WO1999040195A1 - Proteines de recepteurs de mammiferes; reactifs et procedes apparentes - Google Patents

Proteines de recepteurs de mammiferes; reactifs et procedes apparentes Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
protein
leu
dcrsl
pro
gly
Prior art date
Application number
PCT/US1999/002600
Other languages
English (en)
Inventor
Jeanine D. Mattson
Terrill K. Mcclanahan
Robert A. Kastelein
Original Assignee
Schering Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Schering Corporation filed Critical Schering Corporation
Priority to AU26611/99A priority Critical patent/AU2661199A/en
Publication of WO1999040195A1 publication Critical patent/WO1999040195A1/fr

Links

Classifications

    • 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.

Abstract

L'invention concerne des acides nucléiques codant des récepteurs de mammifères, par exemple, de primates ou de rongeurs, des protéines de récepteurs purifiées et des fragments de ces dernières. L'invention traite aussi d'anticorps polyclonaux et monoclonaux. L'invention a aussi pour objet des procédés d'utilisation de compositions pour des applications tant diagnostiques que thérapeutiques.
PCT/US1999/002600 1998-02-06 1999-02-05 Proteines de recepteurs de mammiferes; reactifs et procedes apparentes WO1999040195A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU26611/99A AU2661199A (en) 1998-02-06 1999-02-05 Mammalian receptor proteins; related reagents and methods

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US7394198P 1998-02-06 1998-02-06
US60/073,941 1998-02-06
US7819498A 1998-05-13 1998-05-13
US09/078,194 1998-05-13

Publications (1)

Publication Number Publication Date
WO1999040195A1 true WO1999040195A1 (fr) 1999-08-12

Family

ID=26755077

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/002600 WO1999040195A1 (fr) 1998-02-06 1999-02-05 Proteines de recepteurs de mammiferes; reactifs et procedes apparentes

Country Status (2)

Country Link
AU (1) AU2661199A (fr)
WO (1) WO1999040195A1 (fr)

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053631A1 (fr) * 1999-03-11 2000-09-14 Schering Corporation Cytokines de mammifere : reactifs et procedes associes
EP1072610A1 (fr) * 1998-04-14 2001-01-31 Chugai Research Institute for Molecular Medicine Inc. Nouvelle proteine du type cytokine
WO2001036467A2 (fr) * 1999-11-18 2001-05-25 Schering Corporation Proteines de recepteurs mammaliens, reactifs et procedes s'y rapportant
WO2001055172A2 (fr) * 2000-01-27 2001-08-02 Pierre Fabre Medicament COMPLEXE ISOLE COMPRENANT UNE PROTEINE NNT-1 ET EN OUTRE AU MOINS UNE PROTEINE CLF-1 ET/OU UNE PROTEINE sCNTFR$g(a)
FR2804434A1 (fr) * 2000-01-27 2001-08-03 Pf Medicament Heterocomplexe nnt-1/clf-1 et son utilisation en tant qu'activateur du recepteur complexe cntrfalpha/gp130/lifrbet a
US6800460B1 (en) 1999-03-11 2004-10-05 Schering Corporation Mammalian cytokine complexes
EP1522858A1 (fr) * 2003-10-10 2005-04-13 Jean-Christophe Roegel Procédé de sélection de composés utilisant des anticorps agissant comme agoniste, antagoniste ou modulateur allostériques
US7247711B2 (en) 2003-05-09 2007-07-24 Centocor, Inc. IL-23p40 specific antibody
US7252971B2 (en) 2004-09-24 2007-08-07 Centocor, Inc. IL-23p40 specific immunoglobulin derived proteins
EP1210434B1 (fr) * 1999-09-09 2008-03-12 Schering Corporation Interleukine-12 p40 et interleukine b30. combinations. anticorps. utilisation dans des compositions pharmaceutiques.
US7491391B2 (en) 2005-06-30 2009-02-17 Centocor, Inc. Anti-IL-23 antibodies, compositions, methods and uses
EP1905832A3 (fr) * 1999-09-09 2009-09-09 Schering Corporation Interleukin-12 P40 et interleukin B30 de mammifères, anticorps, utilisation dans des compositions pharmaceutiques
US7790862B2 (en) 2006-06-13 2010-09-07 Zymogenetics, Inc. IL-17 and IL-23 antagonists and methods of using the same
US7883695B2 (en) 1999-09-09 2011-02-08 Schering Corporation IL-12P40 and IL-B30 polypeptide complex
US7935344B2 (en) 2005-12-29 2011-05-03 Centocor Ortho Biotech Inc. Human anti-IL-23 antibodies, compositions, methods and uses
US8778346B2 (en) 2010-11-04 2014-07-15 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US10059763B2 (en) 2014-09-03 2018-08-28 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
US10507241B2 (en) 2014-07-24 2019-12-17 Boehringer Ingelheim International Gmbh Biomarkers useful in the treatment of IL-23A related diseases
US11078265B2 (en) 2012-05-03 2021-08-03 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US11548941B2 (en) 2018-11-20 2023-01-10 Janssen Biotech, Inc. Safe and effective method of treating psoriasis with anti-IL-23 specific antibody
US11780911B2 (en) 2019-05-23 2023-10-10 Janssen Biotech, Inc. Method of treating inflammatory bowel disease with a combination therapy of antibodies to IL-23 and TNF alpha

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044455A1 (fr) * 1996-05-23 1997-11-27 Zymogenetics, Inc. Recepteur de cytokines hematopoietiques
WO1998011225A2 (fr) * 1996-09-11 1998-03-19 Amrad Operations Pty. Ltd. Nouveau recepteur d'hematopoietine et sequences genetiques codant ce dernier
WO1998031811A1 (fr) * 1997-01-16 1998-07-23 Genetics Institute, Inc. Membre de la superfamille des recepteurs d'hematopoietine
WO1998049307A1 (fr) * 1997-05-01 1998-11-05 Zymogenetics, Inc. Recepteurs de mammiferes analogues de la cytokine
WO1999005280A1 (fr) * 1997-07-25 1999-02-04 Schering Corporation Cytokine mammalienne: interleukine-b30 et reactifs correspondants

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044455A1 (fr) * 1996-05-23 1997-11-27 Zymogenetics, Inc. Recepteur de cytokines hematopoietiques
WO1998011225A2 (fr) * 1996-09-11 1998-03-19 Amrad Operations Pty. Ltd. Nouveau recepteur d'hematopoietine et sequences genetiques codant ce dernier
WO1998031811A1 (fr) * 1997-01-16 1998-07-23 Genetics Institute, Inc. Membre de la superfamille des recepteurs d'hematopoietine
WO1998049307A1 (fr) * 1997-05-01 1998-11-05 Zymogenetics, Inc. Recepteurs de mammiferes analogues de la cytokine
WO1999005280A1 (fr) * 1997-07-25 1999-02-04 Schering Corporation Cytokine mammalienne: interleukine-b30 et reactifs correspondants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FISCHER M ET AL: "A BIOACTIVE DESIGNER CYTOKINE FOR HUMAN HEMATOPOIETIC PROGENITOR CELL EXPANSION", NATURE BIOTECHNOLOGY, vol. 15, no. 2, 1 February 1997 (1997-02-01), pages 142 - 145, XP002047603, ISSN: 1087-0156 *

Cited By (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6610285B1 (en) 1998-04-14 2003-08-26 Chugai Seiyaku Kabushiki Kaisha Cytokine-like proteins that promote cell proliferation
EP1072610A1 (fr) * 1998-04-14 2001-01-31 Chugai Research Institute for Molecular Medicine Inc. Nouvelle proteine du type cytokine
US7750126B2 (en) 1998-04-14 2010-07-06 Chugai Seiyaku Kabushiki Kaisha Antibodies that bind to a member of the IL-6/G-CSF/MGF family
US7252967B2 (en) 1998-04-14 2007-08-07 Chugai Seiyaku Kabushiki Kaisha Nucleic acids encoding cytokine-like proteins that promote cell proliferation
EP1072610A4 (fr) * 1998-04-14 2005-01-26 Chugai Pharmaceutical Co Ltd Nouvelle proteine du type cytokine
WO2000053631A1 (fr) * 1999-03-11 2000-09-14 Schering Corporation Cytokines de mammifere : reactifs et procedes associes
US7390481B2 (en) 1999-03-11 2008-06-24 Schering Corporation Mammalian cytokine complexes
US6800460B1 (en) 1999-03-11 2004-10-05 Schering Corporation Mammalian cytokine complexes
EP1210434B1 (fr) * 1999-09-09 2008-03-12 Schering Corporation Interleukine-12 p40 et interleukine b30. combinations. anticorps. utilisation dans des compositions pharmaceutiques.
US7883695B2 (en) 1999-09-09 2011-02-08 Schering Corporation IL-12P40 and IL-B30 polypeptide complex
EP1905832A3 (fr) * 1999-09-09 2009-09-09 Schering Corporation Interleukin-12 P40 et interleukin B30 de mammifères, anticorps, utilisation dans des compositions pharmaceutiques
EP3184637A1 (fr) * 1999-09-09 2017-06-28 Merck Sharp & Dohme Corp. Utilisations de complexes d'interleukine-12 p40 et d'interleukine b30 mammaliennes, anticorps, dans des compositions pharmaceutiques
WO2001036467A3 (fr) * 1999-11-18 2002-05-10 Schering Corp Proteines de recepteurs mammaliens, reactifs et procedes s'y rapportant
WO2001036467A2 (fr) * 1999-11-18 2001-05-25 Schering Corporation Proteines de recepteurs mammaliens, reactifs et procedes s'y rapportant
FR2804434A1 (fr) * 2000-01-27 2001-08-03 Pf Medicament Heterocomplexe nnt-1/clf-1 et son utilisation en tant qu'activateur du recepteur complexe cntrfalpha/gp130/lifrbet a
FR2804435A1 (fr) * 2000-01-27 2001-08-03 Pf Medicament COMPLEXE ISOLE COMPRENANT UNE PROTEINE NNT-1 ET EN OUTRE AU MOINS UNE PROTEINE CLF-1 ET/OU UNE PROTEINE sCNTFRalpha
WO2001055172A2 (fr) * 2000-01-27 2001-08-02 Pierre Fabre Medicament COMPLEXE ISOLE COMPRENANT UNE PROTEINE NNT-1 ET EN OUTRE AU MOINS UNE PROTEINE CLF-1 ET/OU UNE PROTEINE sCNTFR$g(a)
WO2001055172A3 (fr) * 2000-01-27 2001-11-15 Pf Medicament COMPLEXE ISOLE COMPRENANT UNE PROTEINE NNT-1 ET EN OUTRE AU MOINS UNE PROTEINE CLF-1 ET/OU UNE PROTEINE sCNTFR$g(a)
US7247711B2 (en) 2003-05-09 2007-07-24 Centocor, Inc. IL-23p40 specific antibody
WO2005040820A3 (fr) * 2003-10-10 2005-07-21 Jean-Christophe Roegel Procedes de selection de composes au moyen d'anticorps fonctionnels
EP1522858A1 (fr) * 2003-10-10 2005-04-13 Jean-Christophe Roegel Procédé de sélection de composés utilisant des anticorps agissant comme agoniste, antagoniste ou modulateur allostériques
US7252971B2 (en) 2004-09-24 2007-08-07 Centocor, Inc. IL-23p40 specific immunoglobulin derived proteins
US7807471B2 (en) 2004-09-24 2010-10-05 Centocor Ortho Biotech, Inc. IL-23p40 specific immunoglobulin derived proteins, compositions, epitopes, methods and uses
US11185584B2 (en) 2005-06-30 2021-11-30 Janssen Biotech, Inc. Anti-IL-23 antibodies, compositions, methods and uses
US7807414B2 (en) 2005-06-30 2010-10-05 Centocor Ortho Biotech Inc. Anti-IL-23 antibodies, compositions, methods and uses
US10576150B2 (en) 2005-06-30 2020-03-03 Janssen Biotech, Inc. Anti-IL-23 antibodies
US7491391B2 (en) 2005-06-30 2009-02-17 Centocor, Inc. Anti-IL-23 antibodies, compositions, methods and uses
US10272152B2 (en) 2005-06-30 2019-04-30 Janssen Biotech, Inc. Anti-IL-23 antibodies, compositions, methods and uses
US9714287B2 (en) 2005-06-30 2017-07-25 Janssen Biotech, Inc. Anti-IL-23 antibody compositions
US9127058B2 (en) 2005-06-30 2015-09-08 Janssen Biotech, Inc. Anti-IL-23 Antibodies
US8574579B2 (en) 2005-06-30 2013-11-05 Janssen Biotech, Inc. Anti-IL-23 antibodies
US9505837B2 (en) 2005-06-30 2016-11-29 Janssen Biotech, Inc. Anti-IL-23 antibodies
US9096667B2 (en) 2005-06-30 2015-08-04 Janssen Biotech, Inc. Anti-IL-23 antibodies, compositions, methods and uses
US7993645B2 (en) 2005-12-29 2011-08-09 Janssen Biotech, Inc. Human anti-IL-23 antibodies, compositions, methods and uses
US8106177B2 (en) 2005-12-29 2012-01-31 Janssen Biotech, Inc. Nucleic acids encoding human anti-IL-23 antibodies
US10954297B2 (en) 2005-12-29 2021-03-23 Janssen Biotech, Inc. Methods of treatment using human anti-IL-23 antibodies
US8221760B2 (en) 2005-12-29 2012-07-17 Janssen Biotech, Inc. Methods of treatment using human anti-IL-23 antibodies
US9783607B2 (en) 2005-12-29 2017-10-10 Janssen Biotech, Inc. Human anti-IL-23 antibodies, compositions, methods and uses
US10030070B2 (en) 2005-12-29 2018-07-24 Janssen Biotech, Inc. Human anti-IL-23 antibodies, compositions, methods and uses
US7935344B2 (en) 2005-12-29 2011-05-03 Centocor Ortho Biotech Inc. Human anti-IL-23 antibodies, compositions, methods and uses
US9353181B2 (en) 2005-12-29 2016-05-31 Janssen Biotech, Inc. Human anti-IL-23 antibodies, compositions, methods and uses
US7790862B2 (en) 2006-06-13 2010-09-07 Zymogenetics, Inc. IL-17 and IL-23 antagonists and methods of using the same
US8227579B2 (en) 2006-06-13 2012-07-24 Zymogenetics, Inc. IL-23 antagonists
US10202448B2 (en) 2010-11-04 2019-02-12 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US8778346B2 (en) 2010-11-04 2014-07-15 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US9441036B2 (en) 2010-11-04 2016-09-13 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US11078265B2 (en) 2012-05-03 2021-08-03 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US10507241B2 (en) 2014-07-24 2019-12-17 Boehringer Ingelheim International Gmbh Biomarkers useful in the treatment of IL-23A related diseases
US10059763B2 (en) 2014-09-03 2018-08-28 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
US10793629B2 (en) 2014-09-03 2020-10-06 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
US11680096B2 (en) 2014-09-03 2023-06-20 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
US11548941B2 (en) 2018-11-20 2023-01-10 Janssen Biotech, Inc. Safe and effective method of treating psoriasis with anti-IL-23 specific antibody
US11780911B2 (en) 2019-05-23 2023-10-10 Janssen Biotech, Inc. Method of treating inflammatory bowel disease with a combination therapy of antibodies to IL-23 and TNF alpha

Also Published As

Publication number Publication date
AU2661199A (en) 1999-08-23

Similar Documents

Publication Publication Date Title
EP0980429B1 (fr) Recepteur proteique humain du type toll, reactifs connexes et techniques afferentes
US7510853B2 (en) Nucleic acids encoding DCRS5
US20050009145A1 (en) Mammalian receptor proteins; related reagents and methods
WO2002020569A2 (fr) Genes mammaliens, reactifs et methodes associes
AU2001261351A1 (en) Mammalian receptor proteins; related reagents and methods
WO1999040195A1 (fr) Proteines de recepteurs de mammiferes; reactifs et procedes apparentes
JP2015007129A (ja) 哺乳動物レセプタータンパク質；関連する試薬および方法
CA2392109A1 (fr) Proteines de recepteurs mammaliens, reactifs et procedes s'y rapportant
EP1062332A2 (fr) Proteines receptrices humaines; reactifs et procedes associes
US7812126B2 (en) DIRS1 polypeptides
US6326472B1 (en) Human receptor proteins; related reagents and methods
US20050106673A1 (en) Mammalian receptor proteins; related reagents and methods
EP1029043A1 (fr) Proteines receptrices humaines et reactifs et methodes associes
AU2007202362B2 (en) Mammalian receptor proteins; related reagents and methods
MXPA00008848A (en) Human receptor proteins;related reagents and methods

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GD GE HR HU ID IL IN IS JP KG KR KZ LC LK LR LT LU LV MD MG MK MN MX NO NZ PL PT RO RU SE SG SI SK SL TJ TM TR TT UA UZ VN YU

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase