presented to an immune system to generate monoclonal or polyclonal antibodies. See for example Coligan (1991) Current Protocols in Immunoloav Wiley / Greene; and Harlow and Lane (1989) Antibodies: A Laboratorv Manual, Cold Spring Harbor Press.

For example, the specific binding composition could be used for the evaluation of an expression library made from a cell line expressing an IL-BKW. The evaluation of intracellular expression can be performed by various staining or immunofluorescence procedures. The binding compositions could be used to affinity purify or classify cells expressing a surface fusion protein. Peptide segments can also be used to predict the appropriate oligonucleotides to evaluate a library. The genetic code can be used to select the appropriate oligonucleotides useful as probes for evaluation. See for example, SEQ ID NO: 1 or 5. In combination with polymerase chain reaction (PCR) techniques, synthetic oligonucleotides will be useful for selecting the correct clones from a library. The complementary sequences will also be used as probes, primers, or antisense strands. Several fragments should be particularly useful for example, coupled with anchored vectors or PCR techniques complementary to poly-A or with DNA complementary to other peptides. This invention contemplates the use of isolated DNA or fragments thereof to encode a biologically active corresponding IL-BKW polypeptide that is particularly lacking in the portion encoding the 5 'untranslated portion of the described sequence. In addition, this invention covers recombinant or isolated DNA encoding a biologically active protein or polypeptide and which is capable of hybridizing under the appropriate conditions to the DNA sequences described herein. Said biologically active protein or polypeptide can be an intact antigen, or fragment, and have an amino acid sequence described in for example SEQ ID NO: 2 or 6, particularly a mature peptide of SEQ ID NO: 6, or a mature polypeptide secreted from SEQ ID NO: 2. In addition, this invention covers the use of recombinant or isolated DNA, or fragments thereof, which encode proteins that exhibit high identity to a secreted IL-BKW. The isolated DNA can have the respective regulatory sequences on the 5 'and 3' flanks, for example promoters, enhancers, poly-A addition signals and others. An "isolated" nucleic acid is a nucleic acid for example, an RNA or DNA or a mixed polymer, which is substantially separated from other components that naturally accompany it in a native sequence for example, ribosomes, polymerases and / or sequences flanking genomes from the species that originate them. The term encompasses a nucleic acid sequence that has been removed from its naturally occurring media, and which includes cloned or recombinant DNA isolates and chemically synthesized analogs or biologically analogs synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule. Generally, the nucleic acid will be in a vector or fragment of less than about 50 kb, usually less than about 30 kb, typically less than about 10 kb, and preferably less than about 6 kb. An isolated nucleic acid will generally be a homogeneous composition of molecules, but in some embodiments it will contain less heterogeneity. This heterogeneity is typically found at the ends of the polymer or in non-critical portions for a desired biological function or activity. A "recombinant" nucleic acid is defined either by its production method or its structure. In reference to its production method, for example, a product made by a process, in which the method uses recombinant nucleic acid techniques, for example involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, this may be a nucleic acid made by generating a sequence consisting of the fusion of two fragments that are not naturally contiguous with one another, but which are intended to exclude products of nature, for example, mutants that are they present naturally. Thus, for example, products made by transforming cells with any vector that occurs unnaturally are encompassed, as are nucleic acids comprising the sequences derived using any synthetic oligonucleotide method. This is done frequently to replace a codon with a redundant codon encoding the same amino acid or a conservative amino acid, while typically entering or removing a sequence recognition site. Alternatively, this is performed to join together nucleic acid segments of desired functions to generate an individual genetic entity that consists of a desired combination of functions not found in the commonly available natural forms. Restriction enzyme recognition sites are frequently the target of such artificial manipulations, but other site-specific targets can be incorporated by design, for example, promoters, DNA replication sites, regulatory sequences, control sequences or other useful features. A similar concept is intended for a recombinant polypeptide, for example, a fusion polypeptide. Specifically included are the synthetic nucleic acids which, by redundancy of the genetic code, encode polypeptides similar to the fragments of these antigens, and fusions of sequences from several different species or polymorphic variants. A "significant fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least about 22 nucleotides, ordinarily at least about 29 nucleotides, more frequent at least about 35 nucleotides, typically at less than about 41 nucleotides, usually at least about 47 nucleotides, preferably at least about 55 nucleotides, and in particularly preferred embodiments will be at least about 60 or more nucleotides, eg, 67, 73, 81, 89, 95, etc. A DNA encoding an IL-BKW protein will be particularly useful for identifying genes, mRNA species, and cDNAs that code for related or similar proteins, as well as DNAs that code for homologous proteins from different species.

Probably there are similar homologs in other species, including primates, rodents, canines, felines and birds. Several IL-BKW proteins must be homologous and are encompassed herein. However, even proteins that have a more distant evolutionary relationship with the antigen can be easily isolated under the appropriate conditions using these sequences if they are sufficiently homologous. Primate IL-BKW proteins are of particular interest. Recombinant clones derived from genomic sequences such as, for example, containing introns, will be useful for transgenic studies, including for example, transgenic cells and organisms and for gene therapy. See for example, Goodnow (1993) "Transgenic Animáis" in Roitt (ed.) Encvclopedia of Immunoloqy, Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science 256: 1392-1394; Kuhn, and others. (1991) Science 254: 707-710; Capecchi (1989) Science 244: 1288; Robertson (1987) (ed.) Teratocarcinomas and Embrvonic Stem Cells: A Practical Approach. IRL Press, Oxford; and Rosenberg (1992) J. Clinical Oncologv 10: 180-199.

Substantial homology for example, identity, in the context of nucleic acid sequence comparison means either that the segments, or their complementary strands, when compared, are identical when they are aligned optimally, with nucleotide insertions or deletions appropriate, in at least about 50% of the nucleotides, generally at least about 58%, ordinarily at least about 65%, frequently at least about 71%, typically at least about 77, usually at least about 85 %, preferably at least about 95 to 98% or more, and in particular embodiments, as high as about 99% or more of the nucleotides. Alternatively, substantial homology exists when the segments hybridize under conditions of selective hybridization, to a chain or its complement, typically using a sequence of IL-BKW, for example, in SEQ ID NO: 1, or 5. Typically, hybridization selective will be presented when there is at least about 55% identity over a stretch of at least about 30 nucleotides, preferably at least about 75% over a stretch of about 25 nucleotides, and more preferably at least about 90% over a stretch of approximately 20 nucleotides. See, Kanehisa (1984) Nuc. Acids Res. 12: 203-213. The length of the identity comparison, as described, can be over longer stretches, and in certain embodiments will be over a stretch of about 17 nucleotides, usually at least 28 nucleotides, typically at least 40 nucleotides and preferably at least about 75 to 100 or more nucleotides.

The astringent conditions, in terms of homology in the hybridization context, will be conditions of combined astringency of salt, temperature, organic solvents and other parameters, typically those controlled in the hybridization reactions. The astringent temperature conditions will usually include temperatures above about 30 ° C, usually above about 37 ° C, typically above 55 ° C, preferably above 70 ° C. The salt stringent conditions will ordinarily be less than about 1000 mM, usually less than 400 mM, typically less than about 250 mM, preferably less than 150 mM, including about 100, 50 or even 20 mM. However, the combination of the parameters is much more important than the measurement of any individual parameter. See, for example, Wetmur and Davidson (1968) J. Mol. Biol. 31: 349-370. IL-BKW from other mammalian species can be cloned and separated by hybridization between species of closely related species. The homology can be relatively low between species not very related, and therefore the hybridization of closely related species is advisable. Alternatively, the preparation of an antibody preparation exhibiting less species specificity may be useful in the expression of cloning methods.

VIL Elaborating IL-BKW; imitators The DNA encoding the IL-BKW or fragment thereof can be obtained by chemical synthesis, by evaluating cDNA libraries, or by evaluating genomic libraries prepared from a wide variety of cell lines or tissue samples. . See for example, Okayama and Berg (1982) Mol Cell. Biol. 2: 161-170; Guber and Hoffman (1983) Gene 25: 263-269; and Glover (ed.) (1984) ADN Cloninq: A Practical Approach, IRL Press, Oxford. Alternatively, the sequences provided herein provide useful PCR primers or allow for the synthetic preparation or other preparation of appropriate genes encoding an IL-BKW; Including the modalities that occur naturally. This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length IL-BKW or fragments that can in turn be used to generate polyclonal or monoclonal antibodies; for studies of union; for the construction and expression of modified molecules; and for structure / function studies. Vectors, as used herein, include plasmids, viruses, bacteriophages, integrable DNA fragments, and other vehicles that allow the integration of DNA fragments into the host's genome. See for example Pouweis, and others. (1985 and Supplements) Cloninq Vectors: A Laboratorv Manual, Elsevier, N.Y .; and Rodríguez, and others. (1988) (eds.) Vectors: A Survev of Molecular Cloninq Vectors and Their Uses, Buttersworth, Boston, MA.

For the purposes of this invention, the DNA sequences are operably linked when they are functionally related to one another. For example, the DNA for a presequence or secretory guide is operably linked to a polypeptide if it is expressed as a preprotein or participates by directing the polypeptide to the cell membrane or in the secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; A ribosome binding site is operably linked to a coding sequence if it is positioned to allow translation. Usually, operably linked means contiguous and in reading frame, however, in certain genetic elements such as repressor genes are not linked in a continuous manner but remain attached to the operator sequences which in turn control the expression. See for example, Rodríguez, and others., Cap. 10, pp. 205-236; Balbas and Bolívar (1990) Methods in Enzvmoloqy 185: 14-37; and Ausubel, and others. (1993) Current Protocols in Molecular Bioloqy, Greene and Wiley, NY. Representative examples of suitable expression vectors include pCDNAl; pCD, see Okayama and others. (1985) Mol. Cell Biol. 5: 1136-1142; pMCIneo Poli-A, see Thomas, and others. (1987) CEN 51: 503-512; and a baculovirus vector such as pAC 373 or pAC 610. See e.g., Miller (1988) Ann. Rev. Microbiol. 42: 177-199. Frequently it will be desired to express an IL-BKW polypeptide in a system that provides a specific or defined glycosylation pattern. See for example Luckow and Summers (1988) Bio / Technoloqy 6: 47-55; and Kaufman (1990) Meth. Enzymol. 185: 487-511. IL-BKW, or a fragment thereof, can be genetically engineered to be linked by phosphatidyl inositol (Pl) to a cell membrane, but can be removed from the membranes by treatment with a phosphatidylnositol digestion enzyme, for example phosphatidyl inositol phospholipase-C. It releases the antigen in a biologically active form, and allows purification by standard protein chemistry procedures. See for example, Low (1989) Biochim. Biophvs. Acta 988: 427-454; Tse, and others. (1985) Science 230: 1003-1008; and Brunner, and others. (1991) J. Cell Biol. 114: 1275-1283. Once the IL-BKW has been characterized, the fragments or derivatives thereof can be prepared by conventional methods for the synthesis of peptides. These include procedures such as those described in Stewart and Ypung (1984) Solid Phase Peptide Svnthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Svnthesis, Springer-Verlag, New York; Bodanszky (1984) The Principies of Peptide Svnthesis, Springer-Verlag, New York; and Villafranca (ed.) (1991) Techniques in Protein Chemistry II, Academic Press, San Diego, Ca.

VIII. Uses The present invention provides reagents that will find use in diagnostic applications as described elsewhere herein, for example, under conditions mediated by IL-BKW or later in the description of diagnostic equipment. This invention also provides reagents with significant therapeutic potential. IL-BKW (occurring naturally or recombinantly), fragments thereof and antibodies created by it, together with compounds that are identified as having binding affinity to IL-BKW, should be useful in the treatment of associated conditions with physiology or abnormal development, including inflammatory conditions. In particular, modulation of the physiology of lymphoid cells will be achieved by appropriate therapeutic treatment using the compositions provided herein, for example, a disease or disorder associated with abnormal expression or abnormal signaling by an IL-BKW, should be a likely target for an agonist or antagonist. The new cytokine should play a role in the regulation or development of hematopoietic cells, for example, lymphoid cells, which affect the immunological responses for example, inflammation and / or autoimmune disorders. In particular, the cytokine must mediate, in various contexts, the synthesis of cytokine by cells, proliferation, etc. Antagonists of IL-BKW, such as variants of mutein in a naturally occurring form of IL-BKW or blocking antibodies, can provide a selective and powerful way to block immune responses, for example, in situations such as inflammatory or autoimmune responses. including rheumatoid arthritis, systematic lupus erythematosis (SLE), Hashimoto autoimmune thyroiditis, as well as acute and chronic inflammatory responses, for example inflammatory bowel disease. See also Samter, and others (eds.) Immunoloqical Diseases vols. 1 and 2, Little, Brown and Co. The release of cytokine modulated by the naturally occurring secreted form of IL-BKW that can be produced in large quantities by recombinant methods, or by blocking antibodies, should be adjustable by reagents made available by the present, for example, in a transplant rejection situation. In addition, certain combination compositions would be useful, for example, with other inflammation modulators. Such other molecules may include steroids, other versions of IL-10, including variants of cellular species, or viral IL-10s, for example, EBV or EHV, and all their respective antagonists. Several abnormal conditions are known in each of the cell types shown that produce IL-BKW mRNA by Northern blot analysis. See Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J .; Thorn, and others Harrison's Principies of Intemal Medicine, McGraw-Hill, N.Y .; and Weatherall, et al. (eds.) Oxford Textbook of Medicine, Oxford University Press, Oxford. Many other medical conditions and diseases involve activation by macrophages or monocytes, and many of these will respond to treatment by an agonist or antagonist provided herein. See, for example, Stites and Terr (eds). 1991) Basic and Clinical Immunoloqy Appleton and Lange, Norwalk, Connecticut; and Samter, et al., (eds.) Immunoloqical Diseases Little, Brown and Co. These problems should be amenable to prevention or treatment using compositions provided herein. IL-BKW, antagonists, antibodies, etc., can be purified and then administered to a patient, animal or human. These reagents may be combined for therapeutic use with additional active or inert ingredients, for example, in conventional pharmaceutically acceptable carriers or diluents, for example, immunogenic adjuvants, together with physiologically harmless stabilizers, excipients, or conservatives. These combinations can be sterilized by filtering and placed in dosage forms by lyophilization in dosing or storage ampoules in stabilized aqueous preparations. This invention also contemplates the use of antibodies or binding fragments thereof, including forms that are not binding complements. Evaluation of the drug using IL-BKW or fragments thereof can be performed to identify compounds that have binding affinity to or other relevant biological effects on IL-BKW functions, including the isolation of associated components. Subsequent biological assays can then be used to determine whether the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in which it blocks the activity of the cytokine. Likewise, a compound having an intrinsic stimulating activity can activate the signal path and is therefore an agonist in that it mimics the activity of IL-BKW. This invention additionally contemplates the therapeutic use of blocking antibodies to IL-BKW as antagonists and of stimulatory antibodies as agonists. This method should be particularly useful with other variants of IL-BKW species. In addition, IL-BKW may play a role in leukemogenesis or in viral infections by, for example, HTLV or herpes virus. It is induced by infection with herpesvirus saimiri. Herpes viruses also encode a homologue of the cytokine IL-17 (CTLA-8) therefore, the cytokine, or antagonists, may be useful in antitumor therapy. The viral correlation may suggest that the cytokine may be important in viral infection or proliferation procedures, or oncological procedures, for example, oncogenic and proliferative transformation conditions, such as cancers or leukemias. See, for example, Thorn, and others. Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y. In addition, the cytokine appears to be expressed in the kidney cell, and may play a significant role in organ function, for example, the exchange of blood or the regulation of blood pressure. The cytokine can also have important water balance functions. The cytokine seems to have some expression in the kidney.

The amounts of reagents needed for effective therapy will depend on many different factors, including means of administration, target site, physiological state of the patient, and other medications administered. Therefore, treatment doses must be determined by titration to optimize safety and efficiency.

Typically, dosages used in vitro can provide useful guidance in amounts useful for the in situ administration of these reagents. Animal tests of effective dosages for the treatment of particular disorders will provide additional predictive indication for human dosing. Several considerations are described, for example in Gilman, et al. (Eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics. 8th. Ed., Pergamon Press; and Reminqton's Pharmaceutical Sciences, 17 ed. (1990), Mack Publishing Co., Easton, Penn. Methods for administration are discussed therein and then for example, for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others. Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, for example, in the Merck Index, Merck & Co., Rahway, New Jersey. Dosage scales will normally be expected to be in lower amounts than concentrations of 1mM, typically less than concentrations of 10 μM, usually less than 100 nM, preferably less than 10 pM (picomolar), and more preferably less than 1f (femtomolar) , with a suitable carrier. Slow release formulations, or a slow release device will often be used for continuous or long-term administration. See, for example, Langer (1990) Science 249: 1527-1533. IL-BKW, fragments thereof, and antibodies thereof or their fragments, antagonists, and agonists, can be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or albumin serum before administration. Therapeutic formulations can be administered in many conventional dosage formulations. Although it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. The formulations typically consist of at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not harmful to the patient. The formulations include those suitable for oral, rectal, nasal, topical, or parenteral administration (including subcutaneous, intramuscular, intravenous and intradermal administration). The formulations may be conveniently presented in the form of unit doses and may be prepared by any method well known in the art of pharmacy. See, for example Gilman, et al. (Eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Reminqton's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn .; Avis, et al., (Eds.) (1993) Pharmaceutical Dosaqe Forms: Tablets. Dekker, New York; and Lieberman, et al., (eds.) (1990) Pharmaceutical Dosaqe Forms: Disperse Systems, Dekker, New York. The therapy of this invention can be combined with or used in association with other agents, for example other types of IL-10s, or their respective antagonists. Both the recombinant and naturally occurring forms of the IL-BKWs of this invention are particularly useful in equipment and assay methods that are capable of evaluating compounds for binding activity to proteins. Several methods of automation testing have been developed in recent years to allow the evaluation of tens of thousands of compounds in a short period. See, for example, Fodor, et al. (1991) Science 251: 767-773, which describes means for evaluating binding affinity by a plurality of defined polymers synthesized on a solid substrate. The development of suitable assays can be greatly facilitated by the availability of large quantities of purified, soluble IL-BKW such as those provided by this invention. Other methods can be used to determine the critical residues in the interactions of IL-BKW-IL-BKW receptor. Mutational analysis can be performed, for example, see Somoza, et al. (1993) L Exptl. Med. 178: 549-558, to determine specific critical residues in the interaction and / or signaling. However, the residues in the helices A and D are also to be the most important in receptor interaction. For example, antagonists can normally be discovered once the antigen has been structurally defined, for example, by tertiary structure data. The evaluation of potentially interacting analogues is now possible with the development of highly automated assay methods using a purified IL-BKW. In particular, new agonists and antagonists will be discovered through the use of evaluation techniques described herein. Of particular importance are the discovered compounds that have a combined binding affinity for a spectrum of IL-BKW molecules, for example compounds that can serve as antagonists for variants of BKW species. A method of drug evaluation utilizes Eurocariotic or prokaryotic host cells that are stably transformed with recombinant DNA molecules that express an IL-BKW. Cells that express an IL-BKW in isolation from other molecules can be isolated. Such cells, either in a viable or fixed form, can be used for couple binding assays by standard binding. See also, Parce, et al. (1989) Science 246: 243-247, and Owicki, et al. (1990) Proc. Nat Acad. Sci. USA 87: 4007-4011. which describe sensitive methods for detecting cellular responses.

Another technique for drug evaluation includes a method that provides high evaluation performance for compounds that have adequate binding affinity to an IL-BKW and are described in detail in Geysen, European Patent Application 84/03564, September 13, 1994. First, large quantities of peptide test compounds of different sizes are synthesized on a solid substrate, for example plastic pins or some other suitable surface, see Fodor, and others ( 1991). Then all the pins are reacted with IL-BKW, solubilized, doped or solubilized, purified, and washed. The next step includes detecting IL-BKW binding. The rational drug design can also be based on structural studies of the molecular forms of IL-BKW and other effectors or analogues. The effectors may be other proteins that mediate other functions in response to binding, or other proteins that normally interact with IL-BKW, eg, a receptor. A means of determining which sites interact with other specific proteins is a determination of physical structure, for example, x-ray crystallography or 2-dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions, as they are molded, for example, against cellular IL-10. For a detailed description of protein structural determination, see, for example Blundell and Johnson (1976) Protein Crystallograbhv, Academic Press, New York.

IX. Equipment This invention also contemplates the use of IL-BKW proteins, fragments thereof, peptides, and their fusion products in a variety of diagnostic equipment and methods for detecting the presence of other IL-BKW or binding partner. Typically, the kit will have a compartment containing either a defined IL-BKW peptide or a gene segment or a reagent that recognizes one or the other, for example IL-BKW fragments or antibodies. A kit for determining the binding affinity of a test compound to an IL-BKW would typically consist of a test compound; a labeled compound, for example a binding partner or antibody having known binding affinity for IL-BKW; a source of IL.BKW (occurring naturally or recombinantly); and a means for separating links from freely labeled compounds, such as a solid phase to immobilize the molecule, once the compounds are evaluated, those having adequate binding affinity to the antigen can be evaluated in suitable biological assays, as are well known in the art. the technique, to determine if they act as agonists or antagonists to the signal path of IL-BKW. The availability of recombinant IL-BKW polypeptides also provides well-defined standards for the calibration of such assays. A preferred device for determining the concentration of, for example, an IL-BKW in a sample would typically consist of a labeled compound, for example binding partner or antibody, which have known binding affinity for the antigen, a strong cytoxine (which occurs naturally or recombinantly) and means for separating in bond of the free labeled compound, for example, a solid phase to immobilize IL-BKW. Compartments containing reagents, and instructions, will normally be provided. Antibodies, including antigen binding fragments, specific for IL-BKW or fragments are useful in the diagnosis of applications for detecting the presence of high levels of IL-BKW and / or its fragments. Such diagnostic assays can utilize lysates, living cells, fixed cells, proliferation, cell cultures, bodily fluids, and may additionally involve the detection of antigens related to serum antigen, or the like. Diagnostic tests can be homogeneous (without a separation step between the free reagent and the binding partner-antigen complex) or heterogeneous (with a separation step). Several commercial assays exist, such as radioimmunoassay (RIA), immunosorbent enzyme-linked assay (ELISA), enzyme immunoassay (EIA) multiplied enzyme immunoassay technique (EMIT), labeled substrate-labeled fluorescent immunoassay (SLFIA), and the like. See for example Van Vunakis, and others (1980) Meth Enzimol. 70: 1-525: Harlow and Lane (1980) Antibodies: A Laboratorv Manual, CSH Press, NY; and Coligan, et al. (eds.) (1993) Current Protocols in Immunoloqy, Greene and Wiley, NY.

Anti-idiotypic antibodies may have similar use to the presence of antibody diagnosis against an IL-BKW, and such may be the diagnosis for several abnormal states. For example, overproduction of IL-BKW can result in the production of various immunological reactions that can be diagnostic of abnormal physiological states, particularly in proliferative cell conditions such as cancer or abnormal activation or differentiation. Frequently, reagents for diagnostic tests are supplied in equipment, such as to optimize the sensitivity of the assay. For the subject invention, depending on the nature of the assay, the protocol and the label, either a labeled or unlabeled antibody or binding partner, or labeled IL-BKW, is provided. This is normally in conjunction with other additives, such as regulators, stabilizers, materials necessary for signal production as substrates for enzymes, and the like. Preferably, the equipment will also contain instructions for the proper use and disposal of the contents after use. Typically the equipment has compartments for each useful reagent. Desirably, the reagents are provided as a dry lyophilized powder, wherein the reagents can be reconstituted in a harassing environment providing adequate concentrations of reagents to perform the assay. Many of the aforementioned constituents of drug evaluation and diagnostic tests can be used without modification or can be modified in a variety of ways. For example, labeling can be achieved by covalently or non-covalently linking a portion that directly or indirectly provides a detectable signal. In any of these assays, the binding partner, the test compound, IL-BKW or antibodies thereto can be labeled either directly or indirectly. The possibilities for direct dialing include groups of brands: radio brands such as 125 ?, enzymes (Pat.

E.U. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (US Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, change in wavelength, or fluorescence polarization. Possibilities for indirect labeling include biotinization of a constituent followed by avidin binding coupled to one of the groups of earlier labels. There are also numerous methods for separating the binding of the free IL-BKW, or alternatively the binding of the free test compound. The IL-BKW can be immobilized on several matrices followed by washing. Suitable matrices include plastics such as an ELISA plate, filters, and pellets. See, for example, Coligan, et al. (Eds.) (1993) Current Protocols in Immunoloqy, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitable separation techniques include, without limitation, the magnetizable particle method of fluorecin antibody described in Rattie, et al. (1984) Clin. Chem. 30: 1457-1461, and the magnetic particle separation of double antibody as described in U.S. Patent No. 4,659,678.

Methods for linking proteins or subsfragments to different brands have been reported expressly in the literature and do not require detailed discussion here. Many of the techniques include the use of activated carboxyl groups either through the use of carbodimide or active esters to form peptide bonds, the formation of thioethers by the reaction of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin as maleimide, for chaining, or similar. The fusion proteins will also find use in these applications. Another diagnostic aspect of this invention includes the use of oligonucleotide or polynucleotide sequences taken from the sequence of an IL-BKW. These sequences can be used as probes to detect levels of the IL-BKW message in samples from patients who are suspected of having an abnormal condition, for example, inflammatory or autoimmune. Because the cytokine can be a marker or mediator for activation, it can be useful to determine the numbers of activated cells to determine, for example, when additional therapy is needed, for example, in a preventive mode before the effects become and progress to significant. The preparation of RNA and DNA nucleotide sequences, the labeling of the sequences and the preferred size of the sequences have received ample description and discussion in the literature. See, for example, Langer-Safer, et al. (1982) Proc. Nat'l. Acad. Sci. 79; 4381-4385; Caskey (1987) Science 236: 962-967; and Wilchek and others (1988) Anal. Biochem. 171: 1-32.

Diagnostic equipment that also tests the qualitative or quantitative expression of other molecules is also contemplated. The diagnosis or prognosis may depend on the combination of multiple indications used as markers. Therefore, teams can try combinations of markers. See, for example, Viallet, and others. (1989) Proqress in Growth Factor Res. 1: 89-97. Other equipment can be used to evaluate other subsets of cells.

X. Isolation of the IL-BKW receptor Having isolated a ligand from a specific ligand-receptor interaction, there are methods to isolate the receptor. See, Gearing, and others. (1989) EMBO J. 8: 3667-3676. For example, the means for labeling the IL-BKW cytokine without interfering with binding to its receptor can be determined. For example, an affinity tag may be fused to either the amino-or carboxyl-terminus of the ligand, although based on IL-10 the term "amino" is more likely to succeed. Such a marker can be a FLAG epitope tag, or, for example, an Ig or Fe domain. An expression library can be evaluated for specific binding of the cytokine, for example, by cell sorting or other evaluation to detect subpopulations that express such a binding component. See, for example, Ho, and others. (1993) Proc. Nat'l Acad. Sci. USA 90: 11267-11271; and Liu, and others. (1994) J. Immunol. 152: 1821-29. Alternatively, a panning method can be used. See, for example, Seed and Aruffo (1987) Proc. Nat'l. Acad.

Sci. USA 84: 3365-3369. The techniques of protein cross-linking with marker can be applied to isolate cytokine binding partners IL-BKW. This would allow the identification of proteins that interact specifically with the cytokine, for example, in a receptor-ligand-like manner. Initial experiments will be performed to determine if the known IL-10R is involved in the response (s) to IL-BKW.

It is also quite possible that the IL-10 functional receptor complex may share many or all of the components with an IL-BKW receptor complex, either a specific receptor subunit or an accessory receptor subunit. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is limited only by the terms of the appended claims, along with the full scope of the equivalents to which the claims are directed.

EXAMPLES GENERAL METHODS Some of the standard methods are described or referenced, for example, in Maniatis, et al., (1982) Molecular Cloninq, A Laboratorv Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, and others. (1989) Molecular Cloning: A Laboratorv Manual (2d ed.), Vols. 1-3, CSH Press, NY; Ausubel, et al., Bioloqy, Greene Publishing Associates, Brooklyn, NY; or Ausubel, and others. (1987 and Supplements) Current Protocols in Molecular Bioloqy, Greene and Wiley, New York; Innis, et al., (Eds.) (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, N.Y. Methods for protein purification include methods such as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization and others. See, for example, Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) "Guide to Protein Purification" in Methods in Enzymoloqy vol. 182, and other volumes in these series; and the manufacturer's literature on the use of protein purification products, for example, Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA. The combination with recombinant techniques allows fusion to suitable segments, for example, to a FLAG sequence or an equivalent that can be fused through a protease-removable sequence. See, for example, Hochuli (1989) Chemishe Industrie 12: 69-70; Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic Enqineerinq, Pinciple and Methods 12: 87-98, Plenum Press, N.Y .; and Crowe, and others. (1992) QIAexpress: The Hiqh Level Expression & Protein Purification Svstem QUIAGEN, Inc., Chatsworth, CA. Cell culture techniques are described in Doyle, et al. (Eds.) (1994) Cell and Tissue Culture: Laboratorv Procedures, John Wiley and Sons, NY. Normal immunological techniques are described, for example, in Hertzenber, et al., (Eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current Protocols in Immunoloqy and Wiley / Greene, NY; and Methods in Enzymoloqy volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163. FACS analyzes are described in Melamed, et al. (1990) Flow Cvtometrv and Sortinq Wiley-Liss, Inc. , New York, NY; Shapiro (1988) Practical Flow Cytometrv Liss, New York, NY; and Robinson, et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, NY. Fluorescent labeling of suitable reagents was performed by standard methods.

EXAMPLE 1 Cloning of human and mouse IL-BKW.

PBMC was prepared from a healthy human blood donor by conventional Ficoll gradients, as described, for example, in Coligan, and others. Current Protocols in Immunoloqy and Greene / Wiley. The cells, preferably monocytes, of this preparation are stimulated, for example, with PHA. RNA from these activated monocytes is used to isolate a cDNA based on the sequence information about mda7. PCR products are cloned using a cloning kit TA (Invitrogen). The resulting plasmid DNA is sequenced from both termini on an automated sequencer (Applied Biosystems). The mlL-BKW clone was isolated from an activated mouse thymocyte cDNA library of NK1.1 +, CD4 +. (A. Zlotnik, DNAX Research Institute, Palo Alto, CA). The sequencing of this clone revealed a high amino acid sequence identity to hIL-BKW as well as to mlL-10 and hIL-10. In particular, a high degree of identity between the four cytokines was discovered in the domain of the D-helix, involved in receptor binding. Example 2: Cellular Expression of Mammalian IL-BKW Due to the sequence similarity to human IL-10, the distribution will be investigated for similar types of cell type. A specific probe for coding primate IL-BKW cDNA is labeled, for example, by random initiation.

IL-BKW / ak155 is strongly transcribed in various tissues and T-cell clones. Southern Analysis: DNA (5 μg) of primary amplified cDNA library was digested with suitable restriction enzymes to release the inserts, run on an agarose gel at 1% and transferred to a nylon membrane (Schleicher and Schuell, Keene, NH). The Northern blots of multiple tissues were used to determine the size and tissue distribution of hIL-BKW mRNA. A number of mRNA transcripts, varying in size from 1.0 kb to 4.4 kb, were detected in most tissues. Based on the astringency of the hybridization and washing conditions of the spots, these transcripts rather represent mRNAs, separated and not members of different families. A similar Northern blot using cancer cell lines (Clontech) revealed a strongly hybridizing 2.2 RNA transcript in SW480 colorectal carcinoma, but was not present in any of the other cancer lines. The expression profile of hIL-BKW in monocytes / macrophages, dendritic, T, B, and NK cells was analyzed by Southern blot hybridization of cDNA library. The hIL-BKW probe was hybridized to 5μg of each cDNA library following digestion with NotI and SalI to release the cDNA inserts of the pSport vector (Gibco-BRL). Among T, B, and NK cells, expression of hIL-BRW was restricted to an activated ThO clone (Mot72) and to an activated Th1 clone (HY06). By far the highest level of hIL-BKW expression was observed in elutriated monocytes activated with LPS, IFN-α, and anti-IL-10 antibodies. In contrast, when a library made from these same monocytes treated with IL-10 was probed instead of anti-IL10, an acute decrease in the level of IL-BKW expression was observed, suggesting that the expression of IL-BKW it is strongly regulated by IL-10. Samples for mRNA isolation for mlL-BKW analysis include: mouse fibroplastic L cell line (C200) at rest; transfected cells Braf: ER (fusion of Braf to estrogen receptor), control (C201); T cells, TH1 polarized (Mel14 bright, CD4 + spleen cells, polarized for 7 days with IFN-α and anti-IL-4, T200); T cells, TH2 polarized (bright Mell4, CD4 + baso cells, polarized for 7 days with IL-4 and anti-IFN- ?; T201); highly polarized T cells, TH1 (see Openshaw, et al., (1995) J. Exp. Med, 182: 1357-1367; activated with anti-CD3 for 2, 6, 16 hours mixed; T202); highly polarized T2 cells, TH2 (see Openshaw, et al., (1995) J. Exp. Med. 182: 1357-1367; activated with anti-CD3 for 2, 6, 16 h mixed; T203); CD44-CD25 + pre T cells, selected from thymus (T204); T1 cell clone D1.1, at rest for 3 weeks after the last stimulation with antigen (T205); TH1 cell D1.1 clone, 10 μg / ml ConA stimulated 15 h (T206); TH2 cell CDC35 clone, at rest for 3 weeks after the last stimulation with antigen (T207); TH2 cell CDC35 clone, 10μg / ml ConA stimulated 15 h (T208); Mel 14+ simple spleen T cells, at rest (T209); Mel14 + T cells, polarized to TH1 with IFN -? / IL-12 / anti-IL-4 for 6, 12, 24 hrs mixed (T210); Mel14 + T cells, polarized to TH2 with IL-4 / anti-IFN-? for 6, 13, 24 hrs mixed (T211); mature B cell without stimulation of line A20 of leukemia cell (B200); B cell unstimulated CH12 line (B201); large B cells without spleen stimulation (B202); total spleen B cells, activated LPS (B203); enriched dentritic cells of spleen metrizamide, at rest (D200); dendritic cells of bone marrow, at rest (D201); RAW line 264.7 of cell monocyte activated with LPS 4 h (M200); bone marrow macrophages derived with GM and M-CSF (M201); cell line macrophage J774, at rest (M202); cell line macrophage J774 + LPS + anti-IL-10 at 0.5, 1, 3, 6, 12 h mixed (M203); cell line macrophage J774 + LPS + IL-10 at 0.5 1, 3, 5, 12 h mixed (M204); mouse lung tissue attacked with aerosol, initiated with Th2, attacked with OVA aerosol 7, 14, 23 h mixed (see Garlisi, et al. (1995) Clinical Immunoloqy and Immunopatholoqy 75: 75-83; X206); Lung tissue infected by Nippostrongulus (see Coffman, et al. (1989) Science 245: 308-310; X200); Total adult lung, normal (O200); total lung, rag-1 (see Schwarz, et al. (1993) Immunodeficiencv 4: 249-252; O205); IL-10 K.O. spleen (see Kuhn, et al. (1991) Cell 75: 263-274; X201); total adult spleen, normal (O201); total spleen, rag-1 (0207); IL-10 K.O. Peyer's plates (O202); Peyer's total plates, normal (0210); IL-10 K.O. nodules of mesenteric lymph (X203); total mesenteric lymph nodes, normal (0211); colon IL-10 K.O. (X203); Total colon, normal (0212); NOD mouse pancreas (see Makino, et al. (1980) Jikken Dobutsu 29: 1-13; (X205); total thymus, rag-1 (O208); total kidney rag-1 (O209); total heart rag-1 ( O202), rag-1 total brain (O203), rag-1 total testes (O204), rag-1 total liver (O206), rat normal joint tissue (O300), and arthritic rat joint tissue (X300) Expression by Southern analysis of cDNA was very high in polarized TH2 T cells (bright Mel14, spleen CD4 + cells, polarized for 7 days with IL-4 and anti-IFN- ?; T201), high in T cells, highly TH2 polarized (see Openshaw, et al. (1995) J. Exp. Med. 182: 1357-1367; activated with anti-CD3 for 2, 6, 16 h mixed; T203); and significant in: polarized TH1 cells (Mel14). bright, spleen CD4 + cells, polarized for 7 days with IFN-γ and anti IL-4, T200), and T cells Mel14 +, polarized to Th2 with IL-4 / anti-INF-? for 6, 13, 24 h mixed (T211).

EXAMPLE 3 Mapping of mouse and human IL-BKW chromosome The mlL-BKW gene was mapped to mouse chromosome 1 using procedures well known in the art. See, for example, Copeland, et al., (1193) Science 262: 57-66. mlL-10 maps directly adjacent to mlL-BKW on chromosome 1. To map the human counterpart, an isolated human IL-BKW cDNA encoding IL-BKW is used. Chromosome mapping is a standard technique as described above. See, for example, BIOS Laboratories (New Haven, CT) and methods for using a hybrid panel of mouse somatic cell with PCR. The human gene has been mapped to human chromosome 1, and is similarly, from synthetic mouse information, to the 1q32 region.

EXAMPLE 4 Protein purification IL-BKW - Multiple lines of transfected cells are evaluated for one that expresses the cytokine at a high level compared to other cells. 1 Several cell lines are evaluated and selected for their favorable handling properties. Natural IL-BKW can be isolated from natural sources, or by expression of a transformed cell using a suitable expression vector. The purification of the expressed protein is achieved by standard procedure, or it can be combined with engineering means for effective purification at high efficiency from cell lysates or superfluids. The FLAG or His6 segments can be used for such purification characteristics. Alternatively, affinity chromatography can be used with specific antibodies, see below. The protein is produced in coli, insect cell, or mammalian expression systems. The production of mouse fusion protein as an IgG fusion, with an IGase cleavage site, in COP5 cells resulted in secreted product that was N-terminally blocked. Microsequencing analysis indicated that the current N-terminus was QEF the production of similar material with human sequence provides an N-terminus of AQG The recombinant human IL-BKW produced from a standard construct in mammalian cells seems to be found only in small amounts as a soluble form in the medium. There is evidence that the protein binds tightly to the surface of the proteiccane cell, and is difficult to solubilize. The treatment of eparin causes some release of this protein. Proteoglycan surface deficient cells can be used to produce the protein.

EXAMPLE 5 Isolation of IL-BKW genes homologs The IL-BKW cDNA can be used as a hybridization probe to evaluate a library from a desired source, eg, a primate cell cDNA library. Many different species can be evaluated for both the astringency needed for easy hybridization, and for the presence using a probe. Suitable hybridization conditions will be used to select clones that exhibit specificity of between hybridization. Evaluation by hybridization using degenerative probes based on the peptide sequence will also allow the isolation of the appropriate clones. Alternatively, the use of suitable primers for PCR evaluation will yield enrichment of suitable nucleic acid clones. Similar methods are applicable to isolate either species, polymorphic, or allelic variants. Species variants are isolated using cross-species hybridization techniques based on isolation of a full-length isolate or fragment of a species such as a probe. Alternatively, antibodies raised against human IL-BKW will be used to evaluate cells expressing proteins between reactants from a suitable, eg, cDNA library. The purified protein or defined peptides are useful for generating antibodies by standard methods, as described above. The synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, for example, Coligan (1991) Current Protocols in Immunoloqy and Wiley / Greence; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press. The resulting antibodies are used for evaluation, purification, or diagnosis, as described.

EXAMPLE 6 Preparation of antibodies specific for IL-BKW The synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, for example, Coligan (1991) Current Protocols Immunoloqy and Wiley / Greene; Y Harlow and Lane (1989) Antibodies: A Laboratorv Manual Cold Spring Harbor Press. Polyclonal serum, or hybridomas can be prepared. In suitable situations, the binding reagent is either labeled as described above, for example, by fluorescence or otherwise, or immobilized to a substrate for panning methods. A number of monoclonal antibodies was obtained. Of those who were purified; half were IgG and the other half IgM. Most of these were good Western blot reagents. Several of the others of recombinant strain hIL-BKW were expressed on the surface of the cell, and others were suitable for immunohistochemistry. The "positive" cells seen through the latter in tonsil sections appear to be macrophage type.

EXAMPLE 7 Expression of IL-BKW regulated by IL-10 To investigate the regulatory effect of IL-10 directly by Northern blot analysis the mRNA was prepared from elutriated activated monocytes LP and IFN- ?, treated with either IL-10 or an anti-IL-10 antibody. Just as it was measured by Southerns blots of the cDNA libraries, a strong induction of anti-IL-10 was observed in activated monocytes treated with IL-10 while the treatment of those cells under identical conditions in the presence of IL-10 almost turned off completely the synthesis of the IL-BKW message.

EXAMPLE 8 Evaluation of extension of biological functions The biological activities of IL-BKW were tested based on the sequence and structural homology between IL-BKW and IL-10. Initially, trials that had shown biological activities of IL-10 were examined. These include assays on human peripheral blood mononuclear cells, human monocytes and human T cell clones. A) Effects on the expression of cell surface molecules on human monocytes. Monitos were purified by negative selection of peripheral blood mononuclear cells from normal healthy donors. Briefly, 3 x 108 ficol band mononuclear cells were incubated on ice with a mixture of monoclonal antibodies (Becton-Dickenson, Mountain View, CA) consisting of 200 μl of aCD2 (Leu-5A), 200 μl of CD3 (Leu-5A). 4), 100 μl of aCD8 (Leu 2a), 100 μl of CD19 (Leu-12), 100 μl of CD20 (Leu-16), 100 μl of aCD56 (Leu-19), 100 μl of CD67 (IOM 67) (Immunotech, Westbrook, ME), and anti-glycophorin antibody (10F7MN, ATCC, Rockville, MD). The antibody cells were washed and then incubated with sheep anti-mouse IgG coupled magnetic beads (Dynal, Oslo, Norway) at a cell-to-cell ratio of 20: 1. The antibody cells were separated from monocytes by application of a magnetic field. Subsequently, human monocytes were cultured in Yssel's medium (Gemini Biproducts, Calabasas, CA) containing 1% AB serum in the absence or presence of IL-BKW (baculovirus material expressed at 1/100 dilution or 1 μg / ml of mammalian cop 5 expression material) or IL-10 (200 U / ml) in polypropylene plates of 96 wells (Costar) for 40 hours. In addition identical cultures were established in the presence of IFN-? (100 U / ml). Analysis of the expression of cell surface molecules was performed by direct fluorescence immunoassay. Briefly, 2 x 105 purified human monocytes were incubated in phosphate buffered saline (PBS) containing 1% human serum on ice for 20 minutes. The cells were made pill at 200 x g. The cells were resuspended in 20 ml of PE or FITC labeled mAb. Following an additional 20 minute incubation on ice, the cells were washed in PBS containing 1% human serum followed by two washes only in PBS. The cells were fixed in PBS containing 1% paraformaldehyde and analyzed on a FACScan flow cytometer (Becton Dickenson, Mountain View, CA) and the results are expressed as the main fluorescence intensity in Table 3. The following mAbs were used: CD11 b (anti-mad), CD11c (a gpl 50/95), CD14 (Leu-M3), CD54 (Leu 54), CD80 (anti-BB1 / B7), HLA-DR (L243) from Becton Dickenson and CD86 (FUN 1) (Pharmingen), CD64 (32.2) (Medarex), CD40 (mAb89) (Schering-Plow France).

TABLE 3 Effects of IL-BKW on the cell surface phenotype of human monocytes Exp 1 CD11 B CD14 CD54 CD80 CD86 HLA-DR CD40 Med- 261 976 159 16/33 395 1522 251 IL-BKW 202 1850 195 39/69 217 495 249 IFN- 307 365 262 93/87 277 3688 697 IFN + IL-BKW 222 275 342 97/268 375 4157 1299 Exp 2 t = 0 668 1414 37 1 58 805 50 med- 293 1455 239 41/32 202 3130 152 IL-BKW 446 607 325 91/66 299 2190 123 IFN- 513 557 605 96/88 227 5664 481 IFN + IL-BKW 485 529 633 98/181 338 7318 702 IL-BKW improved the expression of D54 (ICAM-1), CD80, and CD86, while the expression of HLA-DR decreased (see Table 3). IL-BKW further improved the positive regulation of CD40, CD54, CD80, and CD86 by IFN- ?. In the presence of IFN- ?, IL-BKW improved the expression of HLA-DR. A variable effect of IL-BKW was only observed on the expression of CD11b, CD11c, and CD14, while in combination with IFN-? reduced cell surface expression compared to IFN-? alone. Because IL-10 negatively regulates CD54, CD80, CD86, and HLA-DR, both in the presence or absence of IFN- ?, these results indicate that IL-20 has different effects on the expression of cell surface molecules on monocytes compared to IL-10. B.- Effects of IL-BKW on cytokine production by human monocytes. Human monocytes were isolated as described and cultured in Yssel's medium (Gemini Bioproducts, Calabasas, CA) containing 1% human serum. AB in the absence or presence of IL-BKW (baculovirus material expressed with 1/100 dilution). In addition, monocytes were stimulated with the indicated amounts of LPS (E. coli 0127: Difco B8) in the absence or presence of IL-BKW for 24 hours and the concentration of cytokines (IL-1 b, IL-6, TNFa, GM -CSF, and IL-10) in the superplant in the cell culture was determined by ELISA. For intracytoplasmic staining by cytokines, monocytes were cultured (1 million / ml) in Yssel's medium in the absence or presence of IL-BKW (baculovirus material expressed with 1/100 dilution or expressed mammalian cop 5 material of 1 mg / ml) and / or 5 mg / ml LPS (E. coli 0127: Difco B8) and 10 mg / ml Brefeldin A (Epientre technologies Madison Wl) for 12 hours. The cells were washed in PBS and incubated in 2% formaldehyde / PBS solution for 20 minutes at RT. Subsequently the cells were washed, resuspended in permeabilization buffer (0.5% saponin (Sigma) in PBS / BSA (0.5%) / Acidic (1 mM) and incubated for 20 minutes at RT. The cells (2 x 105) were centrifuged and resuspended in 20 ml of conjugated anti-cytokine mAbs directly diluted 1: 10 in permeabilization buffer for 20 minutes at RT. The following antibodies were used: IL-1a PE (364-3B3-14); IL-6-PE (MQ2-13A5); TNFa- PE (Mab11); GM-CSF-PE (BVD2-21C11); and IL-12-PE (C11.5.14) (Pharmingen San Diego, CA). Subsequently, the cells were washed twice in permeabilization buffer and once in PBS / BSA / Acidic and analyzed on FACScan flow cytometer (Becton Dickenson; Mountain View, CA). The results are expressed as percentage of positive cells and main fluorescence intensity of the positive population in table 4.

TABLE 4 Effects of IL-BKW on cytokine production by human monocytes (% intracellular staining / main fluorescence intensity) IL-1 IL-6 TNF Medium 5 5 4 IL-BKW (1/100) 79/300 67/182 44/627 LPS (5 μg / ml) 67/456 78/404 78/1524 LPS + IL-BKW 77 / 613 75/458 77/1573 IL-1 IL-6 TNF Medium 9/58 39/65 7/159 IL-BKW (1 μg / ml) 57/93 93/575 70/547 LPS (5 μg / ml) 77/174 94/716 79/498 LPS + IL-BKW 79/172 96/1106 81/725 IL-BKW induced the expression of IÑ-6, TNFα, IL-10, IL-1b, and GM-SF by human monocytes (Table 5). LPS also induced the expression of these cytokines in a dose-dependent manner. The addition of IL-BKW in combination with LPS resulted in an improved production of IL-6, TNFa, IL-10, IL-1b, and GM-CSF compared to either LPS or IL-BKW alone.

TABLE 5 Effects of IL-BKW on cytokine production by human monocytes IL-1 b-IL-6 TNF IL-10 Gl CSF Medium 0 2.229 0 0.007 0 LPS (ng / ml) 0.321 29 4.735 0.867 0.828 1000 100 0 36.78 4,683 1,417 0.371 10 0 37.06 2,156 1,216 0.080 1 0 36.22 0.806 0.918 0 IL-BKW 1,460 27.9 1,808 0.391 0.673 (1/100) + LPS 4.637 93.02 12.09 2.332 2.048 1000 100 3.921 82.56 9.843 3.994 1.309 10 4.024 142.2 5.708 3.096 1.466 1 2.744 85.66 4.006 2.58 0.852 (results expressed in ng / ml) Similar results were obtained when cytokine production was calculated by intracytoplasmic staining. IL-BKW induced the expression of IL-1, IL-6, and TNFa by human monocytes as indicated by an increase in the percentage of positive cells as well as by fluorescence intensity, which reflects the amount of cytokine produced per cell . LPS induced the expression of IL-1, IL-6, and TNFa; and the addition of IL-BKW to these crops further increased this production. This improved production was observed for baculovirus and expressed mammalian material. See table 4 C- Effects of IL-BKW on cytokine production by human peripheral blood mononuclear cells (PBMC). Total PBM was isolated from skin coatings of normal healthy donors by centrifugation through ficoll-hypaque as described (Boyum, et al.) And cultured in Yssel's medium (Gemini Bioproducts, Calabasas, CA) containing 1% human AB serum in the absence or presence of IL-BKW (baculovirus material expressed with 1/100 dilution). The cells were incubated at 2 x 106 cells / ml in medium or activated by PHA (100 ng / ml) or IL-2 (100 U / ml) (R &D Systems). In addition, PBMC were cultured with IL-10 alone (100 U / ml) or IL-10 in combination with IL-BKW (baculovirus material expressed at 1/100 dilution). Cytokine secretion was determined in superplant cultures harvested at 72 hours by specific cytokine ELISA. IL-BKW induced the expression of IL-6, TNFa, IL-10, and IFN-? by PBMC (see table 6). In addition, IL-BKW improved the production of IL-6, TNFa, IL-10, and IFN-? when PBMC was activated by PHA or IL-2. IL-10 inhibited the production of IL-6, TNFa, IL-10, and IFN-? following activation of PBMC by IL-BKW, PHA, IL-2 or combinations of IL-BKW with either PHA or IL-2.

TABLE 6 Effects of IL-BKW on cytokine production by PBMC PBMC IL-6 TNFa IL-10 IFN-? Medium 0.243 0 0.007 0 IL-10 (100 U / ml) 0.196 0 NT 0 IL-BKW (1/100) 24.23 0.029 1.295 0.004 IL-10 + IL-BKW 0.339 0 NT 0 PHA (100 ng / ml) 5,491 0.652 0.637 1.05 IL-10 0.279 0 NT 0.004 IL-BKW 67.06 3.31 4.556 5.742 IL-10 + IL-BKW 1.129 0 NT 0.1 IL-2 (100 U / ml) 41.4 0.216 3.224 0.895 IL-10 0.89 0 NT 0.01 IL-BKW 62.34 0.621 10.04 1.507 IL-10 + IL-BKW 3,456 0 NT 0.081 Results expressed in ng / ml; NT: not tested.

D.- Effects of IL-BKW on the proliferation of human peripheral blood mononuclear cells (PBMC). Total PBMC was isolated from skin coatings of normal healthy donors by centrifugation through ficoll-hypaque as described (Boyum, et al.). PBMC were cultured in 200 ml of medium from Yssel (Gemini Bioproducts, Calabasas, CA) containing 1% human AB serum in 96 well plates (Falcon, Becton, Dickenson, N.J.) in the absence or presence of indicated amounts of IL-BKW (material expressed from mammalian cop 5). The cells were cultured only in medium or in combination, with 100 U / ml IL-2 (R &D Systems) for 120 hours. 3H-Thimidine (0.1 mCi) was added during the last 6 hours of culture and the incorporation of 3H-Thimidine was determined by liquid scintillation counting. The results are expressed as the main cpm of triplicate cultures. IL-BKW induces a low level of PBMC proliferation in a dose-dependent manner. See Table 7. In addition, IL-BKW also improves in a dose dependent manner the proliferative response of PBMC to IL-2.

TABLE 7 Effect of IL-BKW on proliferation by PBMC IL-BKW SD + IL-2 SD MAIN MAIN (ng / ml) (100 U / ml) 1000 5894 2655 43426 12858 333 6430 1877 48187 2470 111 3510 1384 36435 5058 37 1016 331 24859 6014 12 235 60 22738 3379 4 190 75 23323 4266 1.3 175 49 22528 2979 0 100 4 17961 3839 The results are expressed as standard deviation cpm +/- Original, recombinant, and fusion proteins would be tested for agonist and antagonist activity in many other biological assay systems, eg, on T cells, B cells, NK, macrophages, cells dendritic, hematopoietic progenitors, etc. Due to the IL-10 structural relationship, trials related to activity 11-10 would be analyzed. IL-BKW is evaluated for agonist or antagonist activity on transfected cells expressing IL-10 receptor and controls. See, for example, Ho, and others. (1993) Proc. Nat 'I Acad. Sci. USA 90. 11267-11271; Ho, and others, (1995) Mol. Cell. Biol. 15: 5043-5053; and Liu, and others. (1994). J-lmmunol. 152: 1821-1829. Based in part on structural homology to IL-10, IL-BKW is evaluated for effect on macrophage / dendritic cell activation and antigen presentation assays, T-cell cytokine production and proliferation in response to antigen stimuli. alogenics See, for example, Waal Malefyt et al. (1991) J. Exp. Med. 174: 1209-1220; from Waal Malefyt and others. (1991) J. Exp. Med. 174: 915-924; Fiorentino, and others. (1991) J ^ Immunol. 147. 3815-3822; Fiorentino, and others. (1991) J. Immunol. 146: 3444-3451; and Groux, and others. (1996) J. Exp. Med. 184: 19-29. IL-BKW will also be evaluated for the effects on NK cell stimulation. Tests may be based, for example, on Hsu, et al. (1992) Internat, Immunol. 4: 563-569; and Schwarz, and others. (1994) J. Immunother. 16: 95-104. The culture and effects of B cell differentiation will be analyzed, for example, by the methodology described, for example, in Defrance, et al. (1992) J. Exp. Med. 175: 671-682; Rousset, and others (1992) Proc. Nat 'I Acad. Sci. USA 89: 1890-1893: including switch factor assays lgG2 and lgA2. Note that, unlike the COS7 superfluouss, the superfluous NIH3T3 and COP apparently do not interfere with the human B cell assays. All references cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all objects. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of examples only, and the invention should be limited only by the terms of the appended claims, together with the full scope of their equivalents to which such claims are directed.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Schering Corporation (ii) TITLE OF THE INVENTION: Mammalian Citicine; Reagents Related (iii) NUMBER OF SEQUENCES: 7 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Schering Plow Corporation (B) STREET: 200 Galloping Hill Road (C) CITY: Kenilworth (D) STATE: NJ (E) COUNTRY: USA (F) POSTAL CODE: 07033 (v) READABLE FORM IN COMPUTER: (A) TYPE OF MEDIA: Soft disk (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: Macintosh 7.1 (D) SOFTWARE: Microsoft Word 7.5.3 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: PCT (B) APPLICATION DATE: 22-DEC-1997 (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US60 / 034,151 (B) DATE OF APPLICATION: DEC. 23, 1996 (vii) DATA FROM THE PREVIOUS APPLICATION: (A) NUMBER OF APPLICATION: US 08 / 842,659 (B) DATE OF APPLICATION: APR-15-1997 (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: FOULKE, CYNTHIA L. (B) REGISTRATION NUMBER: 32,364 (C) REFERENCE NUMBER / CASE: DX0683K (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: (908) 298-2987 (B) TELEFAX: (908) 298-5388 (2) INFORMATION FOR SEC ID. No. 1 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1700 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: Single (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: cDNA ( ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 275 ... 892 (ix) CHARACTERISTICS: (A) NAME / KEY: mat-peptide (B) LOCATION: 419 ... 892 (xi) SEQUENCE DESCRIPTION: ID. SEC. No. 1: CTTGCCTGCA AACCTTTACT TCTGAAATGA CTTCCACGGC TGGGACGGGA ACCTTCCACC 60 CACAGCTATG CCTCTGATTG GTGAATGGTG AAGGTGCCTG TCTAACTTTT CTGTAAAAAG 120 AACCAGCTGC CTCCAGGCAG CCAGCCCTCA AGCATCACTT ACAGGACCAG AGGGACAAGA 180 CATGACTGTG ATGAGGAGCT GCTTTCGCCA ATTTAACACC AAGAAGAATT GAGGCTGCTT 240 GGGAGGAAGG CCAGGAGGAA CACGAGACTG AGAG ATG AAT TTT CAA CAG AGG 292 Met Asn Phe Gln Gln Arg -48 -45 CTG CAA AGC CTG TGG ACT TTA GCC AGA CCC TTC TGC CCT CCT TTG CTG 340 Leu Gln Ser Leu Trp Thr Leu Ala Arg Pro Phe Cys Pro Pro Leu Leu -40 -35 -30 GCG ACA GCC TCT CAA ATG CAG ATG GTT GTG CTC CCT TGC CTG GGT TTT 388 Ala Thr Ala Ser Gln Met Gln Met Val Val Leu Pro Cys Leu Gly Phe -25 -20 -15 ACC CTG CTT CTC TGG AGC CAG GTA TCA GGG GCC CAG GGC CAA GAA TTC 436 Thr Leu Leu Leu Trp Ser Gln Val Ser Gly Wing Gln Gly Gln Glu Phe -10 -5 1 5 CAC TTT GGG CCC TGC CAA GTG AAG GGG GTT CCC CAG AAA CTG TGG 484 His Phe Gly Pro Cys Gln Val Lys Gly Val Val Pro Gln Lys Leu Trp 10 15 20 GAA GCC TTC TGG GCT GTG AAA GAC ACT ATG CAA GCT CAG GAT AAC ATC 532 Glu Wing Phe Trp Wing Val Lys Asp Thr Met Gln Wing Gln Asp Asn lie 25 30 35 ACG AGT GCC CGG CTG CTG CAG CAG GAG GTT CTG CAG AAC GTC TCG GAT 580 T r Ser Wing Arg Leu Leu Gln Gln Glu Val Leu Gln Asn Val Ser Asp 40 45 50 GCT GAG AGC TGT TAC CTT GTC CAC ACC CTG CTG GAG TTC TAC TTG AAA 628 Wing Glu Ser Cys Tyr Leu Val His Thr Leu Leu Glu Phe Tyr Leu Lys 55 60 65 70 ACT GTT TTC AAA AAC TAC CAC AAT AGA ACÁ GTT GAA GTC AGG ACT CTG 676 Thr Val Phe Lys Asn Tyr His Asn Arg Thr Val Glu Val Arg Thr Leu 75 80 85 AAG TCA TTC TCT ACT CTG GCC AAC AAC TTT GTT CTC ATC GTG TCA CAA 724 Lys Ser Phe Ser Thr Leu Wing Asn Asn Phe Val Leu lie Val Ser Gln 90 95 100 CTG CAA CCC AGT CAA GAA AAT GAG ATG TTT TCC ATC AGA G AC AGT GCA 772 Leu Gln Pro Ser Gln Glu Asn Glu Met Phe Ser lie Arg Asp Ser Wing 105 not H5 CAC AGG CGG TTT CTG CTA TTC CGG AGA GCA TTC AAA CAG TTG GAC GTA 820 His Arg Arg Phe Leu Leu Phe Arg Arg Wing Phe Lys Gln Leu Asp Val 120 125 130 GAA GCT GCT CTG ACC AAA GCC CTT GGG GAA GTG GAC ATT CTT CTG ACC 868 Glu Ala Ala Leu Thr Lys Ala Leu Gly Glu Val Asp lie Leu Leu Thr 135 140 145 150 TGG ATG CAG AAA TTC TAC AAG CTC TGAATGTCTA GACCAGGACC TCCCTCCCCC 922 Trp Met Gln Lys Phe Tyr Lys Leu 155 TGGCACTGGT TTGTTCCCTG TGTCATTTCA AACAGTCTCC CTTCCTATGC TGTTCACTGG 982 ACACTTCACG CCCTTGGCCA TGGGTCCCAT TCTTGGCCCA GGATTATTGT CAAAGAAGTC 1042 ATTCTTTAAG CAGCGCCAGT GACAGTCAGG GAAGGTGCCT CTGGATGCTG TGAAGAGTCT 1102 ACAGAGAAGA TTCTTGTATT TATTACAACT CTATTTAATT AATGTCAGTA TTTCAACTGA 1162 AGTTCTATTT ATTTGTGAGA CTGTAAGTTA CATGAAGGCA GCAGAATATT GTGCCCCATG 1222 CTTCTTTACC CCTCACAATC CTTGCCACAG TGTGGGGCAG TGGATGGGTG CTTAGTAAGT 1282 ACTTAATAAA CTGTGGTGCT TTTTTTGGCC TGTCTTTGGA TTGTTAAAAA ACAGAGAGGG 1342 ATGCTTGGAT GTAAAACTGA ACTTCAGAGC ATGAAAATCA CACTGTCTGC TGATATCTGC 1402 AGGGACAGAG CATTGGGGTG GGGGTATAGGT GCATCTGTTT GAAAAGTAAA CGATAAAATG 1462 TGGATTAAAG TGCCCAGCAC AAAGCAGATC CTCAATAAAC ATTTCATTTC CCACCCACAC 1522 TCGCCAGCTC ACCCCATCAT CCCTTTCCCT TGGTGCCCTC CTTTTTTTTT TATCCTAGTC 1582 ATTCTTCCCT AATCTTCCAC TTGAGTGTCA AGCTGACCTT GCTGATGGTG ACATTGCACC 1642 TGGATGTACT ATCCAATCTG TGATGACATT CCCTGCTAAT AAAAGACAAC ATAACTCA 1700 (2) INFOMATION FOR ID. SEC. No .: 2: (i) SEQUENCE CHARACTERISTOTICS: (A) LENGTH: 206 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Protein (xi) SEQUENCE DESCRIPTION: ID. SEC. No .: 2: Met? Sn Phe Gip Gin Arq Leu Gln Ser Leu Trp Thr Leu Ala Arg Pro -4S - < -40 -35 Phe Cys Pro Pro: - > u L-u Wing Thr Wing Being GIn Met Gln Met Val Val -30 -25 -20 Leu Pr-- Cys Lsu ---- '.and rhe Ti.r Leu Leu Lep Trp Ser Gin Val Ser Gly -15 -10 -5? The Gln Giy G ± n C-iu? Rs His Phe Gly Pro Cys Gln Val Lys Gly Val - 5 10 15 Val Pro Glr. Lys Leu Trp Glu Wing Phe Trp Aia Val Lys Asp Thr Mee 20 25 30 Glr. Wing Gin Asp Asn lie Thr Be Wing Arg Leu Leu Gln Gln Glu Val 35 40 45 Leu Glp Asn Vai Ser Asp Wing Glu Ser Cys Tyr Leu Val His Thr Leu? 0 55 50 Leu Glu Phe Tyr Leu Lys Thr Val Phe Lys Asn Tyr His Asn Arg Thr 65 70 75 80 Val Glu Val Arg Thr Leu Lys Ser Phe Ser Thr Leu Wing Asn Asn Phe 85 90 95 Val Leu He Val Ser Gln Leu Gln Pro Ser Gln Glu Asn Glu Met Phe 100 105 110 Be He Arg Asp Be Wing His Arg Arg Phe Leu Leu Phe Arg Arg Ala 115 120 125 Phe Lys Gln Leu Asp Val Glu Ala Ala Leu Thr Lys Ala Leu Gly Glu 130 135 140 Val Asp lie Leu Leu Thr Trp Met Gln Lys Phe Tyr Lys Leu 145 150 155 (2) INFORMATION FOR ID. SEC. No .: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 178 amino acids (B) TYPE: amino acid (C) CHAIN FORM: NON-RELEVANT (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: Peptide ( xi) SEQUENCE DESCRIPTION: ID. SEC. No .: 3: Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val 1 5 10 15 Arg Ala Pro Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His 20 25 30 Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Wing Phe 35 40 45 Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu 50 55 60 Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys 65 70 75 80 Gln Ala Leu Ser Glu Met lie Gln Phe Tyr Leu Glu Glu Val Met Pro 85 90 95 Gln Ala Glu Asn Gln Asp Pro Asp lie Lys Ala His Val Asn Ser Leu 100 105 110 Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg 115 120 125 Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn 130 135 140 Wing Phe Asn Lys Leu Gln Glu Lys Gly lie Tyr Lys Wing Met Ser Glu 145 150 155 160 Phe Asp He Phe He Asn Tyr He Glu Wing Tyr Met Thr Met Lys He 165 170 175 Arg Asn (2) INFORMATION FOR ID. SEC. No .: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 171 amino acids (B) TYPE: amino acid (C) CHAIN FORM: NON-RELEVANT (D) TOPOLOGY: linear (iii) TYPE OF MOLECULE: Peptide (ii) ) SEQUENCE DESCRIPTION: ID. SEC. No .: 4: Met Leu Val Asn Phe He Leu Arg Cys Gly Leu Leu Leu Val Thr Leu 1 5 10 15 Ser Leu Ala He Ala Lys His Lys Gln Ser Ser Phe Thr Lys Ser Cys 20 25 30 Tyr Pro Arg Gly Thr Leu Ser Gln Ala Val Asp Ala Leu Tyr He Lys 35 40 45, _ Wing Wing Trp Leu Lys Wing Thr He Pro Glu Asp Arg He Lys Asn He b 50 55 60 Arg Leu Leu Lys Lys' Thr Lys Lys Gln Phe Met Lys Asn Cys Gln 65 70 75 80 Phe Gln Glu Gln Leu Leu Be Phe Phe Asn Glu Asp Val Phe Gly Gln 85 90 95 Leu Gln Leu Gln Gly Cys Lys Lys He Arg Phe Val Glu Asp Phe His 100 105 110 0 Thr Leu Arg Gln Lys Leu Ser His Cys He Ser Cys Wing Being Wing 115 120 125 Arg Glu Met Lys Ser He Thr Arg Met Lys Arg He Phe Tyr Arg He 130 135 140 Gly Asn Lys Gly He Tyr Lys Wing He Ser Glu Leu Asp He Leu Leu 145 150 155 160 Ser Trp He Lys Lys Leu Leu Glü Ser Ser Gln 165 170 5 (2) INFORMATION FOR ID. SEC. No .: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1197 base pairs (B) TYPE: amino acid 0 (C) CHAIN FORM: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: CDNA (í?) CHARACTERISTIC: (A) NAME / KEY: CDS (B) LOCATION: 256..798 (x) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 325..798 (xi) DESCRIPTION OF SEQUENCE: ID. SEC. No .: 5 TATCAGGCGA GACGTTTGTT TGGTCCTATG GTAGTCTCAA AGCAGAGTGC ATGGTATCAT 60 GTGGCAGGAA GGCACGGACA AAGCTGAGCT GAAGTGGTTT TCACAAAGTA CCCACTCCAA 120 TGCATACATT CATGGGTTTG TTTAAGAGGG CAGAGATCTG GTAACAGATC TGCGTGTAAG 180 TTCCGAGTCA GAATTTGACT TCAGGGTAAA GCCTTCCTTT CTTCAGCAGG AGCACTGGCC 240 CTTTCTTCAA CACAG ATG AGT TGG GGA CTA CAG ATT CTC CCC TGC CTG AGC 291 Met Ser Trp Gly Leu Gln He Leu Pro Cys Leu Ser -23 -20 - 15 CTA ATC CTT CTT CTT TGG AAC CAA GTG CCA GGG CTT GAG GGT CAA GAG 339 Leu Leu Leu Leu Trp Asn Gln Val Pro Gly Leu Glu Gly Gln Glu -10 -5 1 5 TTC CGA TCG GGG TCT TGC CAA GTG ACH GGG GTG GTT CTC CCA GAA CTG 387 Phe Arg Ser Gly Ser Cys Gln Val Thr Gly Val Val Leu Pro Glu Leu 10 15 20 TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC 435 Trp Glu Wing Phe Trp Thr Val Lys Asn Thr Val Gln Thr Gln Asp Asp 25 30 35 ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG 483 He Thr Ser He Arg Leu Leu Lys Pro Gln Val Leu Arg Asn Val Ser 40 45 50 GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG 531 Gly Wing Glu Ser Cys Tyr Leu Wing His Ser Leu Leu Lys Phe Tyr Leu 55 60 65 AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC 579 Asn Thr Val Phe Lys Asn Tyr His Ser Lys He Wing Lys Phe Lys Val 70 75 80 85 TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA 627 Leu Arg Ser Phe Ser Thr Leu Wing Asn Asn Phe He Val He Met Ser 90 95 10 0 CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT 675 Gln Leu Gln Pro Ser Lys Asp Asn Ser Met Leu Pro He Ser Glu Ser 105 110 115 GAC CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT 723 Wing His Gln Arg Phe Leu Leu Phe Arg Arg Wing Phe Lys Gln Leu Asp 120 125 130 ACÁ GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG 771 Thr Glu Val Wing Leu Val Lys Wing Phe Gly Glu Val Asp He Leu Leu 135 140 145 ACC TGG ATG CAG AAA TTC TAC CAT 'CTC TGACTGCTGA TTGGATAACT 818 Thr Trp Met Gln Lys Phe Tyr His Leu 150 155 TCCTCCTTTG CTCTCCATGC CATTTCAAGG CATTGTGTAC ATCCCTGCTG TCCTCAAGGC 878 ACTTCAGACC CTTGGCCATG GACCCCGTTG TTGGCTCAGG CTTTTCCTCA GACCTCACTC 938 TTCAGTCCAA ATGACAGCCA TAGATGGCAC CTTTGGATGC TCCGACTGAC CCACAAAGTA 998 GATTTGCATA TTTATTACAG CCCTATTAAA TTATTGTCAC CTTCCCTGGA AACCGTATTT 1058 ATTTGTGAGA CCAGAAGTTC CATGAAAGCA TCAGAATTTA GTGCCCCATG CCTCCTCCTC 1118 ACTTCCTGTG ATCTGGCTCA GCATGGGGGC AGTGGATGGT TGCTCAGTAA ATATTTAAAA 1178 TGGAAAAAAA AAAAAAAAA 1197 (2) INFORMATION FOR ID. SEC. No .: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 181 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Protein (xi) SEQUENCE DESCRIPTION: ID. SEC. No .: 6 Met Ser Trp Gly Leu Gln He Leu Pro Cys Leu Ser Leu He Leu Leu -23 -20 -15 -10 Leu Trp Asn Gln Val Pro Gly Leu Glu Gly Gln Glu Phe Arg Ser Gly -5 1 5 Ser Cys Gln Val Thr Gly Val Val Leu Pro Glu Leu Trp Glu Wing Phe 10 15 20 25 Trp Thr Val Lys Asn Thr Val Gln Thr Gln Asp Asp He Thr Ser He 30 35 40 Arg Leu Leu Lys Pro Gln Val Leu Arg Asn Val Ser Gly Wing Glu Ser 45 50 55 Cys Tyr Leu Wing His Ser Leu Leu Lys Phe Tyr Leu Asn Thr Val Phe 60 65 70 Lys Asn Tyr His Ser Lys He Wing Lys Phe Lys Val Leu Arg Ser Phe 75 80 85 Ser Thr Leu Wing Asn Asn Phe He Val He Met Ser Gln Leu Gln Pro 90 95 100 105 Ser Lys Asp Asn Ser Met Leu Pro -He Ser Glu Ser Ala His Gln Arg 110 115 120 Phe Leu Leu Phe Arg Arg Wing Phe Lys Gln Leu Asp Thr Glu Val Wing 125 130 135 Leu Val Lys Wing Phe Gly Glu Val Asp He Leu Leu Thr Trp Met Gln 140 145 150 Lys Phe Tyr His Leu 155 (2) INFORMATION FOR ID. SEC. No .: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 178 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: Simple (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Peptide (xi) SEQUENCE DESCRIPTION: ID. SEC. No .: 7 Met Pro Gly Be Ala Leu Leu Cys Cys Leu Leu Leu Leu Thr Gly Met 1 5 10 15 Arg He Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His 20 25 30 Phe Pro Val Gly Gln Ser His Met Leu Leu Glu Leu Arg Thr Wing Phe 35 40 45 Be Gln Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp Asn He 50 55 60 Leu Leu Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu Gly Cys 65 70 75 80 Gln Ala Leu Ser Glu Met He Gln Phe Tyr Leu Val Glu Val Met Pro 85 90 95 Gln Ala Glu Lys His Gly Pro Glu He Lys Glu His Leu Asn Ser Leu 100 105 110 Gly Glu Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg 115 120 125 Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Ser 130 135 140 Asp Phe Asn Lys Leu Gln Asp Gln Gly Val Tyr Lys Ala Met Asn Glu 145 150 155 160 Phe Asp He Phe He Asn Cys- He Glu Wing Tyr Met Met He Lys Met 165 170 175 Lys Ser

Claims (20)

NOVELTY OF THE INVENTION CLAIMS

1. - A substantially pure or recombinant soluble IL-BKW protein.

2. An antigenic protein or peptide fragment of the IL-BKW of claim 1.

3. The IL-BKW according to claim 2, further characterized in that it is a natural full-length soluble protein from a mammal. , which includes a primate or mouse.

4. The IL-BKW according to claim 2, further characterized in that: a) it is a soluble IL-BKW that lacks the sequence MNFQQRLQSL WTLARPFCPP LLATASQMQM WLPCLGFTL LLWSQVSG of the sequence identification no. 2; b) is a mature polypeptide of the non-sequence identification. 6; or c) is encoded by a nucleic acid of sequence identification no.

5. The IL-BKW according to claim 2, further characterized in that it is a full-length secreted protein that exhibits a post-translational modification pattern distinct from natural IL-BKW.

6. - IL-BKW according to claim 2, further characterized in that it exhibits an immunological activity antagonist of l-10. 1.

A fusion protein comprising a sequence of a protein or peptide according to claim 2, further characterized in that said protein or peptide: a) lacks a sequence of MNFQQRLQSL WTLARPFCPP LLATASQMQM VVLPCLGFTL LLWSQVSG of sequence identification no. 2; b) is a mature polypeptide of the non-sequence identification. 6; or c) is encoded by a nucleic acid of sequence identification no. 5.

A sterile composition comprising a protein or peptide according to claim 2.

9. A method for purifying an IL-BKW protein or peptide according to claim 6 from other materials in a mixture that it comprises contacting the mixture with an antibody of said protein, and separating IL-BKW from other materials.

10. An isolated or recombinant expression vector encoding a soluble IL-BKW according to claim 1.

11. The vector according to claim 10, further characterized in that the nucleic acid encodes a secreted sequence of the sequence identification no. 2 or not. 6

12. - The vector according to claim 10, further characterized in that it consists of a sequence of the identification of sequence no. 1 or 5.

13. A device for detecting that it consists of a positive control which is a substantially pure soluble IL-BKW or fragment according to claim 1.

14.- A method for detecting in a sample the presence of an acid IL-BKW nucleic acid, protein, or antibody, comprising testing the sample with a piece of equipment according to claim 11.

15. The use of a substantially pure soluble IL-BKW according to claim 1, for the manufacture of a medicine to modulate the physiology of a cell.

16. The use according to claim 15, further characterized wherein said cell is a T cell and said modulation or physiology is the inactivation of said T cell.

17. The use according to claim 15, further characterized wherein said cell is in a tissue.

18. A method for manufacturing a soluble IL-BKW comprising expressing a vector according to claim 10.

19. A cell, tissue, or organ comprising a vector according to claim 10.

20. - The use of a substantially pure soluble IL-BKW, for the manufacture of a medicament for treating a mammal that has an abnormal immune response.