WO1999040195A1 - Mammalian receptor proteins; related reagents and methods - Google Patents

Mammalian receptor proteins; related reagents and methods Download PDF

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Publication number
WO1999040195A1
WO1999040195A1 PCT/US1999/002600 US9902600W WO9940195A1 WO 1999040195 A1 WO1999040195 A1 WO 1999040195A1 US 9902600 W US9902600 W US 9902600W WO 9940195 A1 WO9940195 A1 WO 9940195A1
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protein
leu
dcrsl
pro
gly
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PCT/US1999/002600
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French (fr)
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Jeanine D. Mattson
Terrill K. Mcclanahan
Robert A. Kastelein
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Schering Corporation
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Priority to AU26611/99A priority Critical patent/AU2661199A/en
Publication of WO1999040195A1 publication Critical patent/WO1999040195A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to compositions and methods for affecting mammalian physiology, including morphogenesis or immune system function.
  • it provides nucleic acids, proteins, and antibodies which regulate development and/or the immune system.
  • Various subunits of cytokine receptors, and the matching of subunits in a functional complex are described. Diagnostic and therapeutic uses of these materials are also disclosed.
  • BACKGROUND OF THE INVENTION refers generally to techniques of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment.
  • the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product.
  • cDNA complementary DNA
  • mRNA messenger RNA
  • the carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host. See, e.g., Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual . (2d ed. ) vols. 1-3, CSH Press, NY.
  • Lymphokines apparently mediate cellular activities in a variety of ways. See, e.g., Paul (ed. 1996) Fundamental Immunology 3d ed. , Raven Press, New York; and Thomson (ed. 1994) The Cytokine Handbook 2d ed. , Academic Press, San Diego. They have been shown to support the proliferation, growth, and/or differentiation of pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages which make up a complex immune system. Proper and balanced interactions between the cellular components are necessary for a healthy immune response. The different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents .
  • B-cells which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal)
  • T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and various other cells (including other T- cells) making up the immune network.
  • lymphocytes interact with many other cell types .
  • mast cell which has not been positively identified in all mammalian species
  • mast cell is a granule-containing connective tissue cell located proximal to capillaries throughout the body. These cells are found in especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts.
  • Mast cells play a central role in allergy-related disorders, particularly anaphylaxis as follows : when selected antigens crosslink one class of immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases mediators, e.g. , histamine, serotonin, heparin, and prostaglandins, which cause allergic reactions, e.g., anaphylaxis.
  • IL-1 receptors which modulate morphogenetic development. This includes, e.g., the Toll ligands, which signal through binding to receptors which share structural, and mechanistic, features characteristic of the IL-1 receptors. See, e.g., Lemaitre, et al . (1996) Cell 86:973-983; and Belvin and Anderson (1996) Ann. Rev. Cell & Devel . Biol. 12:393- 416. Other receptors for cytokines are also known. Often, there are at least two critical subunits in the functional receptor. See, e.g., Gonda and D 'Andrea (1997) Blood 89:355-369; Presky, et al . (1996) Proc.
  • the present invention provides new receptors for ligands exhibiting similarity to cytokine like compositions and related compounds, and methods for their use.
  • the present invention is directed to a novel receptor related to cytokine receptors , e.g., primate or rodent, cytokine receptor like molecular structures, designated DNAX Cytokine Receptor Subunit 1 (DCRSl) , and their biological activities.
  • DCRSl DNAX Cytokine Receptor Subunit 1
  • the matching of subunits in a functional receptor, and ligand identification are described. It includes nucleic acids coding for the combinations of polypeptides themselves and methods for their production and use.
  • the nucleic acids of the invention are characterized, in part, by their homology to cloned complementary DNA (cDNA) sequences enclosed herein.
  • the invention provides a composition of matter selected from the group of: a substantially pure or recombinant DCRSl protein or peptide exhibiting at least about 85% sequence identity over a length of at least about 12 amino acids to SEQ ID NO: 13 or 4 or 15; a natural sequence DCRSl comprising SEQ ID NO: 13 or 4 or 15; and a fusion protein comprising DCRSl sequence.
  • the substantially pure or isolated protein comprises a segment exhibiting sequence identity to a corresponding portion of a DCRSl, wherein: the homology is at least about 90% identity and the portion is at least about 9 amino acids; the homology is at least about 80% identity and the portion is at least about 17 amino acids; or the homology is at least about 70% identity and the portion is at least about 25 amino acids.
  • the composition of matter is DCRSl, which comprises a mature sequence of Table 1; or exhibits a non-glycosylated DCRSl; or the composition of matter may be a protein or peptide which: is from a warm blooded animal selected from a mammal, including a primate or rodent, such as a human or mouse; comprises at least one polypeptide segment of SEQ ID NO: 13 or 4 or 15; exhibits a plurality of portions exhibiting said identity; is a natural allelic variant of DCRSl; has a length at least about 30 amino acids; exhibits at least two non-overlapping epitopes which are specific for a primate or rodent DCRSl; exhibits a sequence identity at least about 90% over a length of at least about 20 amino acids to a primate or rodent DCRSl; is glycosylated; has a molecular weight of at least 100 kD with natural glycosylation; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another
  • compositions comprising: a sterile DCRSl protein or peptide; or the DCRSl protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
  • the invention provides a fusion protein comprising: mature protein sequence of Table 1; a detection or purification tag, including a FLAG, His6, or Ig sequence; or sequence of another receptor protein.
  • kit embodiments include a kit comprising a DCRSl protein or polypeptide, and: a compartment comprising the protein or polypeptide; and/or instructions for use or disposal of reagents in the kit .
  • Binding compound embodiments include those comprising an antigen binding site from an antibody, which specifically binds to a natural DCRSl protein, wherein: the protein is a primate protein; the binding compound is an Fv, Fab, or Fab2 fragment; the binding b
  • a binding composition kit often comprises the binding compound, and: a compartment comprising said binding compound; and/or instructions for use or disposal of reagents in the kit.
  • kits are capable of making a qualitative or quantitative analysis .
  • Other compositions include a composition comprising: a sterile binding compound, or the binding compound and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
  • Nucleic acid embodiments include an isolated or recombinant nucleic acid encoding a DCRSl protein or peptide or fusion protein, wherein: the DCRSl is from a mammal; or the nucleic acid: encodes an antigenic peptide sequence of Table 1; encodes a plurality of antigenic peptide sequences of Table 1; exhibits at least about 80% identity to a natural cDNA encoding said segment; is an expression vector; further comprises an origin of replication; is from a natural source; comprises a detectable label; comprises synthetic nucleotide sequence; is less than 6 kb, preferably less than 3 kb; is from a mammal, including a primate; comprises a natural full length coding sequence; is a hybridization probe for a gene encoding said DCRSl; or is a PCR primer, PCR product, or mutagenesis primer.
  • a cell, tissue, or organ comprising such a recombinant nucleic acid is also provided.
  • the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell.
  • Kits are provided comprising such nucleic acids, and: a compartment comprising said nucleic acid; a compartment further comprising a primate or rodent DCRSl protein or polypeptide; and/or instructions for use or disposal of reagents in the kit.
  • the kit is capable of making a qualitative or quantitative analysis .
  • nucleic acid which: hybridizes under wash conditions of 30° C and less than
  • 2M salt to SEQ ID NO: 12 or 3 or 14; or exhibits at least about 85% identity over a stretch of at least about 30 nucleotides to a primate DCRSl.
  • such nucleic acid will have such properties, wherein: wash conditions are at 45° C and/or 500 mM salt; or the identity is at least 90% and/or the stretch is at least 55 nucleotides.
  • the wash conditions are at 55° C and/or 150 mM salt; or the identity is at least 95% and/or the stretch is at least 75 nucleotides.
  • the invention also provides a method of modulating physiology or development of a cell or tissue culture cells comprising contacting the cell with an agonist or antagonist of a mammalian, e.g., primate or rodent DCRSl. It also provides cells cotransfected with a nucleic acid encoding DCRSl and another cytokine receptor subunit, e.g., DSRS1. This will allow pairing of subunits to determine the physiological receptor pairs for various cytokine ligands .
  • the present invention provides various compositions, e.g., comprising both a DSRS1 protein and an isolated or recombinant DCRSl protein; both an isolated or recombinant DSRS1 protein and a DCRSl protein; or both a substantially pure or recombinant IL-B30 protein and a DCRSl protein.
  • the DSRS1 protein has sequence of mature SEQ ID NO: 9 or 11; the DCRSl protein has sequence of mature SEQ ID NO: 13 or 15; or the IL-B30 has sequence of mature SEQ ID NO: 17 or 19.
  • at least one of the proteins is unglycosylated; is made with synthetic methods; has a detectable label; is attached to a solid substrate; or is conjugated to another chemical moiety.
  • the invention provides a composition comprising: a substantially pure DCRSl protein and: a DSRSl protein or an IL-B30 cytokine protein; or a DCRSl protein and a substantially pure: DSRSl protein or IL-B30 cytokine protein.
  • Preferred forms combining the DCRSl and the DSRSl proteins are those where the proteins combine to bind IL-B30 with high affinity.
  • Yet other forms include sterile compositions, as described.
  • Kit embodiments include such compositions combined with: a compartment comprising two or more of the proteins; a compartment comprising a soluble receptor alpha subunit; a compartment comprising an IL-B30 cytokine protein; or instructions for use or disposal of reagents in the kit .
  • Binding composition embodiments include those comprising the antigen binding sites from antibodies, which antibodies bind to an epitope found on a composition described above, but not on separate proteins thereof.
  • the DCRSl is: a primate protein, a purified human or mouse DCRSl, or a mature polypeptide of Table 1
  • the DSRSl is: a primate protein, a purified human or mouse DSRSl, or a mature polypeptide of Table 4
  • the IL-B30 is: a primate protein, a purified human or mouse IL-B30, or a mature polypeptide of Table 6.
  • binding composition is in a container; is an Fv, Fab, or Fab2 fragment; is conjugated to another chemical moiety; is immunoselected; is a polyclonal antibody; exhibits a Kd to antigen of at least 30 ⁇ M; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label.
  • Kit embodiments which comprise the binding compositions further include, e.g., a compartment 9
  • nucleic acid composition comprising the binding composition; a compartment comprising the DCRSl, DSRSl, or IL-B30 protein; or instructions for use or disposal of reagents in the kit.
  • Certain nucleic acid composition are provided, e.g., an isolated or recombinant nucleic acid encoding both: a DSRSl protein and a DCRSl protein; a DSRSl protein and an IL-B30 protein; or a DCRSl protein and an IL-B30 protein.
  • Preferred embodiments are those nucleic acids which encode both a DCRSl protein and a DSRSl protein; or both a DSRSl protein and an IL-B30, e.g., in a fusion protein.
  • nucleic acids which are expression vectors are those nucleic acids which are expression vectors.
  • the DSRSl protein has sequence of mature SEQ ID NO: 9 or 11; the DCRSl protein has sequence of mature SEQ ID NO: 13 or 15; or the IL-B30 has sequence of mature SEQ ID NO: 17 or 19.
  • Specific embodiments are those comprising the coding portion of: SEQ ID NO: 8 or 10; SEQ ID NO: 12 or 14; or SEQ ID NO: 16 or 18.
  • Transformed cells with the nucleic acids are provided, including where the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell.
  • a method of producing a receptor complex comprising culturing a described transformed cell of in an environment resulting in expression of the DCRSl and the DSRSl proteins, thereby forming the receptor complex; or of screening for ligands for a receptor complex comprising the DCRSl and the DSRSl proteins, comprising screening a library of compounds for binding to the described cell .
  • the present invention provides the amino acid sequence and DNA sequence of mammalian, herein primate, cytokine receptor-like subunit molecules, this one designated DNAX Cytokine Receptor Subunit 1 (DCRSl) having particular defined properties, both structural and biological .
  • DCRSl DNAX Cytokine Receptor Subunit 1
  • Various cDNAs encoding these molecules were obtained from primate, e.g., human, cDNA sequence libraries . Other primate or other mammalian counterparts would also be desired.
  • Partial nucleotide (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ ID NO: 2) of a human DCRSl coding segment is shown in Table 1, with supplementary sequence provided in SEQ ID NO: 12 and 13.
  • Partial mouse sequence is provided (SEQ ID NO: 3 and 4) , with supplementary sequence in SEQ ID NO: 14 and 15.
  • Table 1 Partial nucleotide and polypeptide sequences of DNAX Cytokine Receptor Subunit like embodiments (DCRSl) .
  • Primate e.g., human embodiment (see SEQ ID NO: 1 and 2) .
  • Partial "downstream" sequences of rodent, e.g., mouse, embodiment of DCRSl (SEQ ID NO: 3 and 4) : aaa gga ggg gtc ccc tat cga att aca gtg act gca gta tac tct gga 48 Lys Gly Gly Val Pro Tyr Arg He Thr Val Thr Ala Val Tyr Ser Gly 1 5 10 15 gga tta get get gca ccc tea gtt tgg gga ttc aga gag gag gag tta gta 96 Gly Leu Ala Ala Ala Pro Ser Val Trp Gly Phe Arg Glu Glu Leu Val 20 25 30 ccc ctt get ggg cca gca gtt tgg cga ctt cca gat gac ccc cca ggg 144 Pro Leu Ala Gly Pro Ala Val Tr
  • supplementary primate e.g., human, DCRSl sequence (SEQ ID NO: 12 and 13 ) : atg egg gga ggc agg ggc gee cct ttc tgg ctg tgg ccg ctg ccc aag 48 Met Arg Gly Gly Arg Gly Ala Pro Phe Trp Leu Trp Pro Leu Pro Lys 1 5 10 15 ctg gcg ctg ctg cct ctg ttg tgg gtg cttttc cag egg acg cgt cccc 96 Leu Ala Leu Leu Pro Leu Leu Trp Val Leu Phe Gin Arg Thr Arg Pro 20 25 30 cag ggc age gec ggg cca ctg cag tgc tac gga gtt gga ccc ttg ggc 144
  • DCRP1 sequences SEQ ID NO: 14 and 15: ggt aag ccc caa gee tgg tgg tgt cac ttg tec ctg gga gec atg aac 48 Gly Lys Pro Gin Ala Trp Trp Cys His Leu Ser Leu Gly Ala Met Asn 1 5 10 15 egg etc ggg ttt gca cgc etc acg ccg ttg gag ctt ctg ctg teg ctg 96 Arg Leu Gly Phe Ala Arg Leu Thr Pro Leu Glu Leu Leu Leu Ser Leu
  • Table 2 Comparison of rodent, e.g., mouse, and primate, e.g., human, DCRSl :
  • Table 3 Alignment of various cytokine receptor subunits.
  • Human gpl30 sequence (hgpl30) is SEQ ID NO: 5 (see GenBank M57230) .
  • Human G-CSF Receptor subunit alpha (hGCSFRa) is SEQ ID NO: 6 (see GenBank X55721) .
  • Human IL-12 Receptor subunit beta (hIL12Rb) is SEQ ID NO: 7 (see GenBank U64198) . Consensus domain boundaries are described in the text .
  • mIL30Rb hIL30Rb EDDPLEA TVHWAPPTWPSHKVLICQF-HYRRCQEAAWTLLEPELKTI human_GCSF EAAPPQAGCLQLCWEPWQPGLHINQKCELRHKPQRGEASWALVGPLP—L human_gpl3 ELSSILK LTWTNPSIKSVIILKYNI-QYRTKDASTWSQIPPEDTAS human_IL12 ASVSRCT LYWRDE GLVLLNRLRYRPSNSRLWNMVNVTK
  • mIL30Rb hIL30Rb RELSPEGITCCCSLIPSGAEWARVSAVNATSWEPLTNLSLVCLDSASAPR human_GCSF GAILPLCNTTELSCTFHLPSEAQEVALVAYNSAGTSRPTPWFSESRGPA human_gpl3 LQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQATHP human IL12 TQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLA
  • Table 4 Sequences of mammalian DNAX Soluble Receptor Subunit 1 (DSRSl).
  • Primate e.g., human, nucleotide and polypeptide sequences (SEQ ID NO: 8 and 9; note WSEWS motif at 327-331): atg ccc gee ggc cgc egg ggc ccc gee gee caa tec gcg egg egg ccg 48 Met Pro Ala Gly Arg Arg Gly Pro Ala Ala Gin Ser Ala Arg Arg Pro 1 5 10 15 ccg ccg ttg ctg cccc ctg ctg ctg etc tgc gtc etc ggg gcg ccg 96 Pro Pro Leu Leu Pro Leu Leu Leu Leu Cys Val Leu Gly Ala Pro
  • Partial rodent e.g., mouse, nucleotide and polypeptide sequences, note WSEWS motif at 321-325 (SEQ ID NO: 10 and 11) : ccc tea eta aag gga ata age ttg egg ccg ctg tec teg ctg tgg teg 48 Pro Ser Leu Lys Gly He Ser Leu Arg Pro Leu Ser Ser Leu Trp Ser 1 5 10 15 cct ctg ttg etc tgt gtc etc ggg gtg cct egg ggc gga teg gga gec 96 Pro Leu Leu Leu Cys Val Leu Gly Val Pro Arg Gly Gly Ser Gly Ala 20 25 30 cac aca get gta ate age ccc cag gac cc ace ctt etc ate ggc tec 144
  • aca tgt gag gag tac cac act gtg ggc cct cac tea tgc cat ate ccc 576 Thr Cys Glu Glu Tyr His Thr Val Gly Pro His Ser Cys His He Pro 180 185 190 aag gac ctg gec etc ttc act ccc tat gag ate tgg gtg gaa gec ace 624 Lys Asp Leu Ala Leu Phe Thr Pro Tyr Glu He Trp Val Glu Ala Thr 195 200 205 aat cgc eta ggc tea gca aga tct gat gtc etc aca ctg gat gtc ctg 672 Asn Arg Leu Gly Ser Ala Arg Ser Asp Val Leu Thr Leu Asp Val Leu 210 215 220 gac gtg gtg ace acg gac ccc 576 Thr
  • ggg gc ctg gag gac cag ctg agt gtg cgc tgg gtc tea cca cca get 768 Gly Gly Leu Glu Asp Gin Leu Ser Val Arg Trp Val Ser Pro Pro Ala 245 250 255 etc aag gat ttc etc ttc caa gec aag tac cag ate cgc tac cgc gtg 816 Leu Lys Asp Phe Leu Phe Gin Ala Lys Tyr Gin He Arg Tyr Arg Val 260 265 270 gag gac age gtg gac tgg aag gtg gtg gat gac gtc age aac cag ace 864 Glu Asp Ser Val Asp Trp Lys Val Val Asp Asp Val Ser Asn Gin Thr 275 280 285 tec tgc cgt etc gcg ggc ctg aag
  • Table 5 Alignment of primate, e.g., human, and rodent, e.g., mouse, DSRSl (SEQ ID NO: 9 and 11): hDSRSl MPAGRRGPAA QSARRPPPLL PLLLLLCVLG APRAGSGAHT AVISPQDPTL mDSRSl RPLSSL WSPLLLCVLG VPRGGSGAHT AVISPQDPTL
  • hDSRSl LTCRWTPGAH GETFLHTNYS LKYKLRWYGQ DNTCEEYHTV GPHSCHIPKD
  • mDSRSl LTCRWTPGAH GETFLHTNYS LKYKLRWYGQ DNTCEEYHTV GPHSCHIPKD
  • Rodent e.g., mouse, nucleotide and polypeptide sequences of IL-B30. Predicted signal cleavage site indicated, but may actually be a residue or more to either side, depending upon the cell (SEQ ID NO: 18 and 19) : cgcttagaag teggactaca gagttagact cagaaccaaa ggaggtggat agggggtcca 60 caggcctggt geagateaca gagceagcea gatctgagaa geagggaaca ag atg ctg 118
  • Table 2 shows comparison of the available sequences of primate and rodent embodiments of DCRSl .
  • Table 3 shows the alignment of the DCRSl with other cytokine receptor subunits .
  • the DCRSl shows particular similarity to the IL-12 receptor subunit beta, though it may be aligned with the gpl30 (IL-6 receptor beta) and G-CSF receptor (alpha) subunits.
  • the similarity to the IL-12 receptor subunit suggests that the functional receptor incorporating the DCRSl may be similar to the IL-12 receptor.
  • the IL-12 receptor alpha subunit is a soluble subunit.
  • the DSRSl is likely to be the corresponding soluble subunit, which would initially interact with ligand, e.g., the IL-B30, and then form a functional complex with the DCRSl. See, e.g., Presky, et al . (1996) Proc. Nat ' 1 Acad. Sci. USA 93:14002-14007.
  • the soluble subunit may interact with the transmembrane receptor subunit, which then could bind ligand.
  • Table 4 provides the nucleotide and polypeptide sequences of two species embodiments of a soluble receptor subunit designated DNAX Soluble Receptor Subunit 1 (DSRSl) . These align with and exhibit features in common with other cytokine receptor alpha type subunits. These subunits are believed to interact with the corresponding DCRSl to form a functional receptor when the receptor ligand is present. Note that relatively close sequence similarity of the DCRSl is with gpl30, which is the beta subunit of the IL-6 receptor. Applicants believe that the ligand for the receptor is likely the ligand designated IL-B30, whose sequence is near to G-CSF and IL-6. See USSN 60/053,765, which is incorporated herein by reference.
  • DSRSl DNAX Soluble Receptor Subunit 1
  • Table 5 shows alignment of the DSRSl from the primate and rodent species. Applicants believe that this soluble subunit forms a dimer, and binds to its dimerized ligand, which then combines with a beta type homo or heterodimer . Alternatively, the soluble subunit may bind to the transmembrane subunit (s), and then bind ligand.
  • Structural features of the human DCRSl include characteristic Ig domains from about (SEQ ID NO: 2) vail to prol33; fibronectin domains corresponding to the DCRSl sequence from about glyl34 to pro232, gly233 to gly306, and pro307 to lys403; a transmembrane segment from about val404 to gly427; and an intraceUular domain from about arg428 to the carboxy terminus .
  • WGEWS motif corresponding to residues trpl04 to serl08.
  • various variants and fragments will be equivalent to the described DSRSl.
  • DCRSl shall be used to describe a protein comprising the amino acid sequence shown in Table 1. In many cases, a substantial fragment thereof will be functionally or structurally equivalent, including, e.g., an extracellular or intraceUular domain.
  • the invention also includes a protein variation of the respective DCRSl allele whose sequence is provided, e.g., a mutein or soluble extracellular construct. Typically, such agonists or antagonists will exhibit less than about 10% sequence differences, and thus will often have between 1- and 11-fold 31
  • substitutions e.g., 2-, 3-, 5-, 7-fold, and others. It also encompasses allelic and other variants, e.g., natural polymorphic, of the protein described. Typically, it will bind to its corresponding biological ligand, perhaps in a dimerized state with an alpha receptor subunit, with high affinity, e.g., at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM.
  • the term shall also be used herein to refer to related naturally occurring forms, e.g., alleles, polymorphic variants, and metabolic variants of the mammalian protein.
  • Preferred forms of the receptor complexes will bind the appropriate ligand with an affinity and selectivity appropriate for a ligand- receptor interaction.
  • This invention also encompasses combinations of proteins or peptides having substantial amino acid sequence identity with the amino acid sequence in Table 1. It will include sequence variants with relatively few substitutions, e.g., preferably less than about 3-5.
  • a substantial polypeptide "fragment”, or “segment” is a stretch of amino acid residues of at least about 8 amino acids, generally at least 10 amino acids, more generally at least 12 amino acids, often at least 14 amino acids, more often at least 16 amino acids, typically at least 18 amino acids, more typically at least 20 amino acids, usually at least 22 amino acids, more usually at least 24 amino acids, preferably at least 26 amino acids, more preferably at least 28 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids. Sequences of segments of different proteins can be compared to one another over appropriate length stretches. In many situations, fragments may exhibit functional properties of the intact subunits, e.g., the extracellular domain of the 32
  • transmembrane receptor may retain the ligand binding features, and may be used to prepare a soluble receptorlike complex.
  • Amino acid sequence homology, or sequence identity is determined by optimizing residue matches. In some comparisons, gaps may be introduces, as required. See, e.g., Needleham, et al . , (1970) J. Mol. Biol. 48:443-453; Sankoff, et al., (1983) chapter one in Time Warps, String Edits , and Macromolecules : The Theory and Practice of Sequence Comparison, Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA; and the University of Wisconsin Genetics Computer Group (GCG) , Madison, WI; each of which is incorporated herein by reference. This changes when considering conservative substitutions as matches .
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • Homologous amino acid sequences are intended to include natural allelic and interspecies variations in the cytokine sequence. Typical homologous proteins or peptides will have from 50-100% homology (if gaps can be introduced) , to 60-100% homology (if conservative substitutions are included) with an amino acid sequence segment of Table 1.
  • Homology measures will be at least about 70%, generally at least 76%, more generally at least 81%, often at least 85%, more often at least 88%, typically at least 90%, more typically at least 92%, usually at least 94%, more usually at least 95%, preferably at least 96%, and more preferably at least 97%, and in particularly preferred embodiments, at least 98% or more.
  • the degree of homology will vary with the length of the compared segments .
  • Homologous proteins or peptides, such as the allelic variants, will share most biological activities with the embodiments described in Table 1. 33
  • biological activity is used to describe, without limitation, effects on inflammatory responses, innate immunity, and/or morphogenic development by cytokine-like ligands .
  • these receptors should mediate phosphatase or phosphorylase activities, which activities are easily measured by standard procedures. See, e.g., Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al . (1991) Meth. Enzvmol. 200:38-62; Hunter, et al . (1992) Cell
  • the receptors, or portions thereof, may be useful as phosphate labeling enzymes to label general or specific substrates .
  • the subunits may also be functional immunogens to elicit recognizing antibodies, or antigens capable of binding antibodies .
  • ligand, agonist, antagonist, and analog of, e.g., a DCRSl include molecules that modulate the characteristic cellular responses to cytokine ligand proteins, as well as molecules possessing the more standard structural binding competition features of ligand-receptor interactions, e.g., where the receptor is a natural receptor or an antibody.
  • the cellular responses likely are typically mediated through receptor tyrosine kinase pathways.
  • a ligand is a molecule which serves either as a natural ligand to which said receptor, or an analog thereof, binds, or a molecule which is a functional analog of the natural ligand.
  • the functional analog may be a ligand with structural modifications, or may be a wholly unrelated molecule which has a molecular shape which interacts with the appropriate ligand binding determinants.
  • the ligands may serve as agonists or antagonists, see, e.g., Goodman, et al. (eds. 1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics , Pergamon Press, New York.
  • Rational drug design may also be based upon structural studies of the molecular shapes of a receptor or antibody and other effectors or ligands. See, e.g., Herz, et al . (1997) J. Recept . Signal Transduct. Res. 17:671-776; and Chaiken, et al . (1996) Trends Biotechnol . 14:369-375. Effectors may be other proteins which mediate other functions in response to ligand binding, or other proteins which normally interact with the receptor.
  • One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., x-ray crystallography or 2 dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions . For a detailed description of protein structural determination, see, e.g., Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York, which is hereby incorporated herein by reference.
  • the cytokine receptor-like proteins will have a number of different biological activities, e.g., modulating cell proliferation, or in phosphate metabolism, being added to or removed from specific substrates, typically proteins. Such will generally result in modulation of an inflammatory function, other innate immunity response, or a morphological effect.
  • the subunit will probably have a specific low affinity binding to the ligand.
  • the DCRSl has the characteristic motifs of a receptor signaling through the JAK pathway. See, e.g., Ihle, et al . (1997) Stem Cells 15(suppl. 1):105-111; Silvennoinen, et al. (1997) APMIS 105:497-509; Levy (1997) Cvtokine Growth Factor Review 8:81-90; Winston and Hunter (1996) Current Biol. 6:668-671; Barrett (1996) Baillieres Clin. Gastroenterol. 10:1-15; and Briscoe, et al. (1996) Philos. Trans. R. Soc . Lond. B. Biol. Sci. 351:167-171.
  • the biological activities of the cytokine receptor subunits will be related to addition or removal of phosphate moieties to substrates, typically in a specific manner, but occasionally in a non specific manner. Substrates may be identified, or conditions for enzymatic activity may be assayed by standard methods, e.g., as described in Hardie, et al . (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al . (1991) Meth. Enzvmol. 200:38-62; Hunter, et al . (1992) Cell 70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al . (1991) Cold Soring Harbor Svmp. Quant. Biol. 56:449-463; and Parker, et al . (1993) Nature 363:736-738.
  • the receptor subunits may combine to form functional complexes, e.g., which may be useful for binding ligand or preparing antibodies . These will have substantial diagnostic uses, including detection or quantitation.
  • nucleic Acids This invention contemplates use of isolated nucleic acid or fragments, e.g., which encode these or closely related proteins, or fragments thereof, e.g., to encode a corresponding polypeptide, preferably one which is biologically active.
  • this invention covers isolated or recombinant DNAs which encode combinations of such proteins or polypeptides having characteristic sequences, e.g., of the DCRSls.
  • the nucleic acid is capable of hybridizing, under appropriate conditions, with a nucleic acid sequence segment shown in Table 1, but preferably not with a corresponding segment of other receptors described in Table 3.
  • Said biologically active protein or polypeptide can be a full length protein, or fragment, and will typically have a segment of amino acid sequence highly homologous, e.g., exhibiting significant stretches of identity, to one shown in Table 1. Further, this invention covers the use of isolated or recombinant nucleic acid, or fragments thereof, which encode proteins having fragments which are 36
  • the isolated nucleic acids can have the respective regulatory sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and others from the natural gene. Combinations, as described, are also provided.
  • an "isolated" nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially pure, e.g., separated from other components which naturally accompany a native sequence, such as ribosomes, polymerases, and flanking genomic sequences from the originating species .
  • the term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, which are thereby distinguishable from naturally occurring compositions, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • a substantially pure molecule includes isolated forms of the molecule, either completely or substantially pure.
  • An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain heterogeneity, preferably minor. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
  • a "recombinant" nucleic acid is typically defined either by its method of production or its structure.
  • the process is use of recombinant nucleic acid techniques, e.g. , involving human intervention in the nucleotide sequence.
  • this intervention involves in vitro manipulation, although under certain circumstances it may involve more classical animal breeding techniques.
  • it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants as 37
  • nucleic acids comprising sequence derived using any synthetic oUgonucleotide process.
  • Such a process is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a restriction enzyme sequence recognition site.
  • the process is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms, e.g., encoding a fusion protein.
  • Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • site specific targets e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • a similar concept is intended for a recombinant, e.g. , fusion, polypeptide. This will include a dimeric repeat.
  • synthetic nucleic acids which, by genetic code redundancy, encode equivalent polypeptides to fragments of DCRSl and fusions of sequences from various different related molecules, e.g., other cytokine receptor family members.
  • a "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least 21 nucleotides, more generally at least 25 nucleotides, ordinarily at least 30 nucleotides, more ordinarily at least 35 nucleotides, often at least 39 nucleotides, more often at least 45 nucleotides, typically at least 50 nucleotides, more typically at least 55 nucleotides, usually at least 60 nucleotides, more usually at least 66 nucleotides, preferably at least 72 nucleotides, more preferably at least 79 nucleotides, and in particularly preferred embodiments will be at least 85 or more nucleotides.
  • fragments of different genetic sequences can be compared to one J o
  • a nucleic acid which codes for the DCRSl will be particularly useful to identify genes, mRNA, and cDNA species which code for itself or closely related proteins, as well as DNAs which code for polymorphic, allelic, or other genetic variants, e.g., from different individuals or related species .
  • Preferred probes for such screens are those regions of the interleukin which are conserved between different polymorphic variants or which contain nucleotides which lack specificity, and will preferably be full length or nearly so. In other situations, polymorphic variant specific sequences will be more useful .
  • Nucleic acids encoding various combinations including, e.g., the DSRSl, the DCRSl, and/or the IL-B30, will be useful for coexpression of the proteins together. Such will be useful, e.g., in producing complexes, for production of antibodies or screening for ligands.
  • This invention further covers recombinant nucleic acid molecules and fragments having a nucleic acid sequence identical to or highly homologous to the isolated DNA set forth herein.
  • the sequences will often be operably linked to DNA segments which control transcription, translation, and DNA replication. These additional segments typically assist in expression of the desired nucleic acid segment.
  • nucleic acid sequences when compared to one another, e.g., DCRSl sequences, exhibit significant similarity.
  • the standards for homology in nucleic acids are either measures for homology generally used in the art by sequence comparison or based upon hybridization conditions . Comparative hybridization conditions are described in greater detail below.
  • nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical 39
  • nucleotide insertions or deletions in at least about 60% of the nucleotides, generally at least 66%, ordinarily at least 71%, often at least 76%, more often at least 80%, usually at least 84%, more usually at least 88%, typically at least 91%, more typically at least about 93%, preferably at least about 95%, more preferably at least about 96 to 98% or more, and in particular embodiments, as high at about 99% or more of the nucleotides, including, e.g., segments encoding structural domains such as the segments described below. Alternatively, substantial identity will exist when the segments will hybridize under selective hybridization conditions, to a strand or its complement, typically using a sequence derived from Table 1.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, more typically at least about 65%, preferably at least about 75%, and more preferably at least about 90%. See, Kanehisa (1984) Nucl. Acids Res . 12:203-213, which is incorporated herein by reference.
  • the length of homology comparison may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, generally at least about 20 nucleotides, ordinarily at least about 24 nucleotides, usually at least about 28 nucleotides, typically at least about 32 nucleotides, more typically at least about 40 nucleotides, preferably at least about 50 nucleotides, and more preferably at least about 75 to 100 or more nucleotides. This includes, e.g., 125, 150, 175, 200, 225, 246, 273, and other lengths.
  • Stringent conditions in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters typically controlled in hybridization reactions .
  • Stringent temperature conditions will usually include temperatures in excess of about 30° C, more usually in excess of about 37° C, typically in excess of about 45° C, more typically in excess of about 55° C, preferably in excess of about 65° C, and more preferably in excess of about 70° C.
  • Stringent salt conditions will ordinarily be less than about 500 mM, usually less than about 400 mM, more usually less than about 300 mM, typically less than about 200 mM, preferably less than about 100 mM, and more preferably less than about 80 mM, even down to less than about 20 M.
  • the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and
  • the isolated DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode this protein or its derivatives . These modified sequences can be used to produce mutant proteins (muteins) or to enhance the expression of variant species. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. Such mutant DCRS1- like derivatives include predetermined or site-specific mutations of the protein or its fragments, including silent mutations using genetic code degeneracy.
  • “Mutant DCRSl” as used herein encompasses a polypeptide otherwise falling within the homology definition of the DCRSl as set forth above, but having an amino acid sequence which differs from that of other cytokine receptor-like proteins as found in nature, whether by way of deletion, substitution, or insertion.
  • site specific mutant DCRSl encompasses a protein having substantial sequence identity with a protein of Table 1, and typically shares most of the biological activities or effects of the forms disclosed herein.
  • Mammalian DCRSl mutagenesis can be achieved by making amino acid insertions or deletions in the gene, coupled with expression. Substitutions, deletions, insertions, or many combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed mammalian DCRSl mutants can then be screened for the desired activity, providing some aspect of a structure-activity relationship. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by M13 primer mutagenesis. See also Sambrook, et al . (1989) and Ausubel, et al . (1987 and periodic Supplements) .
  • the mutations in the DNA normally should not place coding sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins .
  • a double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • PCR Polymerase chain reaction
  • mutagenesis primers are commonly used methods for generating defined mutations at predetermined sites. See, e.g., Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA; and Dieffenbach and Dveksler (1995; eds.) PCR Primer: A Laboratory Manual Cold Spring Harbor Press, CSH, NY.
  • Certain embodiments of the invention are directed to combination compositions comprising the receptor or ligand sequences described.
  • functional portions of the sequences may be joined to encode fusion proteins.
  • variants of the described sequences may be substituted in the combinations .
  • the present invention encompasses primate DCRSl, e.g., whose sequences are disclosed in Table 1, and described above. Allelic and other variants are also contemplated, including, e.g., fusion proteins combining portions of such sequences with others, including, e.g., epitope tags and functional domains .
  • the present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these primate or rodent proteins.
  • a heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner.
  • the fusion product of a DCRSl with another cytokine receptor is a continuous protein molecule having sequences fused in a typical peptide linkage, typically made as a single translation product and exhibiting properties, e.g., sequence or antigenicity, derived from each source peptide.
  • properties e.g., sequence or antigenicity
  • new constructs may be made from combining similar functional or structural domains from other related proteins, e.g., cytokine receptors or Toll- like receptors, including species variants.
  • ligand-binding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, e.g., Cunningham, et al . (1989) Science 243:1330-1336; and O'Dowd, et al . (1988) J. Biol. Chem. 263:15985-15992, each of which is incorporated herein by reference.
  • new chimeric polypeptides exhibiting new combinations of specificities will result from the functional linkage of receptor-binding specificities.
  • a fusion protein may include a targeting domain which may serve to provide sequestering of the fusion protein to a particular subcellular organelle.
  • Candidate fusion partners and sequences can be selected from various sequence data bases, e.g., GenBank, c/o IntelliGenetics, Mountain View, CA; and BCG,
  • the present invention particularly provides muteins which bind cytokine-like ligands, and/or which are affected in signal transduction.
  • Structural alignment of human DCRSl with other members of the cytokine receptor family show conserved features/residues . See Table 3. Alignment of the human DCRSl sequence with other members of the cytokine receptor family indicates various structural and functionally shared features. See also, Bazan, et al. (1996) Nature 379:591; Lodi, et al . (1994) Science 263:1762-1766; Sayle and Milner-White (1995) TIBS 20:374-376; and Gronenberg, et al . (1991) Protein Engineering 4:263-269.
  • DCRSl Primate DCRSl
  • substitutions with either mouse sequences or human sequences are particularly preferred. Conversely, conservative substitutions away from the ligand binding interaction regions will probably preserve most signaling activities; and conservative substitutions away from the intraceUular domains will probably preserve most ligand binding properties .
  • "Derivatives" of the primate DCRSl include amino acid sequence mutants, glycosylation variants, metabolic derivatives and covalent or aggregative conjugates with other chemical moieties . Covalent derivatives can be prepared by linkage of functionalities to groups which are found in the DCRSl amino acid side chains or at the N- or C- termini, e.g., by means which are well known in the art.
  • These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g., lysine or arginine .
  • Acyl groups are selected from the group of alkyl-moieties, including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species .
  • glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. Particularly preferred means for accomplishing this are by exposing the polypeptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes. Deglycosylation enzymes are also contemplated. Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine .
  • a major group of derivatives are covalent conjugates of the receptors or fragments thereof with other proteins of polypeptides. These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups. Preferred derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues. Fusion polypeptides between the receptors and other homologous or heterologous proteins are also provided.
  • Homologous polypeptides may be fusions between different receptors, resulting in, for instance, a hybrid protein exhibiting binding specificity for multiple different cytokine ligands, or a receptor which may have broadened or weakened specificity of substrate effect.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins.
  • Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a receptor, e.g., a ligand-binding segment, so that the presence or location of a desired ligand may be easily determined. See, e.g., Dull, et al . , U.S. Patent No.
  • GST glutathione-S-transferase
  • bacterial ⁇ - galactosidase bacterial ⁇ - galactosidase
  • trpE bacterial ⁇ - galactosidase
  • Protein A ⁇ -lactamase
  • alpha amylase alpha amylase
  • alcohol dehydrogenase yeast alpha mating factor
  • polypeptides may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups.
  • the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands.
  • Fusion proteins will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods. Techniques for nucleic acid manipulation and expression are described generally, for example, in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed. ) , Vols.
  • This invention also contemplates the use of derivatives of a DCRSl other than variations in amino acid sequence or glycosylation.
  • Such derivatives may involve covalent or aggregative association with chemical moieties. These derivatives generally fall into three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes, for example with cell membranes.
  • covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of a receptor or other binding molecule, e.g., an antibody.
  • a cytokine ligand can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated Sepharose, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of a cytokine receptor, antibodies, or other similar molecules.
  • the ligand can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.
  • a combination, e.g., including a DCRSl, of this invention can be used as an immunogen for the production of antisera or antibodies specific, e.g., capable of distinguishing between other cytokine receptor family members, for the combinations described.
  • the complexes can be used to screen monoclonal antibodies or antigen- binding fragments prepared by immunization with various forms of impure preparations containing the protein.
  • the term "antibodies” also encompasses antigen binding fragments of natural antibodies, e.g., Fab, Fab2 , Fv, etc.
  • the purified DCRSl can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of expression, or immunological disorders which lead to antibody production to the endogenous receptor.
  • DCRSl fragments may also serve as immunogens to produce the antibodies of the present invention, as described immediately below.
  • this invention contemplates antibodies having binding affinity to or being raised against the amino acid sequences shown in Table 1, fragments thereof, or various homologous peptides.
  • this invention contemplates antibodies having binding affinity to, or having been raised against, specific fragments which are predicted to be, or actually are, exposed at the exterior protein surface of the native DCRSl. Complexes of combinations of proteins will also be useful, and antibody preparations thereto can be made.
  • the blocking of physiological response to the receptor ligands may result from the inhibition of binding of the ligand to the receptor, likely through competitive inhibition.
  • in vitro assays of the present invention will often use antibodies or antigen binding segments of these antibodies, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either ligand binding region mutations and modifications, or other mutations and modifications, e.g., which affect signaling or enzymatic function.
  • This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to the receptor complexes or fragments compete with a test compound for binding to a ligand or other antibody. In this manner, the neutralizing antibodies or fragments can be used to detect the presence of a polypeptide which shares one or more binding sites to a receptor and can also be used to occupy binding sites on a receptor that might otherwise bind a ligand.
  • DNA which encodes the protein or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples .
  • Natural sequences can be isolated using standard methods and the sequences provided herein, e.g., in Table 1. Other species counterparts can be identified by hybridization techniques, or by various PCR techniques, combined with or by searching in sequence databases, e.g., GenBank.
  • This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length receptor or fragments which can in turn, for example, be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified ligand binding or kinase/phosphatase domains; and for structure/function studies.
  • Variants or fragments can be expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules can be substantially free of protein or cellular contaminants, other than those derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a 49
  • the protein, or portions thereof, may be expressed as fusions with other proteins. Combinations of the described proteins, or nucleic acids encoding them, are particularly interesting.
  • Expression vectors are typically self-replicating DNA or RNA constructs containing the desired receptor gene or its fragments, usually operably linked to suitable genetic control elements that are recognized in a suitable host cell. These control elements are capable of effecting expression within a suitable host.
  • the multiple genes may be coordinately expressed, and may be on a polycistronic message. The specific type of control elements necessary to effect expression will depend upon the eventual host cell used.
  • the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation.
  • Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell.
  • the vectors of this invention include those which contain DNA which encodes a combination of proteins, as described, or a biologically active equivalent polypeptide.
  • the DNA can be under the control of a viral promoter and can encode a selection marker.
  • This invention further contemplates use of such expression vectors which are capable of expressing eukaryotic cDNAs coding for such proteins in a prokaryotic or eukaryotic host, where the vector is compatible with the host and where the eukaryotic cDNAs are inserted into the vector such that growth of the host containing the vector expresses the cDNAs in question.
  • expression 50 usually, expression 50
  • vectors are designed for stable replication in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell, e.g., it is possible to effect transient expression of the protein or its fragments in various hosts using vectors that do not contain a replication origin that is recognized by the host cell. It is also possible to use vectors that cause integration of the protein encoding portions into the host DNA by recombination .
  • Vectors comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host.
  • Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels, et al . (1985 and Supplements) Cloning Vectors : A Laboratory Manual , Elsevier, N.Y. , and Rodriguez, et al . (eds. 1988) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, which are incorporated herein by reference.
  • Transformed cells are cells, preferably mammalian, that have been transformed or transfected with vectors constructed using recombinant DNA techniques .
  • Transformed host cells usually express the desired proteins, but for purposes of cloning, amplifying, and manipulating its DNA, do not need to express the subject proteins .
  • This invention further contemplates culturing transformed cells in a nutrient medium, thus permitting the proteins to accumulate. The proteins can be recovered, either from the culture or, in certain instances, from the culture medium. 51
  • nucleic sequences are operably linked when they are functionally related to each other.
  • DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation.
  • operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression.
  • Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes .
  • Prokaryotes include both gram negative and gram positive organisms, e.g., E. coli and B. subtilis.
  • Lower eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and species of the genus Dictvosteliu .
  • Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents.
  • Prokaryotic host-vector systems include a wide variety of vectors for many different species. As used herein, E. coli and its vectors will be used generically to include equivalent vectors used in other prokaryotes .
  • a representative vector for amplifying DNA is pBR322 or many of its derivatives.
  • Vectors that can be used to express the receptor or its fragments include, but are not limited to, such vectors as those containing the lac promoter (pUC-series) ; trp promoter (pBR322-trp) ; Ipp promoter (the pIN-series) ; lambda-pP or pR promoters (pOTS) ; or hybrid promoters such as ptac (pDR540) . See Brosius, et al. (1988) "Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters", in Vectors : A Survey of Molecular Cloning Vectors and Their 52
  • Lower eukaryotes e.g., yeasts and Dictvostelium
  • DCRSl sequence containing vectors may be transformed with DCRSl sequence containing vectors.
  • the most common lower eukaryotic host is the baker's yeast, Saccharomvces cerevisiae. It will be used to generically represent lower eukaryotes although a number of other strains and species are also available.
  • Yeast vectors typically consist of a replication origin (unless of the integrating type) , a selection gene, a promoter, DNA encoding the receptor or its fragments, and sequences for translation termination, polyadenylation, and transcription termination.
  • Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or such inducible promoters as the alcohol dehydrogenase 2 promoter or metallothionine promoter.
  • Suitable vectors include derivatives of the following types: self-replicating low copy number (such as the YRp-series) , self-replicating high copy number (such as the YEp-series) ; integrating types (such as the Yip-series) , or mini-chromosomes (such as the YCp-series) .
  • Higher eukaryotic tissue culture cells are normally the preferred host cells for expression of the functionally active interleukin or receptor proteins .
  • many higher eukaryotic tissue culture cell lines are workable, e.g., insect baculovirus expression systems, whether from an invertebrate or vertebrate source.
  • mammalian cells are preferred. Transformation or transfection and propagation of such cells has become a routine procedure.
  • useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS) cell lines. Expression vectors for such cell lines usually 53
  • Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, e.g., from such sources as from adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus .
  • suitable expression vectors include pCDNAl; pCD, see Okayama, et al . (1985) Mol. Cell Biol. 5:1136-1142; pMClneo PolyA, see Thomas, et al . (1987) Cell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
  • an open reading frame usually encodes a polypeptide that consists of a mature or secreted product covalently linked at its N-terminus to a signal peptide.
  • the signal peptide is cleaved prior to secretion of the mature, or active, polypeptide.
  • the cleavage site can be predicted with a high degree of accuracy from empirical rules, e.g., von-Heijne (1986) Nucleic Acids Research 14:4683- 4690 and Nielsen, et al . (1997) Protein Eng. 10:1-12, and the precise amino acid composition of the signal peptide often does not appear to be critical to its function, e.g., Randall, et al . (1989) Science 243:1156-1159;
  • polypeptides it will often be desired to express these polypeptides in a system which provides a specific or defined glycosylation pattern.
  • the usual pattern will be that provided naturally by the expression system.
  • the pattern will be modifiable by exposing the polypeptide, e.g., an unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system.
  • the receptor gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes . 54
  • the source of DCRSl can be a eukaryotic or prokaryotic host expressing recombinant DCRSl, such as is described above.
  • the source can also be a cell line such as mouse Swiss 3T3 fibroblasts, but other mammalian cell lines are also contemplated by this invention, with the preferred cell line being from the human species.
  • the primate DCRSl, fragments, or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis, Springer-Verlag, New York; and Bodanszky (1984) The Principles of Peptide Synthesis, Springer-Verlag, New York; all of each which are incorporated herein by reference.
  • an azide process for example, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester) , a carbodiimidazole process, an oxidative-reductive process, or a dicyclohexylcarbodiimide (DCCD) /additive process
  • Solid phase and solution phase syntheses are both applicable to the foregoing processes . Similar techniques can be used with partial DCRSl sequences.
  • DCRSl proteins, fragments, or derivatives are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid.
  • the C-terminal amino acid is bound to an insoluble carrier or support through its carboxyl group.
  • the insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group.
  • examples of such insoluble carriers include halomethyl resins, such as chloromethyl resin or bromomethyl resin, hydroxymethyl resins, phenol resins, tert-alkyloxycarbonylhydrazidated resins, and the like.
  • An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously formed peptide or chain, to synthesize the peptide step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the peptide.
  • This solid-phase approach is generally described by Merrifield, et al . (1963) in CL. Am. Chem. Soc . 85:2149-2156, which is incorporated herein by reference.
  • the prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, precipitation, electrophoresis, various forms of chromatography, and the like.
  • the receptors of this invention can be obtained in varying degrees of purity depending upon desired uses. Purification can be accomplished by use of the protein purification techniques disclosed herein, see below, or by the use of the antibodies herein described in methods of immunoabsorbant affinity chromatography. This immunoabsorbant affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate cells, lysates of other cells expressing the receptor, or lysates or supernatants of cells bo
  • the purified protein will be at least about 40% pure, ordinarily at least about 50% pure, usually at least about 60% pure, typically at least about 70% pure, more typically at least about 80% pure, preferable at least about 90% pure and more preferably at least about 95% pure, and in particular embodiments, 97%- 99% or more.
  • Purity will usually be on a weight basis, but can also be on a molar basis. Different assays will be applied as appropriate . Individual proteins may be purified and thereafter combined.
  • Antibodies can be raised to the various mammalian, e.g., primate DCRSl proteins and fragments thereof, both in naturally occurring native forms and in their recombinant forms, the difference being that antibodies to the active receptor are more likely to recognize epitopes which are only present in the native conformations. Denatured antigen detection can also be useful in, e.g., Western analysis. Anti-idiotypic antibodies are also contemplated, which would be useful as agonists or antagonists of a natural receptor or an antibody.
  • Antibodies including binding fragments and single chain versions, against predetermined fragments of the protein can be raised by immunization of animals with conjugates of the fragments with immunogenic proteins.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective protein, or screened for agonistic or antagonistic activity. These monoclonal antibodies will usually bind with at least a K- of about 1 mM, more usually at least about 300 ⁇ M, typically at least about lOO ⁇ M, more typically at least about 30 ⁇ M, preferably at least about 10 ⁇ M, and more preferably at least about 3 ⁇ M or better.
  • K- K- of about 1 mM, more usually at least about 300 ⁇ M, typically at least about lOO ⁇ M, more typically at least about 30 ⁇ M, preferably at least about 10 ⁇ M, and more preferably at least about 3 ⁇ M or better.
  • the antibodies, including antigen binding fragments, of this invention can have significant diagnostic or therapeutic value. They can be potent antagonists that bind to the receptor and inhibit binding to ligand or inhibit the ability of the receptor to elicit a biological response, e.g., act on its substrate. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides to bind producing cells, or cells localized to the source of the interleukin. Further, these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker.
  • the antibodies of this invention can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they might bind to the receptor without inhibiting ligand or substrate binding. As neutralizing antibodies, they can be useful in competitive binding assays. They will also be useful in detecting or quantifying ligand. They may be used as reagents for Western blot analysis, or for immunoprecipitation or immunopurification of the respective protein. Likewise, nucleic acids and proteins may be immobilized to solid substrates for affinity purification or detection methods. The substrates may be, e.g., solid resin beads or sheets of plastic.
  • Protein fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens.
  • Mammalian cytokine receptors and fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Seroloqical Reactions, Dover Publications, New York; and Williams, et al . (1967) Methods in Immunology and
  • the population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen.
  • the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
  • the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal .
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • recombinant or chimeric immunoglobulins may be produced, see Cabilly, U.S. Patent No. 4,816,567; or made in transgenic mice, see Mendez, et al. (1997) Nature Genetics 15:146-156. These references are incorporated herein by reference.
  • the antibodies of this invention can also be used for affinity chromatography in isolating the DCRSl proteins or peptides . Columns can be prepared where the antibodies are linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate may be passed through the column, the column washed, followed by increasing concentrations of a mild denaturant, whereby the purified protein will be released. Alternatively, the protein may be used to purify antibody. Appropriate cross absorptions or depletions may be applied.
  • the antibodies may also be used to screen expression libraries for particular expression products. Usually the antibodies used in such a procedure will be labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
  • Antibodies raised against a cytokine receptor will also be used to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various 60
  • immunological conditions related to expression of the protein or cells which express the protein They also will be useful as agonists or antagonists of the ligand, which may be competitive inhibitors or substitutes for naturally occurring ligands.
  • a cytokine receptor protein that specifically binds to or that is specifically immunoreactive with an antibody generated against a defined immunogen, such as an immunogen consisting of the amino acid sequence of SEQ ID NO: 13, is typically determined in an immunoassay.
  • the immunoassay typically uses a polyclonal antiserum which was raised, e.g., to a protein of SEQ ID NO: 13. This antiserum is selected to have low crossreactivity against other cytokine receptor family members, e.g., IL- 12 receptor beta or gpl30, preferably from the same species, and any such crossreactivity is removed by immunoabsorption prior to use in the immunoassay.
  • the protein e.g., of SEQ ID NO: 13 is isolated as described herein.
  • recombinant protein may be produced in a mammalian cell line.
  • An appropriate host e.g. , an inbred strain of mice such as Balb/c, is immunized with the selected protein, typically using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and
  • a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
  • Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, e.g., a solid phase immunoassay with the immunogen immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 ⁇ or greater are selected and tested for their cross reactivity against other cytokine receptor family members, e.g., IL-12 receptor beta and/or gpl30, using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573.
  • cytokine receptor family members are used in this determination.
  • These cytokine receptor family members can be produced as recombinant proteins and isolated using standard molecular biology and protein chemistry techniques as described herein.
  • Immunoassays in the competitive binding format can be used for the crossreactivity determinations.
  • the protein of SEQ ID NO: 13 can be immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the proteins of IL-12 receptor beta or gpl30. The percent crossreactivity for the above proteins is calculated, using standard calculations.
  • Those antisera with less than 10% crossreactivity with each of the proteins listed above are selected and pooled.
  • the cross-reacting antibodies are then removed from the pooled antisera by immunoabsorption with the above-listed proteins.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein to the immunogen protein (e.g., the DCRSl like protein of SEQ ID NO: 13).
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than twice the amount of the protein of the selected protein or proteins that is required, then the second protein is said to specifically bind to an antibody generated to the immunogen.
  • these cytokine receptor proteins are members of a family of homologous proteins that comprise at least 6 so far identified genes.
  • the term refers not only to the amino acid sequences disclosed herein, but also to other proteins that are allelic, non- allelic, or species variants.
  • the terms include nonnatural mutations introduced by deliberate mutation using conventional recombinant technology such as single site mutation, or by excising short sections of DNA encoding the respective proteins, or by substituting new amino acids, or adding new amino acids. Such minor alterations typically will substantially maintain the immunoidentity of the original molecule and/or its biological activity.
  • these alterations include proteins that are specifically immunoreactive with a designated naturally occurring DCRSl protein.
  • the biological properties of the altered proteins can be determined by expressing the protein in an appropriate cell line and measuring the appropriate effect, e.g., upon transfected lymphocytes. Particular protein modifications considered minor would include conservative substitution of amino acids with similar chemical properties, as described above for the cytokine receptor family as a whole. By aligning a protein optimally with the protein of the cytokine receptors and by using the conventional immunoassays described herein to determine immunoidentity, one can determine the protein compositions of the invention.
  • kits and assay methods Both naturally occurring and recombinant forms of the cytokine receptor like molecules of this invention are particularly useful in kits and assay methods . For example, these methods would also be applied to screening for binding activity, e.g., ligands for these proteins.
  • Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds per year. See, e.g., a BIOMEK automated workstation, Beckman Instruments, Palo Alto, California, and Fodor, et al . (1991) Science 251:767-773, which is incorporated herein by reference. The latter describes means for testing binding by a plurality of defined polymers synthesized on a solid substrate.
  • suitable assays to screen for a ligand or agonist/antagonist homologous proteins can be greatly facilitated by the availability of large amounts of purified, soluble cytokine receptors in an active state such as is provided by this invention.
  • Purified DCRSl can be coated directly onto plates for use in the aforementioned ligand screening techniques.
  • non-neutralizing antibodies to these proteins can ' be used as capture antibodies to immobilize the respective receptor on the solid phase, useful, e.g., in diagnostic uses.
  • This invention also contemplates use of DCRSl, fragments thereof, peptides, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of the protein or its ligand.
  • antibodies against the molecules may be incorporated into the kits and methods.
  • the kit will have a compartment containing either a DCRSl peptide or gene segment or a reagent which recognizes one or the other.
  • recognition reagents in the case of peptide, would be a receptor or antibody, or in the case of a gene segment, would usually be a hybridization probe.
  • a preferred kit for determining the concentration of DCRSl in a sample would typically comprise a labeled compound, e.g., ligand or antibody, having known binding affinity for DCRSl, a source of DCRSl (naturally occurring or recombinant) as a positive control, and a means for separating the bound from free labeled compound, for example a solid phase for immobilizing the DCRSl in the test sample. Compartments containing reagents, and instructions, will normally be provided. Appropriate nucleic acid or protein containing kits are also provided. 64
  • Antibodies including antigen binding fragments, specific for mammalian DCRSl or a peptide fragment, or receptor fragments are useful in diagnostic applications to detect the presence of elevated levels of ligand and/or its fragments. Diagnostic assays may be homogeneous (without a separation step between free reagent and antibody-antigen complex) or heterogeneous (with a separation step) .
  • Various commercial assays exist, such as radioimmunoassay (RIA) , enzyme-linked immunosorbent assay (ELISA) , enzyme immunoassay (EIA) , enzyme-multiplied immunoassay technique (EMIT) , substrate-labeled fluorescent immunoassay (SLFIA) and the like.
  • unlabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the antibody to a cytokine receptor or to a particular fragment thereof.
  • a second antibody which is labeled and which recognizes the antibody to a cytokine receptor or to a particular fragment thereof.
  • Anti-idiotypic antibodies may have similar use to serve as agonists or antagonists of cytokine receptors. These should be useful as therapeutic reagents under appropriate circumstances. Frequently, the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay.
  • the protocol for the subject invention, depending upon the nature of the assay, the protocol, and the label, either labeled or unlabeled antibody, or labeled ligand is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like.
  • the kit will also contain instructions for proper use and disposal of the contents after use.
  • the kit has compartments for each useful reagent, and will contain instructions for proper use and disposal of reagents.
  • the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium having appropriate concentrations for performing the assay.
  • labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.
  • a test compound, cytokine receptor, or antibodies thereto can be labeled either directly or indirectly.
  • Possibilities for direct labeling include label groups: radiolabels such as 125 ⁇ _ enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization. Both of the patents are incorporated herein by reference.
  • Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups .
  • the cytokine receptor can be immobilized on various matrixes followed by washing.
  • Suitable matrices include plastic such as an ELISA plate, filters, and beads.
  • Methods of immobilizing the receptor to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling, and biotin-avidin.
  • the last step in this approach involves the precipitation of antibody/antigen complex by any of several methods including those utilizing, e.g., an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate.
  • suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al . (1984) Clin. Chem. 30 (9) : 1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678, each of which is incorporated herein by reference.
  • sequences can be used as probes for detecting levels of the respective cytokine receptor in patients suspected of having an immunological disorder.
  • the preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature.
  • an oUgonucleotide probe should have at least about 14 nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes may be up to several kilobases.
  • Various labels may be employed, most commonly radionuclides, particularly 32p # However, other techniques may also be employed, such as using biotin modified nucleotides for introduction into a polynucleotide.
  • the biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like.
  • antibodies may be employed which can recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • probes to the novel anti-sense RNA may be carried out in conventional techniques such as nucleic acid hybridization, plus and minus screening, recombinational probing, hybrid released translation (HRT) , and hybrid arrested translation (HART) .
  • This also includes amplification techniques such as polymerase chain reaction (PCR) .
  • kits which also test for the qualitative or quantitative presence of other markers are also contemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers. Thus, kits may test for combinations of markers. See, e.g., Viallet, et al . (1989) Progress in Growth Factor Res. 1:89-97.
  • This invention provides reagents with significant therapeutic value. See, e.g., Levitzki (1996) Curr . Qpin. Cell Biol. 8:239-244.
  • the cytokine receptors See, e.g., Levitzki (1996) Curr . Qpin. Cell Biol. 8:239-244.
  • the cytokine receptors See, e.g., Levitzki (1996) Curr . Qpin. Cell Biol. 8:239-244.
  • IL-1 ligands have been suggested to be involved in morphologic development, e.g., dorso-ventral polarity determination, and immune responses, particularly the primitive innate responses. See, e.g., Sun, et al . (1991) Eur. J. Biochem. 196:247- 254; and Hultmark (1994) Nature 367:116-117.
  • Recombinant cytokine receptors, muteins, agonist or antagonist antibodies thereto, or antibodies can be 68
  • reagents can be combined for therapeutic use with additional active ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, along with physiologically innocuous stabilizers and excipients. These combinations can be sterile, e.g., filtered, and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations .
  • This invention also contemplates use of antibodies or binding fragments thereof which are not complement binding.
  • Ligand screening using cytokine receptor or fragments thereof can be performed to identify molecules having binding affinity to the receptors. Subsequent biological assays can then be utilized to determine if a putative ligand can provide competitive binding, which can block intrinsic stimulating activity. Receptor fragments can be used as a blocker or antagonist in that it blocks the activity of ligand. Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of ligand, e.g., inducing signaling. This invention further contemplates the therapeutic use of antibodies to cytokine receptors as antagonists.
  • reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, reagent physiological life, pharmacological life, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman, et al . (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics , 8th Ed. , Pergamon 69
  • compositions for administration are discussed therein and below, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others.
  • Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co. , Rahway, New Jersey. Because of the likely high affinity binding, or turnover numbers, between a putative ligand and its receptors, low dosages of these reagents would be initially expected to be effective. And the signaling pathway suggests extremely low amounts of ligand may have effect.
  • dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 ⁇ M concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar) , and most preferably less than about 1 fM (femtomolar) , with an appropriate carrier.
  • Slow release formulations, or slow release apparatus will often be utilized for continuous administration.
  • Cytokine receptors, fragments thereof, and antibodies or its fragments, antagonists, and agonists may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration.
  • Therapeutic formulations may be administered in many conventional dosage formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • Formulations include those suitable for oral , rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. See, e.g., Gilman, et al . (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington ' s Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn.; Avis, et al. (eds. 1993) Pharmaceutical Dosage
  • compositions Parenteral Medications Dekker, NY; Lieberman, et al . (eds. 1990) Pharmaceutical Dosage Forms : Tablets Dekker, NY; and Lieberman, et al . (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker , NY.
  • the therapy of this invention may be combined with or used in association with other therapeutic agents, particularly agonists or antagonists of other cytokine receptor family members.
  • DCRSl or fragments thereof can be performed to identify compounds having binding affinity to the receptor subunit, including isolation of associated components . Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in that it blocks the activity of the ligand. Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of a cytokine ligand. This invention further contemplates the therapeutic use of antibodies to the receptor as cytokine agonists or antagonists.
  • complexes comprising multiple proteins may be used to screen for ligands or reagents capable of recognizing the complex.
  • Most cytokine receptors comprise at least two subunits, which may be the same, or distinct.
  • the transmembrane receptor may 71
  • the DCRSl may bind to a complex of the IL-B30 with the DSRSl.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing the DCRSl in combination with the DSRSl. Cells may be isolated which express a receptor in isolation from other functional receptors. Such cells, either in viable or fixed form, can be used for standard antibody/antigen or ligand/receptor binding assays. See also, Parce, et al . (1989) Science 246:243-247; and Owicki, et al . (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitive methods to detect cellular responses.
  • Viable cells could also be used to screen for the effects of drugs on cytokine mediated functions, e.g., second messenger levels, i.e., Ca ++ ; cell proliferation; inositol phosphate pool changes; and others.
  • second messenger levels i.e., Ca ++
  • cell proliferation i.e., cell proliferation
  • inositol phosphate pool changes e.g., cell proliferation
  • inositol phosphate pool changes e.g., cell proliferation
  • inositol phosphate pool changes e.g., cell proliferation
  • Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system.
  • Calcium sensitive dyes will be useful for detecting Ca ++ levels, with a fluorimeter or a fluorescence cell sorting apparatus.
  • ligands The descriptions of the DCRSl herein provide means to identify ligands, as described above. Such ligand should bind specifically to the respective receptor with reasonably high affinity.
  • Various constructs are made available which allow either labeling of the receptor to detect its ligand. For example, directly labeling cytokine receptor, fusing onto it markers for secondary labeling, e.g., FLAG or other epitope tags, etc., will allow detection of receptor. This can be histological, as an affinity method for biochemical purification, or labeling or selection in an expression cloning approach. A two-hybrid selection system may also be applied making appropriate constructs with the available cytokine receptor sequences.
  • cytokine receptors will be analogously applicable to individual specific embodiments directed to DCRSl reagents and compositions.
  • the DCRSl might bind to a soluble complex of the DSRSl with another ligand.
  • expression cloning of a cotransfectant of a library with the DSRSl may express combinations of the DSRSl with cytokine-like ligand, to form the soluble complex, which binds to the DCRSl .
  • Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel, et al . (1987 and periodic supplements); Coligan, et al . (ed. 1996) and periodic supplements, Current Protocols In Protein Science
  • Standard analysis programs may be used to evaluate structure, e.g., PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (King and Sternberg (1996) Protein Sci. 5:2298-2310).
  • Standard comparison software includes, e.g., Altschul, et al . (1990) J. Mol. Biol. 215:403-10; Waterman (1995) Introduction to Computational Biology: Maps, Sequences, and Genomes Chapman & Hall; Lander and Waterman (eds. 1995) Calculating the Secrets of Life: Applications of the Mathematical Sciences in Molecular Biology National Academy Press; and Speed and Waterman (eds. 1996) Genetic Mapping and DNA Sequencing
  • PCR primers derived from the DCRSl sequence are used to probe a human cDNA library. Sequences may be derived, e.g., from Table 1, preferably those adjacent the ends of incomplete sequences. Full length cDNAs for primate, rodent, or other species DCRSl are cloned, e.g., by DNA hybridization screening of ⁇ gtlO phage. PCR reactions are conducted using T. aquaticus Taqplus DNA polymerase (Stratagene) under appropriate conditions.
  • Chromosome spreads are prepared. In situ hybridization is performed on chromosome preparations obtained from phytohemagglutinin-stimulated human lymphocytes cultured for 72 h. 5-bromodeoxyuridine was added for the final seven hours of culture (60 ⁇ g/ml of 75
  • a PCR fragment, amplified with the help of primers, is cloned into an appropriate vector.
  • the vector is labeled by nick-translation with ⁇ H.
  • the radiolabeled probe is hybridized to metaphase spreads at final concentration of 200 ng/ml of hybridization solution as described in Mattei, et al . (1985) Hum. Genet. 69:327- 331.
  • After coating with nuclear track emulsion (KODAK NTB2) slides are exposed. To avoid any slipping of silver grains during the banding procedure, chromosome spreads are first stained with buffered Giemsa solution and metaphase photographed. R-banding is then performed by the fluorochrome-photolysis-Giemsa (FPG) method and metaphases rephotographed before analysis.
  • FPG fluorochrome-photolysis-Giemsa
  • RT-PCR is used on an appropriate mRNA 76
  • sample selected for the presence of message to produce a cDNA e.g., a sample which expresses the gene.
  • Full length clones may be isolated by hybridization of cDNA libraries from appropriate tissues pre-selected by PCR signal. Northern blots can be performed.
  • DCRSl Message for genes encoding DCRSl will be assayed by appropriate technology, e.g., PCR, immunoassay, hybridization, or otherwise. Tissue and organ cDNA preparations are available, e.g., from Clontech, Mountain View, CA. Identification of sources of natural expression are useful, as described. And the identification of functional receptor subunit pairings will allow for prediction of what cells express the combination of receptor subunits which will result in a physiological responsiveness to each of the cytokine ligands .
  • appropriate technology e.g., PCR, immunoassay, hybridization, or otherwise.
  • Tissue and organ cDNA preparations are available, e.g., from Clontech, Mountain View, CA. Identification of sources of natural expression are useful, as described. And the identification of functional receptor subunit pairings will allow for prediction of what cells express the combination of receptor subunits which will result in a physiological responsiveness to each of the cytokine ligands .
  • DNA 5 ⁇ g
  • DNA 5 ⁇ g
  • a primary amplified cDNA library was digested with appropriate restriction enzymes to release the inserts, run on a 1% agarose gel and transferred to a nylon membrane (Schleicher and Schuell, Keene, NH) .
  • Samples for mouse mRNA isolation may include: resting mouse fibroblastic L cell line (C200) ; Braf :ER
  • TH2 T cell clone CDC35 resting for 3 weeks after last stimulation with antigen (T207) ; TH2 T cell clone CDC35, 10 ⁇ g/ml ConA stimulated 15 h (T208) ; Mell4+ naive T cells from spleen, resting (T209) ; Mell4+ T cells, polarized to Thl with IFN- ⁇ /IL-12/anti-IL-4 for 6, 12, 24 h pooled (T210); Mell4+ T cells, polarized to Th2 with IL-4/anti-IFN- ⁇ for 6, 13, 24 h pooled (T211) ; unstimulated mature B cell leukemia cell line A20 (B200) ; unstimulated B cell line CH12 (B201) ; unstimulated large B cells from spleen (B202); B cells from total spleen, LPS activated (B203); metrizamide enriched dendritic cells from spleen, resting (D200
  • Peyer ' s patches O202; total Peyer 's patches, normal (O210) ; IL- 10 K.O. mesenteric lymph nodes (X203); total mesenteric lymph nodes, normal (0211); IL-10 K.O. colon (X203); total colon, normal (0212); NOD mouse pancreas (see Makino, et al .
  • Samples for human mRNA isolation may include: peripheral blood mononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells), resting (T100) ; peripheral blood mononuclear cells, activated with anti- CD3 for 2, 6, 12 h pooled (TlOl) ; T cell, THO clone Mot 72, resting (T102); T cell, THO clone Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T103); T cell, THO clone Mot 72, anergic treated with specific peptide for 2, 7, 12 h pooled (T104) ; T cell, THl clone HY06, resting (T107); T cell, THl clone HY06, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T108) ; T cell, THl clone HY06, anergic treated
  • IL-10 for 1, 2, 6, 12, 24 h pooled M103
  • elutriated monocytes, activated with LPS, IFN ⁇ , anti-IL-10 for 4, 16 h pooled M106
  • elutriated monocytes activated LPS for 1 h (M108)
  • DC 70% CDla+ from CD34+ GM-CSF, TNF ⁇ 12 days, resting (D101)
  • DC 70% CDla+ from CD34+ GM-CSF, TNF ⁇ 12 days, activated with
  • Various strategies are used to obtain species counterparts of the DCRSl, preferably from other primates or rodents.
  • One method is by cross hybridization using closely related species DNA probes. It may be useful to go into evolutionarily similar species as intermediate steps.
  • Another method is by using specific PCR primers based on the identification of blocks of similarity or difference between genes, e.g., areas of highly conserved or nonconserved polypeptide or nucleotide sequence.
  • An appropriate, e.g., GST, fusion construct is engineered for expression, e.g., in E. coli.
  • a mouse IGIF pGex plasmid is constructed and transformed into E. coli.
  • Freshly transformed cells are grown, e.g., in LB medium containing 50 ⁇ g/ml ampicillin and induced with IPTG (Sigma, St. Louis, MO) . After overnight induction, the bacteria are harvested and the pellets containing the DCRSl protein are isolated. The pellets are homogenized, e.g., in TE buffer (50 mM Tris- base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in 2 liters.
  • TE buffer 50 mM Tris- base pH 8.0, 10 mM EDTA and 2 mM pefabloc
  • This material is passed through a microfluidizer (Microfluidics, Newton, MA) three times.
  • the fluidized supernatant is spun down on a Sorvall GS-3 rotor for 1 h at 13,000 rpm.
  • the resulting supernatant containing the cytokine receptor protein is filtered and passed over a glutathione-SEPHAROSE column equilibrated in 50 mM Tris- base pH 8.0.
  • the fractions containing the DCRSl-GST fusion protein are pooled and cleaved, e.g., with thrombin (Enzyme Research Laboratories, Inc., South Bend, IN) .
  • the cleaved pool is then passed over a Q-SEPHAROSE column equilibrated in 50 mM Tris-base.
  • Fractions containing DCRSl are pooled and diluted in cold distilled H2O, to lower the conductivity, and passed back over a fresh Q-Sepharose column, alone or in succession with an immunoaffinity antibody column.
  • Fractions containing the DCRSl protein are pooled, aliquoted, and stored in the - 70° C freezer.
  • the cellular forms of receptors for ligands can be tested with the various ligands and receptor subunits provided.
  • multiple cytokine receptor like ligands have been identified.
  • the IL-B30 cytokine has been described. See above.
  • Cotransformation of the DCRSl with putative other receptor subunits may be performed.
  • the DSRSl is suggested to be a second receptor subunit needed for functional receptor signaling.
  • Such cells may be used to screen putative cytokine ligands, such as the IL-
  • the DSRSl may combine with the IL-B30 to form a soluble cytokine-receptor subunit complex, which then binds to the DCRSl.
  • cytokine receptors function as heterodimers .
  • the IL-l ⁇ and IL-l ⁇ ligands bind an IL-1R1 as the primary receptor and this complex then forms a high affinity receptor complex with the IL-1R3.
  • the sequence similarity to IL-12 receptor subunits suggests functional similarity of the functional receptor, e.g., a soluble alpha subunit, and transmembrane beta subunit.
  • a soluble alpha subunit e.g., a soluble alpha subunit
  • transmembrane beta subunit e.g., a soluble alpha subunit
  • constructs can be made for transformation or transfection of subunits into cells.
  • Constructs for the alpha chains, e.g., DSRSl forms can be made .
  • Beta subunit DCRSl Combinatorial transfections of transformations can make cells expressing defined subunits, which can be tested for response to the predicted ligands.
  • Appropriate cell types can be used, e.g., 293 T cells, with, e.g., an NFKb reporter construct.
  • Bio assays will generally be directed to the ligand binding feature of the protein or to the kinase/phosphatase activity of the receptor.
  • the activity will typically be reversible, as are many other enzyme reactions, and may mediate phosphatase or phosphorylase activities, which activities are easily measured by standard procedures. See, e.g., Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al . (1991) Meth. Enzvmol. 200:38-62; Hunter, et al .
  • cytokines contains molecules which are important mediators of hematopoiesis or inflammatory disease. See, e.g., Thomson (ed. 1994) The Cytokine Handbook Academic Press, San Diego; and Dinarello (1996) Blood 87:2095-2147.
  • Balb/c mice are immunized intraperitoneally with recombinant forms of the protein, e.g., purified DCRSl or stable transfected NIH-3T3 cells. Animals are boosted at appropriate time points with protein, with or without additional adjuvant, to further stimulate antibody production. Serum is collected, or hybridomas produced with harvested spleens. Alternatively, Balb/c mice are immunized with cells transformed with the gene or fragments thereof, either endogenous or exogenous cells, or with isolated membranes enriched for expression of the antigen. Serum is collected at the appropriate time, typically after numerous further administrations. Various gene therapy techniques may be useful, e.g., in producing protein in situ, for generating an immune response. Serum or antibody preparations may be cross-absorbed or immunoselected to prepare substantially purified antibodies of defined specificity and high affinity.
  • the protein e.g., purified DCRSl or stable transfected NIH-3T3 cells. Animals are boosted at appropriate time points with protein,
  • Monoclonal antibodies may be made. For example, splenocytes are fused with an appropriate fusion partner and hybridomas are selected in growth medium by standard procedures . Hybridoma supernatants are screened for the presence of antibodies which bind to the DCRSl, e.g., by ELISA or other assay. Antibodies which specifically recognize specific DCRSl embodiments may also be selected or prepared.
  • binding reagent is either labeled as described above, e.g., fluorescence or otherwise, or immobilized to a substrate for panning methods.
  • Nucleic acids may also be introduced into cells in an animal to produce the antigen, which serves to elicit an immune response. See, e.g., Wang, et al. (1993) Proc . Nat ' 1. Acad. Sci. 90:4156-4160; Barry, et al . (1994) BioTechnicrues 16:616-619; and Xiang, et al . (1995) Immunity 2: 129-135.
  • antibodies which may be useful to determine the combination of the DCRSl with a functional alpha subunit may be generated.
  • epitopes characteristic of a particular functional alpha/beta combination may be identified with appropriate antibodies .
  • DCRSl Various fusion constructs are made with DCRSl.
  • a portion of the appropriate gene is fused to an epitope tag, e.g., a FLAG tag, or to a two hybrid system construct.
  • the epitope tag may be used in an expression cloning procedure with detection with anti-FLAG antibodies to detect a binding partner, e.g., ligand for the respective cytokine receptor.
  • the two hybrid system may also be used to isolate proteins which specifically bind to DCRSl .
  • Standard mutagenesis analysis is performed, e.g., by generating many different variants at determined positions, e.g., at the positions identified above, and evaluating biological activities of the variants. This may be performed to the extent of determining positions which modify activity, or to focus on specific positions to determine the residues which can be substituted to either retain, block, or modulate biological activity.
  • analysis of natural variants can indicate what positions tolerate natural mutations. This may result from populational analysis of variation among individuals, or across strains or species. Samples from selected individuals are analyzed, e.g., by PCR analysis and sequencing. This allows evaluation of population polymorphisms.
  • a cytokine receptor can be used as a specific binding reagent to identify its binding partner, by taking advantage of its specificity of binding, much like an antibody would be used.
  • the binding receptor may be a heterodimer of receptor subunits; or may involve, e.g., a complex of the DCRSl with DSRSl.
  • a binding reagent is either labeled as described above, e.g., fluorescence or otherwise, or immobilized to a substrate for panning methods .
  • the binding composition is used to screen an expression library made from a cell line which expresses a binding partner, i.e., ligand, preferably membrane associated. Standard staining techniques are used to detect or sort surface expressed ligand, or surface expressing transformed cells are screened by panning.
  • HBSS HBSS.
  • the slides may be stored at -80° C after all liquid is removed.
  • 0.5 ml incubations are performed as follows. Add HBSS/saponin (0.1%) with 32 ⁇ l/ l of 1 M NaN 3 for 20 min. Cells are then washed with HBSS/saponin IX. Add appropriate DCRSl or DCRSl/antibody complex to cells and incubate for 30 min. Wash cells twice with HBSS/saponin. If appropriate, add first antibody for 30 min. Add second antibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, and incubate for 30 min.
  • second antibody e.g., Vector anti-mouse antibody
  • ELISA solution e.g., Vector Elite ABC horseradish peroxidase solution, and preincubate for 30 min.
  • Use e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solution and incubate for 30 min. Wash cells twice with HBSS, second wash for 2 min, which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5 to 10 min.
  • DAB Vector diaminobenzoic acid
  • receptor reagents are used to affinity purify or sort out cells expressing a putative ligand. See, e.g., Sambrook, et al . or Ausubel, et al .
  • Another strategy is to screen for a membrane bound receptor by panning.
  • the receptor cDNA is constructed as described above.
  • the ligand e.g., either IL-B30 alone or a complex of IL-B30 with DSRSl, can be immobilized and used to immobilize expressing cells.
  • Immobilization may be achieved by use of appropriate antibodies which recognize, e.g., a FLAG sequence of a DCRSl fusion construct, or by use of antibodies raised against the first antibodies . Recursive cycles of selection and amplification lead to enrichment of appropriate clones and eventual isolation of receptor expressing clones . Phage expression libraries can be screened by mammalian DCRSl. Appropriate label techniques, e.g., anti-FLAG antibodies, will allow specific labeling of appropriate clones . All citations herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

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Abstract

Nucleic acids encoding mammalian, e.g., primate or rodent receptors, purified receptor proteins and fragments thereof. Antibodies, both polyclonal and monoclonal, are also provided. Methods of using the compositions for both diagnostic and therapeutic utilities are provided.

Description

MAMMALIAN RECEPTOR PROTEINS; RELATED REAGENTS AND METHODS
FIELD OF THE INVENTION
The present invention relates to compositions and methods for affecting mammalian physiology, including morphogenesis or immune system function. In particular, it provides nucleic acids, proteins, and antibodies which regulate development and/or the immune system. Various subunits of cytokine receptors, and the matching of subunits in a functional complex are described. Diagnostic and therapeutic uses of these materials are also disclosed.
BACKGROUND OF THE INVENTION Recombinant DNA technology refers generally to techniques of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product. The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host. See, e.g., Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual . (2d ed. ) vols. 1-3, CSH Press, NY.
For some time, it has been known that the mammalian immune response is based on a series of complex cellular interactions, called the "immune network". Recent research has provided new insights into the inner workings of this network. While it remains clear that much of the immune response does, in fact, revolve around the network-like interactions of lymphocytes, macrophages, granulocytes, and other cells, immunologists now generally hold the opinion that soluble proteins, known as lymphokines, cytokines, or monokines, play critical roles in controlling these cellular interactions. Thus, there is considerable interest in the isolation, characterization, and mechanisms of action of cell modulatory factors, an understanding of which will lead to significant advancements in the diagnosis and therapy of numerous medical abnormalities, e.g., immune system disorders.
Lymphokines apparently mediate cellular activities in a variety of ways. See, e.g., Paul (ed. 1996) Fundamental Immunology 3d ed. , Raven Press, New York; and Thomson (ed. 1994) The Cytokine Handbook 2d ed. , Academic Press, San Diego. They have been shown to support the proliferation, growth, and/or differentiation of pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages which make up a complex immune system. Proper and balanced interactions between the cellular components are necessary for a healthy immune response. The different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents . Cell lineages especially important to the immune response include two classes of lymphocytes: B-cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal) , and T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and various other cells (including other T- cells) making up the immune network. These lymphocytes interact with many other cell types .
Another important cell lineage is the mast cell (which has not been positively identified in all mammalian species) , which is a granule-containing connective tissue cell located proximal to capillaries throughout the body. These cells are found in especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts. Mast cells play a central role in allergy-related disorders, particularly anaphylaxis as follows : when selected antigens crosslink one class of immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases mediators, e.g. , histamine, serotonin, heparin, and prostaglandins, which cause allergic reactions, e.g., anaphylaxis. Research to better understand and treat various immune disorders has been hampered by the general inability to maintain cells of the immune system in vitro. Immunologists have discovered that culturing many of these cells can be accomplished through the use of T- cell and other cell supernatants, which contain various growth factors, including many of the lymphokines.
Various growth and regulatory factors exist which modulate morphogenetic development. This includes, e.g., the Toll ligands, which signal through binding to receptors which share structural, and mechanistic, features characteristic of the IL-1 receptors. See, e.g., Lemaitre, et al . (1996) Cell 86:973-983; and Belvin and Anderson (1996) Ann. Rev. Cell & Devel . Biol. 12:393- 416. Other receptors for cytokines are also known. Often, there are at least two critical subunits in the functional receptor. See, e.g., Gonda and D 'Andrea (1997) Blood 89:355-369; Presky, et al . (1996) Proc. Nat'l Acad. Sci. USA 93:14002-14007; Drachman and Kaushansky (1995) Curr. Qpin. Hematol. 2:22-28; Theze (1994) Eur. Cytokine Netw. 5:353-368; and Lemmon and Schlessinger (1994) Trends Biochem. Sci. 19:459-463.
From the foregoing, it is evident that the discovery and development of new soluble proteins and their receptors, including ones similar to lymphokines, should contribute to new therapies for a wide range of degenerative or abnormal conditions which directly or indirectly involve development, differentiation, or function, e.g., of the immune system and/or hematopoietic 4
cells. In particular, the discovery and understanding of novel receptors for lymphokine-like molecules which enhance or potentiate the beneficial activities of other lymphokines would be highly advantageous . The present invention provides new receptors for ligands exhibiting similarity to cytokine like compositions and related compounds, and methods for their use.
SUMMARY OF THE INVENTION The present invention is directed to a novel receptor related to cytokine receptors , e.g., primate or rodent, cytokine receptor like molecular structures, designated DNAX Cytokine Receptor Subunit 1 (DCRSl) , and their biological activities. The matching of subunits in a functional receptor, and ligand identification are described. It includes nucleic acids coding for the combinations of polypeptides themselves and methods for their production and use. The nucleic acids of the invention are characterized, in part, by their homology to cloned complementary DNA (cDNA) sequences enclosed herein.
In certain embodiments , the invention provides a composition of matter selected from the group of: a substantially pure or recombinant DCRSl protein or peptide exhibiting at least about 85% sequence identity over a length of at least about 12 amino acids to SEQ ID NO: 13 or 4 or 15; a natural sequence DCRSl comprising SEQ ID NO: 13 or 4 or 15; and a fusion protein comprising DCRSl sequence. Preferably, the substantially pure or isolated protein comprises a segment exhibiting sequence identity to a corresponding portion of a DCRSl, wherein: the homology is at least about 90% identity and the portion is at least about 9 amino acids; the homology is at least about 80% identity and the portion is at least about 17 amino acids; or the homology is at least about 70% identity and the portion is at least about 25 amino acids. In specific embodiments, the composition of matter is DCRSl, which comprises a mature sequence of Table 1; or exhibits a non-glycosylated DCRSl; or the composition of matter may be a protein or peptide which: is from a warm blooded animal selected from a mammal, including a primate or rodent, such as a human or mouse; comprises at least one polypeptide segment of SEQ ID NO: 13 or 4 or 15; exhibits a plurality of portions exhibiting said identity; is a natural allelic variant of DCRSl; has a length at least about 30 amino acids; exhibits at least two non-overlapping epitopes which are specific for a primate or rodent DCRSl; exhibits a sequence identity at least about 90% over a length of at least about 20 amino acids to a primate or rodent DCRSl; is glycosylated; has a molecular weight of at least 100 kD with natural glycosylation; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is a 5-fold or less substitution from natural sequence; or is a deletion or insertion variant from a natural sequence .
Other embodiments include a composition comprising: a sterile DCRSl protein or peptide; or the DCRSl protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration. In certain fusion protein embodiments, the invention provides a fusion protein comprising: mature protein sequence of Table 1; a detection or purification tag, including a FLAG, His6, or Ig sequence; or sequence of another receptor protein. Various kit embodiments include a kit comprising a DCRSl protein or polypeptide, and: a compartment comprising the protein or polypeptide; and/or instructions for use or disposal of reagents in the kit . Binding compound embodiments include those comprising an antigen binding site from an antibody, which specifically binds to a natural DCRSl protein, wherein: the protein is a primate protein; the binding compound is an Fv, Fab, or Fab2 fragment; the binding b
compound is conjugated to another chemical moiety; or the antibody: is raised against a peptide sequence of a mature polypeptide of Table 1; is raised against a mature DCRSl; is raised to a purified human or mouse DCRSl; is immunoselected; is a polyclonal antibody; binds to a denatured DCRSl; exhibits a Kd to antigen of at least 30 μM; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label. A binding composition kit often comprises the binding compound, and: a compartment comprising said binding compound; and/or instructions for use or disposal of reagents in the kit. Often the kit is capable of making a qualitative or quantitative analysis . Other compositions include a composition comprising: a sterile binding compound, or the binding compound and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
Nucleic acid embodiments include an isolated or recombinant nucleic acid encoding a DCRSl protein or peptide or fusion protein, wherein: the DCRSl is from a mammal; or the nucleic acid: encodes an antigenic peptide sequence of Table 1; encodes a plurality of antigenic peptide sequences of Table 1; exhibits at least about 80% identity to a natural cDNA encoding said segment; is an expression vector; further comprises an origin of replication; is from a natural source; comprises a detectable label; comprises synthetic nucleotide sequence; is less than 6 kb, preferably less than 3 kb; is from a mammal, including a primate; comprises a natural full length coding sequence; is a hybridization probe for a gene encoding said DCRSl; or is a PCR primer, PCR product, or mutagenesis primer. A cell, tissue, or organ comprising such a recombinant nucleic acid is also provided. Preferably, the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell. Kits are provided comprising such nucleic acids, and: a compartment comprising said nucleic acid; a compartment further comprising a primate or rodent DCRSl protein or polypeptide; and/or instructions for use or disposal of reagents in the kit. Often, the kit is capable of making a qualitative or quantitative analysis .
Other embodiments include a nucleic acid which: hybridizes under wash conditions of 30° C and less than
2M salt to SEQ ID NO: 12 or 3 or 14; or exhibits at least about 85% identity over a stretch of at least about 30 nucleotides to a primate DCRSl. Preferably, such nucleic acid will have such properties, wherein: wash conditions are at 45° C and/or 500 mM salt; or the identity is at least 90% and/or the stretch is at least 55 nucleotides.
More preferably, the wash conditions are at 55° C and/or 150 mM salt; or the identity is at least 95% and/or the stretch is at least 75 nucleotides. The invention also provides a method of modulating physiology or development of a cell or tissue culture cells comprising contacting the cell with an agonist or antagonist of a mammalian, e.g., primate or rodent DCRSl. It also provides cells cotransfected with a nucleic acid encoding DCRSl and another cytokine receptor subunit, e.g., DSRS1. This will allow pairing of subunits to determine the physiological receptor pairs for various cytokine ligands .
The present invention provides various compositions, e.g., comprising both a DSRS1 protein and an isolated or recombinant DCRSl protein; both an isolated or recombinant DSRS1 protein and a DCRSl protein; or both a substantially pure or recombinant IL-B30 protein and a DCRSl protein. In certain embodiments, the DSRS1 protein has sequence of mature SEQ ID NO: 9 or 11; the DCRSl protein has sequence of mature SEQ ID NO: 13 or 15; or the IL-B30 has sequence of mature SEQ ID NO: 17 or 19. In other embodiments, at least one of the proteins: is unglycosylated; is made with synthetic methods; has a detectable label; is attached to a solid substrate; or is conjugated to another chemical moiety.
In other forms, the invention provides a composition comprising: a substantially pure DCRSl protein and: a DSRSl protein or an IL-B30 cytokine protein; or a DCRSl protein and a substantially pure: DSRSl protein or IL-B30 cytokine protein. Preferred forms combining the DCRSl and the DSRSl proteins are those where the proteins combine to bind IL-B30 with high affinity. Yet other forms include sterile compositions, as described.
Kit embodiments include such compositions combined with: a compartment comprising two or more of the proteins; a compartment comprising a soluble receptor alpha subunit; a compartment comprising an IL-B30 cytokine protein; or instructions for use or disposal of reagents in the kit .
Binding composition embodiments include those comprising the antigen binding sites from antibodies, which antibodies bind to an epitope found on a composition described above, but not on separate proteins thereof. Various embodiments include where: the DCRSl is: a primate protein, a purified human or mouse DCRSl, or a mature polypeptide of Table 1; the DSRSl is: a primate protein, a purified human or mouse DSRSl, or a mature polypeptide of Table 4; or the IL-B30 is: a primate protein, a purified human or mouse IL-B30, or a mature polypeptide of Table 6. Other embodiments include those where the binding composition: is in a container; is an Fv, Fab, or Fab2 fragment; is conjugated to another chemical moiety; is immunoselected; is a polyclonal antibody; exhibits a Kd to antigen of at least 30 μM; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label.
Kit embodiments which comprise the binding compositions further include, e.g., a compartment 9
comprising the binding composition; a compartment comprising the DCRSl, DSRSl, or IL-B30 protein; or instructions for use or disposal of reagents in the kit. Certain nucleic acid composition are provided, e.g., an isolated or recombinant nucleic acid encoding both: a DSRSl protein and a DCRSl protein; a DSRSl protein and an IL-B30 protein; or a DCRSl protein and an IL-B30 protein. Preferred embodiments are those nucleic acids which encode both a DCRSl protein and a DSRSl protein; or both a DSRSl protein and an IL-B30, e.g., in a fusion protein. Other preferred embodiments are those nucleic acids which are expression vectors. Preferably, the DSRSl protein has sequence of mature SEQ ID NO: 9 or 11; the DCRSl protein has sequence of mature SEQ ID NO: 13 or 15; or the IL-B30 has sequence of mature SEQ ID NO: 17 or 19. Specific embodiments are those comprising the coding portion of: SEQ ID NO: 8 or 10; SEQ ID NO: 12 or 14; or SEQ ID NO: 16 or 18.
Transformed cells with the nucleic acids are provided, including where the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell.
Various methods are also provided, e.g., a method of producing a receptor complex, comprising culturing a described transformed cell of in an environment resulting in expression of the DCRSl and the DSRSl proteins, thereby forming the receptor complex; or of screening for ligands for a receptor complex comprising the DCRSl and the DSRSl proteins, comprising screening a library of compounds for binding to the described cell .
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. General
The present invention provides the amino acid sequence and DNA sequence of mammalian, herein primate, cytokine receptor-like subunit molecules, this one designated DNAX Cytokine Receptor Subunit 1 (DCRSl) having particular defined properties, both structural and biological . Various cDNAs encoding these molecules were obtained from primate, e.g., human, cDNA sequence libraries . Other primate or other mammalian counterparts would also be desired.
Some of the standard methods applicable are described or referenced, e.g., in Maniatis, et al . (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual, (2d ed. ) , vols. 1-3, CSH Press, NY; Ausubel, et al . , Biology, Greene Publishing Associates, Brooklyn, NY; or Ausubel, et al . (1987 and periodic supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York; each of which is incorporated herein by reference.
Partial nucleotide (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ ID NO: 2) of a human DCRSl coding segment is shown in Table 1, with supplementary sequence provided in SEQ ID NO: 12 and 13. Partial mouse sequence is provided (SEQ ID NO: 3 and 4) , with supplementary sequence in SEQ ID NO: 14 and 15.
Table 1: Partial nucleotide and polypeptide sequences of DNAX Cytokine Receptor Subunit like embodiments (DCRSl) . Primate, e.g., human embodiment (see SEQ ID NO: 1 and 2) . gtc tgg ccc ccc gtc ttc gtg aac eta gaa ace caa atg aag cca aac 48 Val Trp Pro Pro Val Phe Val Asn Leu Glu Thr Gin Met Lys Pro Asn 1 5 10 15 gec ccc egg ctg ggc cct gac gtg gac ttt tec gag gat gac ccc ctg 96 Ala Pro Arg Leu Gly Pro Asp Val Asp Phe Ser Glu Asp Asp Pro Leu 20 25 30 gag gec act gtc cat tgg gec cca cct aca tgg cca tct cat aaa gtt 144 Glu Ala Thr Val His Trp Ala Pro Pro Thr Trp Pro Ser His Lys Val 35 40 45 ctg ate tgc cag ttc cac tac cga aga tgt cag gag gcg gec tgg ace 192 Leu lie Cys Gin Phe His Tyr Arg Arg Cys Gin Glu Ala Ala Trp Thr 50 55 60 ctg ctg gaa ccg gag ctg aag ace ata ccc ctg ace cct gtt gag ate 240 Leu Leu Glu Pro Glu Leu Lys Thr lie Pro Leu Thr Pro Val Glu lie 65 70 75 80 11
caa gat ttg gag eta gec act ggc tac aaa gtg tat ggc cgc tgc egg 288 Gin Asp Leu Glu Leu Ala Thr Gly Tyr Lys Val Tyr Gly Arg Cys Arg 85 90 95 atg gag aaa gaa gag gat ttg tgg ggc gag tgg age ccc att ttg tec 336 Met Glu Lys Glu Glu Asp Leu Trp Gly Glu Trp Ser Pro lie Leu Ser 100 105 110 ttc cag aca ccg cct tct get cca aaa gat gtg tgg gta tea ggg aac 384 Phe Gin Thr Pro Pro Ser Ala Pro Lys Asp Val Trp Val Ser Gly Asn 115 120 125 etc tgt ggg acg cct gga gga gag gaa cct ttg ctt eta tgg aag gec 432 Leu Cys Gly Thr Pro Gly Gly Glu Glu Pro Leu Leu Leu Trp Lys Ala 130 135 140 cca ggg ccc tgt gtg cag gtg age tac aaa gtc tgg ttc tgg gtt gga 480 Pro Gly Pro Cys Val Gin Val Ser Tyr Lys Val Trp Phe Trp Val Gly 145 150 155 160 ggt cgt gag ctg agt cca gaa gga att ace tgc tgc tgc tec eta att 528 Gly Arg Glu Leu Ser Pro Glu Gly lie Thr Cys Cys Cys Ser Leu lie 165 170 175 ccc agt ggg gcg gag tgg gee agg gtg tec get gtc aac gee aca age 576 Pro Ser Gly Ala Glu Trp Ala Arg Val Ser Ala Val Asn Ala Thr Ser 180 185 190 tgg gag cct etc ace aac etc tct ttg gtc tgc ttg gat tea gec tct 624 Trp Glu Pro Leu Thr Asn Leu Ser Leu Val Cys Leu Asp Ser Ala Ser 195 200 205 gee ccc cgt age gtg gca gtc age age ate get ggg age acg gag eta 672 Ala Pro Arg Ser Val Ala Val Ser Ser lie Ala Gly Ser Thr Glu Leu 210 215 220 ctg gtg ace tgg caa ccg ggg cct ggg gaa cca ctg gag cat gta atg 720 Leu Val Thr Trp Gin Pro Gly Pro Gly Glu Pro Leu Glu His Val Met 225 230 235 240 gac tgg get cga gat ggg gac ccc ctg gag aaa etc aac tgg gtc egg 768 Asp Trp Ala Arg Asp Gly Asp Pro Leu Glu Lys Leu Asn Trp Val Arg 245 250 255 ctt ccc cct ggg aac etc agt get ctg tta cca ggg aat ttc act gtc 816 Leu Pro Pro Gly Asn Leu Ser Ala Leu Leu Pro Gly Asn Phe Thr Val 260 265 270 ggg gtc ccc tat cga ate act gtg ace gca gtc tct get tea ggc ttg 864 Gly Val Pro Tyr Arg lie Thr Val Thr Ala Val Ser Ala Ser Gly Leu 275 280 285 gee tct gca tec tec gtc tgg ggg ttc agg gag gaa tta gca ccc eta 912 Ala Ser Ala Ser Ser Val Trp Gly Phe Arg Glu Glu Leu Ala Pro Leu 290 295 300 gtg ggg cca acg ctt tgg cga etc caa gat gec cct cca ggg ace ccc 960 Val Gly Pro Thr Leu Trp Arg Leu Gin Asp Ala Pro Pro Gly Thr Pro 305 310 315 320 gee ata gcg tgg gga gag gtc cca agg cac cag ctt cga ggc cac etc 1008
Ala lie Ala Trp Gly Glu Val Pro Arg His Gin Leu Arg Gly His Leu 325 330 335 ace cac tac ace ttg tgt gca cag agt gga ace age ccc tec gtc tgc 1056
Thr His Tyr Thr Leu Cys Ala Gin Ser Gly Thr Ser Pro Ser Val Cys 340 345 350 atg aat gtg agt ggc aac aca cag agt gtc ace ctg cct gac ctt cct 1104
Met Asn Val Ser Gly Asn Thr Gin Ser Val Thr Leu Pro Asp Leu Pro 355 360 365 tgg ggt ccc tgt gag ctg tgg gtg aca gca tct ace ate get gga cag 1152 Trp Gly Pro Cys Glu Leu Trp Val Thr Ala Ser Thr He Ala Gly Gin
370 375 380 ggc cct cct ggt ccc ate etc egg ctt cat eta cca gat aac ace ctg 1200
Gly Pro Pro Gly Pro He Leu Arg Leu His Leu Pro Asp Asn Thr Leu 385 390 395 400 agg tgg aaa gtt ctg ccg ggc ate eta ttc ttg tgg ggc ttg ttc ctg 1248
Arg Trp Lys Val Leu Pro Gly He Leu Phe Leu Trp Gly Leu Phe Leu 405 410 415 ttg ggg tgt ggc ctg age ctg gec ace tct gga agg tgc tac cac eta 1296
Leu Gly Cys Gly Leu Ser Leu Ala Thr Ser Gly Arg Cys Tyr His Leu 420 425 430 agg cac aaa gtg ctg ccc cgc tgg gtc tgg gag aaa gtt cct gat cct 1344
Arg His Lys Val Leu Pro Arg Trp Val Trp Glu Lys Val Pro Asp Pro 435 440 445 gee aac age agt tea ggc cag ccc cac atg gag caa gta cct gag gee 1392 Ala Asn Ser Ser Ser Gly Gin Pro His Met Glu Gin Val Pro Glu Ala
450 455 460 cag ccc ctt ggg gac ttg ccc ate ctg gaa gtg gag gag atg gag ccc 1440
Gin Pro Leu Gly Asp Leu Pro He Leu Glu Val Glu Glu Met Glu Pro 465 470 475 480 ccg ccg gtt atg gag tec tec cag ccc gee cag gee ace gee ccg ctt 1488 Pro Pro Val Met Glu Ser Ser Gin Pro Ala Gin Ala Thr Ala Pro Leu 485 490 495 gac tct ggg tat gag aag cac ttc ctg ccc aca cct gag gag ctg ggc 1536
Asp Ser Gly Tyr Glu Lys His Phe Leu Pro Thr Pro Glu Glu Leu Gly 500 505 510 ctt ctg ggg ccc ccc agg cca cag gtt ctg gec tgaaccacac gtctggctgg 1589 Leu Leu Gly Pro Pro Arg Pro Gin Val Leu Ala 515 520 gggctgccag ccaggctaga gggatgetea tgcaggttgc accccagtcc tggattagee 1649 ctcttgatgg atgaagacac tgaggactca gagaggctga gtcacttacc tgaggacacc 1709 cageeaggca gagetgggat tgaaggaecc etatagagaa gggettggce cceatgggga 1769 agaeaeggat ggaaggtgga gcaaaggaaa atacatgaaa ttgagagtgg eagetgectg 1829 ceaaaatctg tteegetgta acagaaetga atttggaecc cageaeagtg gctcaegcct 1889 gtaatcecag cactttggea ggceaaggtg gaaggatcac ttagagetag gagtttgaga 1949 ccagcctggg caatatagca agacccctca ctacaaaaat aaaacatcaa aaacaaaaac 2009 aattagctgg geatgatgge acacaectgt agtecgagcc acttgggagg ctgaggtggg 2069 aggateggtt gageccagga gtttgaaget gcagggacet ctgattgeae cactgcacte 2129 caggctgggt aacagaatga gaccttatyt caaaaataaa caaactaat aaaarmaaaa 2189 aaaaaaamwm raraaaaaaa aaaa 2213
Partial "downstream" sequences of rodent, e.g., mouse, embodiment of DCRSl (SEQ ID NO: 3 and 4) : aaa gga ggg gtc ccc tat cga att aca gtg act gca gta tac tct gga 48 Lys Gly Gly Val Pro Tyr Arg He Thr Val Thr Ala Val Tyr Ser Gly 1 5 10 15 gga tta get get gca ccc tea gtt tgg gga ttc aga gag gag tta gta 96 Gly Leu Ala Ala Ala Pro Ser Val Trp Gly Phe Arg Glu Glu Leu Val 20 25 30 ccc ctt get ggg cca gca gtt tgg cga ctt cca gat gac ccc cca ggg 144 Pro Leu Ala Gly Pro Ala Val Trp Arg Leu Pro Asp Asp Pro Pro Gly 35 40 45 aca cct gtt gta gec tgg gga gaa gta cca aga cac cag etc aga ggc 192 Thr Pro Val Val Ala Trp Gly Glu Val Pro Arg His Gin Leu Arg Gly 50 55 60 cag get act cac tac ace ttc tgc ata cag age aga ggc etc tec act 240 Gin Ala Thr His Tyr Thr Phe Cys He Gin Ser Arg Gly Leu Ser Thr 65 70 75 80 gtc tgc agg aac gtg age agt caa ace cag act gec act ctg ccc aac 288 Val Cys Arg Asn Val Ser Ser Gin Thr Gin Thr Ala Thr Leu Pro Asn 85 90 95 ctt cac teg ggt tec ttc aag ctg tgg gtg acg gtg tec ace gtt gca 336
Leu His Ser Gly Ser Phe Lys Leu Trp Val Thr Val Ser Thr Val Ala
100 105 110 gga cag ggc cca cct ggt ccc gac ctt tea ctt cac eta cca gat aat 384
Gly Gin Gly Pro Pro Gly Pro Asp Leu Ser Leu His Leu Pro Asp Asn
115 120 125 agg ate agg tgg aaa get ctg ccc tgg ttt ctg tec ctg tgg ggt ttg 432 Arg He Arg Trp Lys Ala Leu Pro Trp Phe Leu Ser Leu Trp Gly Leu 130 135 140 ctt ctg atg ggc tgt ggc ctg age ctg gec agt ace agg tgc eta cag 480 Leu Leu Met Gly Cys Gly Leu Ser Leu Ala Ser Thr Arg Cys Leu Gin 145 150 155 160 gee agg tgc tta cac tgg cga cac aag ttg ctt ccc cag tgg ate tgg 528 Ala Arg Cys Leu His Trp Arg His Lys Leu Leu Pro Gin Trp He Trp 165 170 175 gag agg gtt cct gat cct gee aac age aat tct ggg caa cct tac ate 576 Glu Arg Val Pro Asp Pro Ala Asn Ser Asn Ser Gly Gin Pro Tyr He 180 185 190 aag gag gtg age ctg ccc caa ccg ccc aag gac gga ccc ate ctg gag 624 Lys Glu Val Ser Leu Pro Gin Pro Pro Lys Asp Gly Pro He Leu Glu 195 200 205 gtg gag gaa gtg gag eta cag cct gtt gtg gag tec cct aaa gee tct 672 Val Glu Glu Val Glu Leu Gin Pro Val Val Glu Ser Pro Lys Ala Ser 210 215 220 gee ccg att tac tct ggg tat gag aaa cac ttc ctg ccc aca cca gag 720 Ala Pro He Tyr Ser Gly Tyr Glu Lys His Phe Leu Pro Thr Pro Glu 225 230 235 240 gag ctg ggc ctt eta gtc tgatctgett aeggctaggg gctgtacccc 768
Glu Leu Gly Leu Leu Val 245 tatcttgggc tagacgtttt tgtattttta gatttttgag acaggatctc actatggctg 828 gectggaact tgatataaca accaggctgg cctggaactc accaagactc acctggtttt 888 gccttccaag gactgagaag aaatgagtgt gccgcctccc gcccaaccag ettttgettt 948 ccttgcctct gggtcttggg catctgtttg ttactgeaga agaatcagtg agctcacagc 1008 ctcgaacttg tgatcctccc tgetgeagea tccccagagc tgggattaca ggtgtgcgtc 1068 acttcatega gtcataaett ttgattctag tgagaataac taccaggcag gctatgaggt 1128 ggtgactcga aagacacatt caaggaccta aagtggttaa gagcctgtgt tttcttgcag 1188 tagaccaaag tttggttccc tgcccttgca aaggacacac gttcagtttc cagcacccac 1248 agggcagtte agaateacet gtaactceag gtecaaggaa tecaatgecc tcttctggct 1308 tctgtgagcc ccgcacacac atggttactt atgcaccgaa aaacacacgc ataaaataaa 1368 aataaataaa taaataaaaa taaattaata aataatettt tttttcttaa aaaaaaaaaa 1428 aaa 1431
supplementary primate, e.g., human, DCRSl sequence (SEQ ID NO: 12 and 13 ) : atg egg gga ggc agg ggc gee cct ttc tgg ctg tgg ccg ctg ccc aag 48 Met Arg Gly Gly Arg Gly Ala Pro Phe Trp Leu Trp Pro Leu Pro Lys 1 5 10 15 ctg gcg ctg ctg cct ctg ttg tgg gtg ctt ttc cag egg acg cgt ccc 96 Leu Ala Leu Leu Pro Leu Leu Trp Val Leu Phe Gin Arg Thr Arg Pro 20 25 30 cag ggc age gec ggg cca ctg cag tgc tac gga gtt gga ccc ttg ggc 144
Gin Gly Ser Ala Gly Pro Leu Gin Cys Tyr Gly Val Gly Pro Leu Gly 35 40 45 ID
gac ttg aac tgc teg tgg gag cct ctt ggg gac ctg gga gee ccc tec 192 Asp Leu Asn Cys Ser Trp Glu Pro Leu Gly Asp Leu Gly Ala Pro Ser 50 55 60 gag tta cac etc cag age caa aag tac cgt tec aac aaa ace cag act 240 Glu Leu His Leu Gin Ser Gin Lys Tyr Arg Ser Asn Lys Thr Gin Thr 65 70 75 80 gtg gca gtg gca gec gga egg age tgg gtg gec att cct egg gaa cag 288 Val Ala Val Ala Ala Gly Arg Ser Trp Val Ala He Pro Arg Glu Gin 85 90 95 etc ace atg tct gac aaa etc ctt gtc tgg ggc ayt aag gca ggc cag 336 Leu Thr Met Ser Asp Lys Leu Leu Val Trp Gly Xaa Lys Ala Gly Gin 100 105 110 cct etc tgg ccc ccc gtc ttc gtg aac eta gaa ace caa atg aag cca 384 Pro Leu Trp Pro Pro Val Phe Val Asn Leu Glu Thr Gin Met Lys Pro 115 120 125 aac gee ccc egg ctg ggc cct gac gtg gac ttt tec gag gat gac ccc 432 Asn Ala Pro Arg Leu Gly Pro Asp Val Asp Phe Ser Glu Asp Asp Pro 130 135 140 ctg gag gec act gtc cat tgg gee cca cct aca tgg cca tct cat aaa 480 Leu Glu Ala Thr Val His Trp Ala Pro Pro Thr Trp Pro Ser His Lys 145 150 155 160 gtt ctg ate tgc cag ttc cac tac cga aga tgt cag gag gcg gec tgg 528 Val Leu He Cys Gin Phe His Tyr Arg Arg Cys Gin Glu Ala Ala Trp 165 170 175 ace ctg ctg gaa ccg gag ctg aag ace ata ccc ctg ace cct gtt gag 576 Thr Leu Leu Glu Pro Glu Leu Lys Thr He Pro Leu Thr Pro Val Glu 180 185 190 ate caa gat ttg gag eta gee act ggc tac aaa gtg tat ggc cgc tgc 624 He Gin Asp Leu Glu Leu Ala Thr Gly Tyr Lys Val Tyr Gly Arg Cys 195 200 205 egg atg gag aaa gaa gag gat ttg tgg ggc gag tgg age ccc att ttg 672
Arg Met Glu Lys Glu Glu Asp Leu Trp Gly Glu Trp Ser Pro He Leu
210 215 220 tec ttc cag aca ccg cct tct get cca aaa gat gtg tgg gta tea ggg 720
Ser Phe Gin Thr Pro Pro Ser Ala Pro Lys Asp Val Trp Val Ser Gly 225 230 235 240 aac etc tgt ggg acg cct gga gga gag gaa cct ttg ctt eta tgg aag 768 Asn Leu Cys Gly Thr Pro Gly Gly Glu Glu Pro Leu Leu Leu Trp Lys 245 250 255 gee cca ggg ccc tgt gtg cag gtg age tac aaa gtc tgg ttc tgg gtt 816 Ala Pro Gly Pro Cys Val Gin Val Ser Tyr Lys Val Trp Phe Trp Val 260 265 270 gga ggt cgt gag ctg agt cca gaa gga att ace tgc tgc tgc tec eta 864 Gly Gly Arg Glu Leu Ser Pro Glu Gly He Thr Cys Cys Cys Ser Leu 275 280 285 16
att ccc agt ggg gcg gag tgg gee agg gtg tec get gtc aac gec aca 912 He Pro Ser Gly Ala Glu Trp Ala Arg Val Ser Ala Val Asn Ala Thr 290 295 300 age tgg gag cct etc ace aac etc tct ttg gtc tgc ttg gat tea gee 960 Ser Trp Glu Pro Leu Thr Asn Leu Ser Leu Val Cys Leu Asp Ser Ala 305 310 315 320 tct gee ccc cgt age gtg gca gtc age age ate get ggg age acg gag 1008 Ser Ala Pro Arg Ser Val Ala Val Ser Ser He Ala Gly Ser Thr Glu 325 330 335 eta ctg gtg ace tgg caa ccg ggg cct ggg gaa cca ctg gag cat gta 1056 Leu Leu Val Thr Trp Gin Pro Gly Pro Gly Glu Pro Leu Glu His Val 340 345 350 atg gac tgg get cga gat ggg gac ccc ctg gag aaa etc aac tgg gtc 1104 Met Asp Trp Ala Arg Asp Gly Asp Pro Leu Glu Lys Leu Asn Trp Val 355 360 365 egg ctt ccc cct ggg aac etc agt get ctg tta cca ggg aat ttc act 1152 Arg Leu Pro Pro Gly Asn Leu Ser Ala Leu Leu Pro Gly Asn Phe Thr 370 375 380 gtc ggg gtc ccc tat cga ate act gtg ace gca gtc tct get tea ggc 1200 Val Gly Val Pro Tyr Arg He Thr Val Thr Ala Val Ser Ala Ser Gly 385 390 395 400 ttg gee tct gca tec tec gtc tgg ggg ttc agg gag gaa tta gca ccc 1248 Leu Ala Ser Ala Ser Ser Val Trp Gly Phe Arg Glu Glu Leu Ala Pro 405 410 415 eta gtg ggg cca acg ctt tgg cga etc caa gat gec cct cca ggg ace 1296 Leu Val Gly Pro Thr Leu Trp Arg Leu Gin Asp Ala Pro Pro Gly Thr 420 425 430 ccc gee ata gcg tgg gga gag gtc cca agg cac cag ctt cga ggc cac 1344 Pro Ala He Ala Trp Gly Glu Val Pro Arg His Gin Leu Arg Gly His 435 440 445 etc ace cac tac ace ttg tgt gca cag agt gga ace age ccc tec gtc 1392 Leu Thr His Tyr Thr Leu Cys Ala Gin Ser Gly Thr Ser Pro Ser Val 450 455 460 tgc atg aat gtg agt ggc aac aca cag agt gtc ace ctg cct gac ctt 1440 Cys Met Asn Val Ser Gly Asn Thr Gin Ser Val Thr Leu Pro Asp Leu 465 470 475 480 cct tgg ggt ccc tgt gag ctg tgg gtg aca gca tct ace ate get gga 1488 Pro Trp Gly Pro Cys Glu Leu Trp Val Thr Ala Ser Thr He Ala Gly 485 490 495 cag ggc cct cct ggt ccc ate etc egg ctt cat eta cca gat aac ace 1536 Gin Gly Pro Pro Gly Pro He Leu Arg Leu His Leu Pro Asp Asn Thr 500 505 510 ctg agg tgg aaa gtt ctg ccg ggc ate eta ttc ttg tgg ggc ttg ttc 1584 Leu Arg Trp Lys Val Leu Pro Gly He Leu Phe Leu Trp Gly Leu Phe 515 520 525 17
ctg ttg ggg tgt ggc ctg age ctg gec ace tct gga agg tgc tac cac 1632 Leu Leu Gly Cys Gly Leu Ser Leu Ala Thr Ser Gly Arg Cys Tyr His 530 535 540 eta agg cac aaa gtg ctg ccc cgc tgg gtc tgg gag aaa gtt cct gat 1680 Leu Arg His Lys Val Leu Pro Arg Trp Val Trp Glu Lys Val Pro Asp 545 550 555 560 cct gee aac age agt tea ggc cag ccc cac atg gag caa gta cct gag 1728 Pro Ala Asn Ser Ser Ser Gly Gin Pro His Met Glu Gin Val Pro Glu
565 570 575 gee cag ccc ctt ggg gac ttg ccc ate ctg gaa gtg gag gag atg gag 1776 Ala Gin Pro Leu Gly Asp Leu Pro He Leu Glu Val Glu Glu Met Glu 580 585 590 ccc ccg ccg gtt atg gag tec tec cag ccc gec cag gee ace gee ccg 1824 Pro Pro Pro Val Met Glu Ser Ser Gin Pro Ala Gin Ala Thr Ala Pro 595 600 605 ctt gac tct ggg tat gag aag cac ttc ctg ccc aca cct gag gag ctg 1872 Leu Asp Ser Gly Tyr Glu Lys His Phe Leu Pro Thr Pro Glu Glu Leu 610 615 620 ggc ctt ctg ggg ccc ccc agg cca cag gtt ctg gec tga 1911 Gly Leu Leu Gly Pro Pro Arg Pro Gin Val Leu Ala 625 630 635
MRGGRGAPFW LWPLPKLALL PLLWVLFQRT RPQGSAGPLQ CYGVGPLGDL NCSWEPLGDL GAPSELHLQS QKYRSNKTQT VAVAAGRSWV AIPREQLTMS DKLLVWG . KA GQPLWPPVFV NLETQMKPNA PRLGPDVDFS EDDPLEATVH WAPPTWPSHK VLICQFHYRR CQEAAWTLLE PELKTIPLTP VEIQDLELAT GYKVYGRCRM EKEEDLWGEW SPILSFQTPP SAPKDVWVSG NLCGTPGGEE PLLLWKAPGP CVQVSYKVWF WVGGRELSPE GITCCCSLIP SGAEWARVSA VNATSWEPLT NLSLVCLDSA SAPRSVAVSS lAGSTELLVT WQPGPGEPLE HVMDWARDGD PLEKLNWVRL PPGNLSALLP GNFTVGVPYR ITVTAVSASG LASASSVWGF REELAPLVGP TLWRLQDAPP GTPAIAWGEV PRHQLRGHLT HYTLCAQSGT SPSVCMNVSG NTQSVTLPDL PWGPCELWVT ASTIAGQGPP GPILRLHLPD NTLRWKVLPG ILFLWGLFLL GCGLSLATSG RCYHLRHKVL PRWVWEKVPD PANSSSGQPH MEQVPEAQPL GDLPILEVEE MEPPPVMESS QPAQATAPLD SGYEKHFLPT PEELGLLGPP RPQVLA
supplementary partial "upstream" rodent, e.g. mouse, DCRP1 sequences (SEQ ID NO: 14 and 15) : ggt aag ccc caa gee tgg tgg tgt cac ttg tec ctg gga gec atg aac 48 Gly Lys Pro Gin Ala Trp Trp Cys His Leu Ser Leu Gly Ala Met Asn 1 5 10 15 egg etc ggg ttt gca cgc etc acg ccg ttg gag ctt ctg ctg teg ctg 96 Arg Leu Gly Phe Ala Arg Leu Thr Pro Leu Glu Leu Leu Leu Ser Leu
20 25 30 atg teg ctg ctg etc ggg acg egg ccc cac ggc agt cca ggc cca ctg 144 Met Ser Leu Leu Leu Gly Thr Arg Pro His Gly Ser Pro Gly Pro Leu 35 40 45 cag tgc tac age gtc ggt ccc ctg gga ate ctg aac tgc tec tgg gaa 192 Gin Cys Tyr Ser Val Gly Pro Leu Gly He Leu Asn Cys Ser Trp Glu 50 55 60 cct ttg ggc gac ctg gag act cca cct gtg ctg tat cac cag agt cag 240 Pro Leu Gly Asp Leu Glu Thr Pro Pro Val Leu Tyr His Gin Ser Gin 65 70 75 80 aaa tac cat ccc aat aga gtc tgg gag gtg aag gtg cct tec aaa cag 288 Lys Tyr His Pro Asn Arg Val Trp Glu Val Lys Val Pro Ser Lys Gin 85 90 95 agt tgg gtg ace att ccc egg gaa cag ttc ace atg get gac aaa etc 336 Ser Trp Val Thr He Pro Arg Glu Gin Phe Thr Met Ala Asp Lys Leu 100 105 110 etc ate tgg ggg aca caa aag gga egg cct ctg tgg tec tct gtc tct 384 Leu He Trp Gly Thr Gin Lys Gly Arg Pro Leu Trp Ser Ser Val Ser 115 120 125 gtg aac ctg gag ace caa atg aag cca gac aca cct cag ate ttc tct 432 Val Asn Leu Glu Thr Gin Met Lys Pro Asp Thr Pro Gin He Phe Ser 130 135 140 caa gtg gat att tct gan 450
Gin Val Asp He Ser Xaa 145 150
Table 2: Comparison of rodent, e.g., mouse, and primate, e.g., human, DCRSl :
mDCRSl MNRLGXARLTPLELLLSLMSLLLGTRPHGSPGPLQCYSVGPLGILNCSWEPLGDL hDCRSl MRGGRGAPFWLWPLPKLALLPLLWVLFQRTRPQGSAGPLQCYGVGPLGDLNCSWEPLGDL
* * ** * *. ***.** ****** ***** ***********
mDCRSl ETPPVLYHQSQKYHPNRVWEVKVPSKQSWVTIPREQFTMADKLLIWGTQKGRPLWSSVSV hDCRSl GAPSELHLQSQKYRSNKTQTVAVAAGRSWVAIPREQLTMSDKLLVWGXKAGQPLWPPVFV .* *. * ***. *. * * . .***.*****.**.****.***. *.*** * *
mDCRSl NLETQMKPDTPQIFSQVDIS hDCRSl NLETQMKPNAPRLGPDVDFSEDDPLEATVHWAPPTWPSHKVLICQFHYRRCQEAAWTLLE
******.
mDCRSl hDCRSl PELKTIPLTPVEIQDLELATGYKVYGRCRMEKEEDLWGEWSPILSFQTPPSAPKDVWVSG
mDCRSl hDCRSl NLCGTPGGEEPLLLWKAPGPCVQVSYKVWFWVGGRELSPEGITCCCSLIPSGAEWARVSA
mDCRSl hDCRSl VNATSWEPLTNLSLVCLDSASAPRSVAVSSIAGSTELLVTWQPGPGEPLEHVMDWARDGD
mDCRSl KGGVPYRITVTAVYSGGLAAAPSVWGFREELVPLAGP hDCRSl PLEKLNWVRLPPGNLSALLPGNFTVGVPYRITVTAVSASGLASASSVWGFREELAPLVGP
*********** *** * ********* ** ** 19
mDCRSl AVWRLPDDPPGTPWAWGEVPRHQLRGQATHYTFCIQSRGLSTVCRNVSSQTQTATLPNL hDCRSl TLWRLQDAPPGTPAIAWGEVPRHQLRGHLTHYTLCAQSGTSPSVCMNVSGNTQSVTLPDL *** * ***** ************ **** * ** ** *** ** *** *
mDCRSl HSGSFKLWVTVSTVAGQGPPGPDLSLHLPDNRIRWKALPWFLSLWGLLLMGCGLSLASTR hDCRSl PWGPCELWVTASTIAGQGPPGPILRLHLPDNTLRWKVLPGILFLWGLFLLGCGLSLATS- * **** ** ******** * ****** *** ** * **** * *******
mDCRSl CLQARCLHWRHKLLPQWIWERVPDPANSNSGQPYIKEVSLPQPPKDGPILEVEEVELQPV hDCRSl GRCYHLRHKVLPRWVWEKVPDPANSSSGQPHMEQVPEAQPLGDLPILEVEEMEPPPV
** * *** ** * ** ******* **** * ** * ******* * **
mDCRSl VESPK ASAPIYSGYEKHFLPTPEELGLLV hDCRSl MESSQPAQATAPLDSGYEKHFLPTPEELGLLGPPRPQVLA
*****************
Table 3: Alignment of various cytokine receptor subunits. Human gpl30 sequence (hgpl30) is SEQ ID NO: 5 (see GenBank M57230) . Human G-CSF Receptor subunit alpha (hGCSFRa) is SEQ ID NO: 6 (see GenBank X55721) . Human IL-12 Receptor subunit beta (hIL12Rb) is SEQ ID NO: 7 (see GenBank U64198) . Consensus domain boundaries are described in the text .
mIL30Rb MNRLGXARLTPLELLLSLMSLLLGTR hIL30Rb MRGGRGAPFWLWPLPKLALLPLLWVLFQRTR human_GCSF MARLGNCSLTWAALIILLLPGSLEECGHISVSAPIVHLGDPIT human_gpl3 MLTLQTWWQALFIFLTTESTGELLDP CGYISPESPWQLHSNFT human_ILl2 MAHTFRGCSLAFMFIITWLLIKAKIDACK—RGDVTVKPSHVILLGSTVN
mIL30Rb hIL30Rb human_GCSF ASCIIK--QNCSHLDPEPQ—ILWRLGAELQPGGRQQRLSDGTQESIITL human_gpl3 AVCVLK--EKCMDYFHVNANYIVWKTNHFTIPKE-QYTIINRTASSVTFT human IL12 ITCSLKPRQGCFHYSRRNK-LILYKFDRRINFHHGHSLNSQVTGLPLG—
mIL30Rb PHGSPGPLQCYSVGPLGI hIL30Rb PQGSAGPLQCYGVGPLGD human_GCSF PHLNHTQAFLSCCLNWGNSLQILDQVELRAGYPPAIPHNLSCLMNLTTSS human_gpl3 DIASLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGK-K human_IL12 TTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGT
mIL30Rb LNCSWEPLGDLETPPVLYHQSQKYHPN RVWEVKVPS-KQSWVTIP hIL3ORb LNCSWEPLGDLGAPSELHLQXQKYRSN KTQTVAVAA-GRSWVAIP human_GCSF LICQWEPGPETHLPTSFTLKSFKSRGNCQTQGDSILDCVPKDGQSHCCIP human_gpl3 MRCEWDGGRETHLETNFTLKS-EWATH KFADCKAKRDTPTSCTVD human_ILl2 VACTWERGRDTHLYTEYTLQL-SGPKN LTWQKQCKDIYCDYLDFGIN 20
mIL3ORb REQFTMADKLLIWGTQK GRPLWSSVSVNLETQMKPDTPQIFSQVDIS hIL3ORb REQLTMSDKLLVWGTKA GQPLWPPVFVNLETQMKPNAPRLGPDVDFS human_GCSF RKHLLLYQNMGIWVQAENALGTSMSPQLCLDPMDWKLEPPMLRTMDPSP human_gpl3 YS-TVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSE human_IL12 LTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQK
mIL30Rb hIL30Rb EDDPLEA TVHWAPPTWPSHKVLICQF-HYRRCQEAAWTLLEPELKTI human_GCSF EAAPPQAGCLQLCWEPWQPGLHINQKCELRHKPQRGEASWALVGPLP—L human_gpl3 ELSSILK LTWTNPSIKSVIILKYNI-QYRTKDASTWSQIPPEDTAS human_IL12 ASVSRCT LYWRDE GLVLLNRLRYRPSNSRLWNMVNVTK
mIL30Rb hIL30Rb PLTPVEIQDLELATGYKVYGRCRMEKEEDLWGEWSPILSFQTPPSAP human_GCSF EALQYELCGLLPATAYTLQIRCIRWPLPGHWSDWSPSLELRTTERAPTVR human_gpl3 TRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKA human IL12 AKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGM
mIL30Rb hIL30Rb KDVWVSG NLCGTPGGEEPLLLWKAPGPCVQVSYKVWFWVG G human_GCSF LDTWWRQR QLDPRTVQLFWKPVPLEEDSG-RIQGYWSWRPSGQ—A human_gpl3 PSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKS H human IL12 LDVWYMKR-HIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAM
mIL30Rb hIL30Rb RELSPEGITCCCSLIPSGAEWARVSAVNATSWEPLTNLSLVCLDSASAPR human_GCSF GAILPLCNTTELSCTFHLPSEAQEVALVAYNSAGTSRPTPWFSESRGPA human_gpl3 LQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQATHP human IL12 TQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLA
mIL30Rb hIL30Rb SVAVSSIAGS-TELLVTWQPGPGEP LEHVMDWARDGD-PLEKLN—W human_GCSF LTRLHAMARDPHSLWVGWEPPNPWP QGYVIEWGLGPP-SASNSNKTW human_gpl3 VMDLKAFPKD-NMLWVEWTTPRESV KKYILEWCVLSD-KAPCIT-DW human IL12 PRQVSANSEGMDNILVTWQPPRKDPSAVQEYWEWRELHPGGDTQVPLNW
mIL30Rb KGGVPYRITVTAVYSGGLAAAPSVWGFREELVP hIL3ORb VRLPPG-NLSALLPGNFTVGVPYRITVTAVSASGLASASSVWGFREELAP human_GCSF RMEQNGRATGFLLKENIRPFQLYEIIVTPLYQDTMGPSQHVYAYSQEMAP human_gpl3 QQE-DGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPP human_ILl2 LRS-RPYNVSALISENIKSYICYEIRVYALSGD-QGGCSSILGNSKHKAP
mIL3ORb LAGPAVWRLPDDPPGTPWAWGEVPRHQLRGQATHYTFCIQ SRGLS hIL3ORb LVGPTLWRLQDAPPGTPAIAWGEVPRHQLRGHLTHYTLCAQ SGTSP human_GCSF SHAP-ELHLKHIGKTWAQLEWVPEPPELGKSPLTHYTIFWT NAQNQ human_gpl3 SKGP-TVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYR TIIGN human_IL12 LSGP-HINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQ 21
mIL3ORb TVCRNVSSQTQTATLPNLHSGSFKLWVTVSTVAGQGPPGPDLSLHLPDNR hIL3ORb SVCMNVSGNTQSVTLPDLPWGPCELWVTASTIAGQGPPGPILRLHLPDNT human_GCSF SFSAILNASSRGFVLHGL—EPASLYHIHLMAASQAGATNSTVLTLMTLT human_gpl3 ETAVNVDSSHTEYTLSSL—TSDTLYMVRMAAYTDEGGKDGPEFTFTTPK human_ILI2 LCEIPYRVSQNSHPINSL—QPRVTYVLWMTALTAAGESSHGNEREFCLQ
mIL3 ORb IRWKALPWFLSLWGLLLMGCGLSLASTRCLQARCLHWRHKLLPQWIWER- hIL30Rb LRWKVLPGILFLWGLFLLGCGLSLATS GRCYHLRHKVLPRWVWEK- human_GCSF PEGSELHIILGLFGLLLLLTCLCGTAW LCCSPNR KNPLWPS- human_gpl3 FAQGEIEAIWPVCLAFLLTTLLG VLFCFNKR-DLIKKHIWPN- human_IL12 GKANWMAFVAPSICIAIIMVGIFSTHY FQQKVFVLLAALRPQWCSR
mIL30Rb -VPDPANSNSG QPYIKEVS LPQPPKDGP ILEVEEVE hIL30Rb -VPDPANSSSG QPHMEQVP EAQPLGDLP ILEVEE— human_GCSF -VPDPAHSSLGSWVPTIMEEDAFQLPG--LGTPPITKLT VLEEDE— human_gpl3 -VPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVS WEIEAND human_IL12 EIPDPANSTCAKKYPIAEEKTQLPLDR-LLIDWPTPEDPEPLVISEVLHQ
mIL30Rb LQPV PWESPK AS hIL30Rb MEPP PVMESSQPAQAT human_GCSF KKPVPWESHNSSET--CGLPTLVQTYVLQGDPRAVSTQPQSQSG TS human_gpl3 KKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESS human_IL12 VTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAE S
mIL30Rb APIYSG YEKHFLPTP hIL30Rb APLDSG YEKHFLPTP human_GCSF DQVLYGQLLGSPTSPGPGHYLRCDSTQPLLAGLTPSPKSYENLW FQ human_gpl3 QNTSSTVQYSTWHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVD human_IL12 RQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLP SN
mIL30Rb -EELGLLV hIL30Rb -EELGLLGPPRP-QVLA human_GCSF ASPLGTLVTPAP-SQEDDCVFG P-LLNFPLLQGIRVHGMEALGSF-- human_gpl3 HVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQ human_IL12 IDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLK
mIL30Rb hIL30Rb humanJSCSF human_gpl3 QISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEG human IL12 MRCDSLML
mIL30Rb hIL30Rb human_GCSF human_gpl3 MPKSYLPQTVRQGGYMPQ human_IL12 22
Table 4: Sequences of mammalian DNAX Soluble Receptor Subunit 1 (DSRSl). Primate, e.g., human, nucleotide and polypeptide sequences (SEQ ID NO: 8 and 9; note WSEWS motif at 327-331): atg ccc gee ggc cgc egg ggc ccc gee gee caa tec gcg egg egg ccg 48 Met Pro Ala Gly Arg Arg Gly Pro Ala Ala Gin Ser Ala Arg Arg Pro 1 5 10 15 ccg ccg ttg ctg ccc ctg ctg ctg ctg etc tgc gtc etc ggg gcg ccg 96 Pro Pro Leu Leu Pro Leu Leu Leu Leu Leu Cys Val Leu Gly Ala Pro
20 25 30 cga gee gga tea gga gee cac aca get gtg ate agt ccc cag gat ccc 144 Arg Ala Gly Ser Gly Ala His Thr Ala Val He Ser Pro Gin Asp Pro 35 40 45 acg ctt etc ate ggc tec tec ctg ctg gec ace tgc tea gtg cac gga 192 Thr Leu Leu He Gly Ser Ser Leu Leu Ala Thr Cys Ser Val His Gly 50 55 60 gac cca cca gga gee ace gec gag ggc etc tac tgg ace etc aac ggg 240 Asp Pro Pro Gly Ala Thr Ala Glu Gly Leu Tyr Trp Thr Leu Asn Gly 65 70 75 80 cgc cgc ctg ccc cct gag etc tec cgt gta etc aac gee tec ace ttg 288 Arg Arg Leu Pro Pro Glu Leu Ser Arg Val Leu Asn Ala Ser Thr Leu 85 90 95 get ctg gee ctg gec aac etc aat ggg tec agg cag egg teg ggg gac 336 Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gin Arg Ser Gly Asp 100 105 110 aac etc gtg tgc cac gee cgt gac ggc age ate ctg get ggc tec tgc 384 Asn Leu Val Cys His Ala Arg Asp Gly Ser He Leu Ala Gly Ser Cys 115 120 125 etc tat gtt ggc ctg ccc cca gag aaa ccc gtc aac ate age tgc tgg 432 Leu Tyr Val Gly Leu Pro Pro Glu Lys Pro Val Asn He Ser Cys Trp 130 135 140 tec aag aac atg aag gac ttg ace tgc cgc tgg acg cca ggg gee cac 480 Ser Lys Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro Gly Ala His 145 150 155 160 ggg gag ace ttc etc cac ace aac tac tec etc aag tac aag ctt agg 528 Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys Tyr Lys Leu Arg 165 170 175 tgg tat ggc cag gac aac aca tgt gag gag tac cac aca gtg ggg ccc 576 Trp Tyr Gly Gin Asp Asn Thr Cys Glu Glu Tyr His Thr Val Gly Pro 180 185 190 cac tec tgc cac ate ccc aag gac ctg get etc ttt acg ccc tat gag 624 His Ser Cys His He Pro Lys Asp Leu Ala Leu Phe Thr Pro Tyr Glu 195 200 205 ate tgg gtg gag gec ace aac cgc ctg ggc tct gee cgc tec gat gta 672 He Trp Val Glu Ala Thr Asn Arg Leu Gly Ser Ala Arg Ser Asp Val 210 215 220 23
etc acg ctg gat ate ctg gat gtg gtg ace acg gac ccc ccg ccc gac 720 Leu Thr Leu Asp He Leu Asp Val Val Thr Thr Asp Pro Pro Pro Asp 225 230 235 240 gtg cac gtg age cgc gtc ggg ggc ctg gag gac cag ctg age gtg cgc 768 Val His Val Ser Arg Val Gly Gly Leu Glu Asp Gin Leu Ser Val Arg 245 250 255 tgg gtg teg cca ccc gee etc aag gat ttc etc ttt caa gec aaa tac 816 Trp Val Ser Pro Pro Ala Leu Lys Asp Phe Leu Phe Gin Ala Lys Tyr 260 265 270 cag ate cgc tac cga gtg gag gac agt gtg gac tgg aag gtg gtg gac 864 Gin He Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys Val Val Asp 275 280 285 gat gtg age aac cag ace tec tgc cgc ctg gee ggc ctg aaa ccc ggc 912
Asp Val Ser Asn Gin Thr Ser Cys Arg Leu Ala Gly Leu Lys Pro Gly
290 295 300 ace gtg tac ttc gtg caa gtg cgc tgc aac ccc ttt ggc ate tat ggc 960
Thr Val Tyr Phe Val Gin Val Arg Cys Asn Pro Phe Gly He Tyr Gly 305 310 315 320 tec aag aaa gee ggg ate tgg agt gag tgg age cac ccc aca gee gec 1008 Ser Lys Lys Ala Gly He Trp Ser Glu Trp Ser His Pro Thr Ala Ala 325 330 335 tec act ccc cgc agt gag cgc ccg ggc ccg ggc ggc ggg gcg tgc gaa 1056 Ser Thr Pro Arg Ser Glu Arg Pro Gly Pro Gly Gly Gly Ala Cys Glu 340 345 350 ccg egg ggc gga gag ccg age teg ggg ccg gtg egg cgc gag etc aag 1104 Pro Arg Gly Gly Glu Pro Ser Ser Gly Pro Val Arg Arg Glu Leu Lys 355 360 365 cag ttc ctg ggc tgg etc aag aag cac gcg tac tgc tec aac etc age 1152
Gin Phe Leu Gly Trp Leu Lys Lys His Ala Tyr Cys Ser Asn Leu Ser
370 375 380 ttc cgc etc tac gac cag tgg cga gec tgg atg cag aag teg cac aag 1200
Phe Arg Leu Tyr Asp Gin Trp Arg Ala Trp Met Gin Lys Ser His Lys
385 390 395 400 ace cgc aac cag gtc ctg cca gat aag ctg tag 1233
Thr Arg Asn Gin Val Leu Pro Asp Lys Leu 405 410
MPAGRRGPAA QSARRPPPLL PLLLLLCVLG APRAGSGAHT AVISPQDPTL LIGSSLLATC SVHGDPPGAT AEGLYWTLNG RRLPPELSRV LNASTLALAL ANLNGSRQRS GDNLVCHARD GSILAGSCLY VGLPPEKPVN ISCWSKNMKD LTCRWTPGAH GETFLHTNYS LKYKLRWYGQ DNTCEEYHTV GPHSCHIPKD LALFTPYEIW VEATNRLGSA RSDVLTLDIL DWTTDPPPD λtHVSRVGGLE DQLSVRWVSP PALKDFLFQA KYQIRYRVED SVDWKWDDV SNQTSCRLAG LKPGTVYFVQ VRCNPFGIYG SKKAGIWSEW SHPTAASTPR SERPGPGGGA CEPRGGEPSS GPVRRELKQF LGWLKKHAYC SNLSFRLYDQ WRAWMQKSHK TRNQVLPDKL 24
Partial rodent, e.g., mouse, nucleotide and polypeptide sequences, note WSEWS motif at 321-325 (SEQ ID NO: 10 and 11) : ccc tea eta aag gga ata age ttg egg ccg ctg tec teg ctg tgg teg 48 Pro Ser Leu Lys Gly He Ser Leu Arg Pro Leu Ser Ser Leu Trp Ser 1 5 10 15 cct ctg ttg etc tgt gtc etc ggg gtg cct egg ggc gga teg gga gec 96 Pro Leu Leu Leu Cys Val Leu Gly Val Pro Arg Gly Gly Ser Gly Ala 20 25 30 cac aca get gta ate age ccc cag gac ccc ace ctt etc ate ggc tec 144
His Thr Ala Val He Ser Pro Gin Asp Pro Thr Leu Leu He Gly Ser 35 40 45 tec ctg caa get ace tgc tct ata cat gga gac aca cct ggg gec ace 192
Ser Leu Gin Ala Thr Cys Ser He His Gly Asp Thr Pro Gly Ala Thr 50 55 60 get gag ggg etc tac tgg ace etc aat ggt cgc cgc ctg ccc tct gag 240
Ala Glu Gly Leu Tyr Trp Thr Leu Asn Gly Arg Arg Leu Pro Ser Glu
65 70 75 80 ctg tec cgc etc ctt aac ace tec ace ctg gec ctg gee ctg get aac 288 Leu Ser Arg Leu Leu Asn Thr Ser Thr Leu Ala Leu Ala Leu Ala Asn
85 90 95 ctt aat ggg tec agg cag cag tea gga gac aat ctg gtg tgt cac gee 336 Leu Asn Gly Ser Arg Gin Gin Ser Gly Asp Asn Leu Val Cys His Ala 100 105 110 cga gat ggc age att ctg get ggc tec tgc etc tat gtt ggc ttg ccc 384
Arg Asp Gly Ser He Leu Ala Gly Ser Cys Leu Tyr Val Gly Leu Pro 115 120 125 cct gag aag cct ttt aac ate age tgc tgg tec egg aac atg aag gat 432
Pro Glu Lys Pro Phe Asn He Ser Cys Trp Ser Arg Asn Met Lys Asp 130 135 140 etc acg tgc cgc tgg aca ccg ggt gca cac ggg gag aca ttc tta cat 480 Leu Thr Cys Arg Trp Thr Pro Gly Ala His Gly Glu Thr Phe Leu His 145 150 155 160 ace aac tac tec etc aag tac aag ctg agg tgg tac ggt cag gat aac 528 Thr Asn Tyr Ser Leu Lys Tyr Lys Leu Arg Trp Tyr Gly Gin Asp Asn
165 170 175 aca tgt gag gag tac cac act gtg ggc cct cac tea tgc cat ate ccc 576 Thr Cys Glu Glu Tyr His Thr Val Gly Pro His Ser Cys His He Pro 180 185 190 aag gac ctg gec etc ttc act ccc tat gag ate tgg gtg gaa gec ace 624 Lys Asp Leu Ala Leu Phe Thr Pro Tyr Glu He Trp Val Glu Ala Thr 195 200 205 aat cgc eta ggc tea gca aga tct gat gtc etc aca ctg gat gtc ctg 672 Asn Arg Leu Gly Ser Ala Arg Ser Asp Val Leu Thr Leu Asp Val Leu 210 215 220 gac gtg gtg ace acg gac ccc cca ccc gac gtg cac gtg age cgc gtt 720 Asp Val Val Thr Thr Asp Pro Pro Pro Asp Val His Val Ser Arg Val 225 230 235 240 25
ggg gc ctg gag gac cag ctg agt gtg cgc tgg gtc tea cca cca get 768 Gly Gly Leu Glu Asp Gin Leu Ser Val Arg Trp Val Ser Pro Pro Ala 245 250 255 etc aag gat ttc etc ttc caa gec aag tac cag ate cgc tac cgc gtg 816 Leu Lys Asp Phe Leu Phe Gin Ala Lys Tyr Gin He Arg Tyr Arg Val 260 265 270 gag gac age gtg gac tgg aag gtg gtg gat gac gtc age aac cag ace 864 Glu Asp Ser Val Asp Trp Lys Val Val Asp Asp Val Ser Asn Gin Thr 275 280 285 tec tgc cgt etc gcg ggc ctg aag ccc ggc ace gtt tac ttc gtc caa 912 Ser Cys Arg Leu Ala Gly Leu Lys Pro Gly Thr Val Tyr Phe Val Gin 290 295 300 gtg cgt tgt aac cca ttc ggg ate tat ggg teg aaa aag gcg gga ate 960
Val Arg Cys Asn Pro Phe Gly He Tyr Gly Ser Lys Lys Ala Gly He
305 310 315 320 tgg age gag tgg age cac ccc ace get gec tec ace cct cga agt gag 1008
Trp Ser Glu Trp Ser His Pro Thr Ala Ala Ser Thr Pro Arg Ser Glu 325 330 335 cgc ccg ggc ccg ggc ggc ggg gtg tgc gag ccg egg ggc ggc gag ccc 1056 Arg Pro Gly Pro Gly Gly Gly Val Cys Glu Pro Arg Gly Gly Glu Pro 340 345 350 age teg ggc ccg gtg egg cgc gag etc aag cag ttc etc ggc tgg etc 1104 Ser Ser Gly Pro Val Arg Arg Glu Leu Lys Gin Phe Leu Gly' Trp Leu 355 360 365 aag aag cac gca tac tgc teg aac ctt agt ttc cgc ctg tac gac cag 1152 Lys Lys His Ala Tyr Cys Ser Asn Leu Ser Phe Arg Leu Tyr Asp Gin 370 375 380 tgg cgt get tgg atg cag aag tea cac aag ace cga aac cag gac gag 1200 Trp Arg Ala Trp Met Gin Lys Ser His Lys Thr Arg Asn Gin Asp Glu 385 390 395 400 ggg ate ctg ccc teg ggc aga egg ggt gcg gcg aga ggt cct gee ggc 1248 Gly He Leu Pro Ser Gly Arg Arg Gly Ala Ala Arg Gly Pro Ala Gly 405 410 415 taaactctaa ggataggeca tcctcctgct gggtcagacc tggaggctca cctgaattgg 1308 ageccctctg taccatctgg geaacaaaga aacetaecag aggctgggge acaatgaget 1368 eccacaaeca eagctttggt ceaeatgatg gtcacaettg gatataccce agtgtgggta 1428 gggttggggt attgeaggge eteeeaagag tttttttaaa taaataaagg agttgtteag 1488 gtcccgatgg naaaaaaaaa aaaaaaaaaa aaaaaa 1524 9/40195 26
Table 5: Alignment of primate, e.g., human, and rodent, e.g., mouse, DSRSl (SEQ ID NO: 9 and 11): hDSRSl MPAGRRGPAA QSARRPPPLL PLLLLLCVLG APRAGSGAHT AVISPQDPTL mDSRSl RPLSSL WSPLLLCVLG VPRGGSGAHT AVISPQDPTL
hDSRSl LIGSSLLATC SVHGDPPGAT AEGLYWTLNG RRLPPELSRV LNASTLALAL mDSRSl LIGSSLQATC SIHGDTPGAT AEGLYWTLNG RRLPSELSRL LNTSTLALAL
hDSRSl ANLNGSRQRS GDNLVCHARD GSILAGSCLY VGLPPEKPVN ISCWSKNMKD mDSRSl ANLNGSRQQS GDNLVCHARD GSILAGSCLY VGLPPEKPFN ISCWSRNMKD
hDSRSl LTCRWTPGAH GETFLHTNYS LKYKLRWYGQ DNTCEEYHTV GPHSCHIPKD mDSRSl LTCRWTPGAH GETFLHTNYS LKYKLRWYGQ DNTCEEYHTV GPHSCHIPKD
hDSRSl LALFTPYEIW VEATNRLGSA RSDVLTLDIL DWTTDPPPD VHVSRVGGLE mDSRSl LALFTPYEIW VEATNRLGSA RSDVLTLDVL DWTTDPPPD VHVSRVGGLE
hDSRSl DQLSVRWVSP PALKDFLFQA KYQIRYRVED SVDWKWDDV SNQTSCRLAG mDSRSl DQLSVRWVSP PALKDFLFQA KYQIRYRVED SVDWKWDDV SNQTSCRLAG
hDSRSl LKPGTVYFVQ VRCNPFGIYG SKKAGIWSEW SHPTAASTPR SERPGPGGGA mDSRSl LKPGTVYFVQ VRCNPFGIYG SKKAGIWSEW SHPTAASTPR SERPGPGGGV
hDSRSl CEPRGGEPSS GPVRRELKQF LGWLKKHAYC SNLSFRLYDQ WRAWMQKSHK mDSRSl CEPRGGEPSS GPVRRELKQF LGWLKKHAYC SNLSFRLYDQ WRAWMQKSHK
hDSRSl TRNQ...VLP DKL mDSRSl TRNQDEGILP SGRRGAARGP AG
Table 6: Primate, e.g., human, IL-B30 nucleotide and polypeptide sequences. Predicted signal cleavage site indicated, but may actually be a residue or more to either side, depending upon the cell (SEQ ID NO: 16 and 17): atg ctg ggg age aga get gta atg ctg ctg ttg ctg ctg ccc tgg aca 48 Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro Trp Thr -20 -15 -10 get cag ggc aga get gtg cct ggg ggc age age cct gee tgg act cag 96 Ala Gin Gly Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gin -5 -1 1 5 10 tgc cag cag ctt tea cag aag etc tgc aca ctg gee tgg agt gca cat 144 Cys Gin Gin Leu Ser Gin Lys Leu Cys Thr Leu Ala Trp Ser Ala His 15 20 25 cca eta gtg gga cac atg gat eta aga gaa gag gga gat gaa gag act 192 Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr 30 35 40 aca aat gat gtt ccc cat ate cag tgt gga gat ggc tgt gac ccc caa 240 Thr Asn Asp Val Pro His He Gin Cys Gly Asp Gly Cys Asp Pro Gin 45 50 55 gga etc agg gac aac agt cag ttc tgc ttg caa agg ate cac cag ggt 288 Gly Leu Arg Asp Asn Ser Gin Phe Cys Leu Gin Arg He His Gin Gly 60 65 70 75 ctg att ttt tat gag aag ctg eta gga teg gat att ttc aca ggg gag 336 Leu He Phe Tyr Glu Lys Leu Leu Gly Ser Asp He Phe Thr Gly Glu
80 85 90 cct tct ctg etc cct gat age cct gtg gcg cag ctt cat gee tec eta 384 Pro Ser Leu Leu Pro Asp Ser Pro Val Ala Gin Leu His Ala Ser Leu 95 100 105 ctg ggc etc age caa etc ctg cag cct gag ggt cac cac tgg gag act 432
Leu Gly Leu Ser Gin Leu Leu Gin Pro Glu Gly His His Trp Glu Thr
110 115 120 cag cag att cca age etc agt ccc age cag cca tgg cag cgt etc ctt 480
Gin Gin He Pro Ser Leu Ser Pro Ser Gin Pro Trp Gin Arg Leu Leu
125 130 135 etc cgc ttc aaa ate ctt cgc age etc cag gec ttt gtg get gta gec 528 Leu Arg Phe Lys He Leu Arg Ser Leu Gin Ala Phe Val Ala Val Ala 140 145 150 155 gec egg gtc ttt gec cat gga gca gca ace ctg agt ccc taa 570 Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser Pro
160 165
MLGSRAVMLL LLLPWTAQGR AVPGGSSPAW TQCQQLSQKL CTLAWSAHPL VGHMDLREEG DEETTNDVPH IQCGDGCDPQ GLRDNSQFCL QRIHQGLIFY EKLLGSDIFT GEPSLLPDSP VAQLHASLLG LSQLLQPEGH HWETQQIPSL SPSQPWQRLL LRFKILRSLQ AFVAVAARVF AHGAATLSP
Rodent, e.g., mouse, nucleotide and polypeptide sequences of IL-B30. Predicted signal cleavage site indicated, but may actually be a residue or more to either side, depending upon the cell (SEQ ID NO: 18 and 19) : cgcttagaag teggactaca gagttagact cagaaccaaa ggaggtggat agggggtcca 60 caggcctggt geagateaca gagceagcea gatctgagaa geagggaaca ag atg ctg 118
Met Leu -20 gat tgc aga gca gta ata atg eta tgg ctg ttg ccc tgg gtc act cag 166 Asp Cys Arg Ala Val He Met Leu Trp Leu Leu Pro Trp Val Thr Gin -15 -10 -5 ggc ctg get gtg cct agg agt age agt cct gac tgg get cag tgc cag 214 Gly Leu Ala Val Pro Arg Ser Ser Ser Pro Asp Trp Ala Gin Cys Gin -1 1 5 10 cag etc tct egg aat etc tgc atg eta gec tgg aac gca cat gca cca 262 Gin Leu Ser Arg Asn Leu Cys Met Leu Ala Trp Asn Ala His Ala Pro 15 20 25 28
gcg gga cat atg aat eta eta aga gaa gaa gag gat gaa gag act aaa 310 Ala Gly His Met Asn Leu Leu Arg Glu Glu Glu Asp Glu Glu Thr Lys 30 35 40 45 aat aat gtg ccc cgt ate cag tgt gaa gat ggt tgt gac cca caa gga 358 Asn Asn Val Pro Arg He Gin Cys Glu Asp Gly Cys Asp Pro Gin Gly 50 55 60 etc aag gac aac age cag ttc tgc ttg caa agg ate cgc caa ggt ctg 406 Leu Lys Asp Asn Ser Gin Phe Cys Leu Gin Arg He Arg Gin Gly Leu
65 70 75 get ttt tat aag cac ctg ctt gac tct gac ate ttc aaa ggg gag cct 454 Ala Phe Tyr Lys His Leu Leu Asp Ser Asp He Phe Lys Gly Glu Pro 80 85 90 get eta etc cct gat age ccc atg gag caa ctt cac ace tec eta eta 502 Ala Leu Leu Pro Asp Ser Pro Met Glu Gin Leu His Thr Ser Leu Leu 95 100 105 gga etc age caa etc etc cag cca gag gat cac ccc egg gag ace caa 550 Gly Leu Ser Gin Leu Leu Gin Pro Glu Asp His Pro Arg Glu Thr Gin 110 115 120 125 cag atg ccc age ctg agt tct agt cag cag tgg cag cgc ccc ctt etc 598 Gin Met Pro Ser Leu Ser Ser Ser Gin Gin Trp Gin Arg Pro Leu Leu 130 135 140 cgt tec aag ate ctt cga age etc cag gee ttt ttg gee ata get gec 646 Arg Ser Lys He Leu Arg Ser Leu Gin Ala Phe Leu Ala He Ala Ala 145 150 155 egg gtc ttt gee cac gga gca gca act ctg act gag ccc tta gtg cca 694 Arg Val Phe Ala His Gly Ala Ala Thr Leu Thr Glu Pro Leu Val Pro 160 165 170 aca get taaggatgee caggttccca tggctaccat gataagacta atctatcagc 750 Thr Ala 175 ccagacatct accagttaat taacccatta ggacttgtgc tgttcttgtt tcgtttgttt 810 tgcgtgaagg gcaaggacac cattattaaa gagaaaagaa acaaacccca gagcaggcag 870 ctggctagag aaaggagctg gagaagaaga ataaagtctc gageccttgg ccttggaagc 930 gggcaagcag ctgcgtggcc tgaggggaag ggggcggtgg catcgagaaa ctgtgagaaa 990 acceagagea tcagaaaaag tgagcecagg etttggceat tatctgtaag aaaaacaaga 1050 aaaggggaac attatacttt cetgggtgge teagggaaat gtgeagatgc acagtactec 1110 agaeageage tctgtacctg cctgctetgt cccteagttc taacagaate tagteactaa 1170 gaactaacag gactaccaat acgaactgac aaa 1203
MLDCRAVIML WLLPWVTQGL AVPRSSSPDW AQCQQLSRNL CMLAWNAHAP AGHMNLLREE
EDEETKNNVP RIQCEDGCDP QGLKDNSQFC LQRIRQGLAF YKHLLDSDIF KGEPALLPDS
PMEQLHTSLL GLSQLLQPED HPRETQQMPS LSSSQQWQRP LLRSKILRSL QAFLAIAARV FAHGAATLTE PLVPTA Partial polypeptide sequence of IL-B30 from pig (SEQ ID NO : 20) :
Ser Cys Leu Gin Arg He His Gin Gly Leu Val Phe Tyr Glu Lys Leu 1 5 10 15
Leu Gly Ser Asp He Phe Thr Gly Glu Pro Ser Leu His Pro Asp Gly 20 25 30
Ser Val Gly Gin Leu His Ala Ser Leu Leu Gly Leu Arg Gin Leu Leu 35 40 45
Gin Pro Glu Gly His His Trp Glu Thr Glu Gin Thr Pro Ser Pro Ser 50 55 60 Pro Ser Gin Pro Trp Gin Arg Leu Leu Leu Arg Leu Lys He Leu Arg 65 70 75 80
Ser Leu Gin Ala Phe Val Ala Val Ala Ala Arg Val Phe Ala His Gly 85 90 95
Ala Ala Thr Leu Ser Gin 100
SCLQRIHQGLVFYEKLLGSDIFTGEPSLHPDGSVGQLHASLLGLRQLLQPEGHHWETEQTPSPSPSQ PWQRLLLRLKILRSLQAFVAVAARVFAHGAATLSQ
Table 2 shows comparison of the available sequences of primate and rodent embodiments of DCRSl . Table 3 shows the alignment of the DCRSl with other cytokine receptor subunits . The DCRSl shows particular similarity to the IL-12 receptor subunit beta, though it may be aligned with the gpl30 (IL-6 receptor beta) and G-CSF receptor (alpha) subunits. The similarity to the IL-12 receptor subunit suggests that the functional receptor incorporating the DCRSl may be similar to the IL-12 receptor. The IL-12 receptor alpha subunit is a soluble subunit. The DSRSl is likely to be the corresponding soluble subunit, which would initially interact with ligand, e.g., the IL-B30, and then form a functional complex with the DCRSl. See, e.g., Presky, et al . (1996) Proc. Nat ' 1 Acad. Sci. USA 93:14002-14007. Alternatively, the soluble subunit may interact with the transmembrane receptor subunit, which then could bind ligand.
Table 4 provides the nucleotide and polypeptide sequences of two species embodiments of a soluble receptor subunit designated DNAX Soluble Receptor Subunit 1 (DSRSl) . These align with and exhibit features in common with other cytokine receptor alpha type subunits. These subunits are believed to interact with the corresponding DCRSl to form a functional receptor when the receptor ligand is present. Note that relatively close sequence similarity of the DCRSl is with gpl30, which is the beta subunit of the IL-6 receptor. Applicants believe that the ligand for the receptor is likely the ligand designated IL-B30, whose sequence is near to G-CSF and IL-6. See USSN 60/053,765, which is incorporated herein by reference.
Table 5 shows alignment of the DSRSl from the primate and rodent species. Applicants believe that this soluble subunit forms a dimer, and binds to its dimerized ligand, which then combines with a beta type homo or heterodimer . Alternatively, the soluble subunit may bind to the transmembrane subunit (s), and then bind ligand.
Structural features of the human DCRSl, and similarly for the other receptors as aligned in Table 3, include characteristic Ig domains from about (SEQ ID NO: 2) vail to prol33; fibronectin domains corresponding to the DCRSl sequence from about glyl34 to pro232, gly233 to gly306, and pro307 to lys403; a transmembrane segment from about val404 to gly427; and an intraceUular domain from about arg428 to the carboxy terminus . Of particular interest is the WGEWS motif corresponding to residues trpl04 to serl08. In many contexts, various variants and fragments will be equivalent to the described DSRSl.
As used herein, the term DCRSl shall be used to describe a protein comprising the amino acid sequence shown in Table 1. In many cases, a substantial fragment thereof will be functionally or structurally equivalent, including, e.g., an extracellular or intraceUular domain. The invention also includes a protein variation of the respective DCRSl allele whose sequence is provided, e.g., a mutein or soluble extracellular construct. Typically, such agonists or antagonists will exhibit less than about 10% sequence differences, and thus will often have between 1- and 11-fold 31
substitutions, e.g., 2-, 3-, 5-, 7-fold, and others. It also encompasses allelic and other variants, e.g., natural polymorphic, of the protein described. Typically, it will bind to its corresponding biological ligand, perhaps in a dimerized state with an alpha receptor subunit, with high affinity, e.g., at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM. The term shall also be used herein to refer to related naturally occurring forms, e.g., alleles, polymorphic variants, and metabolic variants of the mammalian protein. Preferred forms of the receptor complexes will bind the appropriate ligand with an affinity and selectivity appropriate for a ligand- receptor interaction.
This invention also encompasses combinations of proteins or peptides having substantial amino acid sequence identity with the amino acid sequence in Table 1. It will include sequence variants with relatively few substitutions, e.g., preferably less than about 3-5.
Other embodiments include forms in association with an alpha subunit, e.g., a DSRSl, and/or with ligand, e.g., IL-B30.
A substantial polypeptide "fragment", or "segment", is a stretch of amino acid residues of at least about 8 amino acids, generally at least 10 amino acids, more generally at least 12 amino acids, often at least 14 amino acids, more often at least 16 amino acids, typically at least 18 amino acids, more typically at least 20 amino acids, usually at least 22 amino acids, more usually at least 24 amino acids, preferably at least 26 amino acids, more preferably at least 28 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids. Sequences of segments of different proteins can be compared to one another over appropriate length stretches. In many situations, fragments may exhibit functional properties of the intact subunits, e.g., the extracellular domain of the 32
transmembrane receptor may retain the ligand binding features, and may be used to prepare a soluble receptorlike complex.
Amino acid sequence homology, or sequence identity, is determined by optimizing residue matches. In some comparisons, gaps may be introduces, as required. See, e.g., Needleham, et al . , (1970) J. Mol. Biol. 48:443-453; Sankoff, et al., (1983) chapter one in Time Warps, String Edits , and Macromolecules : The Theory and Practice of Sequence Comparison, Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA; and the University of Wisconsin Genetics Computer Group (GCG) , Madison, WI; each of which is incorporated herein by reference. This changes when considering conservative substitutions as matches . Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Homologous amino acid sequences are intended to include natural allelic and interspecies variations in the cytokine sequence. Typical homologous proteins or peptides will have from 50-100% homology (if gaps can be introduced) , to 60-100% homology (if conservative substitutions are included) with an amino acid sequence segment of Table 1. Homology measures will be at least about 70%, generally at least 76%, more generally at least 81%, often at least 85%, more often at least 88%, typically at least 90%, more typically at least 92%, usually at least 94%, more usually at least 95%, preferably at least 96%, and more preferably at least 97%, and in particularly preferred embodiments, at least 98% or more. The degree of homology will vary with the length of the compared segments . Homologous proteins or peptides, such as the allelic variants, will share most biological activities with the embodiments described in Table 1. 33
As used herein, the term "biological activity" is used to describe, without limitation, effects on inflammatory responses, innate immunity, and/or morphogenic development by cytokine-like ligands . For example, these receptors should mediate phosphatase or phosphorylase activities, which activities are easily measured by standard procedures. See, e.g., Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al . (1991) Meth. Enzvmol. 200:38-62; Hunter, et al . (1992) Cell
70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al . (1991) Cold Spring Harbor Svmp. Quant. Biol. 56:449-463; and Parker, et al . (1993) Nature 363:736-738. The receptors, or portions thereof, may be useful as phosphate labeling enzymes to label general or specific substrates . The subunits may also be functional immunogens to elicit recognizing antibodies, or antigens capable of binding antibodies .
The terms ligand, agonist, antagonist, and analog of, e.g., a DCRSl, include molecules that modulate the characteristic cellular responses to cytokine ligand proteins, as well as molecules possessing the more standard structural binding competition features of ligand-receptor interactions, e.g., where the receptor is a natural receptor or an antibody. The cellular responses likely are typically mediated through receptor tyrosine kinase pathways.
Also, a ligand is a molecule which serves either as a natural ligand to which said receptor, or an analog thereof, binds, or a molecule which is a functional analog of the natural ligand. The functional analog may be a ligand with structural modifications, or may be a wholly unrelated molecule which has a molecular shape which interacts with the appropriate ligand binding determinants. The ligands may serve as agonists or antagonists, see, e.g., Goodman, et al. (eds. 1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics , Pergamon Press, New York. Rational drug design may also be based upon structural studies of the molecular shapes of a receptor or antibody and other effectors or ligands. See, e.g., Herz, et al . (1997) J. Recept . Signal Transduct. Res. 17:671-776; and Chaiken, et al . (1996) Trends Biotechnol . 14:369-375. Effectors may be other proteins which mediate other functions in response to ligand binding, or other proteins which normally interact with the receptor. One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., x-ray crystallography or 2 dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions . For a detailed description of protein structural determination, see, e.g., Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York, which is hereby incorporated herein by reference.
II. Activities The cytokine receptor-like proteins will have a number of different biological activities, e.g., modulating cell proliferation, or in phosphate metabolism, being added to or removed from specific substrates, typically proteins. Such will generally result in modulation of an inflammatory function, other innate immunity response, or a morphological effect. The subunit will probably have a specific low affinity binding to the ligand.
The DCRSl has the characteristic motifs of a receptor signaling through the JAK pathway. See, e.g., Ihle, et al . (1997) Stem Cells 15(suppl. 1):105-111; Silvennoinen, et al. (1997) APMIS 105:497-509; Levy (1997) Cvtokine Growth Factor Review 8:81-90; Winston and Hunter (1996) Current Biol. 6:668-671; Barrett (1996) Baillieres Clin. Gastroenterol. 10:1-15; and Briscoe, et al. (1996) Philos. Trans. R. Soc . Lond. B. Biol. Sci. 351:167-171. The biological activities of the cytokine receptor subunits will be related to addition or removal of phosphate moieties to substrates, typically in a specific manner, but occasionally in a non specific manner. Substrates may be identified, or conditions for enzymatic activity may be assayed by standard methods, e.g., as described in Hardie, et al . (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al . (1991) Meth. Enzvmol. 200:38-62; Hunter, et al . (1992) Cell 70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al . (1991) Cold Soring Harbor Svmp. Quant. Biol. 56:449-463; and Parker, et al . (1993) Nature 363:736-738.
The receptor subunits may combine to form functional complexes, e.g., which may be useful for binding ligand or preparing antibodies . These will have substantial diagnostic uses, including detection or quantitation.
Ill . Nucleic Acids This invention contemplates use of isolated nucleic acid or fragments, e.g., which encode these or closely related proteins, or fragments thereof, e.g., to encode a corresponding polypeptide, preferably one which is biologically active. In addition, this invention covers isolated or recombinant DNAs which encode combinations of such proteins or polypeptides having characteristic sequences, e.g., of the DCRSls. Typically, the nucleic acid is capable of hybridizing, under appropriate conditions, with a nucleic acid sequence segment shown in Table 1, but preferably not with a corresponding segment of other receptors described in Table 3. Said biologically active protein or polypeptide can be a full length protein, or fragment, and will typically have a segment of amino acid sequence highly homologous, e.g., exhibiting significant stretches of identity, to one shown in Table 1. Further, this invention covers the use of isolated or recombinant nucleic acid, or fragments thereof, which encode proteins having fragments which are 36
equivalent to the DCRSl proteins. The isolated nucleic acids can have the respective regulatory sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and others from the natural gene. Combinations, as described, are also provided.
An "isolated" nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially pure, e.g., separated from other components which naturally accompany a native sequence, such as ribosomes, polymerases, and flanking genomic sequences from the originating species . The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, which are thereby distinguishable from naturally occurring compositions, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule, either completely or substantially pure. An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain heterogeneity, preferably minor. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
A "recombinant" nucleic acid is typically defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g. , involving human intervention in the nucleotide sequence. Typically this intervention involves in vitro manipulation, although under certain circumstances it may involve more classical animal breeding techniques. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants as 37
found in their natural state. Thus, for example, products made by transforming cells with an unnaturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any synthetic oUgonucleotide process. Such a process is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a restriction enzyme sequence recognition site. Alternatively, the process is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms, e.g., encoding a fusion protein. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design. A similar concept is intended for a recombinant, e.g. , fusion, polypeptide. This will include a dimeric repeat.
Specifically included are synthetic nucleic acids which, by genetic code redundancy, encode equivalent polypeptides to fragments of DCRSl and fusions of sequences from various different related molecules, e.g., other cytokine receptor family members.
A "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least 21 nucleotides, more generally at least 25 nucleotides, ordinarily at least 30 nucleotides, more ordinarily at least 35 nucleotides, often at least 39 nucleotides, more often at least 45 nucleotides, typically at least 50 nucleotides, more typically at least 55 nucleotides, usually at least 60 nucleotides, more usually at least 66 nucleotides, preferably at least 72 nucleotides, more preferably at least 79 nucleotides, and in particularly preferred embodiments will be at least 85 or more nucleotides. Typically, fragments of different genetic sequences can be compared to one J o
another over appropriate length stretches, particularly defined segments such as the domains described below. A nucleic acid which codes for the DCRSl will be particularly useful to identify genes, mRNA, and cDNA species which code for itself or closely related proteins, as well as DNAs which code for polymorphic, allelic, or other genetic variants, e.g., from different individuals or related species . Preferred probes for such screens are those regions of the interleukin which are conserved between different polymorphic variants or which contain nucleotides which lack specificity, and will preferably be full length or nearly so. In other situations, polymorphic variant specific sequences will be more useful . Nucleic acids encoding various combinations including, e.g., the DSRSl, the DCRSl, and/or the IL-B30, will be useful for coexpression of the proteins together. Such will be useful, e.g., in producing complexes, for production of antibodies or screening for ligands. This invention further covers recombinant nucleic acid molecules and fragments having a nucleic acid sequence identical to or highly homologous to the isolated DNA set forth herein. In particular, the sequences will often be operably linked to DNA segments which control transcription, translation, and DNA replication. These additional segments typically assist in expression of the desired nucleic acid segment. Homologous, or highly identical, nucleic acid sequences, when compared to one another, e.g., DCRSl sequences, exhibit significant similarity. The standards for homology in nucleic acids are either measures for homology generally used in the art by sequence comparison or based upon hybridization conditions . Comparative hybridization conditions are described in greater detail below.
Substantial identity in the nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical 39
when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 60% of the nucleotides, generally at least 66%, ordinarily at least 71%, often at least 76%, more often at least 80%, usually at least 84%, more usually at least 88%, typically at least 91%, more typically at least about 93%, preferably at least about 95%, more preferably at least about 96 to 98% or more, and in particular embodiments, as high at about 99% or more of the nucleotides, including, e.g., segments encoding structural domains such as the segments described below. Alternatively, substantial identity will exist when the segments will hybridize under selective hybridization conditions, to a strand or its complement, typically using a sequence derived from Table 1. Typically, selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, more typically at least about 65%, preferably at least about 75%, and more preferably at least about 90%. See, Kanehisa (1984) Nucl. Acids Res . 12:203-213, which is incorporated herein by reference. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, generally at least about 20 nucleotides, ordinarily at least about 24 nucleotides, usually at least about 28 nucleotides, typically at least about 32 nucleotides, more typically at least about 40 nucleotides, preferably at least about 50 nucleotides, and more preferably at least about 75 to 100 or more nucleotides. This includes, e.g., 125, 150, 175, 200, 225, 246, 273, and other lengths.
Stringent conditions, in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters typically controlled in hybridization reactions . Stringent temperature conditions will usually include temperatures in excess of about 30° C, more usually in excess of about 37° C, typically in excess of about 45° C, more typically in excess of about 55° C, preferably in excess of about 65° C, and more preferably in excess of about 70° C. Stringent salt conditions will ordinarily be less than about 500 mM, usually less than about 400 mM, more usually less than about 300 mM, typically less than about 200 mM, preferably less than about 100 mM, and more preferably less than about 80 mM, even down to less than about 20 M. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and
Davidson (1968) J. Mol. Biol. 31:349-370, which is hereby incorporated herein by reference.
The isolated DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode this protein or its derivatives . These modified sequences can be used to produce mutant proteins (muteins) or to enhance the expression of variant species. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. Such mutant DCRS1- like derivatives include predetermined or site-specific mutations of the protein or its fragments, including silent mutations using genetic code degeneracy. "Mutant DCRSl" as used herein encompasses a polypeptide otherwise falling within the homology definition of the DCRSl as set forth above, but having an amino acid sequence which differs from that of other cytokine receptor-like proteins as found in nature, whether by way of deletion, substitution, or insertion. In particular, "site specific mutant DCRSl" encompasses a protein having substantial sequence identity with a protein of Table 1, and typically shares most of the biological activities or effects of the forms disclosed herein.
Although site specific mutation sites are predetermined, mutants need not be site specific. Mammalian DCRSl mutagenesis can be achieved by making amino acid insertions or deletions in the gene, coupled with expression. Substitutions, deletions, insertions, or many combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed mammalian DCRSl mutants can then be screened for the desired activity, providing some aspect of a structure-activity relationship. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by M13 primer mutagenesis. See also Sambrook, et al . (1989) and Ausubel, et al . (1987 and periodic Supplements) .
The mutations in the DNA normally should not place coding sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins .
The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNA fragments . A double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
Polymerase chain reaction (PCR) techniques can often be applied in mutagenesis. Alternatively, mutagenesis primers are commonly used methods for generating defined mutations at predetermined sites. See, e.g., Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA; and Dieffenbach and Dveksler (1995; eds.) PCR Primer: A Laboratory Manual Cold Spring Harbor Press, CSH, NY. Certain embodiments of the invention are directed to combination compositions comprising the receptor or ligand sequences described. In other embodiments, functional portions of the sequences may be joined to encode fusion proteins. In other forms, variants of the described sequences may be substituted in the combinations .
IV. Proteins, Peptides
As described above, the present invention encompasses primate DCRSl, e.g., whose sequences are disclosed in Table 1, and described above. Allelic and other variants are also contemplated, including, e.g., fusion proteins combining portions of such sequences with others, including, e.g., epitope tags and functional domains .
The present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these primate or rodent proteins. A heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner. Thus, the fusion product of a DCRSl with another cytokine receptor is a continuous protein molecule having sequences fused in a typical peptide linkage, typically made as a single translation product and exhibiting properties, e.g., sequence or antigenicity, derived from each source peptide. A similar concept applies to heterologous nucleic acid sequences. Combinations of various designated proteins into complexes are also provided.
In addition, new constructs may be made from combining similar functional or structural domains from other related proteins, e.g., cytokine receptors or Toll- like receptors, including species variants. For example, ligand-binding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, e.g., Cunningham, et al . (1989) Science 243:1330-1336; and O'Dowd, et al . (1988) J. Biol. Chem. 263:15985-15992, each of which is incorporated herein by reference. Thus, new chimeric polypeptides exhibiting new combinations of specificities will result from the functional linkage of receptor-binding specificities. For example, the ligand binding domains from other related receptor molecules may be added or substituted for other domains of this or related proteins. The resulting protein will often have hybrid function and properties. For example, a fusion protein may include a targeting domain which may serve to provide sequestering of the fusion protein to a particular subcellular organelle.
Candidate fusion partners and sequences can be selected from various sequence data bases, e.g., GenBank, c/o IntelliGenetics, Mountain View, CA; and BCG,
University of Wisconsin Biotechnology Computing Group, Madison, WI, which are each incorporated herein by reference. In particular, combinations of polypeptide sequences provided in Tables 1, 4, or 6 are particularly preferred. Variant forms of the proteins may be substituted in the described combinations . In certain embodiments, portions of the DSRSl may be fused to portions of the IL-B30.
The present invention particularly provides muteins which bind cytokine-like ligands, and/or which are affected in signal transduction. Structural alignment of human DCRSl with other members of the cytokine receptor family show conserved features/residues . See Table 3. Alignment of the human DCRSl sequence with other members of the cytokine receptor family indicates various structural and functionally shared features. See also, Bazan, et al. (1996) Nature 379:591; Lodi, et al . (1994) Science 263:1762-1766; Sayle and Milner-White (1995) TIBS 20:374-376; and Gronenberg, et al . (1991) Protein Engineering 4:263-269.
Substitutions with either mouse sequences or human sequences are particularly preferred. Conversely, conservative substitutions away from the ligand binding interaction regions will probably preserve most signaling activities; and conservative substitutions away from the intraceUular domains will probably preserve most ligand binding properties . "Derivatives" of the primate DCRSl include amino acid sequence mutants, glycosylation variants, metabolic derivatives and covalent or aggregative conjugates with other chemical moieties . Covalent derivatives can be prepared by linkage of functionalities to groups which are found in the DCRSl amino acid side chains or at the N- or C- termini, e.g., by means which are well known in the art. These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g., lysine or arginine . Acyl groups are selected from the group of alkyl-moieties, including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species .
In particular, glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. Particularly preferred means for accomplishing this are by exposing the polypeptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes. Deglycosylation enzymes are also contemplated. Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine . A major group of derivatives are covalent conjugates of the receptors or fragments thereof with other proteins of polypeptides. These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups. Preferred derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues. Fusion polypeptides between the receptors and other homologous or heterologous proteins are also provided. Homologous polypeptides may be fusions between different receptors, resulting in, for instance, a hybrid protein exhibiting binding specificity for multiple different cytokine ligands, or a receptor which may have broadened or weakened specificity of substrate effect. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a receptor, e.g., a ligand-binding segment, so that the presence or location of a desired ligand may be easily determined. See, e.g., Dull, et al . , U.S. Patent No. 4,859,609, which is hereby incorporated herein by reference. Other gene fusion partners include glutathione-S-transferase (GST) , bacterial β- galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor. See, e.g., Godowski, et al . (1988) Science
241:812-816. Labeled proteins will often be substituted in the described combinations of proteins .
The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNA fragments. A double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
Such polypeptides may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups. In some embodiments, the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands. Fusion proteins will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods. Techniques for nucleic acid manipulation and expression are described generally, for example, in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed. ) , Vols. 1-3, Cold Spring Harbor Laboratory, and Ausubel, et al . (eds. 1987 and periodic supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York, which are each incorporated herein by reference. Techniques for synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc . 85:2149-2156; Merrifield (1986) Science 232: 341-347; and Atherton, et al . (1989) Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford; each of which is incorporated herein by reference. See also Dawson, et al. (1994) Science 266:776-779 for methods to make larger polypeptides.
This invention also contemplates the use of derivatives of a DCRSl other than variations in amino acid sequence or glycosylation. Such derivatives may involve covalent or aggregative association with chemical moieties. These derivatives generally fall into three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes, for example with cell membranes. Such covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of a receptor or other binding molecule, e.g., an antibody. For example, a cytokine ligand can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated Sepharose, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of a cytokine receptor, antibodies, or other similar molecules. The ligand can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.
A combination, e.g., including a DCRSl, of this invention can be used as an immunogen for the production of antisera or antibodies specific, e.g., capable of distinguishing between other cytokine receptor family members, for the combinations described. The complexes can be used to screen monoclonal antibodies or antigen- binding fragments prepared by immunization with various forms of impure preparations containing the protein. In particular, the term "antibodies" also encompasses antigen binding fragments of natural antibodies, e.g., Fab, Fab2 , Fv, etc. The purified DCRSl can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of expression, or immunological disorders which lead to antibody production to the endogenous receptor. Additionally, DCRSl fragments may also serve as immunogens to produce the antibodies of the present invention, as described immediately below. For example, this invention contemplates antibodies having binding affinity to or being raised against the amino acid sequences shown in Table 1, fragments thereof, or various homologous peptides. In particular, this invention contemplates antibodies having binding affinity to, or having been raised against, specific fragments which are predicted to be, or actually are, exposed at the exterior protein surface of the native DCRSl. Complexes of combinations of proteins will also be useful, and antibody preparations thereto can be made.
The blocking of physiological response to the receptor ligands may result from the inhibition of binding of the ligand to the receptor, likely through competitive inhibition. Thus, in vitro assays of the present invention will often use antibodies or antigen binding segments of these antibodies, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either ligand binding region mutations and modifications, or other mutations and modifications, e.g., which affect signaling or enzymatic function. This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to the receptor complexes or fragments compete with a test compound for binding to a ligand or other antibody. In this manner, the neutralizing antibodies or fragments can be used to detect the presence of a polypeptide which shares one or more binding sites to a receptor and can also be used to occupy binding sites on a receptor that might otherwise bind a ligand.
V. Making Nucleic Acids and Protein
DNA which encodes the protein or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples . Natural sequences can be isolated using standard methods and the sequences provided herein, e.g., in Table 1. Other species counterparts can be identified by hybridization techniques, or by various PCR techniques, combined with or by searching in sequence databases, e.g., GenBank.
This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length receptor or fragments which can in turn, for example, be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified ligand binding or kinase/phosphatase domains; and for structure/function studies. Variants or fragments can be expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules can be substantially free of protein or cellular contaminants, other than those derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a 49
pharmaceutically acceptable carrier and/or diluent. The protein, or portions thereof, may be expressed as fusions with other proteins. Combinations of the described proteins, or nucleic acids encoding them, are particularly interesting.
Expression vectors are typically self-replicating DNA or RNA constructs containing the desired receptor gene or its fragments, usually operably linked to suitable genetic control elements that are recognized in a suitable host cell. These control elements are capable of effecting expression within a suitable host. The multiple genes may be coordinately expressed, and may be on a polycistronic message. The specific type of control elements necessary to effect expression will depend upon the eventual host cell used. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation. Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell.
The vectors of this invention include those which contain DNA which encodes a combination of proteins, as described, or a biologically active equivalent polypeptide. The DNA can be under the control of a viral promoter and can encode a selection marker. This invention further contemplates use of such expression vectors which are capable of expressing eukaryotic cDNAs coding for such proteins in a prokaryotic or eukaryotic host, where the vector is compatible with the host and where the eukaryotic cDNAs are inserted into the vector such that growth of the host containing the vector expresses the cDNAs in question. Usually, expression 50
vectors are designed for stable replication in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell, e.g., it is possible to effect transient expression of the protein or its fragments in various hosts using vectors that do not contain a replication origin that is recognized by the host cell. It is also possible to use vectors that cause integration of the protein encoding portions into the host DNA by recombination .
Vectors, as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host. Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels, et al . (1985 and Supplements) Cloning Vectors : A Laboratory Manual , Elsevier, N.Y. , and Rodriguez, et al . (eds. 1988) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, which are incorporated herein by reference.
Transformed cells are cells, preferably mammalian, that have been transformed or transfected with vectors constructed using recombinant DNA techniques . Transformed host cells usually express the desired proteins, but for purposes of cloning, amplifying, and manipulating its DNA, do not need to express the subject proteins . This invention further contemplates culturing transformed cells in a nutrient medium, thus permitting the proteins to accumulate. The proteins can be recovered, either from the culture or, in certain instances, from the culture medium. 51
For purposes of this invention, nucleic sequences are operably linked when they are functionally related to each other. For example, DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. Usually, operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression.
Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes . Prokaryotes include both gram negative and gram positive organisms, e.g., E. coli and B. subtilis. Lower eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and species of the genus Dictvosteliu . Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents. Prokaryotic host-vector systems include a wide variety of vectors for many different species. As used herein, E. coli and its vectors will be used generically to include equivalent vectors used in other prokaryotes . A representative vector for amplifying DNA is pBR322 or many of its derivatives. Vectors that can be used to express the receptor or its fragments include, but are not limited to, such vectors as those containing the lac promoter (pUC-series) ; trp promoter (pBR322-trp) ; Ipp promoter (the pIN-series) ; lambda-pP or pR promoters (pOTS) ; or hybrid promoters such as ptac (pDR540) . See Brosius, et al. (1988) "Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters", in Vectors : A Survey of Molecular Cloning Vectors and Their 52
Uses, (eds. Rodriguez and Denhardt) , Buttersworth, Boston, Chapter 10, pp. 205-236, which is incorporated herein by reference.
Lower eukaryotes, e.g., yeasts and Dictvostelium, may be transformed with DCRSl sequence containing vectors. For purposes of this invention, the most common lower eukaryotic host is the baker's yeast, Saccharomvces cerevisiae. It will be used to generically represent lower eukaryotes although a number of other strains and species are also available. Yeast vectors typically consist of a replication origin (unless of the integrating type) , a selection gene, a promoter, DNA encoding the receptor or its fragments, and sequences for translation termination, polyadenylation, and transcription termination. Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or such inducible promoters as the alcohol dehydrogenase 2 promoter or metallothionine promoter. Suitable vectors include derivatives of the following types: self-replicating low copy number (such as the YRp-series) , self-replicating high copy number (such as the YEp-series) ; integrating types (such as the Yip-series) , or mini-chromosomes (such as the YCp-series) .
Higher eukaryotic tissue culture cells are normally the preferred host cells for expression of the functionally active interleukin or receptor proteins . In principle, many higher eukaryotic tissue culture cell lines are workable, e.g., insect baculovirus expression systems, whether from an invertebrate or vertebrate source. However, mammalian cells are preferred. Transformation or transfection and propagation of such cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS) cell lines. Expression vectors for such cell lines usually 53
include an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used) , a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, e.g., from such sources as from adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus . Representative examples of suitable expression vectors include pCDNAl; pCD, see Okayama, et al . (1985) Mol. Cell Biol. 5:1136-1142; pMClneo PolyA, see Thomas, et al . (1987) Cell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
For secreted proteins and some membrane proteins, an open reading frame usually encodes a polypeptide that consists of a mature or secreted product covalently linked at its N-terminus to a signal peptide. The signal peptide is cleaved prior to secretion of the mature, or active, polypeptide. The cleavage site can be predicted with a high degree of accuracy from empirical rules, e.g., von-Heijne (1986) Nucleic Acids Research 14:4683- 4690 and Nielsen, et al . (1997) Protein Eng. 10:1-12, and the precise amino acid composition of the signal peptide often does not appear to be critical to its function, e.g., Randall, et al . (1989) Science 243:1156-1159;
Kaiser et al . (1987) Science 235:312-317. The mature proteins of the invention can be readily determined using standard methods .
It will often be desired to express these polypeptides in a system which provides a specific or defined glycosylation pattern. In this case, the usual pattern will be that provided naturally by the expression system. However, the pattern will be modifiable by exposing the polypeptide, e.g., an unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system. For example, the receptor gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes . 54
Using this approach, certain mammalian glycosylation patterns will be achievable in prokaryote or other cells. Expression in prokaryote cells will typically lead to unglycosylated forms of protein. The source of DCRSl can be a eukaryotic or prokaryotic host expressing recombinant DCRSl, such as is described above. The source can also be a cell line such as mouse Swiss 3T3 fibroblasts, but other mammalian cell lines are also contemplated by this invention, with the preferred cell line being from the human species.
Now that the sequences are known, the primate DCRSl, fragments, or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis, Springer-Verlag, New York; and Bodanszky (1984) The Principles of Peptide Synthesis, Springer-Verlag, New York; all of each which are incorporated herein by reference. For example, an azide process, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester) , a carbodiimidazole process, an oxidative-reductive process, or a dicyclohexylcarbodiimide (DCCD) /additive process can be used. Solid phase and solution phase syntheses are both applicable to the foregoing processes . Similar techniques can be used with partial DCRSl sequences.
The DCRSl proteins, fragments, or derivatives are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid. Amino groups that are not being bb
used in the coupling reaction typically must be protected to prevent coupling at an incorrect location.
If a solid phase synthesis is adopted, the C-terminal amino acid is bound to an insoluble carrier or support through its carboxyl group. The insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group. Examples of such insoluble carriers include halomethyl resins, such as chloromethyl resin or bromomethyl resin, hydroxymethyl resins, phenol resins, tert-alkyloxycarbonylhydrazidated resins, and the like.
An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously formed peptide or chain, to synthesize the peptide step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the peptide. This solid-phase approach is generally described by Merrifield, et al . (1963) in CL. Am. Chem. Soc . 85:2149-2156, which is incorporated herein by reference.
The prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, precipitation, electrophoresis, various forms of chromatography, and the like. The receptors of this invention can be obtained in varying degrees of purity depending upon desired uses. Purification can be accomplished by use of the protein purification techniques disclosed herein, see below, or by the use of the antibodies herein described in methods of immunoabsorbant affinity chromatography. This immunoabsorbant affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate cells, lysates of other cells expressing the receptor, or lysates or supernatants of cells bo
producing the protein as a result of DNA techniques, see below.
Generally, the purified protein will be at least about 40% pure, ordinarily at least about 50% pure, usually at least about 60% pure, typically at least about 70% pure, more typically at least about 80% pure, preferable at least about 90% pure and more preferably at least about 95% pure, and in particular embodiments, 97%- 99% or more. Purity will usually be on a weight basis, but can also be on a molar basis. Different assays will be applied as appropriate . Individual proteins may be purified and thereafter combined.
VI. Antibodies Antibodies can be raised to the various mammalian, e.g., primate DCRSl proteins and fragments thereof, both in naturally occurring native forms and in their recombinant forms, the difference being that antibodies to the active receptor are more likely to recognize epitopes which are only present in the native conformations. Denatured antigen detection can also be useful in, e.g., Western analysis. Anti-idiotypic antibodies are also contemplated, which would be useful as agonists or antagonists of a natural receptor or an antibody.
Antibodies, including binding fragments and single chain versions, against predetermined fragments of the protein can be raised by immunization of animals with conjugates of the fragments with immunogenic proteins. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective protein, or screened for agonistic or antagonistic activity. These monoclonal antibodies will usually bind with at least a K- of about 1 mM, more usually at least about 300 μM, typically at least about lOOμM, more typically at least about 30 μM, preferably at least about 10 μM, and more preferably at least about 3 μM or better. b /
The antibodies, including antigen binding fragments, of this invention can have significant diagnostic or therapeutic value. They can be potent antagonists that bind to the receptor and inhibit binding to ligand or inhibit the ability of the receptor to elicit a biological response, e.g., act on its substrate. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides to bind producing cells, or cells localized to the source of the interleukin. Further, these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker.
The antibodies of this invention can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they might bind to the receptor without inhibiting ligand or substrate binding. As neutralizing antibodies, they can be useful in competitive binding assays. They will also be useful in detecting or quantifying ligand. They may be used as reagents for Western blot analysis, or for immunoprecipitation or immunopurification of the respective protein. Likewise, nucleic acids and proteins may be immobilized to solid substrates for affinity purification or detection methods. The substrates may be, e.g., solid resin beads or sheets of plastic.
Protein fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens. Mammalian cytokine receptors and fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Seroloqical Reactions, Dover Publications, New York; and Williams, et al . (1967) Methods in Immunology and
Immunochemistry, Vol. 1, Academic Press, New York; each of which are incorporated herein by reference, for descriptions of methods of preparing polyclonal antisera. A typical method involves hyperimmunization of an animal with an antigen. The blood of the animal is then collected shortly after the repeated immunizations and the gamma globulin is isolated. In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., S ites, et al . (eds.) Basic and Clinical Immunology (4th ed. ) , Lange Medical
Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies : A Laboratory Manual , CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and particularly in Kohler and Milstein (1975) in Nature 256: 495-497, which discusses one method of generating monoclonal antibodies. Each of these references is incorporated herein by reference. Summarized briefly, this method involves injecting an animal with an immunogen. The animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See, Huse, et al . (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda, " Science 246:1275-1281; and Ward, et al . (1989) Nature 341:544- 546, each of which is hereby incorporated herein by reference . The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal . A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant or chimeric immunoglobulins may be produced, see Cabilly, U.S. Patent No. 4,816,567; or made in transgenic mice, see Mendez, et al. (1997) Nature Genetics 15:146-156. These references are incorporated herein by reference. The antibodies of this invention can also be used for affinity chromatography in isolating the DCRSl proteins or peptides . Columns can be prepared where the antibodies are linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate may be passed through the column, the column washed, followed by increasing concentrations of a mild denaturant, whereby the purified protein will be released. Alternatively, the protein may be used to purify antibody. Appropriate cross absorptions or depletions may be applied.
The antibodies may also be used to screen expression libraries for particular expression products. Usually the antibodies used in such a procedure will be labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
Antibodies raised against a cytokine receptor will also be used to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various 60
immunological conditions related to expression of the protein or cells which express the protein. They also will be useful as agonists or antagonists of the ligand, which may be competitive inhibitors or substitutes for naturally occurring ligands.
A cytokine receptor protein that specifically binds to or that is specifically immunoreactive with an antibody generated against a defined immunogen, such as an immunogen consisting of the amino acid sequence of SEQ ID NO: 13, is typically determined in an immunoassay. The immunoassay typically uses a polyclonal antiserum which was raised, e.g., to a protein of SEQ ID NO: 13. This antiserum is selected to have low crossreactivity against other cytokine receptor family members, e.g., IL- 12 receptor beta or gpl30, preferably from the same species, and any such crossreactivity is removed by immunoabsorption prior to use in the immunoassay.
In order to produce antisera for use in an immunoassay, the protein, e.g., of SEQ ID NO: 13, is isolated as described herein. For example, recombinant protein may be produced in a mammalian cell line. An appropriate host, e.g. , an inbred strain of mice such as Balb/c, is immunized with the selected protein, typically using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and
Lane, supra) . Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, e.g., a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 10^ or greater are selected and tested for their cross reactivity against other cytokine receptor family members, e.g., IL-12 receptor beta and/or gpl30, using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573. Preferably at least two cytokine receptor family members are used in this determination. These cytokine receptor family members can be produced as recombinant proteins and isolated using standard molecular biology and protein chemistry techniques as described herein. Immunoassays in the competitive binding format can be used for the crossreactivity determinations. For example, the protein of SEQ ID NO: 13 can be immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the proteins of IL-12 receptor beta or gpl30. The percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorption with the above-listed proteins. The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein to the immunogen protein (e.g., the DCRSl like protein of SEQ ID NO: 13). In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than twice the amount of the protein of the selected protein or proteins that is required, then the second protein is said to specifically bind to an antibody generated to the immunogen.
It is understood that these cytokine receptor proteins are members of a family of homologous proteins that comprise at least 6 so far identified genes. For a particular gene product, such as the DCRSl, the term refers not only to the amino acid sequences disclosed herein, but also to other proteins that are allelic, non- allelic, or species variants. It is also understood that the terms include nonnatural mutations introduced by deliberate mutation using conventional recombinant technology such as single site mutation, or by excising short sections of DNA encoding the respective proteins, or by substituting new amino acids, or adding new amino acids. Such minor alterations typically will substantially maintain the immunoidentity of the original molecule and/or its biological activity. Thus, these alterations include proteins that are specifically immunoreactive with a designated naturally occurring DCRSl protein. The biological properties of the altered proteins can be determined by expressing the protein in an appropriate cell line and measuring the appropriate effect, e.g., upon transfected lymphocytes. Particular protein modifications considered minor would include conservative substitution of amino acids with similar chemical properties, as described above for the cytokine receptor family as a whole. By aligning a protein optimally with the protein of the cytokine receptors and by using the conventional immunoassays described herein to determine immunoidentity, one can determine the protein compositions of the invention.
While many of the descriptions above (and below) are directed to individual proteins, they may be applied to complexes, e.g., natural or functional, of proteins.
VII. Kits and quantitation
Both naturally occurring and recombinant forms of the cytokine receptor like molecules of this invention are particularly useful in kits and assay methods . For example, these methods would also be applied to screening for binding activity, e.g., ligands for these proteins. Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds per year. See, e.g., a BIOMEK automated workstation, Beckman Instruments, Palo Alto, California, and Fodor, et al . (1991) Science 251:767-773, which is incorporated herein by reference. The latter describes means for testing binding by a plurality of defined polymers synthesized on a solid substrate. The development of suitable assays to screen for a ligand or agonist/antagonist homologous proteins can be greatly facilitated by the availability of large amounts of purified, soluble cytokine receptors in an active state such as is provided by this invention.
Purified DCRSl can be coated directly onto plates for use in the aforementioned ligand screening techniques. However, non-neutralizing antibodies to these proteins can' be used as capture antibodies to immobilize the respective receptor on the solid phase, useful, e.g., in diagnostic uses. This invention also contemplates use of DCRSl, fragments thereof, peptides, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of the protein or its ligand. Alternatively, or additionally, antibodies against the molecules may be incorporated into the kits and methods. Typically the kit will have a compartment containing either a DCRSl peptide or gene segment or a reagent which recognizes one or the other. Typically, recognition reagents, in the case of peptide, would be a receptor or antibody, or in the case of a gene segment, would usually be a hybridization probe.
A preferred kit for determining the concentration of DCRSl in a sample would typically comprise a labeled compound, e.g., ligand or antibody, having known binding affinity for DCRSl, a source of DCRSl (naturally occurring or recombinant) as a positive control, and a means for separating the bound from free labeled compound, for example a solid phase for immobilizing the DCRSl in the test sample. Compartments containing reagents, and instructions, will normally be provided. Appropriate nucleic acid or protein containing kits are also provided. 64
Antibodies, including antigen binding fragments, specific for mammalian DCRSl or a peptide fragment, or receptor fragments are useful in diagnostic applications to detect the presence of elevated levels of ligand and/or its fragments. Diagnostic assays may be homogeneous (without a separation step between free reagent and antibody-antigen complex) or heterogeneous (with a separation step) . Various commercial assays exist, such as radioimmunoassay (RIA) , enzyme-linked immunosorbent assay (ELISA) , enzyme immunoassay (EIA) , enzyme-multiplied immunoassay technique (EMIT) , substrate-labeled fluorescent immunoassay (SLFIA) and the like. For example, unlabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the antibody to a cytokine receptor or to a particular fragment thereof. These assays have also been extensively discussed in the literature. See, e.g., Harlow and Lane (1988) Antibodies : A Laboratory Manual , CSH. , and Coligan (ed. 1991 and periodic supplements) Current Protocols In Immunology Greene/Wiley, New York. Anti-idiotypic antibodies may have similar use to serve as agonists or antagonists of cytokine receptors. These should be useful as therapeutic reagents under appropriate circumstances. Frequently, the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay. For the subject invention, depending upon the nature of the assay, the protocol, and the label, either labeled or unlabeled antibody, or labeled ligand is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like. Preferably, the kit will also contain instructions for proper use and disposal of the contents after use. Typically the kit has compartments for each useful reagent, and will contain instructions for proper use and disposal of reagents. Desirably, the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium having appropriate concentrations for performing the assay.
The aforementioned constituents of the diagnostic assays may be used without modification or may be modified in a variety of ways. For example, labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal. In many of these assays, a test compound, cytokine receptor, or antibodies thereto can be labeled either directly or indirectly. Possibilities for direct labeling include label groups: radiolabels such as 125χ _ enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization. Both of the patents are incorporated herein by reference. Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups .
There are also numerous methods of separating the bound from the free ligand, or alternatively the bound from the free test compound. The cytokine receptor can be immobilized on various matrixes followed by washing.
Suitable matrices include plastic such as an ELISA plate, filters, and beads. Methods of immobilizing the receptor to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling, and biotin-avidin. The last step in this approach involves the precipitation of antibody/antigen complex by any of several methods including those utilizing, e.g., an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate. Other suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al . (1984) Clin. Chem. 30 (9) : 1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678, each of which is incorporated herein by reference.
The methods for linking protein or fragments to various labels have been extensively reported in the literature and do not require detailed discussion here. Many of the techniques involve the use of activated carboxyl groups either through the use of carbodiimide or active esters to form peptide bonds, the formation of thioethers by reaction of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin such as maleimide, for linkage, or the like. Fusion proteins will also find use in these applications . Another diagnostic aspect of this invention involves use of oUgonucleotide or polynucleotide sequences taken from the sequence of an cytokine receptor. These sequences can be used as probes for detecting levels of the respective cytokine receptor in patients suspected of having an immunological disorder. The preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature. Normally an oUgonucleotide probe should have at least about 14 nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes may be up to several kilobases. Various labels may be employed, most commonly radionuclides, particularly 32p# However, other techniques may also be employed, such as using biotin modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies may be employed which can recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes. The antibodies in turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected. The use of probes to the novel anti-sense RNA may be carried out in conventional techniques such as nucleic acid hybridization, plus and minus screening, recombinational probing, hybrid released translation (HRT) , and hybrid arrested translation (HART) . This also includes amplification techniques such as polymerase chain reaction (PCR) .
Diagnostic kits which also test for the qualitative or quantitative presence of other markers are also contemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers. Thus, kits may test for combinations of markers. See, e.g., Viallet, et al . (1989) Progress in Growth Factor Res. 1:89-97.
VIII. Therapeutic Utility
This invention provides reagents with significant therapeutic value. See, e.g., Levitzki (1996) Curr . Qpin. Cell Biol. 8:239-244. The cytokine receptors
(naturally occurring or recombinant) , fragments thereof, mutein receptors, and antibodies, along with compounds identified as having binding affinity to the receptors or antibodies, should be useful in the treatment of conditions exhibiting abnormal expression of the receptors of their ligands . Such abnormality will typically be manifested by immunological disorders. Additionally, this invention should provide therapeutic value in various diseases or disorders associated with abnormal expression or abnormal triggering of response to the ligand. For example, the IL-1 ligands have been suggested to be involved in morphologic development, e.g., dorso-ventral polarity determination, and immune responses, particularly the primitive innate responses. See, e.g., Sun, et al . (1991) Eur. J. Biochem. 196:247- 254; and Hultmark (1994) Nature 367:116-117.
Recombinant cytokine receptors, muteins, agonist or antagonist antibodies thereto, or antibodies can be 68
purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, along with physiologically innocuous stabilizers and excipients. These combinations can be sterile, e.g., filtered, and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations . This invention also contemplates use of antibodies or binding fragments thereof which are not complement binding.
Ligand screening using cytokine receptor or fragments thereof can be performed to identify molecules having binding affinity to the receptors. Subsequent biological assays can then be utilized to determine if a putative ligand can provide competitive binding, which can block intrinsic stimulating activity. Receptor fragments can be used as a blocker or antagonist in that it blocks the activity of ligand. Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of ligand, e.g., inducing signaling. This invention further contemplates the therapeutic use of antibodies to cytokine receptors as antagonists. The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, reagent physiological life, pharmacological life, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman, et al . (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics , 8th Ed. , Pergamon 69
Press; and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn.; each of which is hereby incorporated herein by reference. Methods for administration are discussed therein and below, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others. Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co. , Rahway, New Jersey. Because of the likely high affinity binding, or turnover numbers, between a putative ligand and its receptors, low dosages of these reagents would be initially expected to be effective. And the signaling pathway suggests extremely low amounts of ligand may have effect. Thus, dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 μM concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar) , and most preferably less than about 1 fM (femtomolar) , with an appropriate carrier. Slow release formulations, or slow release apparatus will often be utilized for continuous administration.
Cytokine receptors, fragments thereof, and antibodies or its fragments, antagonists, and agonists, may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration. Therapeutic formulations may be administered in many conventional dosage formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral , rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. See, e.g., Gilman, et al . (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington ' s Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn.; Avis, et al. (eds. 1993) Pharmaceutical Dosage
Forms : Parenteral Medications Dekker, NY; Lieberman, et al . (eds. 1990) Pharmaceutical Dosage Forms : Tablets Dekker, NY; and Lieberman, et al . (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker , NY. The therapy of this invention may be combined with or used in association with other therapeutic agents, particularly agonists or antagonists of other cytokine receptor family members.
IX. Screening
Drug screening using DCRSl or fragments thereof can be performed to identify compounds having binding affinity to the receptor subunit, including isolation of associated components . Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in that it blocks the activity of the ligand. Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of a cytokine ligand. This invention further contemplates the therapeutic use of antibodies to the receptor as cytokine agonists or antagonists.
Similarly, complexes comprising multiple proteins may be used to screen for ligands or reagents capable of recognizing the complex. Most cytokine receptors comprise at least two subunits, which may be the same, or distinct. Alternatively, the transmembrane receptor may 71
bind to a complex comprising a cytokine-like ligand associated with another soluble protein serving, e.g., as a second receptor subunit. In such a case, the DCRSl may bind to a complex of the IL-B30 with the DSRSl. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing the DCRSl in combination with the DSRSl. Cells may be isolated which express a receptor in isolation from other functional receptors. Such cells, either in viable or fixed form, can be used for standard antibody/antigen or ligand/receptor binding assays. See also, Parce, et al . (1989) Science 246:243-247; and Owicki, et al . (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitive methods to detect cellular responses.
Competitive assays are particularly useful, where the cells (source of putative ligand) are contacted and incubated with a labeled receptor or antibody having known binding affinity to the ligand, such as 125 _ antibody, and a test sample whose binding affinity to the binding composition is being measured. The bound and free labeled binding compositions are then separated to assess the degree of ligand binding. The amount of test compound bound is inversely proportional to the amount of labeled receptor binding to the known source. Many techniques can be used to separate bound from free ligand to assess the degree of ligand binding. This separation step could typically involve a procedure such as adhesion to filters followed by washing, adhesion to plastic followed by washing, or centrifugation of the cell membranes . Viable cells could also be used to screen for the effects of drugs on cytokine mediated functions, e.g., second messenger levels, i.e., Ca++; cell proliferation; inositol phosphate pool changes; and others. Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system. Calcium sensitive dyes will be useful for detecting Ca++ levels, with a fluorimeter or a fluorescence cell sorting apparatus.
X. Ligands The descriptions of the DCRSl herein provide means to identify ligands, as described above. Such ligand should bind specifically to the respective receptor with reasonably high affinity. Various constructs are made available which allow either labeling of the receptor to detect its ligand. For example, directly labeling cytokine receptor, fusing onto it markers for secondary labeling, e.g., FLAG or other epitope tags, etc., will allow detection of receptor. This can be histological, as an affinity method for biochemical purification, or labeling or selection in an expression cloning approach. A two-hybrid selection system may also be applied making appropriate constructs with the available cytokine receptor sequences. See, e.g., Fields and Song (1989) Nature 340:245-246. Generally, descriptions of cytokine receptors will be analogously applicable to individual specific embodiments directed to DCRSl reagents and compositions. Alternatively, the DCRSl might bind to a soluble complex of the DSRSl with another ligand. Thus, expression cloning of a cotransfectant of a library with the DSRSl may express combinations of the DSRSl with cytokine-like ligand, to form the soluble complex, which binds to the DCRSl .
The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific embodiments . EXAMPLES
I . General Methods
Some of the standard methods are described or referenced, e.g., in Maniatis, et al . (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual, (2d ed. ) , vols. 1-3, CSH Press, NY; Ausubel, et al . , Biology, Greene Publishing Associates, Brooklyn, NY; or Ausubel, et al. (1987 and Supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York. Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel, et al . (1987 and periodic supplements); Coligan, et al . (ed. 1996) and periodic supplements, Current Protocols In Protein Science
Greene/Wiley, New York; Deutscher (1990) "Guide to Protein Purification" in Methods in Enzymology, vol. 182, and other volumes in this series; and manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA. Combination with recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence. See, e.g., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed. ) Genetic Engineering, Principle and Methods 12:87-98,
Plenum Press, N.Y. ; and Crowe, et al . (1992) OIAexpress : The High Level Expression & Protein Purification System QUIAGEN, Inc., Chatsworth, CA.
Computer sequence analysis is performed, e.g., using available software programs, including those from the GCG (U. Wisconsin) and GenBank sources. Public sequence databases were also used, e.g., from GenBank and others. Many techniques applicable to IL-10 or IL-12 receptors may be applied to the DCRSl, as described, e.g., in USSN 08/110,683 (IL-10 receptor), which is incorporated herein by reference.
II . Computational Analysis
Human sequences related to cytokine receptors were identified from genomic sequence database using, e.g., the BLAST server (Altschul, et al . (1994) Nature Genet. 6:119-129) . Standard analysis programs may be used to evaluate structure, e.g., PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (King and Sternberg (1996) Protein Sci. 5:2298-2310). Standard comparison software includes, e.g., Altschul, et al . (1990) J. Mol. Biol. 215:403-10; Waterman (1995) Introduction to Computational Biology: Maps, Sequences, and Genomes Chapman & Hall; Lander and Waterman (eds. 1995) Calculating the Secrets of Life: Applications of the Mathematical Sciences in Molecular Biology National Academy Press; and Speed and Waterman (eds. 1996) Genetic Mapping and DNA Sequencing
(Ima Volumes in Mathematics and Its Applications, Vol 81) Springer Verlag.
III. Cloning of full-length DCRSl cDNAs; Chromosomal localization
PCR primers derived from the DCRSl sequence are used to probe a human cDNA library. Sequences may be derived, e.g., from Table 1, preferably those adjacent the ends of incomplete sequences. Full length cDNAs for primate, rodent, or other species DCRSl are cloned, e.g., by DNA hybridization screening of λgtlO phage. PCR reactions are conducted using T. aquaticus Taqplus DNA polymerase (Stratagene) under appropriate conditions.
Chromosome spreads are prepared. In situ hybridization is performed on chromosome preparations obtained from phytohemagglutinin-stimulated human lymphocytes cultured for 72 h. 5-bromodeoxyuridine was added for the final seven hours of culture (60 μg/ml of 75
medium) , to ensure a posthybridization chromosomal banding of good quality.
A PCR fragment, amplified with the help of primers, is cloned into an appropriate vector. The vector is labeled by nick-translation with ^H. The radiolabeled probe is hybridized to metaphase spreads at final concentration of 200 ng/ml of hybridization solution as described in Mattei, et al . (1985) Hum. Genet. 69:327- 331. After coating with nuclear track emulsion (KODAK NTB2), slides are exposed. To avoid any slipping of silver grains during the banding procedure, chromosome spreads are first stained with buffered Giemsa solution and metaphase photographed. R-banding is then performed by the fluorochrome-photolysis-Giemsa (FPG) method and metaphases rephotographed before analysis.
Similar appropriate methods are used for other species .
IV. Localization of DCRSl mRNA
Human multiple tissue (Cat# 1, 2) and cancer cell line blots (Cat# 7757-1) , containing approximately 2 μg of poly (A) + RNA per lane, are purchased from Clontech (Palo Alto, CA) . Probes are radiolabeled with [α-32p] dATP, e.g., using the Amersham Rediprime random primer labeling kit (RPN1633) . Prehybridization and hybridizations are performed at 65° C in 0.5 M N 2HP04,
7% SDS, 0.5 M EDTA (pH 8.0). High stringency washes are conducted, e.g., at 65° C with two initial washes in 2 x SSC, 0.1% SDS for 40 min followed by a subsequent wash in 0.1 x SSC, 0.1% SDS for 20 min. Membranes are then exposed at -70° C to X-Ray film (Kodak) in the presence of intensifying screens. More detailed studies by cDNA library Southerns are performed with selected human DCRSl clones to examine their expression in hemopoietic or other cell subsets .
Alternatively, two appropriate primers are selected from Table 1. RT-PCR is used on an appropriate mRNA 76
sample selected for the presence of message to produce a cDNA, e.g., a sample which expresses the gene.
Full length clones may be isolated by hybridization of cDNA libraries from appropriate tissues pre-selected by PCR signal. Northern blots can be performed.
Message for genes encoding DCRSl will be assayed by appropriate technology, e.g., PCR, immunoassay, hybridization, or otherwise. Tissue and organ cDNA preparations are available, e.g., from Clontech, Mountain View, CA. Identification of sources of natural expression are useful, as described. And the identification of functional receptor subunit pairings will allow for prediction of what cells express the combination of receptor subunits which will result in a physiological responsiveness to each of the cytokine ligands .
For mouse distribution, e.g., Southern Analysis can be performed: DNA (5 μg) from a primary amplified cDNA library was digested with appropriate restriction enzymes to release the inserts, run on a 1% agarose gel and transferred to a nylon membrane (Schleicher and Schuell, Keene, NH) . Samples for mouse mRNA isolation may include: resting mouse fibroblastic L cell line (C200) ; Braf :ER
(Braf fusion to estrogen receptor) transfected cells, control (C201) ; T cells, THl polarized (Mell4 bright,
CD4+ cells from spleen, polarized for 7 days with IFN-γ and anti IL-4; T200) ; T cells, TH2 polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IL-4 and anti-IFN-γ; T201) ; T cells, highly THl polarized (see Openshaw, et al . (1995) J. Exp. Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 16 h pooled; T202); T cells, highly TH2 polarized (see Openshaw, et al . (1995) J. EXP. Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 16 h pooled; T203); CD44- CD25+ pre T cells, sorted from thymus (T204) ; THl T cell clone Dl.l, resting for 3 weeks after last stimulation with antigen (T205) ; THl T cell clone Dl.l, 10 μg/ml ConA stimulated 15 h (T206) ;
TH2 T cell clone CDC35, resting for 3 weeks after last stimulation with antigen (T207) ; TH2 T cell clone CDC35, 10 μg/ml ConA stimulated 15 h (T208) ; Mell4+ naive T cells from spleen, resting (T209) ; Mell4+ T cells, polarized to Thl with IFN-γ/IL-12/anti-IL-4 for 6, 12, 24 h pooled (T210); Mell4+ T cells, polarized to Th2 with IL-4/anti-IFN-γ for 6, 13, 24 h pooled (T211) ; unstimulated mature B cell leukemia cell line A20 (B200) ; unstimulated B cell line CH12 (B201) ; unstimulated large B cells from spleen (B202); B cells from total spleen, LPS activated (B203); metrizamide enriched dendritic cells from spleen, resting (D200) ; dendritic cells from bone marrow, resting (D201) ; monocyte cell line RAW 264.7 activated with LPS 4 h (M200) ; bone-marrow macrophages derived with GM and M-CSF (M201) ; macrophage cell line J774, resting (M202); macrophage cell line J774 + LPS + anti-IL-10 at 0.5, 1, 3, 6, 12 h pooled (M203); macrophage cell line J774 + LPS + IL-10 at 0.5, 1, 3, 5, 12 h pooled (M204) ; aerosol challenged mouse lung tissue, Th2 primers, aerosol OVA challenge 7, 14, 23 h pooled (see Garlisi, et al . (1995) Clinical Immunology and
Immunopathology 75:75-83; X206) ; Nippostrongulus-infected lung tissue (see Coffman, et al . (1989) Science 245:308- 310; X200); total adult lung, normal (O200) ; total lung, rag-1 (see Schwarz, et al . (1993) Immunodeficiency 4:249- 252; O205); IL-10 K.O. spleen (see Kuhn, et al . (1991) Cell 75:263-274; X201) ; total adult spleen, normal (O201) ; total spleen, rag-1 (O207) ; IL-10 K.O. Peyer ' s patches (O202); total Peyer 's patches, normal (O210) ; IL- 10 K.O. mesenteric lymph nodes (X203); total mesenteric lymph nodes, normal (0211); IL-10 K.O. colon (X203); total colon, normal (0212); NOD mouse pancreas (see Makino, et al . (1980) Jikken Dobutsu 29:1-13; X205) ; total thymus, rag-1 (O208) ; total kidney, rag-1 (O209); total heart, rag-1 (O202); total brain, rag-1 (O203); total testes, rag-1 (O204) ; total liver, rag-1 (O206) ; rat normal joint tissue (0300) ; and rat arthritic joint tissue (X300) . 78
Samples for human mRNA isolation may include: peripheral blood mononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells), resting (T100) ; peripheral blood mononuclear cells, activated with anti- CD3 for 2, 6, 12 h pooled (TlOl) ; T cell, THO clone Mot 72, resting (T102); T cell, THO clone Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T103); T cell, THO clone Mot 72, anergic treated with specific peptide for 2, 7, 12 h pooled (T104) ; T cell, THl clone HY06, resting (T107); T cell, THl clone HY06, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T108) ; T cell, THl clone HY06, anergic treated with specific peptide for 2, 6, 12 h pooled (T109) ; T cell, TH2 clone HY935, resting (T110) ; T cell, TH2 clone HY935, activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (Till) ; T cells CD4+CD45RO- T cells polarized 27 days in anti- CD28, IL-4, and anti IFN-γ, TH2 polarized, activated with anti-CD3 and anti-CD28 4 h (T116) ; T cell tumor lines Jurkat and Hut78, resting (T117); T cell clones, pooled AD130.2, TC783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118) ; T cell random γδ T cell clones, resting (T119) ;
Splenocytes, resting (B100) ; Splenocytes, activated with anti-CD40 and IL-4 (B101) ; B cell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3 , HSY, resting (B102); B cell line JY, activated with PMA and ionomycin for 1, 6 h pooled (B103); NK 20 clones pooled, resting (K100) ; NK 20 clones pooled, activated with PMA and ionomycin for 6 h (K101) ; NKL clone, derived from peripheral blood of LGL leukemia patient, IL-2 treated (K106) ; NK cytotoxic clone 640-A30-1, resting (K107) ; hematopoietic precursor line TF1, activated with PMA and ionomycin for 1, 6 h pooled (C100) ; U937 premonocytic line, resting (M100) ; U937 premonocytic line, activated with PMA and ionomycin for 1, 6 h pooled (M101) ; elutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes, activated with LPS, IFNγ,
IL-10 for 1, 2, 6, 12, 24 h pooled (M103); elutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for 4, 16 h pooled (M106) ; elutriated monocytes, activated with LPS, IFNγ, IL-10 for 4, 16 h pooled (M107); elutriated monocytes, activated LPS for 1 h (M108) ; elutriated monocytes, activated LPS for 6 h (M109) ; DC 70% CDla+, from CD34+ GM-CSF, TNFα 12 days, resting (D101) ; DC 70% CDla+, from CD34+ GM-CSF, TNFα 12 days, activated with
PMA and ionomycin for 1 hr (D102); DC 70% CDla+, from CD34+ GM-CSF, TNFα 12 days, activated with PMA and ionomycin for 6 hr (D103); DC 95% CDla+, from CD34+ GM- CSF, TNFα 12 days FACS sorted, activated with PMA and ionomycin for 1, 6 h pooled (D104) ; DC 95% CD14+, ex CD34+ GM-CSF, TNFα 12 days FACS sorted, activated with
PMA and ionomycin 1, 6 hr pooled (D105) ; DC CDla+ CD86+, from CD34+ GM-CSF, TNFα 12 days FACS sorted, activated with PMA and ionomycin for 1, 6 h pooled (D106) ; DC from monocytes GM-CSF, IL-4 5 days, resting (D107) ; DC from monocytes GM-CSF, IL-4 5 days, resting (D108); DC from monocytes GM-CSF, IL-4 5 days, activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF, IL-4 5 days, activated TNFα, monocyte supe for 4, 16 h pooled (DUO) ; leiomyoma Lll benign tumor (X101) ; normal myometrium M5 (0115); malignant leiomyosarcoma GSl (X103); lung fibroblast sarcoma line MRC5, activated with PMA and ionomycin for 1, 6 h pooled (C101) ; kidney epithelial carcinoma cell line CHA, activated with PMA and ionomycin for 1, 6 h pooled (C102); kidney fetal 28 wk male (O100) ; lung fetal 28 wk male (O101) ; liver fetal 28 wk male (O102); heart fetal 28 wk male (O103); brain fetal 28 wk male (O104) ; gallbladder fetal 28 wk male (O106) ; small intestine fetal 28 wk male (O107) ; adipose tissue fetal 28 wk male (O108) ; ovary fetal 25 wk female (O109) ; uterus fetal 25 wk female (0110) ; testes fetal 28 wk male (0111); spleen fetal 28 wk male (0112); adult placenta 28 wk (0113) ; and tonsil inflamed, from 12 year old (X100) . Similar samples may isolated in other species for evaluation. V. Cloning of species counterparts of DCRSl
Various strategies are used to obtain species counterparts of the DCRSl, preferably from other primates or rodents. One method is by cross hybridization using closely related species DNA probes. It may be useful to go into evolutionarily similar species as intermediate steps. Another method is by using specific PCR primers based on the identification of blocks of similarity or difference between genes, e.g., areas of highly conserved or nonconserved polypeptide or nucleotide sequence.
VI . Production of mammalian DCRSl protein
An appropriate, e.g., GST, fusion construct is engineered for expression, e.g., in E. coli. For example, a mouse IGIF pGex plasmid is constructed and transformed into E. coli. Freshly transformed cells are grown, e.g., in LB medium containing 50 μg/ml ampicillin and induced with IPTG (Sigma, St. Louis, MO) . After overnight induction, the bacteria are harvested and the pellets containing the DCRSl protein are isolated. The pellets are homogenized, e.g., in TE buffer (50 mM Tris- base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in 2 liters. This material is passed through a microfluidizer (Microfluidics, Newton, MA) three times. The fluidized supernatant is spun down on a Sorvall GS-3 rotor for 1 h at 13,000 rpm. The resulting supernatant containing the cytokine receptor protein is filtered and passed over a glutathione-SEPHAROSE column equilibrated in 50 mM Tris- base pH 8.0. The fractions containing the DCRSl-GST fusion protein are pooled and cleaved, e.g., with thrombin (Enzyme Research Laboratories, Inc., South Bend, IN) . The cleaved pool is then passed over a Q-SEPHAROSE column equilibrated in 50 mM Tris-base. Fractions containing DCRSl are pooled and diluted in cold distilled H2O, to lower the conductivity, and passed back over a fresh Q-Sepharose column, alone or in succession with an immunoaffinity antibody column. Fractions containing the DCRSl protein are pooled, aliquoted, and stored in the - 70° C freezer.
Comparison of the CD spectrum with cytokine receptor protein may suggest that the protein is correctly folded. See Hazuda, et al . (1969) J. Biol. Chem. 264:1689-1693.
VII. Determining physiological forms of receptors
The cellular forms of receptors for ligands can be tested with the various ligands and receptor subunits provided. In particular, multiple cytokine receptor like ligands have been identified. The IL-B30 cytokine has been described. See above.
Cotransformation of the DCRSl with putative other receptor subunits may be performed. In particular, the DSRSl is suggested to be a second receptor subunit needed for functional receptor signaling. Such cells may be used to screen putative cytokine ligands, such as the IL-
B30, for signaling. A cell proliferation assay may be used. In fact, the DSRSl may combine with the IL-B30 to form a soluble cytokine-receptor subunit complex, which then binds to the DCRSl.
In addition, it has been known that many cytokine receptors function as heterodimers . The IL-lα and IL-lβ ligands bind an IL-1R1 as the primary receptor and this complex then forms a high affinity receptor complex with the IL-1R3. As indicated above, the sequence similarity to IL-12 receptor subunits suggests functional similarity of the functional receptor, e.g., a soluble alpha subunit, and transmembrane beta subunit. These subunit combinations can be tested now with the provided reagents. In particular, appropriate constructs can be made for transformation or transfection of subunits into cells. Constructs for the alpha chains, e.g., DSRSl forms , can be made . Likewise for the beta subunit DCRSl. Combinatorial transfections of transformations can make cells expressing defined subunits, which can be tested for response to the predicted ligands. Appropriate cell types can be used, e.g., 293 T cells, with, e.g., an NFKb reporter construct.
Biological assays will generally be directed to the ligand binding feature of the protein or to the kinase/phosphatase activity of the receptor. The activity will typically be reversible, as are many other enzyme reactions, and may mediate phosphatase or phosphorylase activities, which activities are easily measured by standard procedures. See, e.g., Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al . (1991) Meth. Enzvmol. 200:38-62; Hunter, et al . (1992) Cell 70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al . (1991) Cold Spring Harbor Sy p. Quant. Biol. 56:449-463; and Parker, et al . (1993) Nature 363:736-738.
The family of cytokines contains molecules which are important mediators of hematopoiesis or inflammatory disease. See, e.g., Thomson (ed. 1994) The Cytokine Handbook Academic Press, San Diego; and Dinarello (1996) Blood 87:2095-2147.
VIII. Preparation of antibodies specific for DCRSl
Inbred Balb/c mice are immunized intraperitoneally with recombinant forms of the protein, e.g., purified DCRSl or stable transfected NIH-3T3 cells. Animals are boosted at appropriate time points with protein, with or without additional adjuvant, to further stimulate antibody production. Serum is collected, or hybridomas produced with harvested spleens. Alternatively, Balb/c mice are immunized with cells transformed with the gene or fragments thereof, either endogenous or exogenous cells, or with isolated membranes enriched for expression of the antigen. Serum is collected at the appropriate time, typically after numerous further administrations. Various gene therapy techniques may be useful, e.g., in producing protein in situ, for generating an immune response. Serum or antibody preparations may be cross-absorbed or immunoselected to prepare substantially purified antibodies of defined specificity and high affinity.
Monoclonal antibodies may be made. For example, splenocytes are fused with an appropriate fusion partner and hybridomas are selected in growth medium by standard procedures . Hybridoma supernatants are screened for the presence of antibodies which bind to the DCRSl, e.g., by ELISA or other assay. Antibodies which specifically recognize specific DCRSl embodiments may also be selected or prepared.
In another method, synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g., Coligan (ed. 1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies : A Laboratory Manual Cold Spring Harbor Press . In appropriate situations, the binding reagent is either labeled as described above, e.g., fluorescence or otherwise, or immobilized to a substrate for panning methods. Nucleic acids may also be introduced into cells in an animal to produce the antigen, which serves to elicit an immune response. See, e.g., Wang, et al. (1993) Proc . Nat ' 1. Acad. Sci. 90:4156-4160; Barry, et al . (1994) BioTechnicrues 16:616-619; and Xiang, et al . (1995) Immunity 2: 129-135.
Moreover, antibodies which may be useful to determine the combination of the DCRSl with a functional alpha subunit may be generated. Thus, e.g., epitopes characteristic of a particular functional alpha/beta combination may be identified with appropriate antibodies .
IX. Production of fusion proteins with DCRSl
Various fusion constructs are made with DCRSl. A portion of the appropriate gene is fused to an epitope tag, e.g., a FLAG tag, or to a two hybrid system construct. See, e.g., Fields and Song (1989) Nature 340:245-246. The epitope tag may be used in an expression cloning procedure with detection with anti-FLAG antibodies to detect a binding partner, e.g., ligand for the respective cytokine receptor. The two hybrid system may also be used to isolate proteins which specifically bind to DCRSl .
X. Structure activity relationship
Information on the criticality of particular residues is determined using standard procedures and analysis. Standard mutagenesis analysis is performed, e.g., by generating many different variants at determined positions, e.g., at the positions identified above, and evaluating biological activities of the variants. This may be performed to the extent of determining positions which modify activity, or to focus on specific positions to determine the residues which can be substituted to either retain, block, or modulate biological activity. Alternatively, analysis of natural variants can indicate what positions tolerate natural mutations. This may result from populational analysis of variation among individuals, or across strains or species. Samples from selected individuals are analyzed, e.g., by PCR analysis and sequencing. This allows evaluation of population polymorphisms.
XI. Isolation of a ligand for DCRSl
A cytokine receptor can be used as a specific binding reagent to identify its binding partner, by taking advantage of its specificity of binding, much like an antibody would be used. The binding receptor may be a heterodimer of receptor subunits; or may involve, e.g., a complex of the DCRSl with DSRSl. A binding reagent is either labeled as described above, e.g., fluorescence or otherwise, or immobilized to a substrate for panning methods . The binding composition is used to screen an expression library made from a cell line which expresses a binding partner, i.e., ligand, preferably membrane associated. Standard staining techniques are used to detect or sort surface expressed ligand, or surface expressing transformed cells are screened by panning. Screening of intraceUular expression is performed by various staining or immunofluorescence procedures. See also McMahan, et al . (1991) EMBO J. 10:2821-2832. For example, on day 0, precoat 2-chamber permanox slides with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min at room temperature. Rinse once with PBS. Then plate COS cells at 2-3 x 105 cells per chamber in 1.5 ml of growth media. Incubate overnight at 37° C. On day 1 for each sample, prepare 0.5 ml of a solution of 66 μg/ml DEAE-dextran, 66 μM chloroquine, and 4 μg DNA in serum free DME. For each set, a positive control is prepared, e.g., of DCRS1-FLAG cDNA at 1 and 1/200 dilution, and a negative mock. Rinse cells with serum free DME. Add the DNA solution and incubate 5 hr at 37° C. Remove the medium and add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and wash once with DME. Add 1.5 ml growth medium and incubate overnight .
On day 2 , change the medium. On days 3 or 4 , the cells are fixed and stained. Rinse the cells twice with
Hank's Buffered Saline Solution (HBSS) and fix in 4% paraformaldehyde (PFA) /glucose for 5 min. Wash 3X with
HBSS. The slides may be stored at -80° C after all liquid is removed. For each chamber, 0.5 ml incubations are performed as follows. Add HBSS/saponin (0.1%) with 32 μl/ l of 1 M NaN3 for 20 min. Cells are then washed with HBSS/saponin IX. Add appropriate DCRSl or DCRSl/antibody complex to cells and incubate for 30 min. Wash cells twice with HBSS/saponin. If appropriate, add first antibody for 30 min. Add second antibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, and incubate for 30 min. Prepare ELISA solution, e.g., Vector Elite ABC horseradish peroxidase solution, and preincubate for 30 min. Use, e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solution and incubate for 30 min. Wash cells twice with HBSS, second wash for 2 min, which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5 to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2 drops of H2O2 Per 5 ml of glass distilled water.
Carefully remove chamber and rinse slide in water. Air dry for a few minutes, then add 1 drop of Crystal Mount and a cover slip. Bake for 5 min at 85-90° C.
Evaluate positive staining of pools and progressively subclone to isolation of single genes responsible for the binding. Alternatively, receptor reagents are used to affinity purify or sort out cells expressing a putative ligand. See, e.g., Sambrook, et al . or Ausubel, et al . Another strategy is to screen for a membrane bound receptor by panning. The receptor cDNA is constructed as described above. The ligand, e.g., either IL-B30 alone or a complex of IL-B30 with DSRSl, can be immobilized and used to immobilize expressing cells. Immobilization may be achieved by use of appropriate antibodies which recognize, e.g., a FLAG sequence of a DCRSl fusion construct, or by use of antibodies raised against the first antibodies . Recursive cycles of selection and amplification lead to enrichment of appropriate clones and eventual isolation of receptor expressing clones . Phage expression libraries can be screened by mammalian DCRSl. Appropriate label techniques, e.g., anti-FLAG antibodies, will allow specific labeling of appropriate clones . All citations herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled; and the invention is not to be limited by the specific embodiments that have been presented herein by way of example.

Claims

WHAT IS CLAIMED IS:
1. A composition comprising: a) both: i) a DSRSl protein; and ii) an isolated or recombinant DCRSl protein; b) both: i) an isolated or recombinant DSRSl protein; and ii) a DCRSl protein; or c) both: i) a substantially pure or recombinant IL-B30 protein; and ii) a DCRSl protein.
2. The composition of Claim 1, wherein: a) said DSRSl protein has sequence of mature SEQ ID
NO: 9 or 11; b) said DCRSl protein has sequence of mature SEQ ID NO: 13 or 15; or c) said IL-B30 has sequence of mature SEQ ID NO: 17 or 19; or d) at least one of said proteins : i) is unglycosylated; ii) is made with synthetic methods; iii) has a detectable label; iv) is attached to a solid substrate; or v) is conjugated to another chemical moiety.
3. A composition comprising: a) a substantially pure DCRSl protein and: i) a DSRSl protein; or ii) an IL-B30 cytokine protein; or b) a DCRSl protein and a substantially pure: i) DSRSl protein; or ii) IL-B30 cytokine protein.
4. The composition of Claim 3 comprising said DCRSl and said DSRSl proteins, wherein said proteins combine to bind IL-B30 with high affinity.
5. A sterile composition of Claim 3.
6. A kit comprising said proteins of Claim 3, and: a) a compartment comprising two or more of said proteins ; b) a compartment comprising a soluble receptor alpha subunit; c) a compartment comprising an IL-B30 cytokine protein; or d) instructions for use or disposal of reagents in said kit.
7. A binding composition comprising the antigen binding sites from antibodies, which antibodies bind to an epitope found on a composition of Claim 1, but not on separate proteins thereof.
8. The binding composition of Claim 7, wherein: a) said DCRSl is: i) a primate protein; ii) a purified human or mouse DCRSl; or iii) a mature polypeptide of Table 1; b) said DSRSl is: i) a primate protein; ii) a purified human or mouse DSRSl; or iii) a mature polypeptide of Table 4; or c) said IL-B30 is: i) a primate protein; ii) a purified human or mouse IL-B30; or iii) a mature polypeptide of Table 6.
9. The binding composition of Claim 7, wherein said binding composition: a) is in a container; b) is an Fv, Fab, or Fab2 fragment; c) is conjugated to another chemical moiety; d) is immunoselected; e) is a polyclonal antibody; f) exhibits a Kd to antigen of at least 30 ╬╝M; g) is attached to a solid substrate, including a bead or plastic membrane; h) is in a sterile composition; or i) is detectably labeled, including a radioactive or fluorescent label .
10. A kit comprising said binding composition of Claim 7, and: a) a compartment comprising said binding composition; b) a compartment comprising said DCRSl, DSRSl, or
IL-B30 protein; or c) instructions for use or disposal of reagents in said kit.
11. An isolated or recombinant nucleic acid encoding : a) both: i) a DSRSl protein; and ii) a DCRSl protein; b) both: i) a DSRSl protein; and ii) an IL-B30 protein; or c) both: i) a DCRSl protein; and ii) an IL-B30 protein.
12. The nucleic acid of Claim 11, which encodes both a DCRSl protein and a DSRSl protein.
13. The nucleic acid of Claim 11, which encodes both a DSRSl protein and an IL-B30.
14. The nucleic acid of Claim 11, which is an expression vector.
15. The nucleic acid of Claim 11, wherein: a) said DSRSl protein has sequence of mature SEQ ID
NO: 9 or 11; b) said DCRSl protein has sequence of mature SEQ ID NO: 13 or 15; or c) said IL-B30 has sequence of mature SEQ ID NO: 17 or 19.
16. The nucleic acid of Claim 11, comprising the coding portion of: a) SEQ ID NO 8 or 10; b) SEQ ID NO 12 or 14; or c) SEQ ID NO 16 or 18.
17. A cell comprising said recombinant nucleic acid of Claim 11.
18. The cell of Claim 17, wherein said cell is: a) a prokaryotic cell; b) a eukaryotic cell; c) a bacterial cell; d) a yeast cell; e) an insect cell; f) a mammalian cell; g) a mouse cell; h) a primate cell; or i) a human cell.
19. A method of producing a receptor complex, comprising culturing a cell of Claim 17 in an environment resulting in expression of said DCRSl and said DSRSl proteins, thereby forming said receptor complex.
20. A method of screening for ligands for a receptor complex comprising said DCRSl and said DSRSl proteins, comprising screening a library of compounds for binding to said cell of Claim 17.
PCT/US1999/002600 1998-02-06 1999-02-05 Mammalian receptor proteins; related reagents and methods WO1999040195A1 (en)

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