WO2001023569A1 - Cytokine receptor materials and methods - Google Patents

Abstract

The present invention provides novel nucleic acids encoding human cytokine receptor proteins, the novel polypeptides encoded by these nucleic acids and uses of these and related products.

Description

CYTOKINE RECEPTOR MATERIALS AND METHODS

RELATED APPLICATIONS

This patent application is a contmuation-m-part of U S patent application Serial No 09/408,027 filed September 29, 1999

FIELD OF THE INVENTION

The present invention relates to a novel polynucleotide encoding a protein called CG92, which is structurally related to the class II cytokme receptors, along with therapeutic, diagnostic and lesearch utilities for these and related products

BACKGROUND

Cytokmes are secreted signaling molecules that mediate immune responses by binding to specific receptors expressed on the surface of target cells The cytokme receptor family consists of a heterogeneous group of non-phosphotyrosme kinase receptors which are classified into several distinct superfamihes based on conserved sequence and structural features shared by member proteins The class II cytokme receptor (CRF2) family members share a characteristic 200 amino acid extracellular domain designated IRH1 In addition, some of the CRF2 member proteins contain a conserved intracellular domain (IRH2) that interacts with downstream signaling pathways [Mullersman et al (1995) Trends Biochem Sci 20 55-56] The extracellular domain contains several conserved cysteine, prolme, and tryptophan residues, and is structurally similar to the constant domains of immunoglobulin proteins The CRF2 family includes mterleukin- 10 receptoi Rl (IL-10R1), mterleukm-10 receptor R2 (IL-10R2) also known as CRF2-4, interferon-γ receptor Rl (IFN-γRl ), interferon-γ receptor R2 (IFN-γR2) also known as Accessory Factor (AF-1 ), interferon-α receptor Rl (IFN-αRl ), mterferon-ot receptor R2 (IFN-αR2), and tissue factor (TF) [Bazan (1990) PNAS 87 6934-6938, Mullersman et al (1995) Trends Biochem Sci 20 55-56] Intπgumgly, the genes encoding IFN-αRl , IFN-αR2, IFN-γR2, and IL-10R2/CRF2-4 form a cluster on human chromosome 2 [Lutfalla et al (1995) EMBO I 14 5100-5108] The human IL-10 receptor complex consists of two subunits: IL-10R1 and IL- 10R2, both of which are members of the CRF2 family. IL-10R1 encodes the ligand- binding portion of the IL-10 receptor whereas IL-10R2, encoded by the CRFB4 gene, associates with IL-10R1. Although IL-10R1 alone is able to bind IL-10, IL-10R2 is necessary for proper IL-10 signaling [Kotenko et al. (1997) EMBO J. 19: 5894-5903]. Signal transduction of the IL-10 receptor complex is mediated by the Janus kinases (Jak)/signal transducers and activators of transcription (STAT) pathway, and results in the transcription of specific downstream target genes [for review see: Selzman et al.(l 998) SHOCK 10: 309-318]. IL-10 functions to promote anti-inflammatory effects in immune responses [for review see: O'Garra and Murphy (1994) Curr Opin Immunol 6:458-466]. It was originally identified as a T helper cell 2 (Th2) secreted product that inhibited T helper cell 1 (Thl) cytokine production (Fiorentino et al. (1989), J Exp Med, 170:2081-2095). Th2 cells promote humoral immune responses while Thl cells up regulate pro-inflammatory cell-mediated immune reactions. Thus, inhibiting Thl cytokine production is one mechanism by which IL-10 attenuates inflammation. 11-10 is a pleiotropic immunoregulatory cytokine produced by monocytes, macrophages, eosinophils, mast cells, keratinocytes, and B cells in addition to T cells, and is associated with several disease states including meningococcal septic shock, malaria, bacterial sepsis, hepatitis, pancreatitis, severe trauma, cardiopulmonary bypass, fungal infection, systemic vascuhtis, general surgery, and other systemic inflammatory syndromes. IL-10 therapy is currently in clinical trials for the treatment of a variety of inflammatory diseases including inflammatory bowel disease, rheumatoid arthritis, thoracic-abdominal aortic surgery, acute lung injury, multiple sclerosis, psoriasis, and human immunodeficiency virus (HIV) infection [Opal et al. (1998) Clin Infect Dis 27: 1497-1507; Selzman et al.(1998) SHOCK 10: 309-318].

The IFN-α receptor complex contains two subunits, IFN-αRl and IFN-αR2, both of which are required for activity. Similarly, the IFN-γ receptor complex consists of IFN- γRl and IFN-γR2. All four proteins share structural features and are members of the CRF2 family. The IFN-α receptor complex binds all type I IFNs including IFN-α, IFN-β, IFN-co, and IFN-τ, while type II IFNs, of which IFN-γ is the only member, binds to the IFN-γ receptor complex. Both IFN receptor complexes are coupled to the Jak/STAT pathway to transduce intracellular signals from the receptor to the nucleus and thereby activate IFN-stimulated genes [Pestka (1997) Sem One 24:S9-18-S9-40; Zav'yalov et al (1997) APMIS 105:161 -186]. IFNs were originally identified as proteins with anti-viral, anti-tumor and immunomodulatory activities. They are divided into two groups based on their biological and physiochemical characteristics. Type I IFNs are produced by numerous cell types and function to combat viral infection by inhibiting viral replication and cell proliferation, increasing the lytic activity of natural killer cells, and modulating major histocompatibility complex (MHC) expression on T cells. IFN-γ, the only member of type II IFNs, is produced predominantly by natural killer cells and specific subsets of T cells. IFN-γ also induces an anti-viral state, and in addition, is a potent activator of mononuclear phagocytes, promotes differentiation of lymphocytes, activates neutrophils, and activates vascular endothelial cells. Acute inflammation during the early phase of host defense is mediated primarily through IFN-γ production by natural killer cells. In the subsequent antigen-specific phase of the immune response, IFN-γ produced mainly by Thl cells results in a delayed-type hypersensitivity inflammatory response [Heremans et al. (1998) Ann N Y Acad Sci 856:22-32; Kontsek and Kontsekova (1997) Acta Virol 41 :349-353]. IFN-γ also plays a role in the development and pathology of the central nervous system (CNS), and can contribute to CNS disorders such as multiple sclerosis, Alzheimer's disease and HIV-associated dementia complex. Many of its actions in the CNS have been shown to be mediated by nitric oxide production [Munoz-Fernandez and Fresno ( 1998) Prog Neurobiol 56:307-340]. IFN-α has been used as a highly potent drug for treating malignant, viral, immunological, angiogenic, inflammatory, and fibrotic diseases although such treatment can, in some cases, lead to deleterious production of anti-IFN antibodies [Hanley and Haydon (1998) Leuk Lymphoma 29:257-268; Zav'yalov et al. (1997) APMIS 105: 161 -186].

The 11-10, IFN-γ, and IFN-α receptor complexes are structurally similar in that each consists of ligand binding Rl chains and R2 accessory chains that are involved in intracellular signal transduction. In addition, the Rl and R2 chains of all three receptor complexes are members of the CRF2 family. Both IL-10 and IFNs utilize the Jak-STAT pathway for transducing the cytokine signal from the cell surface to the nucleus m order to activate specific genes Jaks are a distinct class of cytoplasmic protein tyrosine kinases that associate with cytokine receptors To date, there are four mammalian Jaks, Jakl , Jak2, Jak3, and Tyk2, that have been identified [Heim (1999) 1 Rec Sig Trans Res 19 75-120] Ligand binding by any of the three receptor complexes leads to the activation of Statl as well as one or more additional STATs, which are a family of transcπption factors that are activated through phosphorylation on a single tyrosine Once activated, the STATs dimeπze, translocate into the nucleus, and induce the transcπption of target genes There are seven mammalian STAT proteins, Statl, Stat2, Stat3, Stat4, Stat5, Statό, and Stat7, most of which are ubiquitously expressed Statl has been shown to be involved in IFN but not IL-10 signaling [Heim (1999) J Rec Sig Trans Res 19 75-120] IL-10 and IFNs have been shown to interact with each other in immune responses Both INFα and INFβ are able to up-regulate IL-10 production in vitro m specific cell types [Lalani et al (1997) Annals Allergy Asthma Immunol] However, in spite of the structural similarity of their ligand-receptor complexes, IL-10 and IFN-γ function antagonistically m the immune system, particularly in the regulation of Thl and Th2 dependent immune responses [Lalani et al (1997) Annals Allergy Asthma Immunol, Selzman et al (1998) SHOCK 10 309-318]

The last member of the CRF2 family, tissue factor (TF), plays an important role in the initiation of the blood coagulation process Specifically, it acts as a high affinity receptor for the seπne protease factor Vila and is also able to activate factor IX The TF- mediated signaling pathways remain poorly understood, however, the cytoplasmic domain of TF consists of a mere 20 amino acids, suggesting that it may associate with a co- receptor to initiate intracellular signaling Aberrant expression of TF can lead to intravascular coagulations disorders associated with endotoxemia, malignancies, immunological diseases, and atherosclerosis [Morrisey et al ( 1987) Cell 50 129-135, Camici and Sagnpanti ( 1999) Minerva Med 90 25-32]

SUMMARY OF THE INVENTION

The compositions of the present invention include novel isolated polypeptides, in particulai, nov el human cytokine leceptor proteins and activ e variants thereof, isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies

The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides A nucleotide sequence encoding a cytokine receptor protein designated CG92 is set forth in SEQ ID NO 1 , and its deduced amino acid sequence is set forth in SEQ ID NO 2 This cytokine receptor protein is believed to be a new member of the class II cytokine receptor (CRF2) family The polypeptide set out in SEQ ID NO 2 is 31 1 ammo acids in length where amino acid 1 is believed to represent the initiating methionme, although amino acids 6 or 17 are alternative possibilities The mature polypeptide of SEQ ID NO 2 is predicted to start at amino acid 30 and the true N terminus may be confirmed by methods known in the art (e g through expression of the DNA in a mammalian or other host cell that correctly piocesses the signal sequence) The polypeptide of SEQ ID NO 2 displays amino acid homology with the human IL-10R2 (28% identity) and human JFN-γR2 (28% identity) An alignment of CG92 with human IL-10R2 and IFN-γR2 is shown in Figure 1A-1C Results of ematπx analysis of the amino acid sequence set forth in SEQ ID NO 2 indicated that it shows high homology to bacterial chemotaxis sensory transducer signatures at about amino acids 235-255, tissue factor signatures at about ammo acid 38-57, and tissue factor domains located at about amino acids 84-119 Three- dimensional structure analyses using HTM and SeqFold methods of the GeneAtlas program predicted a tissue factor structure at about ammo acids 35 to 224 or at about ammo acids 40 to 226 Clones containing CG092 sequences were observed in DNA libraries prepared from ovary, cervix and fetal skin tissues Additional family members can be identified using SEQ ID NO 1 as a molecular probe The polynucleotides of the invention include naturally occuπing or wholly or partially synthetic DNA, e g , cD A and genomic DNA, and RNA, e g , mRNA The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO 2 or a portion thereof corresponding to the full length or mature protein The isolated polynucleotides of the invention further include, but are not limited to, a polynucleotide comprising the nucleotide sequence of SEQ ID NO 1 , a polynucleotide comprising the full length protein coding sequence of SEQ ID NO 1 , and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NO 1 The polynucleotides of the present invention also include, but are not limited to, polynucleotides that encode polypeptides with cytokine receptor activity and that hybridize under stπngent hybridization conditions to the complement of (a) the nucleotide sequence of SEQ ID NO 1 , or (b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO 2, a polynucleotide which is an allelic vaπant of any polynucleotide recited above, a polynucleotide which encodes a species homolog (e g orthologs) of any of the proteins recited above, or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide having an ammo acid sequence of SEQ ID NO 2 (e g the IRH1, IRH2, intracellular, transmembrane or extracellular domain, or the tissue factor structure domains at about ammo acids 35-224 of SEQ ID NO 2 or at about amino acids 40-226 of SEQ ID NO 2, or any of the tissue factor or bacteπal chemotaxis transducer signatures) The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above

The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising the amino acid sequence of SEQ ID NO 2 or a portion thereof corresponding to the full length or mature protein Polypeptides of the invention also include polypeptides with cytokme receptor activity that are encoded by (a) polynucleotides set out in SEQ ID NO 1 , or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stπngent hybridization conditions Biologically or immunologically acti e variants of the cytokine receptor protein sequence of SEQ ID NO 2 and "substantial equivalents" thereof (e g , with 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that retain cytokine receptor activity are also contemplated The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e g host cells) of the invention

Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophihc, e g , pharmaceutically acceptable, carrier The invention also relates to methods for producing polypeptides of the invention comprising growing a culture of the cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and punfymg the protein from the cells or the culture medium in which the cells are grown Preferred embodiments include those in which the protein produced by such process is a mature form of the protein

Polynucleotides according to the invention have numerous applications m a vaπety of techniques known to those skilled in the art of molecular biology These techniques include use as hybπdization probes, use as oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect or quantify the presence of the particular cell or tissue mRNA in a sample using, e g , in situ hybπdization In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al , Science 258 52-59 (1992), as expressed sequence tags for physical mapping of the human genome

The polypeptides according to the invention can be used in a vaπety of conventional procedures and methods that are currently applied to other proteins For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitatmg the polypeptide in tissue The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable earner

The methods of the present invention further relate to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions The invention also provides kits compnsmg polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited herein

The invention also provides methods for the identification of compounds that modulate (l e , increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention Exemplary methods of identifying a modulator of the polypeptides of the invention comprise the steps of contacting a test compound with the polypeptide of the invention and measunng the hgand-binding or biological activity of the polypeptide in the presence and absence of the test compound Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders, as recited herein Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e g , bind to) the polypeptides of the invention

The methods of the invention also include methods for the treatment of disorders as recited herein which may involve the administration of such compounds to individuals exhibiting symptoms or tendencies related to disorders, as recited herein In addition, the invention encompasses methods for treating diseases or disorders, including disorders related to inflammation and blood coagulation, as recited herein comprising the step of administenng compounds and other substances that modulate the overall activity of the target CG92 gene products Compounds and other substances can effect such modulation either on the level of target gene/protem expression or taiget protein activity The polynucleotides and polypeptides of the invention may function in innate

(nonspecific) or acquired (specific) immunity Innate immunity differs from acquired immunity, which includes defense mechanisms involving the activation of lymphocytes that are induced upon specific infection Innate immunity includes phagocytes, eosinophils, and natural killer cells as well as soluble factors including interferons, lysozyme, and complement that serve as a primary defense against pathogens Dunng the initial phase of an infection, phagocytic cells of the innate immune system, i.e , neutrophils and macrophages, secrete cytokines including IL-10, which tπgger an inflammatory response that includes increased blood flow, increased capillary permeability, and the influx of phagocytic cells to the site of infection Enzyme systems including the clotting system, the kinm system, the fibnnolytic system, and the complement system also produce molecules that cause inflammation Innate immunity is especially important dunng the early phase of infection before the host is able to mount an acquired immune response

Acquired immunity constitutes defense mechanisms that are induced upon exposure to specific foreign substances The major components of acquired immunity are the humoral and cell-mediated responses, which selectively recognize and eliminate the invading pathogen These functions are earned out by B- and T-lymphocytes and antigen presenting cells Additional features of acquired immunity include the ability to distinguish self from non-self and the capability of "remembering" a foreign antigen that has previously been encountered by the host Thus, a second infection with the same antigen will trigger a stronger immune response The memory displayed by acquired immunity can also confer life-long immunity to various pathogens

The polynucleotides and polypeptides of the invention may also play a role in the process of blood coagulation The function of CG92 in blood coagulation is confirmed by the discovery of tissue factor signatures and tissue factor structures in this protein as descnbed above

In particular, the polypeptides and polynucleotides of the invention may play a role in disorders involving sepsis (and associated conditions such as fever, tachycardia, tachypnea, cytokine overstimulation, increased vascular permeability, hypotension, complement activation, disseminated intravascular coagulation, anemia, thrombocytopenia, leukopenia, pulmonary edema, adult respiratory distress syndrome, intestinal ischemia, renal insufficiency and failure, metabolic acidosis and multiorgan dysfunction syndrome), endotoxic shock, cytokine induced shock, thrombosis, acute pancreatitis, rheumatoid or reactive arthritis, chronic inflammatory arthritis, vascuhtis, lupus, immune complex glomerulonephπtis, pancreatic cell damage from diabetes melhtus type 1 , allograft and xenograft transplantation, graft versus host disease, inflammatory bowel disease, inflammation associated with pulmonary disease, multiple sclerosis, hepatitis, sarcoidosis, Dupuytren's disease, HIV-dementia complex, stroke, Alzheimer's disease, other autoimmune disease or inflammatory disease, prohferative disorders such as acute or chronic myelogenous leukemia, hairy cell leukemia, breast cancer, prostate cancer, non-Hodgkm's lymphoma, ovarian carcinoma, lymphomas, or premature labor (e g premature labor secondary to intrauteπne infections), and bone degenerative diseases such as osteoporosis

Concurrent administration of other anti-mflammatory agents such as GM-CSF, IL- 4, IL-10, IL-13, transforming growth factor-beta, IL-lRa, IL-lRa- ke proteins described in co-owned, co-pending U S application senal nos 09/287, 210 [Attorney Docket No 20411-773] filed Apnl 5, 1999, and 09/316, 081 [Attorney Docket No 35659] filed May 20, 1999, incorporated herein by reference, anti-TNF, corticosteroids, and immunosuppressive agents is also contemplated according to the invention

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A-1C shows an alignment of the CG92 amino acid sequence (SEQ ID NO 2) with human IL-10R2 (SEQ ID NO 3) (28% identity) and human IFN-γR2 (SEQ ID NO 4) (28% identity) The alignment was generated using the Clustal Method with PAM250 residue weight table Amino acid numbers for each sequence are labeled accordingly Gaps are presented as dashes, and shaded residues match the consensus exactly

DETAILED DESCRIPTION OF THE INVENTION

1. DEFINITIONS

The term "nucleotide sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides The terms "nucleic acid" and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.

The terms "oligonucleotide fragment" or a "polynucleotide fragment", "portion," or "segment" is a stretch of polypeptide nucleotide residues which is long enough to use in polymerase chain reaction (PCR) or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules. The terms "oligonucleotides" or "nucleic acid probes" are prepared based on the polynucleotide sequences provided in the present invention. Oligonucleotides comprise portions of such a polynucleotide sequence having at least about 15 nucleotides and usually at least about 20 nucleotides. Nucleic acid probes comprise portions of such a polynucleotide sequence having fewer nucleotides than about 6 kb, usually fewer than about 1 kb. After appropriate testing to eliminate false positives, these probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P.S. et al, 1992, PCR Methods Appl 1:241-250).

The term "probes" includes naturally occurring or recombinant or chemically synthesized single- or double-stranded nucleic acids. They may be labeled by nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F.M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, both of which are incorporated herein by reference in their entirety. The term "stringent" is used to refer to conditions that are commonly understood in the art as stringent. Stringent conditions can include highly stringent conditions (e.g., hybridization to filter-bound DNA in 0.5 M NaHP0₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.1 X SSC/0.1 % SDS at 68°C), and moderately stringent conditions (e.g., washing in 0.2X SSC/0.1 % SDS at 42°C). Other exemplary hybridization conditions are described herein in the examples. In instances wherein hybridization of deoxyohgonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6X SSC/0 05% sodium pyrophosphate at 37°C (for 14-base ohgos), 48°C (for 17-base ohgos), 55°C (for 20-base ohgos), and 60°C (for 23-base ohgos) The term "recombinant," when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e g , microbial, insect, or mammalian) expression systems "Microbial" refers to recombinant polypeptides or proteins made in bacterial or fungal (e g , yeast) expression systems As a product, "recombinant microbial" defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation

Polypeptides or proteins expressed in most bactenal cultures, e g , E coli, will be free of glycosylation modifications, polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells

The term "recombinant expression vehicle or vector" refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence An expression vehicle can compnse a transcnptional unit compnsmg an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcπbed into mRNA and translated into protein, and (3) appropnate transcπption initiation and termination sequences Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionme residue This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product

The term "recombinant expression system" means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomall Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed The cells can be prokaryotic or eukaryotic

The term "open reading frame," ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein

The term "expression modulating fragment," EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF

As used herein, a sequence is said to "modulate the expression of an operably linked sequence" when the expression of the sequence is altered by the presence of the EMF EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements) One class of EMFs are fragments which induce the expression or an operably linked ORF m response to a specific regulatory factor or physiological event

As used herein, an "uptake modulating fragment," UMF, means a senes of nucleotides which mediate the uptake of a linked DNA fragment into a cell UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below

The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence The resulting nucleic acid molecule is then incubated with an appropriate host under appropnate conditions and the uptake of the marker sequence is determined As descnbed above, a UMF will increase the frequency of uptake of a linked marker sequence

The term "active" refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurnng polypeptide According to the invention, the term "biologically active" with reference to the cytokine receptor polypeptide of the invention means that the polypeptide retains at least one of the biological activities of CG92, preferably the cytokine receptor activity, while the term "lmmunologically active" with reference to the cytokine receptor polypeptide of the invention means that the polypeptide retains at least one of the immunologic or antigenic activities of CG92

The term "naturally occurring polypeptide" refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, pidation and acylation

The term "derivative" refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e g , with radionuchdes or various enzymes). pegylation (deπvatization with polyethylene glycol) and insertion or substitution by chemical synthesis of ammo acids such as ornithme, which do not normally occur in human proteins

The term "vaπant" (or "analog") refers to any polypeptide diffenng from naturally occurnng polypeptides by ammo acid insertions, deletions, and substitutions, created using, for example, recombinant DNA techniques Guidance m determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, such as cytokine receptor activity, may be found by companng the sequence of the particular polypeptide with that of homologous human or other mammalian class II cytokine receptor polypeptides and minimizing the number of ammo acid sequence changes made m regions of high homology (conserved regions) or by replacing amino acids with consensus sequence

Preferably, amino acid "substitutions" are the result of replacing one amino acid with another ammo acid having similar structural and/or chemical properties, I e , conservative amino acid replacements "Conservative" ammo acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues involved For example, nonpolar (hydrophobic) amino acids include alanme, leucine, isoleucme, valme, prolme, phenylalanine, tryptophan, and methionme, polar neutral amino acids include glycine, serine, threonme, cysteine, tyrosine, asparagine, and glutamine, positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include aspartic acid and glutamic acid "Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant vaπants for activity Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides Such alterations can, for example, alter one or more of the biological functions or biochemical charactenstics of the polypeptides of the invention For example, such alterations may change polypeptide charactenstics such as hgand-bmding affinities, interchain affinities, or degradation/turnover rate Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression For example, cysteine residues can be deleted or substituted with another ammo acid residue in order to eliminate disulfide bndges

As used herein, "substantially equivalent" can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that vanes from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 20% (l e , the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues m the substantially equivalent sequence is about 0 2 or less) Such a sequence is said to have 80% sequence identity to the listed sequence In one embodiment, a substantially equivalent, e g , mutant, sequence of the invention varies from a listed sequence by no more than 10% (90% sequence identity), in a variation of this embodiment, by no more than 5% (95% sequence identity), and in a further variation of this embodiment, by no more than 2% (98% sequence identity) Substantially equivalent, e g , mutant, ammo acid sequences according to the invention generally have at least 95% sequence identity with a listed amino acid sequence, whereas substantially equivalent nucleotide sequence of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent For the purposes of determining equivalence, truncation of the mature sequence (e g , via a mutation which creates a spurious stop codon) should be disregarded Sequence identity may be determined, e g , using the Jotun Hein method

Nucleic acid sequences encoding such substantially equivalent sequences, e g , sequences of the recited percent identities, can routinely be isolated and identified via standard hybridization procedures well known to those of skill in the art

Where desired, an expression vector may be designed to contain a "signal or leader sequence" which will direct the polypeptide through the membrane of a cell Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques

A polypeptide "fragment," "portion," or "segment" is a stretch of amino acid residues of at least about 5 ammo acids, often at least about 7 ammo acids, typically at least about 9 to 13 amino acids, and, in vanous embodiments, at least about 17 or more amino acids To be active, any polypeptide must have sufficient length to display biologic and/or immunologic activity

Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code Various codon substitutions, such as the silent changes which produce vanous restπction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression m a particular prokaryotic or eukaryotic system Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as hgand-bmding affinities, interchain affinities, or degradation/turnover rate

The term "activated" cells as used in this application are those which are engaged in extracellular or intracellular membrane trafficking, including the export of neurosecretory or enzymatic molecules as part of a normal or disease process The term "puπfied" as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules. e g , polynucleotides, proteins, and the like In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99 8% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present)

The term "isolated" as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e g , nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same The terms "isolated" and "purified" do not encompass nucleic acids or polypeptides present in their natural source

The term "infection" refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector

The term "transformation" means introducing DNA into a suitable host cell so that the DNA is rephcable, either as an extrachromosomal element, or by chromosomal integration

The term "transfection" refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed

The term "intermediate fragment" means a nucleic acid between 5 and 1000 bases in length, and preferably between 10 and 40 bp in length

The term "secreted" includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed m a suitable host cell "Secreted" proteins include without limitation proteins secreted wholly (e g , soluble proteins) or partially (e g , receptors) from the cell in which they are expressed "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum "Secreted" proteins are also intended to include proteins containing non-typical signal sequences (e g Interleukin- 1 Beta, see Krasney, P A and Y oung, P R ( 1992) Cytokine 4(2) 134 -143) and factors released from damaged cells (e g Interleukin- 1 Receptor Antagonist, see Arend W P et al ( 1998) Annu Rev Immunol 16 27-55) Each of the above terms is meant to encompasses all that is described for each, unless the context dictates otherwise

NUCLEIC ACIDS AND POLYPEPTIDES OF THE INVENTION

Nucleotide and amino acid sequences of the invention are reported below Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention Fragments of the protein may be linear form or they may be cychzed using known methods, for example, as descπbed in H U Saragovi, et al , Bio/Technology 10, 773-778 (1992) and in R S McDowell, et al J Amer Chem Soc 114, 9245-9253 (1992), both of which are incoφorated herein by reference Such fragments may be fused to carπei molecules such as lmmunoglobulms for many puφoses, including increasing the valency of protein binding sites For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule Other immunoglobulin isotypes may also be used to generate such fusions For example, a protem-IgM fusion would generate a decavalent form of the protein of the invention

The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins The full-length form of the such proteins may be determined by translation of the nucleotide sequence of each disclosed clone The mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide m a suitable mammalian cell or other host cell The sequence of the mature form of the protein is also determmable from the ammo acid sequence of the full-length form Where protein of the present invention is membrane bound, soluble forms of the protein are also provided In such forms part or all of the regions causing the protein to be membrane bound are deleted so that the protein is fully secreted from the cell in which it is expressed

The present invention also provides genes corresponding to the cDNA sequences disclosed herein The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein Such methods include the preparation of probes or pπmers from the disclosed sequence information for identification and/or amplification of genes in appropnate genomic libraries or other sources of genomic mateπals Species homologs (e g orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention Species homologs may be isolated and identified by making suitable probes or pπmers from the sequences provided herein and screening a suitable nucleic acid source from the desired species The invention also encompasses allelic variants of the disclosed polynucleotides or proteins, that is, naturally-occurπng alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides The compositions of the present invention include isolated polynucleotides, including recombinant DNA molecules, cloned genes or degenerate vanants thereof, especially naturally occurnng variants such as allelic vaπants, novel isolated polypeptides, and antibodies that specifically recognize one or more epitopes present on such polypeptides Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention Species homologs may be isolated and identified by making suitable probes or pnmers from the sequences provided herein and screening a suitable nucleic acid source from the desired species The invention also encompasses allelic variants of the disclosed polynucleotides or proteins, that is, naturally-occurπng alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides

2. NUCLEIC ACIDS OF THE INVENTION

The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide encoding a polypeptide compnsmg the ammo acid sequence of SEQ ID NO 2 or the mature protein portion thereof A preferred nucleic acid sequence is set forth in SEQ ID NO 1

The isolated polynucleotides of the invention further include, but are not limited to a polynucleotide comprising the nucleotide sequence of SEQ ID NO 1 , a polynucleotide comprising the full length protein coding sequence of SEQ ID NO 1 , and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NO 1 The polynucleotides of the present invention also include, but are not limited to, polynucleotides that encode polypeptides (preferably with cytokine receptor activity) and that hybridize under stringent hybridization conditions to the complement of either (a) the nucleotide sequence of SEQ ID NO 1 or (b) a nucleotide sequence encoding the ammo acid sequence of SEQ ID NO 2, a polynucleotide which is an allelic variant of any polynucleotide recited above, a polynucleotide which encodes a species homolog of any of the proteins recited above, or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide of SEQ ID NO 2

The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above

The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above Polynucleotides according to the invention can have at least about 65%, more typically at least about 70%, at least about 75%, at least about 80%, at least about 85% or at least about 90%>, and even more typically at least about 95%, sequence identity to a polynucleotide recited above The invention also provides the complement of the polynucleotides including a nucleotide sequence that has at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide recited above The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions which can routinely isolate polynucleotides of the desired sequence identities

A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see

Sambrook J et al (1989) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, NY) Useful nucleotide sequences for joining to polypeptides include an assortment of vectors, e g , plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide In general the vector contains an origin of replication functional in at least one organism. convenient restπction endonuclease sites, and a selectable marker for the host cell Vectors according to the invention include expiession vectors, replication vectors, probe generation vectors, and sequencing vectors A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism

The sequences falling within the scope of the present invention are not limited to the specific sequences herein described, but also include allelic variations thereof Allelic variations can be routinely determined by companng the sequence provided in SEQ ID NO 1 , or a representative fragment thereof, or a nucleotide sequence at least 99 9% identical to SEQ ID NO 1 with a sequence from another isolate of the same species

To accommodate codon vaπabihty, the invention includes nucleic acid molecules coding for the same ammo acid sequences as do the specific ORFs disclosed herein In other words, in the coding region of an ORF, substitution of one codon for another which encodes the same amino acid is expressly contemplated Any specific sequence disclosed herein can be readily screened for errors by resequencing a particular fragment, such as an ORF, in both directions (l e , sequence both strands)

The present invention further provides recombinant constructs compnsmg a nucleic acid having the sequence of SEQ ID NO 1 or a fragment thereof or any other polynucleotides of the invention In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having the sequence of SEQ ID NO 1 or a fragment thereof is inserted, m a forward or reverse onentation In the case of a vector comprising one of the ORFs of the present invention, the vector may further compnse regulatory sequences, including for example, a promoter, operably linked to the ORF For vectors compnsmg the EMFs and UMFs of the present invention, the vector may further comprise a marker sequence or heterologous ORF operably linked to the EMF or UMF Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention The following vectors are provided by way of example Bacterial pBs, phagescπpt, PsιX174, pBluescπpt SK, pBs KS, pNH8a, pNH 16a, pNH 18a, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia) Eukaryotic pWLneo pSV2cat, pOG44, PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia)

The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al , Nucleic Acids Res 19, 4485-4490 (1991 ), in order to produce the protein recombmantly Many suitable expression control sequences are known in the art General methods of expressing recombinant proteins are also known and are exemplified in R Kaufman, Methods in Enzymology 185, 537-566 (1990) As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the hgated polynucleotide/expression control sequence

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers Two appropriate vectors are pKK232-8 and pCM7 Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P_R, and trc Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallofhιoneιn-I Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art Generally, recombinant expression vectors will include ongins of replication and selectable markers permitting transformation of the host cell, e g , the ampicilhn resistance gene of E coli and S cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence Such promoters can be deπved from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others The heterologous structural sequence is assembled m appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the peπplasmic space or extracellular medium Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e g stabilization or simplified purification of expressed recombinant product Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter The vector will comprise one or more phenotypic selectable markers and an oπgin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host Suitable prokaiyotic hosts for transformation include E coli, Bacillus subtilis. Salmonella tvphimurium and various species within the genera Pseudomonas, Streptomvces, and Staphvlococcus. although others may also be employed as a matter of choice

As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropnate cell density, the selected promoter is induced or derepressed by appropnate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further punfication.

Included withm the scope of the nucleic acid sequences of the invention are nucleic acid sequences that hybπdize under stringent conditions to a fragment of the DNA sequence of SEQ ID NO' 1 , which fragment is greater than about 10 bp, preferably 20-50 bp, greater than 100 bp, greater than 300 bp, or greater than 500 bp. In accordance with the invention, polynucleotide sequences which encode the novel nucleic acids, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells

The nucleic acid sequences of the invention are further directed to sequences which encode vaπants of the descπbed nucleic acids These amino acid sequence vaπants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide There are two vanables in the construction of amino acid sequence variants the location of the mutation and the nature of the mutation The ammo acid sequence variants of the nucleic acids are preferably constructed by mutating the polynucleotide to give an ammo acid sequence that does not occur in nature These amino acid alterations can be made at sites that differ m the nucleic acids from different species or other family members (variable positions) or in highly conserved regions (constant regions) Sites at such locations will typically be modified in series, e g , by substituting first with conservative choices (e g , hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e g , hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous Ammo acid insertions include ammo- and/or carboxyl-termmal fusions ranging in length from one to one hundred or more residues, as well as mtrasequence insertions of single or multiple amino acid residues mtrasequence insertions may range geneially from about 1 to 10 ammo residues, preferably from 1 to 5 residues Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells, and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein In a preferred method, polynucleotides encoding the novel nucleic acids are changed via site-directed mutagenesis This method uses oligonucleotide sequences that encode the polynucleotide sequence of the desired amino acid variant, as well as a sufficient adjacent nucleotide on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al , DNA 2 183 (1983) A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res 10 6487-6500 (1982) PCR may also be used to create amino acid sequence variants ot the novel nucleic acids When small amounts of template DNA are used as starting matenal, pπmer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired ammo acid vaπant PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the pπmer The product DNA fragments replace the conesponding region m the plasmid and this gives the desired amino acid vanant A further technique for generating amino acid vaπants is the cassette mutagenesis technique descnbed in Wells et al , Gene 34 315 (1985), and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al , supra, and Current Protocols in Molecular Biology, Ausubel et al Due to the inherent degeneiacy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions

3. HOSTS The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell

Knowledge of cytokme receptor DNA sequences allows for modification of cells to permit, or increase, expression of endogenous cytokine receptors Cells can be modified (e g , by homologous recombination) to provide increased cytokme receptor expression by replacing, m whole or in part, the naturally occurnng promoter with all or part of a heterologous promoter so that the cells express cytokine receptor protein at higher levels The heterologous promoter is inserted in such a manner that it is operatively linked to cytokine receptor encoding sequences See, for example, PCT International Publication No WO 94/12650, PCT International Publication No WO 92/20808, and PCT International Publication No WO 91/09955 It is also contemplated that, in addition to heterologous promoter DNA, amphfiable marker DNA (e g , ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or mtron DNA may be inserted along with the heterologous promoter DNA If linked to the cytokine receptor coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the cytokine receptor coding sequences in the cells

The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L et al , Basic Methods in Molecular Biology (1986)) The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF Any host/vector system can be used to express one or more of the ORFs of the present invention These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as well as prokaryotic host such as E coli and B subtilis The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropnate promoters Cell-free translation systems can also be employed to produce such proteins using RNAs deπved from the DNA constructs of the present invention Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are descπbed by Sambrook, et al , in Molecular Cloning A Laboratory Manual, Second Edition, Cold Spπng Harbor, New York (1989), the disclosure of which is hereby incoφorated by reference

Various mammalian cell culture systems can also be employed to express recombinant protein Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, descπbed by Gluzman, Cell 23 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary πbosome binding sites, polyadenylation site, splice donor and acceptor sites, transcπptional termination sequences, and 5' flanking nontranscπbed sequences DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscnbed genetic elements Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps Protein refolding steps can be used, as necessary, in completing configuration of the mature protein Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysmg agents

A number of types of cells may act as suitable host cells for expression of the protein Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed pπmate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, pπmary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast, insects or in prokaryotes such as bacteria Potentially suitable yeast strains include Saccharomvces cerevisiae, Schizosaccharomvces pombe, Kluvveromvces strains, Candida, or any yeast strain capable of expressing heterologous proteins Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bactenal strain capable of expressing heterologous proteins If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein Such covalent attachments may be accomplished using known chemical or enzymatic methods In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination As descnbed herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting, including polyadenylation signals mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element Alternatively, the targeting event may replace an existing element, for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurnng elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells m which the exogenous DNA has integrated into the host cell genome The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a coπect homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker Markers useful for this puφose include the Heφes Simplex Virus thymid e kinase (TK) gene or the bacterial xanthine-guanme phosphoπbosyl-transferase (gpt) gene

Exemplary gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Patent No. 5,272,071 to Chappel; U.S Patent No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al ; and International Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al, each of which is incoφorated by reference herein in its entirety.

4. POLYPEPTIDES OF THE INVENTION The isolated polypeptides of the invention include, but are not limited to, a polypeptide compnsmg the ammo acid sequence of SEQ ID NO: 2 or the ammo acid sequence encoded by the DNA of SEQ ID NO 1 or a portion thereof corresponding to the full length or mature protein. Polypeptides of the invention also include polypeptides (preferably with cytokine receptor activity) that are encoded by (a) the polynucleotide of SEQ ID NO: 1 , or (b) polynucleotides encoding SEQ ID NO: 2 or (b) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybndization conditions. Biologically active or immunologically active variants of the cytokine receptor protein sequence of SEQ ID NO 2 and "substantial equivalents" thereof (e.g., with 65%, 70%, 75%, 80%, 85%, 90%, typically 95%, more typically 98% or most typically 99% amino acid identity) that retain biological activity, preferably cytokine receptor activity, are also contemplated Polypeptides encoded by allelic variants may have a similar or increased or decreased activity compared to the polypeptides of SEQ ID NO: 2

Protein compositions of the present invention may further comprise an acceptable earner, such as a hydrophihc, e.g., pharmaceutically acceptable, earner.

The invention also relates to methods for producing a polypeptide comprising growing a culture of the cells of the invention in a suitable culture medium, and puπfying the protein from the cells or the culture in which the cells are grown For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide The polypeptide can be recovered from the cells or the culture medium, and further purified Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein. The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. By "degenerate vaπant" is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention At the simplest level, the ammo acid sequence can be synthesized using commercially available peptide synthesizers. This is particularly useful m producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide In an alternative method, the polypeptide or protein is puπfied from host cells which produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins order to obtain one of the isolated polypeptides or proteins of the present invention These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and lmmuno-affmity chromatography See, e.g , Scopes, Protein Purification: Pnncφles and Practice, Spπnger-Verlag (1994), Sambrook, et al , in Molecular Cloning: A Laboratory Manual; Ausubel et al , Current Protocols in Molecular Biology Polypeptide fragments that retain biological/immunological activity include fragments encoding greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains

The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides

Sources for test compounds that may be screened for ability to bind to or modulate (1 e , increase or decrease) the activity of polypeptides of the invention include (1 ) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatonal libraries compnsed of either random or mimetic peptides, oligonucleotides or organic molecules

Chemical hbranes may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as "hits" or "leads" via natural product screening The sources of natural product hbranes are microorganisms (including bactena and fungi), animals, plants or other vegetation, insect, or marine organisms, and hbranes of mixtures for screening may be created by (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves Natural product libraries include polyketides, non-nbosomal peptides, and (non-naturally occurring) variants thereof For a review, see Science 282 63-68 (1998)

Combinatorial hbranes are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or propπetary synthetic methods Of particular interest are peptide and oligonucleotide combinatorial hbranes Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatoπal, and polypeptide hbranes For a review of combinatonal chemistry and hbranes created therefrom, see Myers, Curr Opin Bwtechnol 8 701-707 ( 1997) For reviews and examples of peptidomimetic libraries see Al-Obeidi et al , Mol Bwtechnol, 9(3) 205-23 ( 1998), Hruby et al , Cuπ Op Chem Biol, 1(1) 1 14-19 ( 1997), Dorner et al , Biooig Med Chem. 4(5) 709-15 (1996) (alkylated dipeptides) Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit" (or "lead") to optimize the capacity of the "hit" to bind a polypeptide of the invention The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art In bnef, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells

In addition, the binding molecules may be complexed with toxins, e g , πcm or cholera, or with other compounds that are toxic to cells such as radioisotopes The toxm-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention Alternatively, the polypeptide of the invention or binding molecules may be complexed with imaging agents for targeting and imaging puφoses

The protein of the invention may also be expressed as a product of transgenic animals, e g , as a component of the milk of transgenic cows, goats, pigs, or sheep which are charactenzed by somatic or germ cells containing a nucleotide sequence encoding the protein

The protein may also be produced by known conventional chemical synthesis Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art The synthetically-constructed protein sequences, by virtue of shanng pnmary, secondary or tertiary structural and/or conformational charactenstics with proteins may possess biological properties in common therewith, including protein activity Thus, they may be employed as biologically active or immunological substitutes for natural, puπfied proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies

The proteins provided herein also include proteins characterized by ammo acid sequences similar to those of puπfied proteins but into which modification are naturally provided or deliberately engineered For example, modifications the peptide or DNA sequences can be made by those skilled in the art using known techniques Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected ammo acid residue in the coding sequence For example, one or more of the cysteine residues may be deleted or replaced with another ammo acid to alter the conformation of the molecule 1 echmques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e g , U S Pat No 4,518,584) Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein

Other fragments and denvatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein Regions of the protein that are important for its function can be determined by vanous methods known in the art including the alanine-scanning method which involves systematic substitution of each ammo acid residue by alanine, followed by tesdting of the alanme-substituted variants of the protein for receptor activity This type of analysis determines the importance of the substituted amino acid residue for activity The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e g , Invitrogen, San Diego, Calif , U S A (the MaxBat RTM kit), and such methods are well known in the art, as descnbed in Summers and Smith, Texas Agricultural Expenment Station Bulletin No 1555 (1987), incoφorated herein by reference As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed "

The protein of the invention may be prepared by cultunng transformed host cells under culture conditions suitable to express the recombinant protein The resulting expressed protein may then be puπfied from such culture (l e , from culture medium or cell extracts) using known puπfication processes, such as gel filtration and ion exchange chromatography The punfication of the protein may also include an affinity column containing agents which will bind to the protein, one or more column steps over such affinity resins as concanavalm A-agarose, heparm-toyopearl ™ or Cibacrom blue 3GA Sepharose™, one or more steps involving hydrophobic interaction chromatography using such resms as phenyl ether, butyl ether, or propyl ether, or immunoaffinity chromatography

Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification For example, it may be expressed as a fusion protein, such as fused with maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxm (TRX), or as a His tag Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass ), Pharmacia (Piscataway, N J ), Invitrogen, and Qiagen respectively The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope One such epitope ("Flag") is commercially available from Kodak (New Haven, Conn )

Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e g , silica gel having pendant methyl or other aliphatic groups, can be employed to further puπfy the protein Some or all of the foregoing puπfication steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein The protein thus punfied is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein "

The polypeptides of the invention include CG92 analogs (variants) This embraces fragments of CG92, as well as analogs (variants) thereof m which one or more ammo acids has been deleted, inserted, or substituted Analogs of the invention also embrace fusions or modifications of CG92 wherein the protein or analog is fused to another moiety or moieties, e g , targeting moiety or another therapeutic agent Such analogs may exhibit improved properties such as activity and/or stability Examples of moieties which may be fused to CG92 or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to desired cell types Other moieties which may be fused to CG92 or an analog include therapeutic agents which are used for treatment for example, immunosuppressive drugs such as cyclosporm, FK506, azathiopπne, CD3 antibodies and steroids, or immunostimulants, immune modulators, and cytokmes such as alpha or beta mterferon 5. GENE THERAPY

Mutations in the CG92 gene may lesult in loss ol nom al function of the encoded piotein The invention thus provides gene therapy to lestore nomial CG92 activity or to treat disease states involving CG92 Delivery of a functional CG92 gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e g , adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e g , hposomes or chemical treatments) See, for example, Anderson, Natuie, supplement to vol 392, no 6679, pp 25-20 (1998) For additional leviews of gene therapy technology see Fnedmann, Science, 244 1275-1281 (1989), Verma, Scientific American 68-84 (1990), and Miller, Nature, 357 455-460 (1992) Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of CG92 will be useful in treating the disease states It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of CG92

5.1 TRANSGENIC ANIMALS

In methods to determine biological functions of CG92 in vivo, one or more cytokine receptor genes are either over expressed or inactivated in the germ line ol animals using homologous recombination [Capecchi, Science 244 1288-1292 (1989)] Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals Knockout animals, preferably non-human mammals, can be prepared as described in U S Patent No 5,557,032, incoφorated herein by reference Transgenic animals are useful to determine the role(s) CG92 plays in biological processes, and preferably in disease states Transgenic animals are useful as model systems to identify compounds that modulate growth factor activity Transgenic animals, preferably non-human mammals, are produced using methods as descπbed in U S Patent No 5,489,743 and PCT Publication No W094/28122, incoφorated heiein by leference Transgenic animals can be prepared wherein all oi part of an CG92 promotei is either activated or inactiv ated to alter the level of expiession of the CG92 piotein Inactiv ation can be caπ ied out using homologous lecomb ation methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.

6. USES AND BIOLOGICAL ACTIVITY

The biological activity of a polypeptide of the invention may manifest as, e.g., cytokine receptor signaling activity. The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA). The mechanism underlying the particular condition or pathology will dictate whether CG92 polypeptides, polynucleotides or modulators (activators and inhibitors) thereof would be beneficial to the subject in need of treatment. Thus, therapeutic "compositions of the invention" include compositions comprising polynucleotides or polypeptides of the invention or compounds and other substances that modulate the overall activity of the target CG92 gene products, either at the level of target gene/protein expression or target protein activity. Such modulators include polypeptide analogs (variants), including fragments and fusion proteins; compounds that directly or indirectly activate or inhibit the receptor polypeptides of the invention; antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies that recognize the receptor and other proteins that bind to the receptor, that can be selected for stimulatory or inhibitory activity of the cytokine receptor. CG92 is related to members of the CRF2 family which include the Rl and R2 chains of the IL-10 receptor complex, the Rl and R2 chains of the IFN-α receptor complex, the Rl and R2 chains of the IFN-γ receptor complex, and TF. The intracellular signaling pathway of TF remains poorly understood, however, all of the other known CRF2 family receptors utilize the Jak/STAT signal transduction cascade which results in the transcription of specific target genes. Ligands that activate the Jak/STAT pathway include numerous cytokines, hormones, and growth factors which bind members of various receptor families including receptor tyrosine kinases (RTKs), and all four classes of the cytokine receptor superfamily Thus, CG92 may similarly be coupled to components of the Jak/STAT pathway, or other intracellular signal transduction cascades used by cytokine receptors, e g the mitogen activated protein (MAP) kinase pathway, the Src or other tyrosine kinase pathways, the msulm-receptor substrates 1 and 2 and phosphatιdylmosιtol-3-kιnase pathways, or the phosphohpase C/ protein kinase C pathway [Moutoussamy et al (1998) Eur J Biochem 255 1-1 1] Since CG92 is a new member of the CRF2 family, its activity may be related to the biological activities of IL- 10, IFN-α, IFN-β, IFN-γ, or TF IL-10 and IFNs play important roles m regulating immune responses in vanous disease states IL-10 is a potent anti-inflammatory cytokine whose expression is tightly regulated dunng immune responses IL-10 suppresses Thl dependent cellular immunity while stimulating Th2 mediated humoral responses Because of its pleiotropic immunomodulatory effects, IL-10 has been associated with numerous disease states including menmgococcal septic shock, malana, bactenal sepsis, hepatitis, pancreatitis, severe trauma, cardiopulmonary bypass, fungal infection, systemic vascuhtis, general surgery, and other systemic inflammatory syndromes IL- 10 therapy is currently in clinical tnals for the treatment of a variety of inflammatory diseases including inflammatory bowel disease, rheumatoid arthritis, thoracic-abdominal aortic surgery, acute lung injury, multiple sclerosis, psoπasis, and HIV infection [Opal et al (1998) Chn Infect Dis 27 1497-1507, Selzman et al ( 1998) SHOCK 10 309-318]

IFNs are a family of cytokines which exhibit anti-viral, anti-prohferative, and immunomodulatory activities Although the IFNs appear to have overlapping functions, type I IFNs are essential for immune defense against most viral infections while type II IFN is more important in immune responses against intracellular bacteπa and parasites In addition, type I IFNs function immediately after infection in preventing spread of virus while type II IFN is activated later and is involved in Thl dependent inflammatory cell- mediated immunity Type II IFN is also invol ed in the development of the CNS and is associated with diseases such as multiple sclerosis, Alzheimer's disease, and HIV- associated dementia complex IFNs, primarily IFN-α, have been used for treating a vaπety of malignant, viral, immunological, angiogenic, inflammatory, and fibrotic diseases [Hanley and Haydon ( 1998) Leuk Lymphoma 29 257-268, Zav'yalov et al (1997) APMIS 105 161-186]

Another member of the CRF2 family, TF, plays an essential role in blood coagulation by activating factor Vila TF-induced coagulation is associated with diseases such as sepsis, endotoxemia, coronary thrombosis, disseminated intravascular coagulation, atherosclerosis, stroke, cancer, acute respiratory distress syndrome, neointimal hypeφlasia after angioplasty, and ischemia-reperfusion injury. In animal studies, anti-TF monoclonal antibodies and recombinant tissue factor pathway inhibitor (rTFPI) have been shown to ameliorate some of the clinical conditions exhibited in the above diseases Clinical tnals in patients suffering from sepsis, and those who have undergone microvascular surgery using rTFPI are also currently being carried out [Bajaj et al. (1997) Thromb Haemost 78: 471-7].

CG92 may also be a receptor or co-receptor belonging to other classes of the cytokine receptor family not mentioned above such as the class I cytokme receptor family which includes IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, granulocyte-macrophage colony- stimulating factor, erythropoietm, and growth hormone receptors, the class III cytokme receptor family which includes TNF and nerve growth factor receptors, Fas, and CD40, and the class IV cytokine receptor family which includes the IL-1 receptor [Taniguchi (1995) Science 268:251-255]. Functions of these cytokines and other cytokines are known in the art, and CG92 may be assayed for ability to bind to any cytokines known in the art

6.1. RESEARCH USES AND UTILITIES

The polynucleotides provided by the present invention can be used by the research community for vanous puφoses. The polynucleotides can be used to express recombinant protein for analysis, characteπzation or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states), as molecular weight markers on e.g. Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions, to compare with endogenous DNA sequences in patients to identify potential genetic disorders, as probes to hybπdize and thus discover novel, related DNA sequences, as a source of information to derive PCR pπmers for genetic fingeφπnting, as a probe to "subtract-out" known sequences m the piocess of discovering othei novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns, to raise anti-protein antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit another immune response Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-hgand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that descπbed in Gyuπs et al , Cell 75 791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction

The proteins provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening, to raise antibodies or to elicit another immune response, as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids, as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or m a disease state), and, of course, to isolate correlative receptors or ligands Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-hgand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction

Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products

Methods for performing the uses listed above are well known to those skilled the art References disclosing such methods include without limitation "Molecular Cloning A Laboratory Manual", 2d ed , Cold Spπng Harbor Laboratory Press, Sambrook, J , E F Fπtsch and T Mamatis eds , 1989, and "Methods in Enzymology Guide to Molecular Cloning Techniques", Academic Press, Berger, S L and A R Kimmel eds , 1987 6.2. NUTRITIONAL USES

Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements Such uses include without limitation use as a protein or ammo acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured

6.3. CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION ACTIVITY

A protein of the present invention may exhibit receptor signaling activity relating to cytokme, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations A polynucleotide of the invention can encode a polypeptide exhibiting such attπbutes Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/1 1, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DAI, 123, Tl 165, HT2, CTLL2, TF-1, Mo7e and CMK Therapeutic compositions of the invention can be used m the following

Assays for T-cell or thymocyte proliferation include without limitation those descnbed m Current Protocols in Immunology, Ed by J E Coligan, A M Kruisbeek, D H Marguhes, E M Shevach, W Strober, Pub Greene Publishing Associates and Wiley-lnterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3 1-3 19, Chapter 7, Immunologic studies in Humans), Takai et al , J Immunol 137 3494-3500 1986, Bertagnolh et al , J Immunol 145 1706-1712, 1990, Bertagnolh et al , Cellular Immunology 133 327-341 , 1991 , Bertagnolh, et al , I Immunol 149 3778-3783, 1992, Bowman et al , 1 Immunol 152 1756-1761 , 1994 Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in Polyclonal T cell stimulation, Kruisbeek, A M. and Shevach, E. M. In Current Protocols in Immunology J E e.a Coligan eds. Vol 1 pp. 3 12.1-3 12 14, John Wiley and Sons, Toronto. 1994, and Measurement of mouse and human mterleukin-γ, Schreiber, R. D In Current Protocols in Immunology J E. e.a. Coligan eds Vol 1 pp. 6 8 1-6 8 8, John Wiley and Sons, Toronto 1994.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in Measurement of Human and Murme Interleukin 2 and Interleukin 4, Bottomly, K , Davis, L S. and Lipsky, P. E. In Current Protocols m Immunology. J. E. e a. Coligan eds Vol 1 pp. 6.3 1-6 3.12, John Wiley and Sons, Toronto 1991 ; deVπes et al., J. Exp. Med 173 1205-1211, 1991 ; Moreau et al , Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U S.A 80:2931-2938, 1983; Measurement of mouse and human mterleukin 6— Nordan, R. In Current Protocols in Immunology J E. e.a Coligan eds. Vol 1 pp 6.6.1-6.6.5, John

Wiley and Sons, Toronto. 1991 ; Smith et al, Proc Natl. Aced. Sci U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11— Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology J E e.a. Coligan eds Vol 1 pp 6 15.1 John Wiley and Sons, Toronto. 1991 ; Measurement of mouse and human Interleukin 9— Ciarletta, A., Giannotti, J., Clark, S C. and Turner, K J. In Current Protocols in Immunology J. E. e.a Coligan eds Vol 1 pp 6.13 1, John Wiley and Sons, Toronto 1991

Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuπng proliferation and cytokine production) include, without limitation, those descπbed in Current Protocols in Immunology, Ed by J. E Coligan, A M. Kruisbeek, D. H. Marguhes, E. M. Shevach, W Strober, Pub Greene Publishing Associates and Wiley-lnterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function, Chapter 6, Cytokines and their cellular receptors, Chapter 7, Immunologic studies m Humans), Weinberger et al , Proc Natl Acad. Sci USA 77-6091-6095, 1980, Weinberger et al , Eur J Immun 1 1 405-41 1 , 1981 ; Takai et al., J Immunol 137 3494-3500, 1986, Takai et al , J Immunol 140 508-512, 1988 6.4. IMMUNE STIMULATING OR SUPPRESSING ACTIVITY

Compositions of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein A polynucleotide of the invention can encode a polypeptide involved in such activities A protein or antibody, other binding partner, or other modulator of the invention may be useful in the treatment of vanous immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e g , in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations These immune deficiencies may be genetic or be caused by viral (e g , HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders More specifically, infectious diseases caused by viral, bacterial, fungal or other infection may be treatable using a protein, antibody, binding partner, or other modulator of the invention, including infections by HIV, hepatitis viruses, heφesviruses, mycobacteπa, Leishmama spp , malaria spp and vanous fungal infections such as candidiasis, as well as other conditions where a boost to the immune system generally may be desirable, e g , in the treatment of cancer

Autoimmune disorders which may involve a receptor protein of the present invention include, for example, connective tissue disease, multiple sclei osis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillam-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes melhtis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease Such a receptor protein of the present invention may also to be involved m allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems Using the proteins, antibody, binding partners, or other modulators of the invention it may also be possible to modulate immune lesponses, in a number of ways The immune response may be enhanced or suppressed Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressiv e agent Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from nnmunosuppression in that it is generally antigen-specific and persists after exposure to the toleπzing agent has ceased Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the toleπzing agent

Down regulating or preventing the immune response, e g , preventing high level lymphokine synthesis by activated T cells, will be useful m situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD) For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant The administration of a molecule which inhibits or blocks the immune response (e g a receptor fragment, binding partner, or other modulator such as antisense polynucleotides) may act as an lmmunosuppressant The efficacy of particular immune response modulators in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans Examples of appropnate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as descnbed in Lenschow et al , Science 257 789-792 (1992) and Turka et al , Proc Natl Acad Sci USA, 89 11102-11105 (1992) In addition, muπne models of GVHD (see Paul ed , Fundamental Immunology, Raven Press, New York, 1989, pp 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease Blocking the inflammatory response may also be therapeutically useful for treating autoimmune diseases Many autoimmune disorders are the result of inappropπate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms Administration of reagents which block costimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-teπn relief from the disease The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases Examples include munne experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, munne autoimmune collagen arthntis, diabetes melhtus in NOD mice and BB rats, and munne expeπmental myasthema gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response For example, enhancing an immune response may be useful m cases of viral infection such as influenza, the common cold, and encephalitis

Alternatively, anti-viral immune responses may be enhanced m an infected patient by removing T cells from the patient, costimulating the T cells in vitro and remtroducing the in vitro activated T cells into the patient.

The activity of therapeutic compositions of the invention may, among other means, be measured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those descπbed in: Current Protocols in Immunology, Ed by J. E. Coligan, A M. Kruisbeek, D. H. Marguhes, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-lnterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19, Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc Natl. Acad. Sci. USA 78.2488-2492, 1981 ; Herrmann et al, J. Immunol 128.1968-1974, 1982, Handa et al., J Immunol. 135- 1564-1572, 1985, Takai et al., I Immunol 137.3494-3500, 1986, Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al , Proc Natl. Acad. Sci USA 78:2488-2492, 1981 , Herrmann et al., J. Immunol 128:1968-1974, 1982, Handa et al, J Immunol. 135:1564-1572, 1985, Takai et al, J Immunol 137 3494-3500, 1986, Bowmanet al., J. Virology 61 ' 1992-1998; Takai et al., J Immunol 140.508-512, 1988; Bertagnolh et al., Cellular Immunology 133.327-341 , 1991 , Brown et al., J Immunol 153.3079-3092, 1994.

Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those descπbed in. Mahszewski, J. Immunol 144:3028-3033, 1990, and Assays for B cell function. In vitro antibody production, Mond, J J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a. Coligan eds Vol 1 pp. 3 8.1-3.8.16, John Wiley and Sons, Toronto. 1994

Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in' Current Protocols in Immunology, Ed by J. E. Coligan, A. M Kruisbeek, D. H. Marguhes, E. M Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-lnterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al , J. Immunol 137:3494-3500, 1986; Takai et al., J. Immunol 140:508-512, 1988; Bertagnolh et al., J Immunol. 149:3778-3783, 1992

Dendntic cell-dependent assays (which will identify, among others, proteins expressed by dendntic cells that activate naive T-cells) include, without limitation, those descnbed in: Guery et al., J. Immunol. 134 536-544, 1995; Inaba et al., Journal of Expeπmental Medicine 173:549-559, 1991 ; Macatoma et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al , Journal of Expeπmental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67'4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatoma et al., Journal of Experimental Medicine 169.1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94.797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172-631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in- Darzynkiewicz et al., Cytometry 13.795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al , Cancer Research 53.1945-1951 , 1993; Itoh et al , Cell 66.233-243, 1991 , Zacharchuk, Journal of Immunology 145 4037-4045, 1990, Zamai et al., Cytometry 14:891-897, 1993, Gorczyca et al., International Journal of Oncology 1 639-648. 1992 Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in Antica et al , Blood 84 1 1 1-117, 1994, Fine et al , Cellular Immunology 155- 1 1 1 -122, 1994, Galy et al., Blood 85.2770-2778, 1995, Toki et al , Proc Nat Acad Sci USA 88 7548-7551 , 1991

6.5. HEMATOPOIESIS REGULATING ACTIVITY

A protein of the present invention may be involved in regulation of hematopoiesis and, consequently, m the treatment of myeloid or lymphoid cell deficiencies Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating vanous anemias or for use in conjunction with irradiation chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes'macrophages (i.e , traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; m supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of vanous platelet disorders such as thrombocytopenia, and generally for use m place of or complimentary to platelet transfusions; and/or m supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuπa), as well as in repopulating the stem cell compartment post irradiation chemotherapy, either m-vivo or ex-vivo (i.e., m conjunction with bone marrow transplantation or with peπpheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy

Therapeutic compositions of the invention can be used in the following Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above

Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in Johansson et al Cellulai Biology 15 141 -151, 1995, Keller et al , Molecular and Cellular Biology 13 473-486, 1993, McClanahan et al., Blood 81 .2903-2915, 1993

Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in Methylcellulose colony forming assays, Freshney, M. G In Culture of Hematopoietic Cells R I Freshney, et al. eds Vol pp 265-268, Wiley-Liss, Inc , New York, N Y 1994; Hirayama et al., Proc Natl. Acad Sci USA 89-5907-591 1 , 1992; Pnmitive hematopoietic colony forming cells with high prohferative potential, McNiece, I. K and Bπddell, R A In Culture of Hematopoietic Cells R I Freshney, et al eds Vol pp 23-39, Wiley-Liss, Inc., New York, N Y 1994, Neben et al , Expeπmental

Hematology 22:353-359, 1994, Cobblestone area forming cell assay, Ploemacher, R E. In Culture of Hematopoietic Cells. R. I Freshney, et al eds Vol pp 1-21, Wiley-Liss, Inc , New York, N Y. 1994, Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I Freshney, et al. eds Vol pp 163-179, Wiley-Liss, Inc., New York, N Y. 1994, Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R I Freshney, et al. eds Vol pp 139-162, Wiley-Liss, Inc , New York, N Y 1994

6.6. TISSUE GROWTH ACTIVITY

A protein of the present invention also may be involved m bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers

For example, induction of cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals Compositions of a protein, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced cramofacial defects, and also is useful in cosmetic plastic surgery A protein of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells Treatment of osteopoiosis, osteoarthπtis, bone degenerative disordeis, or penodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc ) mediated by inflammatory processes may also be possible using the composition of the invention

Another category of tissue regeneration activity that may involve the protein of the present invention is tendon/ligament formation Induction of tendon/hgament-hke tissue or other tissue formation m circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects m humans and other animals Such a preparation employing a tendon hgament-hke tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repainng defects to tendon or ligament tissue De novo tendon/hgament-hke tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other oπgm, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or hgament-formmg cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors e\ vivo for return in vivo to effect tissue repair The compositions of the invention may also be useful in the treatment of tendinitis, caφal tunnel syndrome and other tendon or ligament defects The compositions may also include an appropnate matrix and/or sequestering agent as a carrier as is well known in the art The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, I e for the treatment of central and peπpheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue More specifically, a composition may be used in the treatment of diseases of the peπpheral nervous system, such as peripheral nerve injuries, peripheral neuropathv and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateial sclerosis, and Shv-Drager syndrome Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders head trauma and cerebrovascular diseases such as stroke Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention

Compositions of the invention may also be useful to promote better or faster closure of non-heahng wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like

Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver intestine, kidney, skm, endofhehum), muscle (smooth, skeletal or cardiac) and vascular (including vascular endofhehum) tissue, or for promoting the growth of cells comprising such tissues Inhibition or modulation of fibrotic scarπng may allow normal tissue to regenerate A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis. reperfusion injury in vanous tissues, and conditions resulting from systemic cytokine damage

A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells, or for inhibiting the growth of tissues descπbed above

Therapeutic compositions of the invention can be used m the following

Assays for tissue generation activity include, without limitation, those descnbed in International Patent Publication No WO95/16035 (bone, cartilage, tendon), International Patent Publication No WO95/05846 (nerve, neuronal), International Patent Publication No W091/07491 (skin, endothehum)

Assays for wound healing activity include, without limitation, those described in Winter, Epidermal Wound Healing, pps 71 -1 12 (Maibach, H I and Rovee, D T , eds ), Year Book Medical Publishers, Inc , Chicago, as modified by Eaglstem and Mertz, J Invest Dermatol 71 382-84 ( 1978) 6.7. CHEMOTACTIC/CHEMOKINETIC ACTIVITY

A protein of the present invention may be involved in chemotactic or chemokinetic activity (e g , act as a chemokme receptor) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothehal cells A polynucleotide of the invention can encode a polypeptide exhibiting such attributes Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action Chemotactic or chemokinetic compositions (e g proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages m treatment of wounds and other trauma to tissues, as well as in treatment of localized infections For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent

A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis

Therapeutic compositions of the invention can be used in the following Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population Suitable assays for movement and adhesion include, without limitation, those described m Current Protocols m Immunology, Ed by J E Coligan, A M Kruisbeek, D H Marguiles, E M Shevach, W Strober, Pub Greene Publishing Associates and Wiley- Interscience (Chapter 6 12, Measurement of alpha and beta Chemokmes 6 12 1-6 12 28, Taub et al J Clin Invest 95 1370-1376, 1995, Lind et al APMIS 103 140-146, 1995. Muller et al Eur J Immunol 25 1744-1748, Gruber et al J of Immunol 152 5860-5867, 1994, Johnston et al J of Immunol 153 1762-1768, 1994 6.8. HEMOSTATIC AND THROMBOLYTIC ACTIVITY

A protein of the invention may also be involved in hemostatis or thrombolysis or thrombosis A polynucleotide of the invention can encode a polypeptide exhibiting such attributes Compositions may be useful m treatment of vanous coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e g , stroke) Therapeutic compositions of the invention can be used in the following

Assay for hemostatic and thrombolytic activity include, without limitation, those descnbed in Lmet et al , J Clm Pharmacol 26 131 -140, 1986, Burdick et al , Thrombosis Res 45 413-419, 1987, Humphrey et al , Fibπnolysis 5 71-79 (1991), Schaub, Prostaglandins 35 467-474, 1988

6.10. RECEPTOR/LIGAND ACTIVITY

A protein of the present invention may also demonstrate activity as receptors , co- receptors, receptor ligands, or inhibitors or agonists of receptor/hgand interactions A polynucleotide of the invention can encode a polypeptide exhibiting such charactenstics Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, mtegnns and their ligands) and receptor/hgand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses) Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/hgand interaction A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/hgand interactions

The activity of a therapeutic compositions of the invention may, among other means, be measured by the following methods Suitable assays for receptoi -ligand activity include without limitation those described in Current Protocols in Immunology, Ed by J E Coligan, A M Kruisbeek, D H Marguhes, E M Shevach, W Strober, Pub Greene Publishing Associates and Wiley- Interscience (Chapter 7 28, Measurement of Cellular Adhesion under static conditions 7 28 1 -7 28 22), Takai et al , Proc Natl Acad Sci USA 84 6864-6868, 1987, Bιerer et al , J Exp Med 168 1 145-1 156, 1988, Rosenstein et al , J Exp Med 169 149-160 1989, Stoltenborg et al , J Immunol Methods 175 59-68, 1994, Stitt et al , Cell 80 661-670, 1995

By way of example, the CG92 polypeptides of the invention mav be used as a cytokine receptor for a hgand(s) thereby transmitting the biological activity of that hgand(s) Ligands may be identified through binding assays, affinity chromatography, dihybπd screening assays, BIAcore assays, gel overlay assays, or other methods known in the art Fragments of the CG92 polypeptides of the invention may act as a ligand itself, or may bind circulating ligand and thus prevent access of the hgand to the cell-bound receptor

Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands The polypeptides of the present invention or hgand(s) thereof may be labeled by being coupled to radioisotopes, colonmetπc molecules or a toxin molecules by conventional methods ("Guide to Protein Punfication" Murray P Deutscher (ed) Methods in Enzymology Vol 182 (1990) Academic Press, Inc San Diego) Examples of radioisotopes include, but are not limited to, tπtium and carbon- 14 Examples of coloπmetnc molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colonmetπc molecules Examples of toxins include, but are not limited, to πcm

6.11 DRUG SCREENING WITH CYTOKINE RECEPTOR POLYPEPTIDES

This invention is particularly useful for screening compounds by using the cytokine receptor polypeptides of the invention, particularly binding fragments, in any of a variety of drug screening techniques The polypeptides employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly One method of diug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the desired polypeptide Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays One may measure, for example, the formation of complexes between polypeptides of the invention and the agent being tested or examine the diminution m complex formation between the polypeptides and an appropriate cell line, which are well known in the art

6.11.1 ASSAY FOR RECEPTOR ACTIVITY

The invention also provides methods to detect specific binding of a cytokine receptor of the invention to a binding partner polypeptide. The art provides numerous assays particularly useful for identifying binding partners for cytokine receptor polypeptides of the invention. For example, dihybπd screening assays can be used to identify polynucleotides encoding binding partners. As another example, affinity chromatography with an immobilized cytokine receptor polypeptide can be used to isolate polypeptides that recognize and bind a polypeptide of the invention Ligands for CG92 can also be identified by adding one or more cytokines, e.g IL-10, IFN(s), or other cytokines (or e g , serum) to two cell populations that are genetically identical except for the expression of CG92 one cell population expresses CG92 whereas the other does not The response of the two cell populations to the addition of cytokme(s) are then compared Alternatively, an expression library can be co-expressed with CG92 in cells and assayed for an autocπne response to identify potential lιgand(s). As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides

For members of the CRF2 family for which the conesponding intracellular signaling cascade has been identified, it has been shown that the Jak STAT signal transduction pathway is used Since CG92 is a member of this family, it is possible that CG92 also utilizes the Jak/STAT pathway, or other signal transduction pathway used by cytokine receptors e g the mitogen activated protein (MAP) kinase pathway, the Src or other tyrosine kinase pathways, the insulin-receptor substrates 1 and 2 and phosphatιdyhnosιtol-3-kιnase pathways, or the phosphohpase C/ protein kinase C pathway These signaling pathways are well charactenzed and components of these pathways which are modified upon leceptor activation have been identified Thus, the role of these downstream intracellular signaling molecules in the signaling cascade of CG92 can be determined For example, a chimeric protein in which the cytoplasmic domain of CG92 is fused to the extracellular portion of a different receptor which has a known ligand, is produced in a host cell The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor The activation of known downstream proteins involved in intracellular signaling can then be determined e g through measuring phosphorylation As another example, various cocktails of cytokines, or serum, can be added to cells expressing CG92 and activation of downstream molecules can be determined Other methods known to those in the art can also be used to identify signaling molecules involved in CG92 receptor activity

6.12. ANTI-INFLAMMATORY ACTIVITY

Compositions of the present invention may also exhibit anti-inflammatory activity The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), lschemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephπtis, cytokine or chemokme-mduced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1 Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or matenal Compositions of this invention may be utilized to prevent or treat condition such as, but not limited to, utilized, for example, as part of methods for the prevention and/or treatment of disorders involving sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes melhtus type 1 , graft versus host disease, inflammatory bowel disease, lnflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiprohferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to lntrauteπne infections

6.13. LEUKEMIAS Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monotypic. erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chrome lymphocytic leukemia (for a review of such disorders, see Fishman et al , 1985, Medicine, 2d Ed , J B Lippmcott Co , Philadelphia)

6.14. NERVOUS SYSTEM DISORDERS

Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result m either a disconnection of axons, a diminution or degeneration of neurons, or demyelmation Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peπpheral nervous systems

(I) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries,

(n) lschemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia,

(in) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, bv an abscess or associated with infection by human immunodeficiency virus, heφes zoster, or heφes simplex virus or with Lyme disease, tuberculosis, syphilis,

(IV) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis,

(v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, fohc acid deficiency, Wernicke disease, tobacco-alcohol amblvopia, Marchiafava-Bignami disease (pπmary degeneration of the coφus callosum), and alcoholic cerebellar degeneration,

(vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis, (vn) lesions caused by toxic substances including alcohol, lead, or particular neurotoxms, and

(vm) demyehnated lesions in which a portion of the nervous system is destroyed or injured by a demyelmatmg disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, trans erse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelmolysis

Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention

(l) increased survival time of neurons in culture,

(n) increased sprouting of neurons in culture or in vivo,

(m) increased production of a neuron-associated molecule in culture or in \ ιvo, e g , choline acetyltransferase or acetylcholmesterase with respect to motor neurons, or (iv) decreased symptoms of neuron dysfunction in vivo

Such effects may be measured bv any method known in the art In preferred, non-hmiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al (1990, .! Neurosci 10 3507-3515), increased sprouting of neurons may be detected by methods set forth in Pestronk et al (1980, Exp Neurol 70 65-82) or Brown et al ( 1981, Ann Rev Neurosci 4 17-42), increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding. Northern blot assay, etc . depending on the molecule to be measured, and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e g , weakness, motor neuron conduction velocity, or functional disability

In a specific embodiment, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spmal muscular atrophy, progressive bulbar palsy, pπmary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Maπe-Tooth Disease)

6.15. OTHER ACTIVITIES

A protein of the invention may also exhibit or be involved in one or more of the following additional activities or effects inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites, effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skm, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape), effecting biorhythms or cancadic cycles or rhythms, effecting the fertility of male or female subjects, effecting the metabolism, catabohsm, anabohsm, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s), effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors, providing analgesic effects or other pam reducing effects, piomoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages, hormonal or endocrine activity, in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases, treatment of hypeφrohferative disorders (such as, for example, psonasis), lmmunoglobuhn-like activity (such as, for example, the ability to bind antigens or complement), and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein

6.16 IDENTIFICATION OF POLYMORPHISMS

The demonstration of polymoφhisms makes possible the identification of such polymoφhisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment Such polymoφhisms may be associated with, e g , differential predisposition or susceptibility to vanous disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropnately For example, the existence of a polymoφhism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymoφhism

Polymoφhisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymoφhism in the DNA For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybndized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybπdizes immediately adjacent to the position of the polymoφhism is extended with one or more labeled nucleotides) In addition, traditional restriction fragment length polymoφhism analysis (using restπction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymoφhism) may be performed Alternatively a polymoφhism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e g . by an antibody specific to the variant sequence

7. THERAPEUTIC METHODS The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods Examples of therapeutic applications include, but are not limited to, those exemplified below

7.1 SEPSIS One embodiment of the invention is the administration of an effective amount of compositions of the invention to individuals that are at a high πsk of developing sepsis, or that have developed sepsis An example of the former category are patients about to undergo surgery While the mode of administration is not particularly important, parenteral administration is preferred because of the rapid progression of sepsis, and thus, the need to have the inhibitor disseminate quickly throughout the body Thus, the preferred mode of administration is to deliver an I V bolus slightly before, dunng, or after surgery The dosage of the active ingredient of the invention will normally be determined by the prescnbing physician It is to be expected that the dosage will vary according to the age, weight and response of the individual patient Typically, where a protein is being administered, the amount of inhibitor administered per dose will be m the range of about 0 1 to 25 mg/kg of body weight, with the preferred dose being about 0 1 to 10 mg/kg of patient body weight For parenteral administration, the active ingredient of the invention may be formulated in an injectable form that includes a pharmaceutically acceptable parenteral vehicle Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the inhibitor The preparation of such solutions is within the skill of the art Typically, the cytokine inhibitor will be formulated in such vehicles at a concentration of about 1 -8 mg/ml to about 10 mg/ml 7.2 ARTHRITIS AND INFLAMMATION

The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis is determined in an experimental animal model system The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J Holoshitz, et at , 1983, Science, 219 56, or by B Waksman et al , 1963, Int Arch Allergy Appl Immunol , 23 129 Induction of the disease can be caused by a single injection, generally mtradermally, of a suspension of killed Mycobacteπum tuberculosis in complete Freund's adjuvant (CFA) The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture The inhibitor is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg The control consists of administering PBS only

The procedure for testing the effects of the test compound would consist of mtradermally injecting killed Mycobacteπum tuberculosis in CFA followed by immediately administering the inhibitor and subsequent treatment every other day until day 24 At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacteπum CFA, an overall arthritis score may be obtained as described by J Holoskitz above An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthntis score

7.4 PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

A composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable earners or excιpιent(s) at doses to treat or ameliorate a variety of disorders Such a composition may also contain (m addition to protein and a earner) diluents, fillers, salts, buffers, stabilizers, solubihzeis, and other matenals well known in the art The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active mgredιent(s) The characteristics of the carrier will depend on the route of administration The phamiaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFO, TNF1 , TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietm, stem cell factor, and erythropoietm In further compositions, compositions of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in questions These agents include vanous growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect, or to minimize side effects Conversely, compositions of the present invention may be included in formulations of the particular cytokine, lymphokme, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokme, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent A composition of the present invention may be active m multimers (e g , heterodimers or homodimers) or complexes with itself or other proteins As a result, pharmaceutical compositions of the invention may compnse a composition of the invention m such multimenc or complexed form

As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein Techniques for formulation and administration of the compounds of the instant application may be found m "Remington's Pharmaceutical Sciences," Mack Publishing Co , Easton, PA, latest edition A therapeutically effective dose further refers to that amount of the compound sufficient to result m amelioration of symptoms, e g , treatment, healing, prevention or amelioration of the relevant medical condition, or an increase rate of treatment, healing, prevention or amelioration of such conditions When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingiedient alone When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously

In practicing the method of treatment or use of the present invention, a therapeutically effective amount of compositions of the present invention is administered to a mammal having a condition to be treated Active ingredients of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, compositions of the present invention may be administered either simultaneously with the cytokιne(s), lymphokme(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially If administered sequentially, the attending physician will decide on the appropnate sequence of admimstenng active ingredient of the present invention in combination with cytokme(s), lymphokme(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors

7.5. ROUTES OF ADMINISTRATION

Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as mtrathecal, direct mtraventπcular, intravenous, intrapentoneal, mtranasal, or intraocular injections Administration of active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intrapentoneal, parenteral or intravenous injection Intravenous administration to the patient is preferred

Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthπtic lomts or in fibrotic tissue, often in a depot or sustained release formulation In order to prevent the scarπng process frequently occurnng as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a hposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue The hposomes will be targeted to and taken up selectively by the afflicted tissue

7.6. COMPOSITIONS/FORMULATIONS

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers compnsmg excipients and auxihanes which facilitate processing of the active compounds into preparations which can be used pharmaceutically These pharmaceutical compositions may be manufactured m a manner that is itself known, e g , by means of conventional mixing, dissolving, granulating, dragee-makmg, levigating, emulsifying, encapsulating, entrapping or lyophihzing processes Proper formulation is dependent upon the route of administration chosen When a therapeutically effective amount of composition of the present invention is administered orally, active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid earner such as a gelatin or an adjuvant The tablet, capsule, and powder contain from about 5 to 95% active ingredient of the present invention, and preferably from about 25 to 90% active ingredient of the present invention When administered m liquid form, a liquid earner such as water, petroleum, oils of animal or plant ongm such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added The liquid form of the pharmaceutical composition may further contain physiological salme solution, dextrose or other sacchaπde solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol When administered m liquid form, the pharmaceutical composition contains from about 0 5 to 90% by weight of active ingredient of the present invention, and preferably from about 1 to 50% active ingredient of the present invention

When a therapeutically effective amount of composition of the present invention is administered by intravenous, cutaneous or subcutaneous injection, active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art A prefened pharmaceutical composition for intravenous, cutaneous, or subcutaneous nηection should contain, in addition to active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection. Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill m the art For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological salme buffer For transmucosal administration, penetrants appropnate to the barrier to be permeated are used in the formulation Such penetrants are generally known in the art

For oral administration, the compounds can be formulated readilv by combining the active compounds with pharmaceutically acceptable earners well known in the art Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurnes, suspensions and the like, for oral mgestion by a patient to be treated Pharmaceutical preparations for oral use can be obtained solid excipient, optionally gπndmg a resulting mixture, and processing the mixture of granules, after adding suitable auxihanes, if desired, to obtain tablets or dragee cores Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, manmtol, or sorbitol, cellulose preparations such as, for example, maize starch, wheat starch, nee starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrohdone (PVP) If desired, disintegrating agents may be added, such as the cross-linked polyvmyl pyrrohdone, agar, or algimc acid or a salt thereof such as sodium algmate Dragee cores are provided with suitable coatings For this puφose, concentrated sugar solutions may be used, which may optionally contain gum arable, talc, polyvmyl pyrrohdone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol oi sorbitol The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols In addition, stabilizers may be added All formulations for oral administration should be in dosages suitable for such administration For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuhser, with the use of a suitable propellant, e g , dichlorodifluoromethane, tπchlorofluoromethane, dichlorotet afluoroethane, carbon dioxide or other suitable gas In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount Capsules and cartridges of, e g , gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch The compounds may be formulated for parenteral administration by injection, e g , by bolus injection or continuous infusion Formulations for injection may be presented in unit dosage form, e g , in ampules or in multi-dose containers, with an added preservative The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form Additionally, suspensions of the active compounds may be prepared as appropnate oily injection suspensions Suitable hpophihc solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or tπglycendes, or hposomes Aqueous injection suspensions may contain substances which inciease the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e g sterile pyrogen-free watei . before use The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e g , containing conventional suppository bases such as cocoa butter or other glyceπdes In addition to the formulations descπbed previously, the compounds may also be formulated as a depot preparation Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resms, or as sparingly soluble denvatives, for example, as a sparingly soluble salt A pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system compnsmg benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase The cosolvent system may be the VPD co-solvent system VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume m absolute ethanol The VPD co-solvent system (VPD 5W) consists of VPD diluted 1 1 with a 5% dextrose in water solution This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity charactenstics Furthermore, the identity of the co-solvent components may be varied for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80, the fraction size of polyethylene glycol may be varied, other biocompatible polymers may replace polyethylene glycol, e g polyvmyl pyrrohdone, and other sugars or polysacchaπdes may substitute for dextrose Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent Vanous types of sustained-release matenals have been established and are well known by those skilled in the art Susta ed-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose denvatives, gelatin, and polymers such as polyethylene glycols Many of the compounds of the invention may be provided as salts with pharmaceutically compatible counteπons Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, tπalkylamme, dialkylamine, monoalkylamine, dibasic ammo acids, sodium acetate, potassium benzoate, tπethanol amine and the like

The pharmaceutical composition of the invention may be m the form of a complex of the active ιngredιent(s) of present invention along with protein or peptide antigens The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes B lymphocytes will respond to antigen through their surface immunoglobulin receptor T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antιgen(s) to T lymphocytes The antigen components could also be supplied as puπfied MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention The pharmaceutical composition of the invention may be in the form of a hposome in which active ingredient of the present invention is combined, m addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution Suitable lipids for hposomal formulation include, without limitation, monoglyceπdes, diglycendes, sulfatides, lysolecithm, phosphohpids, saponm, bile acids, and the like Preparation of such hposomal formulations is within the level of skill in the art, as disclosed, for example, in U S Patent Nos 4.235,871 , 4,501 ,728, 4.837,028, and 4,737,323, all of which are incoφorated herein by refeience

The amount of composition of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and seventy of the condition being treated, and on the nature of prior treatments which the patient has undergone Ultimately, the attending physician will decide the amount of composition of the present invention with which to treat each individual patient Initially, the attending physician will administer low doses of active ingredient of the present invention and observe the patient's response Larger doses of active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0 01 μg to about 100 mg (preferably about 0 1 μg to about 10 mg, more preferably about 0 1 μg to about 1 mg) of active ingredient of the present invention per kg body weight If desired, the therapeutic method includes administenng the composition topically, systematically, or locally as an implant or device When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage Topical administration may be suitable for wound healing and tissue repair

Therapeutically useful agents other than a composition of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention Preferably for bone and/or cartilage formation, the composition would include a matnx capable of delivering the protem-contaming composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body Such matrices may be formed of materials presently in use for other implanted medical applications

The choice of matrix material is based on biocompatibility, biodegradabihty, mechanical properties, cosmetic appearance and interface properties The particular application of the compositions will define the appropriate formulation Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate. tπcalcium phosphate, hydroxyapatite, polylactic acid, polyglycohc acid and polyanhydπdes Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen Further matrices are comprised of pure proteins or extracellular matnx components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matπces may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tπcalcium phosphate The bioceramics may be altered in composition, such as in calcium-alummate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabihty. Presently preferred is a 50-50 (mole weight) copolymer of lactic acid and glycohc acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequesteπng agent, such as carboxymefhyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matnx A preferred family of sequestering agents is cellulosic matenals such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most prefened being cationic salts of carboxymethylcellulose (CMC). Other prefened sequestering agents include hyaluromc acid, sodium algmate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvmyl polymer and poly(vmyl alcohol) The amount of sequesteπng agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matnx and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matnx, thereby providing the protein the opportunity to assist the fracture repair activity of the progenitor cells

The therapeutic compositions are also presently valuable for veterinary applications Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with compositions of the present invention The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e g , amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e g , bone), the patient's age sex, and diet, the seventy of any infection, time of administration and other clinical factors The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage Progress can be monitored by penodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomoφhometnc determinations and tetracyclme labeling

7.7. EFFECTIVE DOSAGE Pharmaceutical compositions suitable for use m the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended puφose More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being heated Determination of the effective amounts is well within the capability of those skilled m the art, especially in light of the detailed disclosure provided herein For any compound used the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays Such information can be used to more accurately determine useful doses in humans

A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e g , for determining the LD,_f) (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population) The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD,₀ and ED,₀

Compounds which exhibit high therapeutic indices are prefened The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human The dosage of such compounds lies preferably w ithin a range of circulating concentrations that include the ED_S0 with little or no toxicity The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition See, e g , Fingl et al , 1975, in "The Pharmacological Basis of Therapeutics", Ch 1 p 1 Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain the desired effects, or minimal effective concentration (MEC) The MEC will vary for each compound but can be estimated from in vitro Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration However, HPLC assays or bioassays can be used to determine plasma concentrations

Dosage intervals can also be determined using MEC value Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90% In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration

An exemplary dosage regimen for the human polypeptides of the invention will be m the range of about 0 01 to 100 mg/kg of body weight daily, with the preferred dose being about 0 1 to 25 mg/kg of patient body weight daily, varying in adults and children Dosmg may be once daily, or equivalent doses may be delivered at longer or shorter intervals

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the seventy of the affliction, the manner of administration and the judgment of the prescnbing physician

7.8. PACKAGING

The compositions may, if desired, be presented m a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient The pack may, for example, comprise metal or plastic foil, such as a blister pack The pack or dispenser device may be accompanied by instructions for administration Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition 8. ANTIBODIES

Another aspect of the invention is an antibody that specifically binds the cytokine receptor polypeptide of the invention Such antibodies include monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR and/or antigen-binding sequences, which specifically recognize a polypeptide of the invention Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in W093/1 1236, published June 20, 1993, which is incoφorated herein by reference in its entirety Antibody fragments, including Fab, Fab , F(ab ),, and F_%, are also provided by the invention The term "specific for" indicates that the variable regions of the antibodies of the invention recognize and bind CG92 polypeptides exclusively (i e , able to distinguish a CG92 polypeptide from other cytokine receptor polypeptides despite sequence identity, homology, or similaπty found in the family of polypeptides), but may also interact with other proteins (for example, S aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, m the constant region of the molecule Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art For a comprehensive discussion of such assays, see Harlov/ et al (Eds), Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor , NY (1988), Chapter 6 Antibodies that recognize and bind fragments of the CG92 polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, CG92 polypeptides As with antibodies that are specific for full length cytokine receptor polypeptides, antibodies of the invention that recognize CG92 fragments are those which can distinguish CG92 polypeptides from the family of cytokine receptor polypeptides despite inherent sequence identity, homology, or similarity found m the family of proteins Antibodies of the invention can be produced using any method well known and routinely practiced in the art Non-human antibodies may be humanized by any methods known in the art In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity

Antibodies of the invention are useful for, for example, therapeutic puφoses (by modulating activity of a polypeptide of the invention), diagnostic puφoses to detect or quantitate a polypeptide of the invention, as well as purification of a polypeptide of the invention Kits comprising an antibody of the invention for any of the puφoses descnbed herein are also comprehended In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific The invention further provides a hybridoma that produces an antibody according to the invention Antibodies of the invention are useful for detection and/or punfication of the polypeptides of the invention Proteins of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanm

(KLH) Methods for synthesizing such peptides are known in the art, for example, as in R P Merrifield, J Amer. Chem. Soc 85, 2149-2154 (1963), J L Krstenansky, et al., FEBS Lett 21 1. 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the lmmunodetection of the protein Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein In general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (Campbell, A.M . Monoclonal Antibodies Technology Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984), St Groth et al , J Immunol 35 1 -21 (1990), Kohler and Milstein, Nature 256 495-497 (1975)). the tπoma technique, the human B-cell hybridoma technique (Kozbor et al , Immunology Today 4 72 ( 1983 ), Cole et al , in Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc ( 1985), pp 77-96) Any animal (mouse, rabbit, etc.) which is known to produce antibodies can be immunized with a peptide or polypeptide of the invention. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of the protein encoded by the ORF of the present invention used for immunization will vary based on the animal which is immunized, the antigenicity of the peptide and the site of injection. The protein that is used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicity. Methods of increasing the antigenicity of a protein are well known in the art and include, but are not limited to, coupling the antigen with a heterologous protein (such as globulin or β-galactosidase) or through the inclusion of an adjuvant during immunization.

For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Research. 175:109-124 (1988)). Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and

Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)). Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to proteins of the present invention. For polyclonal antibodies, antibody containing antiserum is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures. The present invention further provides the above-described antibodies in delectably labeled form. Antibodies can be delectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labeling are well-known in the art, for example, see (Sternberger, L A et al , J Histochem Cytochem 18 315 (1970), Bayer, E A et al , Meth Enzym 62 308 ( 1979), Engval, E et al , Immunol 109 129 ( 1972), Godmg, J W J Immunol Meth 13 215 ( 1976))

The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the polypeptide of interest is expressed The antibodies may also be used directly in therapies or other diagnostics The present invention further provides the above-described antibodies immobilized on a solid support Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose , acrylic resins and such as polyacrylamide and latex beads Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D M et al , "Handbook of Experimental Immunology" 4th Ed , Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986), Jacoby, W D et al , Meth Enzym 34 Academic Press, N Y (1974)) The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for lmmuno-affinity punfication of the proteins of the present invention

9. COMPUTER READABLE SEQUENCES

In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media As used herein, "computer readable media" refers to any medium which can be read and accessed directly by a computer Such media include, but are not limited to magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape, optical storage media such as CD-ROM, electrical storage media such as RAM and ROM, and hybπds of these categories such as magnetic/optical storage media A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention As used herein, "recorded" refers to a process for storing information on computer readable medium A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures compnsmg the nucleotide sequence information of the piesent invention A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention The choice of the data storage structure will generally be based on the means chosen to access the stored information In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e.g text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention By providing the nucleotide sequence of SEQ ID NO: 1 or a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NO: 1 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of puφoses Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J Mol Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem 17:203-207 ( 1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) withm a nucleic acid sequence Such ORFs may be protein encoding fragments and may be useful m producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites

As used herein, "a computer-based system" refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention As stated above, the computer-based systems of the present invention compnse a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means As used herein, "data storage means" refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention As used herein, "search means" refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif A vaπety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention Examples of such software includes, but is not limited to, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA) A skilled artisan can readily recognize that any one of the available algonthms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems As used herein, a "target sequence" can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database The most preferred sequence length of a target sequence is from about 10 to 100 ammo acids or from about 30 to 300 nucleotide residues However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length

As used herein, "a target structural motif" or "target motif," refers to any rationally selected sequence or combination of sequences m which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif There are a variety of target motifs known m the art Protein target motifs include, but are not limited to, enzyme active sites and signal sequences Nucleic acid target motifs include, but are not limited to, promoter sequences, haiφm structures and inducible expression elements (protein binding sequences) 10. TRIPLE HELIX FORMATION

In addition, gene expression can be controlled through triple helix formation or antisense DNA or RNA, both of which methods aie based on the binding of a polynucleotide sequence to DNA or RNA Polynucleotides suitable for use m these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al , Nucl Acids Res 6 3073 (1979), Cooney et al , Science 15241 456 ( 1988), and Dervan et al , Science 251 1360 (1991)) or to the mRNA itself (antisense - Olmno, J Neurochem 56 560 (1991 ), Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press. Boca Raton, FL (1988)) Tnple hehx- formation optimally results in a shut-off of RNA transcπption from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide Both techniques have been demonstrated to be effective in model systems Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide

11. DIAGNOSTIC ASSAYS AND KITS

The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label In general, methods for detecting a polynucleotide of the invention can compnse contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample Such methods can also compnse contacting a sample under stringent hybπdization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample

In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample

Conditions for incubating a nucleic acid probe or antibody with a test sample vary Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention Examples of such assays can be found in Chard, T , An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands ( 1986), Bullock, G R et al , Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol 1 (1982), Vol 2 (1983), Vol 3 (1985), Tijssen, P , Practice and Theory of immunoassays Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985) The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or unne The test sample used in the above-descπbed method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed Methods for prepaπng protein extracts or membrane extracts of cells are well known m the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises (a) a first container comprising one of the probes or antibodies of the present invention, and (b) one or more other containers comprising one or more of the following wash reagents, reagents capable of detecting presence of a bound probe or antibody

In detail, a compartment kit includes any kit in which reagents are contained in separate containers Such containers include small glass containers, plastic containers or strips of plastic or paper Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered salme, Tπs-buffers, etc ), and containers which contain the reagents used to detect the bound antibody or probe Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the pπmary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incoφorated into one of the established kit formats which are well known in the art

12. MEDICAL IMAGING

The novel polypeptides of the invention are useful in medical imaging, e g , imaging the site of infection, inflammation, and other sites expressing CG92 receptor molecules See, e.g , Kunkel et al , U S Pat NO 5,413,778 Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable earner, and imaging the labeled polypeptide in vivo at the target site

13. SCREENING ASSAYS Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by the ORF from a polynucleotide of the invention to a specific domain of the polypeptide encoded by a polypeptide of the invention In detail, said method compπses the steps of (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention, and

(b) determining whether the agent binds to said protein or said nucleic acid In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified

Likewise, m general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified

Methods for identifying compounds that bind to a polypeptide of the invention can also compnse contacting a compound with a polypeptide of the invention m a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex dnves expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified

Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound) Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound) Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression

The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin denvatives, or other pharmaceutical agents The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques

For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention Alternatively, agents may be rationally selected or designed As used herein, an agent is said to be "rationally selected or designed" when the agent is chosen based on the configuration of the particular protein For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, for example see Hurby et al , Application of Synthetic Peptides Antisense Peptides," In Synthetic Peptides, A User's Guide, W H Freeman, NY ( 1992), pp 289-307, and Kaspczak et al , Biochemistry 28 9230-8 (1989), or pharmaceutical agents, or the like In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention As described above, such agents can be randomly screened or rationally designed/selected Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control One class of DNA binding agents are agents which contain base residues which hybridize or form a tnple helix formation by binding to DNA or RNA Such agents can be based on the classic phosphodiester, nbonucleic acid backbone, or can be a variety of sulfhydryl or polymenc denvatives which have base attachment capacity Agents suitable for use in these methods usually contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcπption (tπple helix - see Lee et al , Nucl Acids Res 6 3073 (1979), Cooney et al , Science 241 456 (1988), and Dervan et al , Science 251 1360 (1991)) or to the mRNA itself (antisense - Okano, J Neurochem 56 560 (1991), Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Triple helix- formation optimally results in a shut-off of RNA transcπption from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide Both techniques have been demonstrated to be effective in model systems Information contained in the sequences of the present invention is necessary for the design of an antisense or tnple helix oligonucleotide and other DNA binding agents Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent, in the control of bacterial infection by modulating the activity of the protein encoded by the ORF Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition 14. USE OF NUCLEIC ACIDS AS PROBES

Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences The hybridization probes of the subject invention may be derived from the nucleotide sequence of the SEQ ID NO 1 Because the conesponding gene is only expressed in a limited number of tissues, a hybπdization probe derived from SEQ ID NO 1 can be used as an indicator of the presence of RNA of cell type of such a tissue a sample

Any suitable hybridization technique can be employed, such as, for example, in situ hybridization PCR as described US Patent Nos 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both The probe will compnse a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences

Other means for producing specific hybndization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes Such vectors are known m the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al ( 1988) Human Chromosomes A Manual of Basic Techniques, Pergamon Press, New York NY Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data Examples of genetic map data can be found in the 1994 Genome Issue of Science (265 1981 f) Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals The nucleotide sequence may be used to produce puπfied polypeptides using well known methods of recombinant DNA technology Among the many publications that teach methods for the expression of genes after they have been isolated is Goeddel (1990) Gene Expression Technology, Methods and Enzymology, Vol 185, Academic Press, San Diego Polypeptides may be expressed in a vaπety of host cells, either prokaryotic or eukaryotic Host cells may be from the same species from which a particular polypeptide nucleotide sequence was isolated or from a different species Advantages of producing polypeptides by recombinant DNA technology include obtaining adequate amounts of the protein for punfication and the availability of simplified purification procedures Each sequence so obtained was compared to sequences in GenBank using a search algoπthm developed by Applied Biosystems and incoφorated into the INHERIT™ 670 Sequence Analysis System In this algorithm, Pattern Specification Language (developed by TRW Inc., Los Angeles, CA) was used to determine regions of homology The three parameters that determine how the sequence compaπsons run were window size, window offset, and error tolerance Using a combination of these three parameters, the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches Smith- Waterman alignments were used to display the results of the homology search Peptide and protein sequence homologies were ascertained using the INHERIT™ 670 Sequence Analysis System in a way similar to that used in DNA sequence homologies Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology that were scored with an initial value Dot-matrix homology plots were examined to distinguish regions of significant homology from chance matches

Alternatively, BLAST, which stands for Basic Local Alignment Search Tool, is used to search for local sequence alignments (Altschul SF (1993) J Mol Evol 36 290-300, Altschul, SF et al ( 1990) J Mol Biol 215:403-10). BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. Whereas it is ideal for matches which do not contain gaps, it is inappropriate for performing motif-style searching. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is inteφreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

In addition, BLAST analysis was used to search for related molecules within the libraries of the LIFESEQ™ database. This process, an "electronic northern" analysis is analogous to northern blot analysis in that it uses one cellubrevin sequence at a time to search for identical or homologous molecules at a set stringency. The stringency of the electronic northern is based on "product score". The product score is defined as (% nucleotide or amino acid [between the query and reference sequences] in Blast multiplied by the % maximum possible BLAST score [based on the lengths of query and reference sequences]) divided by 100. At a product score of 40, the match will be exact within a 1-2% enor; and at 70, the match will be exact. Homologous or related molecules can be identified by selecting those which show product scores between approximately 15 and 30.

The present invention is illustrated in the following examples. Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples. EXAMPLE 1 Cloning of Cytokine Receptor cDNA

Novel nucleic acids were obtained from various cDNA libraries (prepared from human mRNA purchased from Invitrogen, San Diego, CA) using standard PCR, sequencing by hybridization (SBH) sequence signature analysis and Sanger sequencing techniques The inserts of the library were amplified with PCR using primers specific for pSportl (GIBCO BRL, Grand Island, NY) vector sequences which flank the inserts These samples were spotted onto nylon membranes and hybπdized with oligonucleotide probes to give sequence signatures The clones were clustered into groups of similar or identical sequences, and single representative clones were selected from each group for gel sequencing The 5' sequence of the amplified inserts was then deduced using the reverse M13 sequencing pπmer m a typical Sanger sequencing protocol PCR products were purified and subjected to flourescent dye terminator cycle sequencing Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer

Sequence analysis identified a polynucleotide encoding a novel polypeptide designated CG92 The 5 sequence was determined as described in Example 2 The contig encoding CG92 was deduced from numerous clones identified below in Table 1

Table 1

No

Libiary Clones Clone ID Sequence ID

AOV001 (adult o\ ary) 1 8471458 RTA00001958F ι 17

FSKOOl (fetal skin) 2 18124717 RTA00003400F e 21 18146385 RTA00003542F a l4

EXAMPLE 2 5' RACE Extension of CG92 Gene

5' RACE reactions were performed using pans of nested gene-specific pnmeis (GSP) and v ector primers (VP) in sequential PCR leactions on a panel of cDNA libiaπes The cDNA libianes used for RACE ere prepared from mRNA using a ιandom-pι lined, 5' capture method to ennch foi the 5' ends of genes (Carnmci et al Genomics 37, 327-336, 1996) and cloned into the pSPORT v ectoi (BRL Life Technologies) previously digested with Notl and Sail The human mRNAs (Invitrogen) included message from adult brain, adult thymus, fetal muscle, fetal skm. fetal heart, fetal brain, fetal spleen, fetal liver, and fetal lung In addition, adaptor-hgated cDNA pools (Marathon cDNAs, Clontech) made from human fetal kidney, fetal brain and adult ovary mRNAs were used m the RACE expenments

The pπmers used are shown m Table 2 below Generally, in the first reaction, a first GSP (T_m ~80°C) and VP (T_m ~72°C) are mixed in a 5 1 ratio Touchdown PCR was carried out as follows, an initial incubation at 96°C for one minute, followed by five cycles of 96°C for 30 seconds and 72°C for four minutes, five cycles of 96°C for 30 seconds and 70°C for four minutes; and 15 cycles of 96°C for 30 seconds and 68°C for four minutes The products of the first reaction were diluted 1 20 and used as template for the second reaction. Second nested GSP and VP (both T_m ~60°C) were mixed m a 1 ^• 1 ratio and PCR was earned out as follows: an initial incubation at 96°C for one mmute, and 30 cycles of 96°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds This step was sometimes repeated with a third or more nested GSP and VP primer Final RACE products were separated and identified using agarose gel electrophoresis Selected fragments were subcloned into a TA cloning vector and the inserts were sequenced Sequences used to complete the 5' end of CG92 were obtained from the following cDNA sources: human fetal skin cDNA.

Table 2

GSP3 (CG92R3) SEQ ID NO

5 AC AGGCTCTCGTACTCCCCCTGGTATTCG3 '

EXAMPLE 3 Tissue Expression Study

PCR Analysis

Gene expression of the human cytokine receptors is analyzed using a semi-quantitative PCR-based technique A panel of cDNA libraries deriv ed from human tissue (fiom Clontech and Invitiogen) is screened with CG92 specific pπmeis to examine the mRNA expression of CG92 in human tissues and cell types PCR assays (For example, 94 °C for 30 sec , 58 °C for 30 sec , 72 °C for 30 sec , for 30 cycles) are performed with 20 ng of cDNA derived from human tissues and cell lines and 10 picomoles of the CG92 gene-specific primers The PCR pioduct is identified thiough gel electrophoresis Amplified products are separated on an agaiose gel, transferred and chemically linked to a nylon filter The filter is then hybridized with a radioactively labeled ( Pα-dCTP) double-stranded probe generated from the full-length sequence using a Klenow polymerase, random prime method The filters are washed (high stringency) and used to expose a phosphoπmaging screen for several hours Bands of the appropriate size indicate the presence of cDNA sequences m a specific hbiary, and thus mRNA expression in the conesponding cell type or tissue

Expression analysis can also be conducted using Northern blot techniques

EXAMPLE 4 Chromosomal Localization Study

Chromosome mapping technologies allow investigators to link genes to specific regions of chromosomes Chromosomal mapping is performed using the NIGMS human/rodent somatic cell hybrid mapping panel as described by Drwmga, H L et al , Genomics, 16, 31 1 - 314, 1993 (human/rodent somatic cell hybrid mapping panel #2 purchased fiom the Conell Institute for Medical Reseaich, Camden, New Jersey) 60 ng of DNA from each sample in the panel is used as template, and 10 picomoles of the same CG92 gene-specific oligonucleotides are used as primers in a PCR assay (foi example, 94°C foi 30 sec , 58°C foi 30 sec , 72°C for 30 sec , foi 30 cycles) PCR products were analyzed by gel electrophoresis The genomic PCR product is detected in a human/rodent somatic cell hybrid DNA contaming a specific human chromosome

EXAMPLE 5 Expression of Cytokine Receptors in E. coli SEQ ID NO 1 is expressed in E coli by subclonmg the entire coding region into a prokaryotic expression vector The expression vector (pQEl ό) used is from the

® QIAexpression prokaryotic protein expression system (QIAGEN) The features of this vector that make it useful for protein expression include an efficient promoter (phage

T5) to drive transcnption, expression control provided by the lac operator system, which can be induced by addition of IPTG (isopropyl-β-D-thiogalactopyranoside), and an encoded Hιs₆ tag The latter is a stretch of 6 histidine amino acid residues which can bind very tightly to a nickel atom The vector can be used to express a recombinant protein with a Hιs₆ tag fused to its carboxyl terminus, allowing rapid and efficient punfication using Ni-coupled affinity columns PCR is used to amplify the coding region which is then hgated into digested pQEl 6 vector The ligation product is transformed by electroporation into electrocompetent E coli cells (strain M15[pREP4] from QIAGEN), and the transformed cells are plated on ampicillm-containmg plates Colonies are screened for the correct insert in the proper oπentation using a PCR reaction employing a gene-specific pπmer and a vector-specific primer Positives are then sequenced to ensure correct oπentation and sequence To express cytokine receptor polypeptides, a colony containing a correct recombinant clone is inoculated into L-Broth containing 100 μg/ml of ampicillm, 25 μg/ml of kanamycm, and the culture was allowed to grow overnight at 37°C The saturated culture is then diluted 20-fold m the same medium and allowed to grow to an optical density at 600 nm of 0 5 At this point, IPTG is added to a final concentration of 1 mM to induce protein expression The culture is allowed to grow for 5 more hours, and then the cells are harvested by centnfugation at 3000 x g for 15 minutes

The resultant pellet is lysed using a mild, nonio c detergent in 20 mM Tris HCl (pH 7 5) (B-PER™ Reagent from Pierce), or by somcation until the turbid cell suspension turned translucent The lysate obtained is further purified using a nickel containing column (Ni-NTA spin column fiom QIAGEN) under non-denaturing conditions Briefly. the lysate is brought up to 300 mM NaCI and 10 mM lmidazole and centπfuged at 700 x g through the spin column to allow the His-tagged recombinant protein to bind to the nickel column The column is then washed twice with Wash Buffer (50 mM NaH,P0₄, pH 8 0, 300 mM NaCI, 20 mM imidazole) and is eluted with Elution Buffer (50 mM NaH,P0 , pH 8 0, 300 mM NaCI, 250 mM imidazole) All the above procedures are performed at 4°C The presence of a purified protein of the predicted size is confirmed with SDS-PAGE

EXAMPLE 6 Evaluation of Activities In Vitro and In Vivo The activity of polypeptides of the invention is assayed by monitoπng the effect of such polypeptides on the activity of various signal transduction pathways One commercially available system for monitoπng signal transduction is the Dual- Luciferase™ Reporter Assay System (Promega Coφ , Madison, WI) Briefly, mammalian cells are co-trans fected with (1) a construct expressing the cvtokme receptor polypeptide to be tested (e g , CG92, or an active fragment or an active fusion protein), (2) a first reporter construct utilizing a constitutive promoter (as a control for momtonng transfection efficiency), and (3) a second reporter construct that is dependent on a transcπption factor or an enhancer element involved in the signal transduction pathway of interest (which serves to monitor the activity of one of several signal transduction pathways)

Various second reporter constructs are available in both cis- and trans- configurations (from, e g , Stratagene, La Jolla, CA) The trans-configuration involves two constructs, and is used to monitor direct or indirect effects on signal transduction pathways which activate one of several transcription factors Second reporter constructs for the following transcription factors are currently available from Stratagene the Elkl transcnption factor for the mitogen-activated protein kinase (MAPK) signaling pathway, the c-Jun transcription factor for the c-Jun N-terminal kinase (JNK) signaling path ay, the CREB transcπption factor for the cAMP-dependent kinase (PKA) signaling pathway, the CHOP transcription factor for the p38 kinase signaling pathway, and the c-Fos and ATF2 transcπption factors The cis-configuration is used to monitor direct or indirect effects on six different enhancer elements Second reporter constructs for the following enhancer elements are currently available from Stratagene AP-1 , CRE, NF-kappaB, SRE, SRF and p53 Othei similar set of constructs may be prepared to monitor other transcription factors and enhancer elements known in the art

Cytokines, or other exogenous ligand, either alone or in combination with other cytokines. or serum, can be added to the transfected cells to determine the effects on candidate signal transduction pathways Comparison of the effects on different pathways will show specificity of the cytokine receptor's biological effects

In addition, this system can be used to screen libraries for small molecule drug candidates or lead compounds that disrupt or enhance the effects of the cytokine receptor

EXAMPLE 7

In vitro Proliferation Assay for CG92

In vitro proliferation assays are performed to detect or measure the activity of CG92 polypeptide Mammalian cells that preferably express little or no endogenous CG92 are transfected with either DNA encoding CG92 or vector DNA alone as the control Expression of CG92 by transfected cells can be confirmed by methods known in the art, e g , Western blot analysis At a suitable time (e g , 24 hours, 48 hours, 72 hours, or longer) after transfection, cells from each well are counted using a hemocytometer If cells transfected with DNA expressing CG92 proliferate at a different rate, l e , faster or slower, than cells transfected with vector alone, then it can be concluded that proliferation is an indirect measure of the biological activity of CG92 This assay can thus be used to screen for inhibitors or enhancers of CG92 activity by contacting the cells expressing CG92 with various test compounds, and detecting or measuπng proliferation of the cells in the presence and absence of test compounds Optionally, as a control, cells expressing control vector can also be contacted with the test compound and proliferation of these cells can be detected or measured in the presence and absence of the same test compounds

The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention Indeed, numerous modifications and variations in the practice of the inv ention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments. Consequently, the only limitations which should be placed upon the scope of the invention are those which appear the appended claims. All references cited within the body of the instant specification are hereby incoφorated by reference in their entirety.

Claims

WHAT IS CLAIMED IS

1 An isolated polynucleotide comprising a polynucleotide selected from the group consisting of

(a) a polynucleotide having the nucleotide sequence of SEQ ID NO 1 ,

(b) a polynucleotide having the cytokine receptor protein coding nucleotide sequence of a polynucleotide of (a), and

(c) a polynucleotide having the mature cytokine receptor protein coding nucleotide sequence of a polynucleotide of (a)

2 An isolated polynucleotide encoding a polypeptide with cytokine receptor activity, comprising a polynucleotide that encodes the ammo acid sequence of SEQ ID NO 2 or the mature protein sequence thereof

3 An isolated polynucleotide encoding a polypeptide with cytokine receptor activity that hybndizes under stringent conditions to the complement of a polynucleotide of any one of claims 1 or 2

4 An isolated polynucleotide encoding a polypeptide with cytokine receptor activity, said polynucleotide having greater than about 90% sequence identity with the polynucleotide of claim 1 or 2

5 The polynucleotide of claim 1 or 2 which is a DNA

6 An isolated polynucleotide which comprises a complement of the polynucleotide of claim 1

7 An expression vector comprising the DNA of claim 5

8 A host cell genetically engineered to expiess the DNA of claim 5 9 A host cell genetically engineered to contain the DNA of claim 5 in operative association with a regulatory sequence that controls expression of the DNA in the host cell

10 An isolated polypeptide with cytokine receptor activity comprising the ammo acid sequence of SEQ ID NO 2 or the mature protein sequence thereof

1 1 An isolated polypeptide with cytokine receptor activity selected from the group consisting of a) a polypeptide having greater than about 90% sequence identity with the polypeptide of claim 10, and b) a polypeptide encoded by the polynucleotide of claim 3

12 A composition compnsmg the polypeptide of claim 10 or 1 1 and a carrier

13 An antibody directed against the polypeptide of claim 10 or 1 1

14 A method for detecting a polynucleotide of claim 3 in a sample, compnsmg the steps of a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and b) detecting the complex, so that if a complex is detected, a polynucleotide of claim 3 is detected

15 A method for detecting a polynucleotide of claim 3 in a sample, comprising the steps of a) contacting the sample undei stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of claim 3 under such conditions, and b) amplifying the polynucleotides of claim 3 so that if a polynucleotide is amplified, a polynucleotide of claim 3 is detected 16 The method of claim 15, wheiein the polynucleotide is an RNA molecule that encodes a polypeptide of claim 1 1 , and the method further comprises reverse transcnbmg an annealed RNA molecule into a cDNA polynucleotide

17 A method for detectmg a polypeptide of claim 1 1 in a sample, comprising a) contacting the sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and b) detecting the complex, so that if a complex is detected, a polypeptide of claim 1 1 is detected

18 A method for identifying a compound that binds to a polypeptide of claim 1 1, comprising a) contacting a compound with a polypeptide of claim 1 1 for a time sufficient to form a polypeptide/compound complex, and b) detectmg the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polypeptide of claim 1 1 is identified

19 A method for identifying a compound that binds to a polypeptide of claim 1 1 , comprising a) contacting a compound with a polypeptide of claim 1 1 , in a cell, for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence m the cell, and b) detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds to a polypeptide of claim 1 1 is identified

20 A method of producing the polypeptide of claim 1 1. comprising, a) cultuπng the host cell of claim 8 for a period of time sufficient to express the polypeptide, and b) isolating the polypeptide from the cell or culture media in which the cell is mown 21 A method of identifying a candidate modulator of activity of the polypeptide of claim 1 1 comprising the steps of a) contacting a test compound with the polypeptide, and b) measuring the gand-bindmg or biological activity of the polypeptide in the presence and absence of the test compound

22 A method of modulating inflammation or coagulation in a subject in need thereof comprising the step of administering a modulator of the polypeptide of claim 1 1 to the subject