WO1998032858A2 - Chemokines de mammiferes, recepteurs, reactifs, et modes d'utilisation - Google Patents

Chemokines de mammiferes, recepteurs, reactifs, et modes d'utilisation Download PDF

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WO1998032858A2
WO1998032858A2 PCT/US1998/000902 US9800902W WO9832858A2 WO 1998032858 A2 WO1998032858 A2 WO 1998032858A2 US 9800902 W US9800902 W US 9800902W WO 9832858 A2 WO9832858 A2 WO 9832858A2
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leu
val
ser
protein
lys
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WO1998032858A3 (fr
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Jeanine D. Mattson
Hortensia Soto-Trejo
Joseph A. Hedrick
Daniel M. Gorman
Albert Zlotnik
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Merck Sharp and Dohme LLC
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Schering Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • 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
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • 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 related to proteins which function in controlling physiology, development, and/or differentiation of mammalian cells.
  • it provides proteins which are implicated in the regulation of physiology, development, differentiation, or function of various cell types, e.g., chemokines, 7 transmembrane receptors, reagents related to each, e.g., antibodies or nucleic acids encoding them, and uses thereof.
  • the circulating component of the mammalian circulatory system comprises various cell types, including red and white blood cells of the erythroid and myeloid cell lineages. See, e.g., Rapaport (1987) Introduction to Hematology (2d ed.) Lippincott, Philadelphia, PA; Jandl (1987) Blood: Textbook of Hematology. Little, Brown and Co., Boston, MA.; and Paul (ed.) (1993) Fundamental Immunology (3d ed.) Raven Press, N.Y.
  • Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to support the proliferation, growth, and differentiation of the pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. These interactions between the cellular components are necessary for a healthy immune response. These different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents.
  • the chemokines are a large and diverse superfamily of proteins.
  • the superfamily is subdivided into two classical branches, based upon whether the first two cysteines in the chemokine motif are adjacent (termed the "C-C” branch), or spaced by an intervening residue ("C-X-C").
  • a more recently identified branch of chemokines lacks two cysteines in the corresponding motif, and is represented by the chemokines known as lymphotactins.
  • Another recently identified branch has three intervening residues between the two cysteines, e.g., CX3C chemokines. See, e.g., Schall and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy & Immunol. 109:97-109.
  • the chemokine receptors are typically members of the superfamily of G-protein coupled (or linked) receptors (GPCR, or GPLR). As a class, these receptors are integral membrane proteins characterized by amino acid sequences which contain seven hydrophobic domains. See, e.g., Ruffolo and Hollinger (eds. 1995) G- Protein Coupled Transmembrane Signaling Mechanisms CRC Press, Boca Raton, FL; Watson and Arkinstall (1994) The G-Protein Linked Receptor FactsBook Academic Press, San Diego, CA; Peroutka (ed.
  • GPCRs are found in a wide range of organisms and are typically involved in the transmission of signals to the interior of the cell, e.g., through interaction, e.g., with heterotrimeric G-proteins. They respond to a wide and diverse range of agents including lipid analogs, amino acid derivatives, small peptides, and other molecules.
  • the presumed transmembrane segments are typically 20-25 amino acids in length. Based upon models and data on bacteriorhodopsin, these regions are predicted to be a-helices and be oriented to form a ligand binding pocket. See, e.g., Findley, et al. (1990) Trends Pharmacol. Sci. 11:492-499. Other data suggest that the amino termini of the proteins are extracellular, and the carboxy termini are intracellular. See, e.g., Lodish, et al. (1995) Molecular Cell Biology 3d ed., Scientific American, New York; and Watson and Arkinstall (1994) The G-Protein Linked Receptor FactsBook Academic Press, San Diego, CA. Phosphorylation cascades have been implicated in the signal transduction pathway of these receptors.
  • Chemokine receptors are notable members of the GPCR family. See, e.g., Samson, et al. (1996) Biochemistry 35:3362-3367; and Rapport, et al. (1996) T. Leukocyte Biology 59:18-23. The best known biological functions of these chemokine molecules relate to chemoattraction of leukocytes. However, new chemokines and receptors are being discovered, and their biological effects on the various cells responsible for immunological responses are topics of continued study.
  • the present invention is based, in part, upon the discovery of new genes encoding various chemokines, e.g., those designated
  • IBICK which encodes primate CXC chemokines
  • ILINCK which encodes primate CXC chemokines
  • CXC-143 which encodes rodent
  • CXC chemokines which encodes a mouse chemokine
  • MCP243 which encodes a mouse chemokine
  • 7 transmembrane receptors e.g., those designated R277, which encode primate receptors
  • HST01.1 which encode rodent receptors
  • GPCR G-protein coupled receptors
  • the invention also provides mutations (muteins) of the respective natural sequences, fusion proteins, chemical mimetics, antibodies, and other structural or functional analogs. It is also directed to isolated nucleic acids, e.g., genes encoding respective proteins of the invention. Various uses of these different protein, antibody, or nucleic acid compositions are also provided.
  • the present invention provides a composition selected from the group of: a substantially pure antigenic polypeptide comprising sequence from an IBICK; an ILINCK; a CXC-143; an MCP243; an R277; an HST01.1; or a 941D12; a binding composition comprising an antigen binding portion of an antibody specific for binding to such an antigenic polypeptide; a nucleic acid encoding such an antigenic polypeptide; and a fusion protein comprising at least two non- overlapping segments of at least 10 amino acids of such an antigenic polypeptide.
  • the antigenic polypeptide is from a warm blooded animal, e.g., a rodent or primate; it comprises a sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; it exhibits a post-translational modification pattern distinct from a natural form of said polypeptide; it is detectably labeled; or it is made by expression of a recombinant nucleic acid.
  • a sterile form is provided, including, e.g., composition comprising the polypeptide and an acceptable carrier.
  • a detection kit comprising a compartment or container holding such an antigenic polypeptide is also provided.
  • the polypeptide is a mouse or human protein; the antibody is raised against a peptide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; the antibody is a monoclonal antibody; the binding composition is fused to a heterologous protein, or is detectably labeled.
  • An alternative embodiment is a binding compound comprising an antigen binding fragment of the antibody described.
  • a detection kit comprising such a binding compound.
  • Nucleic acid embodiments are provided, e.g., where the nucleic acid is in an expression vector and: encodes a polypeptide from a mouse or human; encodes a mature protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; or comprises a deoxy rib onucleic acid nucleotide.
  • the invention also provides a kit with such nucleic acids, e.g., which include PCR primers for amplifying such sequences.
  • nucleic acids comprising: a sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; and/or sequence of another chemokine or 7 transmembrane receptor, as appropriate.
  • a cell comprising a recombinant nucleic acid, as described, and methods of producing a polypeptide comprising expressing the nucleic acid in an expression system.
  • inventions include methods of modulating physiology or development of a cell, with a step of contacting that cell with a composition comprising an agonist or antagonist of the chemokine or receptor.
  • the cell is a neuron, macrophage, or lymphocyte.
  • Various physiological effects to be modulated include a cellular calcium flux, a chemoattractant response, cellular morphology modification responses, phosphoinositide lipid turnover, or an antiviral response.
  • the present invention provides DNA sequences encoding various mammalian proteins, including chemokines, or which exhibit structural properties characteristic of a 7 transmembrane receptor. See, e.g., Ruffolo and Hollinger (eds. 1995) G-Protein Coupled Transmembrane Signaling Mechanisms CRC Press, Boca Raton, FL; Watson and Arkinstall (1994) The G-Protein Linked Receptor FactsBook Academic Press, San Diego, CA; Peroutka (ed. 1994) G Protein-Coupled Receptors CRC Press, Boca Raton, FL; Houslay and Milligan (1990) G-Proteins as Mediators of Cellular Signaling Processes Wiley and Sons, New York, NY. Certain human and mouse embodiments are described herein.
  • Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes. See, e.g., Schall (1991) Cytokine 3:165-183; and Thomson (ed.) The Cytokine Handbook Academic Press, NY. Chemokines are secreted by activated leukocytes and act as a chemoattractant for a variety of cells which are involved in inflammation.
  • chemokines have been shown to induce other biological responses, e.g., modulation of second messenger levels such as Ca ++ ; inositol phosphate pool changes (see, e.g., Berridge (1993) Nature 361:315-325 or Billah and Anthes (1990) Biochem. T. 269:281- 291); cellular morphology modification responses; phosphoinositide lipid turnover; possible antiviral responses; and others.
  • the chemokines provided herein may, alone or in combination with other therapeutic reagents, have advantageous combination effects.
  • chemokines may have effects on other cell types, e.g., attraction or activation of monocytes, dendritic cells, T cells, eosinophils, and /or perhaps on basophils and/or neutrophils. They may also have chemoattractive effects on various neural cells including, e.g., dorsal root ganglia neurons in the peripheral nervous system and/or central nervous system neurons.
  • G-protein coupled receptors e.g., chemokine receptors
  • chemokine receptors are important in the signal transduction mechanisms mediated by their ligands. They are useful markers for distinguishing cell populations, and have been implicated as specific receptors for retroviral infections.
  • the chemokine superfamily was classically divided into two groups exhibiting characteristic structural motifs, the Cys-X-Cys (C-X- C) and Cys-Cys (C-C) families. These were distinguished on the basis of a single amino acid insertion between the NH-proximal pair of cysteine residues and sequence similarity.
  • C-X-C chemokines i.e., IL-8 and MGSA/Gro-a act on neutrophils but not on monocytes
  • the C-C chemokines i.e., MlP-la and RANTES, are potent chemoattractants for monocytes and lymphocytes but not neutrophils.
  • lymphotactin does not belong to either group and may constitute a first member of a third chemokine family, the C family. Lymphotactin does not have a characteristic CC or CXC motif, and acts on lymphocytes but not neutrophils and monocytes. See, e.g., Kelner et al. (1994) Science 266:1395-1399. This chemokine defines a new C-C chemokine family. Even more recently, another chemokine exhibiting a CX3C motif has been identified, which establishes a fourth structural class.
  • the present invention provides additional chemokine reagents, e.g., nucleic acids, proteins and peptides, antibodies, etc., related to the newly discovered chemokines designated primate IBICK; primate ILLNCK; rodent CXC-143; or rodent MCP243.
  • the invention provides genes encoding novel G-protein coupled receptors, designated primate R277, rodent HST01.1, and rodent 941D12. Their ligands have not yet specifically been identified. However, the receptors exhibit structural features typical of known 7 transmembrane spanning receptors, which receptors include chemokine receptors.
  • the receptors may exhibit properties of binding many different cytokines at varying specificities (shared or promiscuous binding specificity) or may exhibit high affinity for one (specific) or a subset (shared) of chemokines.
  • the ligands may be other molecules, including molecules such as epinephrine, serotonin, or glucagon.
  • the described chemokines or receptors should be important for mediating various aspects of cellular, organ, tissue, or organismal physiology or development.
  • IBICK Purified Chemokines; Receptors Nucleotide and derived amino acid sequences of a human embodiment of a primate CXC chemokine, designated IBICK are shown in SEQ ID NO: 1 and 2.
  • the term "IBICK" will encompass other primate counterparts.
  • the gene encodes a novel protein exhibiting structure and motifs characteristic of a chemokine.
  • the predicted signal cleavage site is around the gly(-l)-phel peptide bond.
  • Complementary nucleic acid sequences may be used for many purposes, e.g., in a PCR primer pair or as a mutagenesis primer.
  • Fragments of the nucleotide sequence may be used as hybridization probes, or PCR primers, or to encode antigenic peptides. Fragments of the polypeptide will be useful as antigenic peptides. Likewise for the other genes.
  • the closest reported chemokines to the IBICK sequences are the MIG and IP10 chemokines, both of which are IFN- ⁇ regulated. See, e.g., Faubert (1993) Biochem. Biophys. Res. Commun. 192:223-230; and Luster, et al. (1985) Nature 315:672-676.
  • Nucleotide and derived amino acid sequences of a novel primate CC chemokine, e.g., from human, designated ILLNCK are shown in SEQ ID NO: 3 and 4.
  • the term "ILINCK” as used in this filing will encompass other primate counterparts.
  • the predicted signal cleavage site is around the ser(-l)-glnl peptide bond.
  • Two different messages have been detected which encode the chemokine, and the larger one, a 1.5 kB message, is upregula ed by IL-10. This is an unusual property of chemokine messages, which implies that the chemokine has a role in anti-inflammatory responses.
  • Partial nucleotide and derived amino acid sequences of a novel rodent CXC chemokine, e.g., from mouse, designated CXC-143, are shown in SEQ ID NO: 5, 6, 7, 8, 9 and 10.
  • CXC-143 will typically encompass rodent counterparts.
  • Sequence analysis shows closest sequence homology to the LP10, MIG, and IBICK chemokines, described above. It may well be that the chemokine will be similarly regulated, e.g., by LFN- ⁇ .
  • Nucleotide and derived amino acid sequences of a novel rodent chemokine, e.g., from mouse, designated MCP243 are shown in SEQ ID NO: 11, 12, 13 and 14. The term may encompass other rodent counterparts. Cys residues 14 and 30 correspond to conserved chemokine Cys3 and Cys4. Sequence analysis shows closest sequence homology to other chemokines.
  • Nucleotide and derived amino acid sequences of a novel rodent GPCR are shown in SEQ ID NO: 15, 16, 17 and 18.
  • the term "R277” may encompass other primate counterparts.
  • nucleotide 447 is designated C, but may be C or T
  • nucleotides 489 and 640 are each designated C, but may be A, C, G, or T
  • nucleotides 480-510 may contain various sequence errors, but which will retain reading frame.
  • Sequence analysis shows closest sequence homology to a human GPCR designated TDAG8.
  • Partial nucleotide and derived amino acid sequences of a novel rodent GPCR, e.g., from mouse, designated HST01.1, are shown in SEQ ID NO: 19 and 20.
  • the term "R277” may encompass other primate counterparts.
  • nucleotide 447 is designated C, but may be C or T
  • nucleotides 489 and 640 are each designated C, but may be A, C, G, or T
  • nucleotides 480-510 may contain various sequence errors, but which will retain reading frame. The sequence is supplemented with more complete sequence in SEQ ID NO: 21 and 22.
  • a DRY box motif runs from about aspl47 to alal55; transmembrane segments run from about ala57 to leu78; phe90 to valllO; vall25 to phel46; vall67 to leul89; phe223 to val243; leu255 to leu279; and val301 to val322. Sequence analysis shows sequence homology to various GPCR family members.
  • Partial nucleotide and derived amino acid sequences of a novel rodent GPCR, e.g., from mouse, designated 941D12, are shown in SEQ ID NO: 23 and 24.
  • the term "941D12" may encompass other rodent counterparts.
  • the nucleotides at positions 169, 178, 217, 287, 290, 382, 386, 395, 411, 484, 512, 515, 517, and 521 are each indicated as C, but may be A, C, G, or T.
  • a complete rodent 941D12 is provided in SEQ ID NO: 25 and 26.
  • Nucleotide 942 is designated C, but may be C or T; nucleotides 1412 and 1422 each designated C, but may be A, C, G, or T. Sequence analysis shows sequence homology to various GPCR family members.
  • amino acid sequences are important in providing sequence information on the chemokine ligand or receptor, allowing for distinguishing the protein from other proteins, particularly naturally occurring versions. Moreover, the sequences allow preparation of peptides to generate antibodies to recognize and distinguish such segments, and allow preparation of oligonucleotide probes, both of which are strategies for isolation, e.g., cloning, of genes encoding such sequences, or related sequences, e.g., natural polymorphic or other variants, including fusion proteins. Similarities of the chemokines have been observed with other cytokines. See, e.g., Bosenberg, et al. (1992) Cell 71:1157-1165; Huang, et. al.
  • IBICK shall encompass, when used in a protein context, a protein having mature amino acid sequence, as shown in SEQ ID NO: 2.
  • the invention also embraces a polypeptide comprising a significant fragment of such protein.
  • the invention also encompasses a polypeptide which is a primate species counterpart, e.g., which exhibits similar sequence, and is more homologous in natural encoding sequence than other genes from a primate species.
  • such chemokine will also interact with its specific binding components, e.g., receptor, or antibodies which bind to it.
  • binding components e.g., antibodies
  • bind to the chemokine 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.
  • Similar concepts apply to the primate embodiments for the chemokine ILINCK and the GPCR R277.
  • rodent embodiments for the chemokines CXC-143 and MCP243, and the GPCRs HST01.1 and 941D12 encompass other rodent species counterparts.
  • polypeptide as used herein includes a significant fragment or segment, and encompasses a stretch of amino acid residues of at least about 8 amino acids, generally at least 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, e.g., about 35, 40, 45, 50, 60, 75, 80, 100, 120, etc. Similar proteins will likely comprise a plurality of such segments.
  • Such fragments may have ends which begin and /or end at virtually all positions, e.g., beginning at residues 1, 2, 3, etc., and ending at, e.g., 69, 68, 67, 66, etc., in all combinatorial pairs in the coding segment.
  • Particularly interesting peptides have ends corresponding to structural domain boundaries, e.g., intracellular or extracellular loops of the receptor embodiments.
  • Such peptides will typically be immunogenic peptides, or may be concatenated to generate larger polypeptides. Short peptides may be attached or coupled to a larger carrier.
  • binding composition refers to molecules that bind with specificity to the respective chemokine or receptor, e.g., in a ligand-receptor type fashion or an antibody-antigen interaction.
  • These compositions may be compounds, e.g., proteins, which specifically associate with the chemokine or receptor, including natural physiologically relevant protein-protein interactions, either covalent or non-covalent.
  • the binding composition may be a polymer, or another chemical reagent. No implication as to whether the chemokine presents a concave or convex shape in its ligand- receptor interaction is necessarily represented, other than the interaction exhibit similar specificity, e.g., specific affinity.
  • a functional analog may be a ligand with structural modifications, or may be a wholly unrelated molecule, e.g., which has a molecular shape which interacts with the appropriate ligand binding determinants.
  • the ligands may serve as agonists or antagonists of a physiological or natural receptor, see, e.g., Goodman, et al. (eds.) (1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics (8th ed.), Pergamon Press.
  • the term expressly includes antibodies, polyclonal or monoclonal, which specifically bind to the respective antigen.
  • Substantially pure means that the protein is free from other contaminating proteins, nucleic acids, and /or other biologicals typically derived from the original source organism. Purity may be assayed by standard methods, and will ordinarily be at least about 40% pure, more ordinarily at least about 50% pure, generally at least about 60% pure, more generally at least about 70% pure, often at least about 75% pure, more often at least about 80% pure, typically at least about 85% pure, more typically at least about 90% pure, preferably at least about 95% pure, more preferably at least about 98% pure, and in most preferred embodiments, at least 99% pure. Analyses will typically be by weight, but may be by molar amounts.
  • Solubility of a polypeptide or fragment depends upon the environment and the polypeptide. Many parameters affect polypeptide solubility, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and nature of the solvent. Typically, the temperature at which the polypeptide is used ranges from about 4° C to about 65° C. Usually the temperature at use is greater than about 18° C and more usually greater than about 22° C. For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturation temperature of components in the assay. For therapeutic purposes, the temperature will usually be body temperature, typically about 37° C for humans, though under certain situations the temperature may be raised or lowered in situ or in vitro.
  • the electrolytes will usually approximate in situ physiological conditions, but may be modified to higher or lower ionic strength where advantageous.
  • the actual ions may be modified, e.g., to conform to standard buffers used in physiological or analytical contexts.
  • the size and structure of the polypeptide should generally be in a substantially stable state, and usually not in a denatured state, though in certain circumstances denatured protein will be important.
  • the polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to confer solubility, or associated with lipids or detergents in a manner which approximates natural lipid bilayer interactions.
  • the solvent will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological solvent. Usually the solvent will have a neutral pH, typically at least about 5, preferably at least 6, and typically less than 10, preferably less than 9, and more preferably about 7.5.
  • a detergent will be added, typically a mild non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS (3-([3-cholamido-propyl] dimethylammonio)-l-propane sulfonate), or a low enough concentration as to avoid significant disruption of structural or physiological properties of the protein.
  • Solubility is reflected by sedimentation measured in Svedberg units, which are a measure of the sedimentation velocity of a molecule under particular conditions. The determination of the sedimentation velocity was classically performed in an analytical ultracentrifuge, but is typically now performed in a standard ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d ed.), W.H.
  • a soluble particle or polypeptide will typically be less than about 30S, more typically less than about 15S, usually less than about 10S, more usually less than about 6S, and, in particular embodiments, preferably less than about 4S, and more preferably less than about 3S.
  • This invention also encompasses proteins or peptides having substantial amino acid sequence homology with the amino acid sequence of each respective receptor.
  • the variants include species or polymorphic variants.
  • Amino acid sequence homology, or sequence identity is determined by optimizing residue matches, if necessary, by introducing gaps as required. 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 typically intended to include natural allelic and interspecies variations in each respective protein sequence.
  • Typical homologous proteins or peptides will have from 25-100% homology (if gaps can be introduced), to 50-100% homology (if conservative substitutions are included) with the amino acid sequence of the appropriate chemokine or receptor.
  • Homology measures will be at least about 35%, generally at least 40%, more generally at least 45%, often at least 50%, more often at least 55%, typically at least 60%, more typically at least 65%, usually at least 70%, more usually at least 75%, preferably at least 80%, and more preferably at least 80%, and in particularly preferred embodiments, at least 85% or more. See also Needleham, et al. (1970) T. Mol. Biol. 48:443-453; Sankoff, et al.
  • Each of the isolated chemokine or GPC receptor DNAs can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications may result in novel DNA sequences which encode these antigens, their derivatives, or proteins having similar physiological, immunogenic, or antigenic activity. These modified sequences can be used to produce mutant antigens or to enhance expression, or to introduce convenient enzyme recognition sites into the nucleotide sequence without significantly affecting the encoded protein sequence. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. Such mutant receptor derivatives include predetermined or site-specific mutations of the respective protein or its fragments.
  • “Mutant chemokine” encompasses a polypeptide otherwise falling within the homology definition of the chemokine as set forth above, but having an amino acid sequence which differs from that of the chemokine as found in nature, whether by way of deletion, substitution, or insertion. Likewise for the GPCRs. These include amino acid residue substitution levels from none, one, two, three, five, seven, ten, twelve, fifteen, etc.
  • site specific mutant generally includes proteins having significant homology with a protein having sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, and as sharing various biological activities, e.g., antigenic or immunogenic, with those sequences, and in preferred embodiments contain most of the disclosed sequences, particularly those found in various groups of animals.
  • descriptions are generally meant to encompass the various chemokine or receptor proteins from other members of related groups, not limited to the mouse or human embodiments specifically discussed.
  • site specific mutation sites are often predetermined, mutants need not be site specific. Chemokine or receptor mutagenesis can be conducted by making amino acid insertions or deletions.
  • Insertions include amino- or carboxy- terminal fusions.
  • Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity.
  • 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 or polymerase chain reaction (PCR) techniques. See also Sambrook, et al. (1989) and Ausubel, et al. (1987 and Supplements).
  • chemokines and GPCRs which allow determination of whether specific residues are embedded into the core of the secondary or tertiary structures, or whether the residues will have relatively little effect on protein folding.
  • Preferred positions for mutagenesis are those which do not prevent functional folding of the resulting protein.
  • 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. But certain situations exist where such problems are compensated. See, e.g., Gesteland and Atkins (1996) Ann. Rev. Biochem. 65:741-768.
  • the present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins, or antibodies.
  • 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 an immunoglobulin with a receptor polypeptide is a continuous protein molecule having sequences fused in a typical peptide linkage, typically made as a single translation product and exhibiting properties derived from each source peptide.
  • a similar chimeric concept applies to heterologous nucleic acid sequences.
  • new constructs may be made from combining similar functional or structural domains from other proteins.
  • 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) T. Biol. Chem. 263:15985-15992.
  • new chimeric polypeptides exhibiting new combinations of specificities will result from the functional linkage of ligand-binding specificities and other functional domains.
  • Such may be chimeric molecules with mixing or matching of the various structural segments, e.g., the ⁇ -sheet or ⁇ - helix structural domains for the chemokine, or receptor segments corresponding to each of the transmembrane segments (TM1-TM7), or the intracellular (cytosolic, C1-C4) or extracellular (E1-E4) loops from the various receptor types.
  • the C3 loop is particularly important.
  • 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, e.g., PCR techniques.
  • chemokines or GPCRs may result from the inhibition of binding of the ligand to its receptor, likely through competitive inhibition.
  • in vitro assays of the present invention will often use isolated protein, membranes from cells expressing a recombinant membrane associated receptor, e.g., ligand binding segments, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either binding segment mutations and modifications, or ligand mutations and modifications, e.g., ligand analogs.
  • This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing binding compositions, e.g., antibodies, to antigen or receptor fragments compete with a test compound for binding to the protein.
  • neutralizing binding compositions e.g., antibodies
  • the antibodies can be used to detect the presence of polypeptides which share one or more antigenic binding sites of the ligand and can also be used to occupy binding sites on the protein that might otherwise interact with a receptor.
  • neutralizing antibodies against a specific chemokine embodiment and soluble fragments of the chemokine which contain a high affinity receptor binding site can be used to inhibit chemokine activity in tissues, e.g., tissues experiencing abnormal physiology.
  • “Derivatives” of chemokine antigens include amino acid sequence mutants, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties.
  • Covalent derivatives can be prepared by linkage of functionalities to groups which are found in chemokine amino acid side chains or at the N- or C- termini, 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. Covalent attachment to carrier proteins may be important when immunogenic moieties are hap tens.
  • 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.
  • phosphorylated amino acid residues e.g., phospho tyrosine, phosphoserine, or phosphothreonine
  • nucleoside or nucleotide derivatives e.g., guanyl derivatized.
  • a major group of derivatives are covalent conjugates of the respective chemokine or receptor or fragments thereof with other proteins or 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 chemokine derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues.
  • Fusion polypeptides between these chemokines or receptors and other homologous or heterologous proteins, e.g., other chemokines or receptors, are also provided.
  • Many growth factors and cytokines are homodimeric entities, and a repeat construct may have various advantages, including lessened susceptibility to proteolytic cleavage.
  • many cytokine receptors require dimerization to transduce a signal, and various dimeric ligands or domain repeats can be desirable.
  • Homologous polypeptides may be fusions between different surface markers, resulting in, e.g., a hybrid protein exhibiting receptor binding specificity.
  • 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 ligand, e.g., a receptor- binding segment, so that the presence or location of the fused ligand, or a binding composition, may be easily determined.
  • a reporter polypeptide e.g., luciferase
  • a segment or domain of a ligand e.g., a receptor- binding segment
  • Other gene fusion partners include bacterial ⁇ -galactosidase, trpE, Protein A, ⁇ -lactamase, alpha amylase, alcohol dehydrogenase, a FLAG fusion, and yeast alpha mating factor. See, e.g., Godowski, et al. (1988) Science 241:812-816.
  • a double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Such polypeptides may also have amino acid residues which have been chemically modified by phosphorylation, guanylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate or guanyl groups.
  • the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity tags as FLAG.
  • Fusion proteins will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods. Techniques for nucleic acid manipulation and expression are described generally, for example, in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.), Vols. 1-3, Cold Spring Harbor Laboratory. Techniques for synthesis of polypeptides are described, for example, in Merrifield (1963) T. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986) Science 232: 341-347; and Atherton, et al. (1989) Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Oxford; and chemical ligation, e.g., Dawson, et al. (1994) Science 266:776-779, a method of linking long synthetic peptides by a peptide bond.
  • This invention also contemplates the use of derivatives of these chemokines or receptors other than variations in amino acid sequence or glycosylation.
  • Such derivatives may involve covalent or aggregative association with chemical moieties.
  • These derivatives generally include: (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 ligands or other binding ligands.
  • a chemokine antigen 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 anti-chemokine antibodies or its receptor.
  • chemokines 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 a fluorescent moiety for use in diagnostic assays.
  • Purification of chemokine, receptor, or binding compositions may be effected by immobilized antibodies or receptor.
  • a solubilized chemokine or receptor or appropriate fragment of this invention can be used as an immunogen for the production of antisera or antibodies specific for the ligand, receptor, or fragments thereof.
  • the purified proteins can be used to screen monoclonal antibodies or chemokine-binding fragments prepared by immunization with various forms of impure preparations containing the protein.
  • antibody equivalents include antigen binding fragments of natural antibodies, e.g., Fv, Fab, or F(ab)2- Purified chemokines can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of the protein or cell fragments containing the protein, both of which may be diagnostic of an abnormal or specific physiological or disease condition.
  • chemokine protein fragments, or their concatenates may also serve as immunogens to produce binding compositions, e.g., antibodies of the present invention, as described immediately below.
  • this invention contemplates antibodies raised against certain amino acid sequences, e.g., in in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or proteins containing them.
  • this invention contemplates antibodies having binding affinity to or being raised against specific fragments, e.g., those which are predicted to lie on the outside surfaces of protein tertiary structure. Similar concepts apply to antibodies specific for receptors of the invention.
  • the present invention contemplates the isolation of additional closely related species variants. Southern and Northern blot analysis should establish that similar genetic entities exist in other related mammals, and establish the stringency of hybridization conditions to isolate such. It is likely that these chemokines and receptors are widespread in species variants, e.g., among the rodents and the primates.
  • the invention also provides means to isolate a group of related chemokines or receptors displaying both distinctness and similarities in structure, expression, and function. Elucidation of many of the physiological effects of the proteins will be greatly accelerated by the isolation and characterization of distinct species variants of the ligands. Related genes found, e.g., in various computer databases will also be useful, in many instances, for similar purposes with structurally related proteins. In particular, the present invention provides useful probes or search features for identifying additional homologous genetic entities in different species.
  • the isolated genes will allow transformation of cells lacking expression of a corresponding chemokine or receptor, e.g., either species types or cells which lack corresponding antigens and exhibit negative background activity. Expression of transformed genes will allow isolation of antigenically pure cell lines, with defined or single specie variants. This approach will allow for more sensitive detection and discrimination of the physiological effects of chemokine or receptor proteins.
  • Subcellular fragments e.g., cytoplasts or membrane fragments, can be isolated and used. Dissection of critical structural elements which effect the various differentiation functions provided by ligands is possible using standard techniques of modern molecular biology, particularly in comparing members of the related class. See, e.g., the homolog- scanning mutagenesis technique described in Cunningham, et al.
  • various segments can be substituted between species variants to determine what structural features are important in both receptor binding affinity and specificity, as well as signal transduction.
  • An array of different chemokine or receptor variants will be used to screen for variants exhibiting combined properties of interaction with different species variants.
  • Intracellular functions would probably involve segments of the receptor which are normally accessible to the cytosol. However, ligand internalization may occur under certain circumstances, and interaction between intracellular components and "extracellular" segments may occur.
  • the specific segments of interaction of a particular chemokine with other intracellular components may be identified by mutagenesis or direct biochemical means, e.g., cross- linking or affinity methods. Structural analysis by crystallographic or other physical methods will also be applicable. Further investigation of the mechanism of signal transduction will include study of associated components which may be isolatable by affinity methods or by genetic means, e.g., complementation analysis of mutants.
  • the controlling elements associated with the proteins may exhibit differential developmental, tissue specific, or other expression patterns. Upstream or downstream genetic regions, e.g., control elements, are of interest. Differential splicing of message may lead to membrane bound forms, soluble forms, and modified versions of ligand.
  • the present invention provides important reagents related to a physiological ligand-receptor interaction.
  • the foregoing description has focused primarily upon the mouse and human embodiments of the chemokines or receptors specifically described, those of skill in the art will immediately recognize that the invention provides other counterparts, e.g., from related species, rodents or primates.
  • Antibodies can be raised to these chemokines or receptors, including species or polymorphic variants, and fragments thereof, both in their naturally occurring forms and in their recombinant forms. Additionally, antibodies can be raised to chemokines or receptors in either their active or inactive forms, or in their native or denatured forms. Anti-idiotypic antibodies are also contemplated.
  • Antibodies including binding fragments and single chain versions, against predetermined fragments of the ligands can be raised by immunization of animals with concatemers or 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 chemokines or receptors, 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 10 ⁇ 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 preparative, diagnostic, or therapeutic value. They can be useful to purify or label the desired antigen in a sample, or may be potent antagonists that bind to ligand and inhibit binding to receptor or inhibit the ability of a ligand to elicit a biological response.
  • antibodies can be useful as non- neutralizing antibodies and can be coupled to, or as fusion proteins with, toxins or radionuclides so that when the antibody binds to antigen, a cell expressing it, e.g., on its surface via receptor, is killed.
  • these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker, and may effect drug targeting.
  • Antibodies to receptors may be more easily used to block ligand binding and /or signal transduction.
  • the antibodies of this invention can also be useful in diagnostic or reagent purification applications.
  • capture or non- neutralizing antibodies they can be screened for ability to bind to the chemokines or receptors without inhibiting ligand-receptor binding.
  • neutralizing antibodies they can be useful in competitive binding assays. They will also be useful in detecting or quantifying chemokine or receptors, e.g., in immunoassays. They may be used as purification reagents in immunoaffinity columns or as immunohistochemistry reagents.
  • Ligand or receptor fragments may be concatenated or joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens.
  • Short peptides will preferably be made as repeat structures to increase size.
  • a ligand and its 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 Serological Reactions, Dover Publications, New York, and Williams, et al. (1967) Methods in Immunology and Immunochemistry. Vol. 1, Academic Press, New York, for descriptions of methods of preparing polyclonal antisera.
  • a typical method involves hyperimmunization of an animal with an antigen. The blood of the animal is then collected shortly after the repeated immunizations and the gamma globulin fraction is isolated.
  • monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology (4th ed.), Lange Medical Publications, Los
  • 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. Large amounts of antibody may be derived from ascites fluid from an animal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly, U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. NatT. Acad. Sci. 86:10029-10033.
  • the antibodies of this invention can also be used for affinity chromatography in isolating the protein.
  • 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 chemokine protein will be released.
  • 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 these chemokines or receptors will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens.
  • the described peptide sequences and the related reagents are useful in isolating a DNA clone encoding these chemokines or receptors, e.g., from a natural source. Typically, it will be useful in isolating a gene from another individual, and similar procedures will be applied to isolate genes from related species, e.g., rodents or primates. Cross hybridization will allow isolation of ligand from other closely related species. A number of different approaches should be available to successfully isolate a suitable nucleic acid clone. Similar concepts apply to the receptor embodiments.
  • the purified protein or defined peptides are useful for generating antibodies by standard methods, as described above.
  • Synthetic peptides or purified protein can be presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley /Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press.
  • a chemokine or receptor may be used as a specific binding reagent, and advantage can be taken of its specificity of binding, much like an antibody would be used.
  • the chemokine receptors are typically 7 transmembrane proteins, which could be sensitive to appropriate interaction with lipid or membrane.
  • the signal transduction typically is mediated through a G-protein, through interaction with a G-protein coupled receptor.
  • the specific binding composition could be used for screening of an expression library made from a cell line which expresses a particular chemokine.
  • the screening can be standard staining of surface expressed ligand, or by panning. Screening of intracellular expression can also be performed by various staining or immunofluorescence procedures.
  • the binding compositions could be used to affinity purify or sort out cells expressing the ligand.
  • the peptide segments can also be used to predict appropriate oligonucleotides to screen a library, e.g., to isolate species variants.
  • the genetic code can be used to select appropriate oligonucleotides useful as probes for screening, n combination with polymerase chain reaction (PCR) techniques, synthetic oligonucleotides will be useful in selecting correct clones from a library. Complementary sequences will also be used as probes or primers. Anchored vector or poly-A complementary PCR techniques or complementary DNA of other peptides may be useful.
  • PCR polymerase chain reaction
  • This invention contemplates use of isolated DNA or fragments to encode a biologically active corresponding chemokine polypeptide.
  • this invention covers isolated or recombinant DNA which encodes a biologically active protein or polypeptide which is capable of hybridizing under appropriate conditions with the DNA sequences described herein.
  • Said biologically active protein or polypeptide can be an intact ligand. receptor, or fragment, and have an amino acid sequence as disclosed in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • this invention covers the use of isolated or recombinant DNA, or fragments thereof, which encode proteins which are homologous to a chemokine or receptor or which was isolated using such a cDNA encoding a chemokine or receptor as a probe.
  • the isolated DNA can have the respective regulatory sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and others.
  • nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other components which naturally accompany a native sequence, e.g., 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 and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • a substantially pure molecule includes isolated forms of the molecule.
  • An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain minor heterogeneity. 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 defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring purified forms.
  • nucleic acids comprising sequence derived using a synthetic oligonucleotide process. Such 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 sequence recognition site. Alternatively, it 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. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • a similar concept is intended for a recombinant, e.g., fusion, polypeptide.
  • synthetic nucleic acids which, by genetic code redundancy, encode polypeptides similar to fragments of these antigens, and fusions of sequences from various different species variants.
  • a significant "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least about 20 nucleotides, more generally at least about 23 nucleotides, ordinarily at least about 26 nucleotides, more ordinarily at least about 29 nucleotides, often at least about 32 nucleotides, more often at least about 35 nucleotides, typically at least about 38 nucleotides, more typically at least about 41 nucleotides, usually at least about 44 nucleotides, more usually at least about 47 nucleotides, preferably at least about 50 nucleotides, more preferably at least about 53 nucleotides, and in particularly preferred embodiments will be at least about 56 or more nucleotides, e.g., 60, 65, 75, 85, 100, 120, 150, 200, 250, 300, 400, etc.
  • Such fragments may have ends which begin and/or end at virtually all positions, e.g., beginning at nucleotides 1, 2, 3, etc., and ending at, e.g., 300, 299, 298, 287, etc., in combinatorial pairs.
  • Particularly interesting polynucleotides have ends corresponding to structural domain boundaries.
  • a DNA which codes for a particular chemokine or receptor protein or peptide will be very useful to identify genes, mRNA, and cDNA species which code for related or homologous ligands or receptors, as well as DNAs which code for homologous proteins from different species. There are likely homologs in closely related species, e.g., rodents or primates.
  • Narious chemokine proteins should be homologous and are encompassed herein, as would be receptors. However, proteins can readily be isolated under appropriate conditions using these sequences if they are sufficiently homologous. Typically, primate chemokines or receptors are of particular interest.
  • This invention further covers recombinant D ⁇ A molecules and fragments having a D ⁇ A sequence identical to or highly homologous to the isolated D ⁇ As set forth herein.
  • the sequences will often be operably linked to D ⁇ A segments which control transcription, translation, and D ⁇ A replication.
  • recombinant clones derived from the genomic sequences e.g., containing introns, will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow (1992) "Transgenic Animals” in Roitt (ed.) Encyclopedia of Immunology Academic Press, San Diego, pp.
  • Substantial homology in the nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generally at least about 56%, more generally at least about 59%, ordinarily at least about 62%, more ordinarily at least about 65%, often at least about 68%, more often at least about 71%, typically at least about 74%, more typically at least about 77%, usually at least about 80%, more usually at least about 85%, preferably at least about 90%, more preferably at least about 95 to 98% or more, and in particular embodiments, as high at about 99% or more of the nucleotides.
  • substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence derived from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 30 nucleotides, preferably at least about 65% over a stretch of at least about 25 nucleotides, more preferably at least about 75%, and most preferably at least about 90% over about 20 nucleotides. See,
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 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.
  • PCR primers will generally have high levels of matches over potentially shorter 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 those 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 1000 mM, usually less than about 500 mM, more usually less than about 400 mM, typically less than about 300 mM, preferably less than about 200 mM, and more preferably less than about 150 mM, e.g., 20-50 mM.
  • the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson Q968 ⁇ I. Mol. Biol. 31:349-370.
  • Corresponding chemokines or receptors from other closely related species can be cloned and isolated by cross-species hybridization.
  • sequences from a sequence data base may be recognized as having similarity. Homology may be very low between distantly related species, and thus hybridization of relatively closely related species is advisable.
  • preparation of an antibody preparation which exhibits less species specificity may be useful in expression cloning approaches. PCR approaches using segments of conserved sequences will also be used.
  • NIL Making Chemokines or Receptors; Mimetics D ⁇ A which encodes each respective chemokine, receptor, or fragments thereof can be obtained by chemical synthesis, screening cD ⁇ A libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples.
  • This D ⁇ A can be expressed in a wide variety of host cells for the synthesis of a full-length ligand 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 molecules; for expression cloning or purification; and for structure /function studies.
  • Each antigen or its fragments can be expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules can be substantially purified to be 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 pharmaceutically acceptable carrier and/or diluent.
  • the antigens or antibodies, or portions thereof, may be expressed as fusions with other proteins.
  • Expression vectors are typically self-replicating DNA or RNA constructs containing the desired antigen 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 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 contain DNA which encode embodiments of a chemokine, receptor, or a fragment thereof, typically encoding a biologically active 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 cDNA coding for each chemokine or receptor in a prokaryotic or eukaryotic host, where the vector is compatible with the host and where the eukaryotic cDNA coding for the protein is inserted into the vector such that growth of the host containing the vector expresses the cDNA in question.
  • expression 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 ligand 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 a chemokine or receptor gene or its fragments into the host DNA by recombination, or to integrate a promoter which controls expression of an endogenous gene.
  • Vectors as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles, including those 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 many 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 Rodriquez, et al. (1988)(eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, MA.
  • Transformed cells include cells, preferably mammalian, that have been transformed or transfected with a chemokine or receptor gene containing vector constructed using recombinant DNA techniques.
  • Transformed host cells usually express the ligand, receptor, or its fragments, but for purposes of cloning, amplifying, and manipulating its DNA, do not need to express the protein.
  • This invention further contemplates culturing transformed cells in a nutrient medium, thus permitting the protein to accumulate in the culture.
  • the protein can be recovered, from the culture or from the culture medium, or from cell membranes.
  • DNA sequences are operably linked when they are functionally related to each other.
  • DNA for a presequence or secretory signal 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 Dictyostelium.
  • 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 these chemokines or their 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.
  • Lower eukaryotes e.g., yeasts and Dictyostelium, may be transformed with chemokine or receptor sequence containing nucleic acids.
  • the most common lower eukaryotic host is the baker's yeast, Saccharomyces 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 desired protein 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 the preferred host cells for expression of the functionally active chemokine or receptor proteins.
  • most any higher eukaryotic tissue culture cell line is workable, e.g., insect baculovirus expression systems, whether from an invertebrate or vertebrate source.
  • mammalian cells are preferred, in that the processing, both cotranslationally and posttranslationally, will be typically most like natural. 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 include an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, e.g., from such sources as from adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalo virus. Representative examples of suitable expression vectors include pCDNAl; pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneo Poly-A, see Thomas, et al. (1987) Cell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
  • chemokine or receptor polypeptide 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.
  • a chemokine or receptor gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes. Using this approach, certain mammalian glycosylation patterns will be achievable or approximated in prokaryote or other cells.
  • a chemokine, receptor, or a fragment thereof may be engineered to be phosphatidyl inositol (PI) linked to a cell membrane, but can be removed from membranes by treatment with a phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C.
  • PI phosphatidyl inositol
  • 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 dicyclohexyl-carbodiimide (DCCD)/ additive process can be used.
  • Solid phase and solution phase syntheses are both applicable to the foregoing processes.
  • chemokines, receptors, fragments, or derivatives are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid. Amino groups that are not being used in the coupling reaction are typically protected to prevent coupling at an incorrect location.
  • the C-terminal amino acid is typically 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-alkyloxycarbonyl-hydrazidated 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, e.g., by Merrifield, et al. (1963) in I. Am. Chem. Soc. 85:2149-2156.
  • the prepared ligand and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, e.g., by extraction, precipitation, electrophoresis, and various forms of chromatography, and the like.
  • the various chemokines or receptors of this invention can be obtained in varying degrees of purity depending upon its desired use. Purification can be accomplished by use of the protein purification techniques disclosed herein or by the use of the antibodies herein described, e.g., in immunoabsorbant affinity chromatography.
  • This immunoabsorbant affinity chromatography is typically carried out, e.g., by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate source cells, lysates of other cells expressing the ligand or receptor, or lysates or supematants of cells producing the desired proteins as a result of DNA techniques, see below. V ⁇ i. Uses
  • the present invention provides reagents which will find use in diagnostic applications as described elsewhere herein, e.g., in the general description for developmental abnormalities, or below in the description of kits for diagnosis.
  • This invention also provides reagents with significant therapeutic potential.
  • These chemokines and receptors naturally occurring or recombinant, fragments thereof, and binding compositions, e.g., antibodies thereto, along with compounds identified as having binding affinity to them, should be useful in the treatment of conditions associated with abnormal physiology or development, including inflammatory conditions, e.g., asthma.
  • modulation of trafficking of leukocytes is one likely biological activity, but a wider tissue distribution might suggest broader biological activity, including, e.g., antiviral effects.
  • Abnormal proliferation, regeneration, degeneration, and atrophy may be modulated by appropriate therapeutic treatment using the compositions provided herein.
  • a disease or disorder associated with abnormal expression or abnormal signaling by a chemokine or ligand for a receptor should be a likely target for an agonist or antagonist of the ligand.
  • Antibodies to the chemokines or receptors can be purified and then used diagnostically or therapeutically, alone or in combination with other chemokines, cytokines, or antagonists thereof.
  • These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients. These combinations can be sterile 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, including forms which are not complement binding.
  • modifications to the antibody molecules or antigen binding fragments thereof may be adopted which affect the pharmacokinetics or pharmacodynamics of the therapeutic entity.
  • Drug screening using antibodies or receptor or fragments thereof can be performed to identify compounds having binding affinity to each chemokine or receptor, 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 ligand.
  • This invention further contemplates the therapeutic use of antibodies to these chemokines as antagonists, or to the receptors as antagonists or agonists. This approach should be particularly useful with other chemokine or receptor species variants.
  • reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy in various populations, including racial subgroups, age, gender, etc. 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.
  • 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 a slow release apparatus will often be utilized for continuous administration.
  • a chemokine, fragments thereof, or antibodies to it 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 often preferable to present it as a pharmaceutical formulation.
  • Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Carriers may improve storage life, stability, etc.
  • 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 any 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, Perm.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York; Lieberman, et al.
  • kits and assay methods which are capable of screening compounds for binding activity to the proteins.
  • automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. See, e.g., Fodor, et al. (1991) Science 251:767-773, which describes means for testing of binding affinity by a plurality of defined polymers synthesized on a solid substrate.
  • suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble chemokine as provided by this invention.
  • antagonists can normally be found once a ligand has been structurally defined. Testing of potential ligand analogs is now possible upon the development of highly automated assay methods using physiologically responsive cells. In particular, new agonists and antagonists will be discovered by using screening techniques described herein.
  • Viable cells could also be used to screen for the effects of drugs on respective chemokine or G-protein coupled receptor mediated functions, e.g., second messenger levels, i.e., Ca ++ ; inositol phosphate pool changes (see, e.g., Berridge (1993) Nature 361:315-325 or Billah and Anthes (1990) Biochem. T. 269:281-291); cellular morphology modification responses; phosphoinositide lipid turnover; an antiviral response, and others.
  • Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system. Calcium sensitive dyes will be useful for detecting Ca ++ levels, with a fluorimeter or a fluorescence cell sorting apparatus.
  • Rational drug design may also be based upon structural studies of the molecular shapes of the chemokines, other effectors or analogs, or the receptors. 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.
  • x-ray crystallography or 2 dimensional NMR techniques.
  • Purified chemokine or receptor can be coated directly onto plates for use in the aforementioned drug screening techniques, and may be associated with detergents or lipids.
  • non- neutralizing antibodies e.g., to the chemokines or receptors can be used as capture antibodies to immobilize the respective protein on the solid phase.
  • Kits This invention also contemplates use of chemokine or receptor proteins, fragments thereof, peptides, binding compositions, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of ligand, antibodies, or receptors.
  • the kit will have a compartment containing a defined chemokine or receptor peptide or gene segment or a reagent which recognizes one or the other, e.g., binding reagents.
  • a kit for determining the binding affinity of a test compound to a chemokine or receptor would typically comprise a test compound; a labeled compound, for example an antibody having known binding affinity for the protein; a source of chemokine or receptor (naturally occurring or recombinant); and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the ligand or receptor.
  • a labeled compound for example an antibody having known binding affinity for the protein
  • a source of chemokine or receptor naturally occurring or recombinant
  • a means for separating bound from free labeled compound such as a solid phase for immobilizing the ligand or receptor.
  • a preferred kit for determining the concentration of, for example, a chemokine or receptor in a sample would typically comprise a labeled compound, e.g., antibody, having known binding affinity for the target, a source of ligand or receptor (naturally occurring or recombinant) and a means for separating the bound from free labeled compound, for example, a solid phase for immobilizing the chemokine or receptor. Compartments containing reagents, and instructions for use or disposal, will normally be provided.
  • Antibodies including antigen binding fragments, specific for the chemokine or receptor, or fragments are useful in diagnostic applications to detect the presence of elevated levels of chemokine, receptor, and/or its fragments.
  • diagnostic assays can employ lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and further can involve the detection of antigens related to the ligand or receptor in serum, or the like. Diagnostic assays may be homogeneous (without a separation step between free reagent and antigen complex) or heterogeneous (with a separation step).
  • RIA radioimmunoassay
  • ELISA enzyme- linked immunosorbent assay
  • EIA enzyme immunoassay
  • EMIT enzyme-multiplied immunoassay technique
  • SFIA substrate- labeled fluorescent immunoassay
  • unlabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the primary antibody to a chemokine or receptor or to a particular fragment thereof.
  • Similar assays have also been extensively discussed in the literature. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH.
  • Anti-idiotypic antibodies may have similar uses to diagnose presence of antibodies against a chemokine or receptor, as such may be diagnostic of various abnormal states. For example, overproduction of a chemokine or receptor may result in production of various immunological reactions which may be diagnostic of abnormal physiological states, particularly in various inflammatory or asthma conditions.
  • the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay.
  • the protocol, and the label either labeled or unlabeled antibody or labeled chemokine or receptor 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 or containers for each useful reagent.
  • the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.
  • labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.
  • the ligand, test compound, chemokine, 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.
  • Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups. There are also numerous methods of separating bound from the free ligand, or alternatively bound from free test compound.
  • the chemokine or receptor can be immobilized on various matrixes, perhaps with detergents or associated lipids, followed by washing. Suitable matrixes include plastic such as an ELISA plate, filters, and beads. Methods of immobilizing the chemokine or 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 may involve the precipitation of antigen/ antibody 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. 30:1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678. Methods for linking proteins or their fragments to the various labels have been extensively reported in the literature and do not require detailed discussion here.
  • oligonucleotide or polynucleotide sequences taken from the sequence of the chemokine or receptor. These sequences can be used as probes for detecting levels of the ligand message in samples from patients suspected of having an abnormal condition, e.g., an inflammatory, physiological, or developmental problem.
  • an abnormal condition e.g., an inflammatory, physiological, or developmental problem.
  • 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 oligonucleotide 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
  • 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.
  • chemokine without interfering with the binding to its receptor can be determined.
  • an affinity label can be fused to either the amino- or carboxy-terminus of the ligand.
  • An expression library can be screened for specific binding of chemokine, e.g., by cell sorting, or other screening to detect subpopulations which express such a binding component. See, e.g., Ho, et al.
  • a receptor means to identify the ligand exist.
  • Methods for using the receptor e.g., on the cell membrane, can be used to screen for ligand by, e.g., assaying for a common G-protein linked signal such as Ca++ flux. See Lerner (1994) Trends in Neurosciences 17:142-146. It is likely that the ligands for these receptors are chemokines.
  • Protein cross-linking techniques with label can be applied to a isolate binding partners of a chemokine. This would allow identification of protein which specifically interacts with a chemokine, e.g., in a ligand-receptor like manner.
  • 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); Deutscher (1990) "Guide to Protein Purification” in Methods in Enzymology, vol. 182, and other volumes in this series; and manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA.
  • Combination with recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease- removable sequence.
  • appropriate segments e.g., to a FLAG sequence or an equivalent which can be fused via a protease- removable sequence.
  • the IBICK was isolated from a cDNA library made from a human astrocytoma cell line. See, e.g., Rani, et al. (1996) T. Biol. Chem. 271:22878-22884. There is reported a gene which is not inducible by LFN- ⁇ , but inducible by IFN- ⁇ and/or IFN- ⁇ . Applicants have identified this gene as a chemokine, and designated it Interferon Beta Induced ChemoKine (IBICK), which is described in SEQ ID NO: 2. Individual cDNA clones are sequenced using standard methods, e.g., the Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA), and the sequence is further characterized.
  • IBICK Interferon Beta Induced ChemoKine
  • the predicted signal sequence corresponds to amino acids metl to about gly21, so the mature form should begin with phe22 and run about 74 amino acids. Additional processing may occur in a physiological system.
  • Computer analysis and alignments for related genes indicates the closest match is to the two IFN- ⁇ regulated chemokines MIG and LP10, but other related molecules are chemoknes. See, e.g., Faubert (1993) Biochem. Bioph s. Res. Commun. 192:223-230; and Luster, et al. (1985) Nature 315:672-676. This similarity in sequence may well correlate with similarity in regulation, which suggests related functions.
  • the rarity of related sequences in the existing sequence databases suggests low message levels, tight negative regulation, and /or a distribution pattern in cell types not yet analysed.
  • the IFN- ⁇ regulatable nature of this chemokine suggests a role as an antiviral or antitumor agent. Its non-ELR chemokine structure suggests angiostatic, in contrast to angiogenic, activity, which may be important in tumor therapy.
  • ILINCK SEQ ID NO: 3 and 4
  • Total RNA can be isolated, e.g., using the guanidine thiocyanate/CsCl gradient procedure as described by Chirgwin, et al. (1978) Biochem. 18:5294- 5299.
  • RNA is isolated using, e.g., the OLIGOTEX mRNA isolation kit (QIAGEN). Such RNA from these cells is used to synthesize first strand cDNA, e.g., by using Notl/Oligo-dT primer (Gibco-BRL, Gaithersburg, MD). Double-stranded cDNA is synthesized, ligated with BstXI adaptors, digested with NotI, size fractionated for > 0.5 kilobase pairs (kb) and ligated into the Notl/BstXI sites of pJFE-14, a derivative of the pCDSRa vector. See Takebe, et al. (1985) Mol. Cell Biol. 8:466-472. Electro-competent E. coli DHlOa cells (Gibco-BRL) are used for transformation.
  • Notl/Oligo-dT primer Gaithersburg, MD
  • Double-stranded cDNA is synthesized, ligated with
  • the gene apparently produces at least two different sized transcripts, 0.6 kB and a 1.5 kB, which are differently regulated.
  • the larger transcript is inducible by IL-10, which is unusual for a chemokine, so it has been designated InterLeukin 10 INduced ChemoKine (ILINCK), which is described in SEQ ID NO: 4.
  • ILINCK InterLeukin 10 INduced ChemoKine
  • Individual cDNA clones were sequenced using standard methods, e.g., the Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA), and the sequence was further characterized.
  • the predicted signal sequence corresponds to amino acids metl to about ser23, so the mature form should begin with gln24 and run about 73 amino acids. Additional processing may occur in a physiological system. Computer analysis and alignments for related genes indicates the closest match is to other chemokines.
  • a Southern blot may indicate the extent of homology across species, and either a cDNA library or mRNA can be screened to identify an appropriate cell source. The physiological state of many different cell types may also be evaluated for increased expression of the gene.
  • ILINCK mRNA is induced in monocytes by IL-10, a most notable feature. This observation strongly suggests that ILINCK has anti-inflammatory properties. It is postulated that ILINCK will be a potential therapeutic in autoimmune or other inflammatory disorders. See, e.g., Samter, et al. (eds) Immunological Diseases vols. 1 and 2, Little, Brown and Co. C. Rodent CXC-143
  • the CXC-143 (SEQ ID NO: 5) was isolated from a cDNA library made from a mouse placenta cDNA library. The partial sequence provided lacks an identifiable initiation codon and termination codon. This chemokine has been designated CXC-143, and is described in SEQ ID NO: 6, 8 and 10. Individual cDNA clones were sequenced using standard methods, e.g., the Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA), and the sequence was further characterized, but the sequence remains incomplete. Clearly the chemokine is a non-ELR class CXC chemokine.
  • the MCP243 (SEQ ID NO: 11) was isolated from a cDNA library made from a mouse cDNA library.
  • the partial sequence provided lacks an identifiable initiation codon and various upstream chemokine motifs. This chemokine has been designated MCP23, and is described in SEQ ID NO: 12 and 14.
  • Individual cDNA clones are sequenced using standard methods, e.g., the Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA), and the sequence further characterized, but the sequence remains incomplete.
  • the encoded protein is a chemokine, and will have many similar biological activities related to the other members of the MCPs.
  • a Southern blot may indicate the extent of homology across species, and either a cDNA library or mRNA can be screened to identify an appropriate cell source. The physiological state of many different cell types may also be evaluated for increased expression of the gene.
  • the primate R277 clone was derived from human fetal tissue cDNA library.
  • the nucleotide and amino acid sequences are provided in SEQ ID NO: 15, 16, 17 and 18.
  • chemokine receptors include the chemokine receptors, and protease, e.g., thrombin, receptors.
  • Structural motifs suggest that the receptor may contain motifs characteristic of the chemokine receptor family, and of the protease receptor family.
  • the transmembrane segments should be about as follows for SEQ ID NO: 16: TM3 to vall7; TM4 from arg36 to leu57; TM5 from asn89 to arglll; TM6 from leul34 to leul ⁇ l; and TM7 from metl78 to val200.
  • a DRY motif is found, e.g., near residue 18.
  • the amino terminal segment is probably an extracellular segment (El), and the others would be E2 between TM2 and TM3; E3 between TM4 and TM5; and E4 between TM6 and TM7.
  • the intracellular segments should then run II between TMl and TM2; 12 between TM3 and TM4, 13 between TM5 and TM6, and 14 the carboxy terminus from the end of TM7. Additional processing may occur in a physiological system.
  • GPCRs conserved residues among the GPCRs would include, e.g., arg33, cys67, and gly217, among others in the transmembrane segments.
  • a computer analysis of GPCR sequences will indicate residues characteristic of the family members.
  • Core transmembrane segments for the R277 receptor sequence are predicted, using SEQ ID NO: 18 numbering, about: TMl leul ⁇ to leu41; TM2 ile ⁇ l to ile71; TM3 leu93 to leul25; TM4 leul32 to leul50; TM5 leul83 to val206; TM6 ile224 to ala256; and TM7 ile274 to val293.
  • the rodent HST01.1 clone was derived from a cDNA library made from mouse TcR ab+ CD4- CD8- T cells. See Zlotnik, et al. (1992) T. Immunol. 149:1211-1215. Individual cDNA clones are sequenced using standard methods, and the sequence identified and further characterized. The partial nucleotide sequence is provided in SEQ ID NO: 19, encoding a polypeptide fragment of about 74 amino acids (SEQ ID NO: 20). Complete rodent HSTOl.l nucleotide and amino acid sequences are prtovided in SEQ ID NO: 21 and 22.
  • the closest related genes are various G-protein coupled receptors. Structural motifs suggest that the receptor may contain motifs characteristic of the chemokine receptor family, and of the protease receptor family.
  • the transmembrane segments based upon hydrophobicity plots and comparisons with other similar GPCRs, should be about as follows: TMl from leu58 to leu78; TM2 from phe90 to valllO; TM3 from alal26 to phel46; TM4 from leul69 to leul89; TM5 from phe223 to val243; TM6 from val256 to asp277; and TM7 from val301 to fly321.
  • a DRY box motif runs from about aspl47 to alal55.
  • Chemokine receptors are generally considered useful targets for novel drug discovery, where the therapeutics would agonise or antagonise the binding of natural ligand(s) of the receptor. These receptor-ligand interactions may result in inflammation, cell recruitment, an/or cell activation processes. Some of these receptors are the portal of entry of infectious agents, e.g., viruses. Therefore, therapeutics directed against the chomokine receptor may find application in these diseases. In addition, the receptors may be important in determining fundamental structure or physiological responses. Other rodent counterparts should be isolatable using the entire coding portion of this mouse clone as a hybridization probe.
  • a Southern blot may indicate the extent of homology across species, and either a cDNA library or mRNA can be screened to identify an appropriate cell source. The physiological state of many different cell types may also be evaluated for increased expression of the gene.
  • MIG and LP-10 as it is incapable of desensitizing the response of the receptor to the other chemokines. MIG can desensitize the response to 6Ckine, but IP-10 does not. These results imply that 6Ckine may have angiostatic and antitumor activities similar to those of MIG and IP-10. See, e.g., Sgadari, et al. (1997) Blood 89:2635-2643; Arenberg, et al. (1996) T. Exp. Med. 184:981-992; Loetscher, et al. (1996) T. Exp. Med. 184:963-969; Sgadari, et al. (1996) Proc. Natl Acad. Sci.
  • the rodent 941D12 clone was derived from a cDNA library made from mouse Th3 polarized cells. Production of 3W Thl or Th2 cells is described in Openshaw, et al. (1995) T. Exp. Med. 182:1357- 1367. Briefly, Thl or Th2 populations were derived from CD4+ T cells stimulated with antigen and antigen presenting cells in the presence of IL-12 or IL-4. Cells were stimulated once each week for 3 weeks, then harvested and restimulated, e.g., with PMA and ionmycin for 4 h. See Murphy, et al. (1996) T. Exp. Med. 183:901-913. A subtraction step was introduced to remove sequences found in mouse L cells. See, e.g., Hara, et al. (1994) Blood 84:189-199.
  • the predicted core transmembrane segments are about TMl leu48 to ile64; TM2 val77 to leu93; TM3 vall24 to vall40; TM4 ilel55 to vall71; TM5 leu201 to ile217; TM6 ser238 to thr254; and TM7 ile279 to leu295.
  • Other rodent counterparts should be isolatable using the entire coding portion of this mouse clone as a hybridization probe.
  • a Southern blot may indicate the extent of homology across species, and either a cDNA library or mRNA can be screened to identify an appropriate cell source. The physiological state of many different cell types may also be evaluated for increased expression of the gene.
  • synthetic peptides may be prepared to be used as antigen, administered to an appropriate animal, and either polyclonal or monoclonal antibodies prepared. Short peptides, e.g., less than about 10 amino acids may be expressed as repeated sequences, while longer peptides may be used alone or conjugated to a carrier.
  • animals are immunized with peptides or complete proteins from SEQ ID NO: 12, 14, 16, or 18. Highest specificity will result when the polypeptides are selected from portions which are most unique, e.g., not from conserved sequence regions. The animals may be used to collect antiserum, or may be used to generate monoclonal antibodies.
  • Antiserum is evaluated for use, e.g., in an ELISA, and will be evaluated for utility in immunoprecipitation, e.g., typically native, or Western blot, e.g., denatured antigen, analysis. Monoclonal antibodies will also be evaluated for those same uses.
  • the antibodies provided will be useful as immunoaffinity reagents, as detection reagents, for immunohistochemistry, and as potential therapeutic reagents, either as agonist or antagonist reagents.
  • Chemokine proteins are produced, e.g., in COS cells transfected with a plasmid carrying the chemokine cDNA by electroporation. See, Hara, et al. (1992) EMBO T. 10:1875-1884. Physical analytical methods may be applied, e.g., CD analysis, to compare tertiary structure to other chemokines to evaluate whether the protein has likely folded into an active conformation. After transfection, a culture supernatant is collected and subjected to bioassays. A mock control, e.g., a plasmid carrying the luciferase cDNA, is used.
  • a positive control e.g., recombinant murine MLP-la from R&D Systems (Minneapolis, MN) is typically used.
  • antibodies may be used to block the biological activities, e.g., as a control.
  • Lymphocyte migration assays are performed as previously described, e.g., in Bacon, et al. (1988) Br. T. Pharmacol. 95:966-974.
  • Murine Th2 T cell clones, CDC-25 see Tony, et al. (1985) T. Exp. Med. 161:223-241
  • HDK-1 see Cherwinski, et al. (1987) T. Exp. Med. 166:1229-1244
  • R. Coffman and A. O'Garra DNAX, Palo Alto, CA
  • Ca2+ flux upon chemokine stimulation is measured, e.g., according to the published procedure described in Bacon, et al. (1995) T. Immunol. 154:3654-3666.
  • MLP-la e.g., is capable of inducing immediate transients of calcium mobilization.
  • the levels of chemokine used in these assays will be comparable to those used for the chemotaxis assays (1/1000 dilution of conditioned supematants).
  • Retroviral infection assays have also been described, and recent description of certain chemokine receptors in retroviral infection processes may indicate that similar roles may apply these receptors. See, e.g., Baiter (1996) Science 272:1740 (describing evidence for chemokine receptors as coreceptors for HIV); and Deng, et al. (1996) Nature 381:661-666.
  • biological activity may be measured in response to an appropriate ligand.
  • the receptors are transfected into an assortment of cell types, each of which is likely to possess the intracellular signaling components compatible with the expressed receptor. Narious ligand sources are tested to find a source of ligand which results in a G-protein coupled response. Alternatively, the cells are tested for Ca++ flux in response to such ligands. Flux may be conveniently measured by electrophysiological means, or by Ca++ sensitive dyes.
  • a robust and sensitive assay is selected as described above, e.g., on immune cells, neuronal cells, or stem cells.
  • Chemokine is added to the assay in increasing doses to see if a dose response is detected.
  • a proliferation assay cells are plated out in plates. Appropriate culture medium is provided, and chemokine is added to the cells in varying amounts. Growth is monitored over a period of time which will detect either a direct effect on the cells, or an indirect effect of the chemokine.
  • an activation assay or attraction assay is used.
  • An appropriate cell type is selected, e.g, hematopoietic cells, myeloid (macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid (T cell, B cell, or NK cells), neural cells (neurons, neuroglia, oligodendrocytes, astrocytes, etc.), or stem cells, e.g., progenitor cells which differentiate to other cell types, e.g., gut crypt cells and undifferentiated cell types.
  • Other assays will be those which have been demonstrated with other chemokines. See, e.g., Schall and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy & Immunol. 109:97-109.
  • Standard mutagenesis analysis is performed, e.g., by generating many different variants at determined positions, e.g., at the structural 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.
  • the cDNA is labeled, e.g., nick-translated with biotin-14 dATP and hybridized in situ at a final concentration of 5 ng/ ⁇ l to metaphases from two normal males.
  • Fluorescence in situ hybridization (FISH) method may be modified from that described by Callen, et al. (1990). Ann. Genet. 33:219-221, in that chromosomes are stained before analysis with both prodidium iodide (as counter stain) and DAPI (for chromosome identification). Images of metaphase preparations are captured by a CCD camera and computer enhanced. Identification of the approapriate labeled chromosomes is determined.
  • RNA blot and hybridization are performed according to the standard methods in Maniatis, et al. (1982) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. An appropriate fragment or the whole coding sequence of a cDNA fragment is selected for use as a probe.
  • a control cDNA e.g., glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA (Clontech, Palo Alto CA).
  • G3PDH glyceraldehyde 3-phosphate dehydrogenase
  • mRNA from the appropriate cell source using the probe will determine the natural size of message. It will also indicate whether different sized messages exist.
  • the messages will be subject to analysis after isolation, e.g., by PCR or hybridization techniques.
  • Northern blot analysis may be performed on many different mRNA sources, e.g., different tissues, different species, or cells exhibiting defined physiological responses, e.g., activation conditions or developmental conditions.
  • cDNA libraries may be used to evaluate sources which are difficult to prepare.
  • a "reverse Northern” uses cDNA inserts removed from vector, but multiplicity of bands may reflect either different sized messages, or may be artifact due to incomplete reverse transcription in the preparation of the cDNA library. In such instances, verification may be appropriate by standard Northern analysis.
  • Southern blots may be used to evaluate species distribution of a gene.
  • the stringency of washes of the blot will also provide information as to the extent of homology of various species counterparts.
  • Tissue distribution, and cell distribution may be evaluated by immunohistochemistry using antibodies.
  • in situ nucleic acid hybridization may also be used in such analysis.
  • Certain distribution data may be ascertained by the frequency and tissue types where messages have been found and collected in sequence databases, e.g., GenBank or proprietary collections.
  • the IBICK was isolated from a human astrocyte cell. There is little distribution data generated at this time.
  • the IBICK was isolated from a human liver library. It is expressed in NK cells, gd T cells, and activated and resting monocytes. Libraries from human T cell lines, e.g., Mot ⁇ l, HY106, and Mut72 show expression. Northern blots of adult spleen, thymus, prostate, testis, yterus, small intestine, colon, and peripheral blood leukocytes give no detectable signal. These data suggest that the expression is in a limited subset of specialized cells. Neither activated splenocytes not activated PBLs give detectable signals.
  • CXC-143 The CXC-143 gene was identified from a cDNA library made from mouse placenta. The sequence has appeared in cDNA libraries from varous C57BL/6J mouse embryo or placenta. Notably, early embryo libraries, e.g., from 8.5 days post conception, exhibited signal. A homologous, human gene is found in placenta, fetal heart, breast, and fetal liver/spleen. This distibution suggests a role of the molecule in early stages of development. The molecule, or its antagonist, should be useful in various early developmental conditions.
  • R277 GPCR The R277 gene was identified from a cDNA library made from
  • the HST01.1 gene was isolated from an ab TCR+ CD4+ CD8+ cell library. Distribution analysis showed a strong positive signal by Northern analysis in spleen and lung. Southern analysis showed strong positive signals in Thl clones, CD4+NK1.1+ cells, abTcR double negative cells, Dl.l resting Thl T cells, and mesentary lymph nodes. Positive signals were detected in macrophages, Th2 cells, and CD3+abTcR double negative cells, Dl.l ConA stimulated Thl T cells, resting J774 cells, and LPS and IL-10 stimulated J774 cells. Weaker signals were detected in thymus, activated pro-T cells, and LPS and anti-IL-10 treated J774 cells.
  • the 941D12 GPCR was isolated from a mouse 3 week polarized Th2 cell cDNA library, subtracted with cDNA sequences from a mouse L cell. See, e.g., Openshaw, et al. (1995) T. Exp. Med. 182:1357-1367; and Murphy, et al. (1996) T. Exp. Med. 183:901-913.
  • DNA (5 mg) from a primary amplified cDNA library was digested with appropriate restriction enzymes to release the inserts, run on a 1% agarose gel and transferred to a nylon membrane (Schleicher and Schuell, Keene, NH).
  • Samples for mouse mRNA isolation include, e.g.: resting mouse fibroblastic L cell line (C200); Braf:ER (Braf fusion to estrogen receptor) transfected cells, control (C201); T cells, TH1 polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IFN- g and anti IL-4; T200); T cells, TH2 polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IL-4 and anti-IFN- ⁇ ; T201); T cells, highly TH1 polarized (see Openshaw, et al. (1995) _ Exp. Med.
  • T cells highly T ⁇ 2 polarized (see Openshaw, et al. (1995) L Exp. Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 16 h pooled; T203); CD44- CD25+ pre T cells, sorted from thymus (T204); TH1 T cell clone Dl.l, resting for 3 weeks after last stimulation with antigen (T205); TH1 T cell clone Dl.l, 10 mg/ml ConA stimulated 15 h (T206); TH2 T cell clone CDC35, resting for 3 weeks after last stimulation with antigen (T207); TH2 T cell clone CDC35, 10 mg/ml ConA stimulated 15 h (T208); Mel 14+ naive T cells from spleen, resting (T209); Mell4+ T cells, polarized to
  • Labeled reagent is useful for screening of an expression library made from a cell line which expresses a chemokine or receptor, as appropriate.
  • Standard staining techniques are used to detect or sort intracellular or surface expressed ligand, or surface expressing transformed cells are screened by panning. Screening of intracellular expression is performed by various staining or immunofluorescence procedures. See also, e.g., McMahan, et al. (1991) EMBO T. 10:2821- 2832. For example, on day 0, precoat 2-chamber permanox slides with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min at room temperature. Rinse once with PBS.
  • DAB Vector diaminobenzoic acid
  • the binding compositions are used to affinity purify or sort out cells expressing the ligand or receptor. See, e.g., Sambrook, et al. or Ausubel et al.
  • SEQ ID NO: 1 is primate IBICK nucleotide sequence
  • SEQ ID NO: 2 is primate IBICK amino acid sequence
  • SEQ ID NO: 3 is primate ILINCK nucleotide sequence
  • SEQ ID NO: 4 is primate ILINCK amino acid sequence
  • SEQ ID NO: 5 is partial rodent CXC-143 nucleotide sequence
  • SEQ ID NO: 6 is partial rodent CXC-143 amino acid sequence.
  • SEQ ID NC ⁇ : 7 is partial alternative rodent CXC-143 nucleotide sequence.
  • SEQ ID NO : is partial alternative rodent CXC-143 amino acid sequence.
  • SEQ ID NO: 9 is revised partial rodent CXC-143 nucleotide sequence.
  • SEQ ID NO: 10 is revised partial rodent CXC-143 amino acid sequence.
  • SEQ ID NO: 11 is partial rodent MCP243 nucleotide sequence.
  • SEQ ID NO: 12 is partial rodent MCP243 amino acid sequence.
  • SEQ ID NO: 13 is revised complete rodent MCP243 nucleotide sequence.
  • SEQ ID NO: 14 is revised complete rodent MCP243 amino acid sequence.
  • SEQ ID NO: 15 is primate R277 nucleotide sequence.
  • SEQ ID NO: 16 is primate R277 amino acid sequence.
  • SEQ ID NO: 17 is revised primate R277 nucleotide sequence.
  • SEQ ID NO: 18 is revised primate R277 amino acid sequence.
  • SEQ ID NO: 19 is partial rodent HST01.1 nucleotide sequence.
  • SEQ ID NO: 20 is partial rodent HST01.1 amino acid sequence.
  • SEQ ID NO: 21 is rodent HST01.1 nucleotide sequence.
  • SEQ ID NO: 22 is rodent HST01.1 amino acid sequence.
  • SEQ ID NO: 23 is partial rodent 941D12 nucleotide sequence.
  • SEQ ID NO: 24 is partial rodent 941D12 amino acid sequence.
  • SEQ ID NO: 25 is rodent 941D12 nucleotide sequence.
  • SEQ ID NO: 26 is rodent 941D12 amino acid sequence.
  • nucleotide 246 designated C may be A, C, G, or T"
  • MOLECULE TYPE protein
  • MOLECULE TYPE protein
  • nucleotide designated C may be A, C, G, or T"
  • nucleotide designated A may be A, C, G, or T"
  • Trp Asn Ile Leu Lys Ile Cys Thr Gly Arg Cys Asn Thr Ser Gin Arg 210 215 220 Gin Arg Lys Arg Ile Leu Ser Val Ser Thr Lys Asp Thr Met Glu Leu 225 230 235 240
  • GAG AAA TGG CAA ATC AAC CTC AAC TTG TTC AGG ACG TGT ACA GGC TAT 576 Glu Lys Trp Gin Ile Asn Leu Asn Leu Phe Arg Thr Cys Thr Gly Tyr 180 185 190
  • MOLECULE TYPE protein
  • AAGCC ATG TAC CTT GAG GTT AGT GAA CGT CAA GTG CTA GAT GCC TCG 107
  • nucleotides 1412 and 1422 each designated C may be A, C, G, or T"
  • MOLECULE TYPE protein

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Abstract

L'invention concerne de nouvelles chémokines et 7 récepteurs transmembranaires de mammifères, des réactifs correspondants, y compris des protéines purifiées, des anticorps spécifiques, et des acides nucléiques codant lesdites chémokines ou lesdits récepteurs. L'invention concerne aussi des modes d'utilisation desdits réactifs et de trousses diagnostiques.
PCT/US1998/000902 1997-01-23 1998-01-22 Chemokines de mammiferes, recepteurs, reactifs, et modes d'utilisation Ceased WO1998032858A2 (fr)

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Cited By (12)

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WO1999013082A1 (fr) * 1997-09-12 1999-03-18 Incyte Pharmaceuticals, Inc. Chimiokine cxc
WO1999033990A1 (fr) * 1997-12-30 1999-07-08 Chiron Corporation CHEMOKINE HUMAINE CXC (Tim-1)
US6184358B1 (en) 1996-09-10 2001-02-06 Millennium Pharmaceuticals, Inc. IP-10/Mig receptor designated CXCR3, antibodies, nucleic acids, and methods of use therefor
US6329159B1 (en) 1999-03-11 2001-12-11 Millennium Pharmaceuticals, Inc. Anti-GPR-9-6 antibodies and methods of identifying agents which modulate GPR-9-6 function
EP1066310A4 (fr) * 1998-02-10 2002-11-27 Millennium Pharm Inc Proteines et molecules d'acides nucleiques de neokine et leur utilisation
US6573095B1 (en) 1998-04-29 2003-06-03 Genesis Research & Development Corporation Limited Polynucleotides isolated from skin cells
US6576445B1 (en) 1996-09-12 2003-06-10 Human Genome Sciences, Inc. Chemokine α-4
WO2003065045A3 (fr) * 2002-02-01 2004-02-19 Bayer Healthcare Ag Diagnostic et traitement de maladies associees au recepteur gpr65
US6919176B2 (en) 2001-05-07 2005-07-19 Amgen Inc. Polypeptides and nucleic acids associated with cancer
EP1676861A3 (fr) * 1998-10-13 2006-07-19 Arena Pharmaceuticals, Inc. Récepteurs non-endogènes de la protéine G humaine ayant une activité constitutive
US7888466B2 (en) 1996-01-11 2011-02-15 Human Genome Sciences, Inc. Human G-protein chemokine receptor HSATU68
US8440391B2 (en) 1998-10-13 2013-05-14 Arena Phamaceuticals, Inc. Constitutively activated human G protein coupled receptors

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* Cited by examiner, † Cited by third party
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US5602008A (en) * 1994-11-29 1997-02-11 Incyte Pharmaceuticals, Inc. DNA encoding a liver expressed chemokine
US6806061B1 (en) * 1995-01-19 2004-10-19 Children's Medical Center Corporation G protein-coupled receptor gene and methods of use therefor
AU2698795A (en) * 1995-06-06 1996-12-24 Human Genome Sciences, Inc. Human chemokine beta-12
US7265201B1 (en) * 1995-06-23 2007-09-04 Millennium Pharmaceuticals, Inc. Human chemotactic cytokine
NZ332318A (en) * 1996-03-19 2000-10-27 Human Genome Sciences Inc Chemokine alpha 2 and it's therapeutic and diagnostic use in treating illnesses

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8937169B2 (en) 1996-01-11 2015-01-20 Human Genome Sciences, Inc. Human G-protein chemokine receptor HSATU68
US7888466B2 (en) 1996-01-11 2011-02-15 Human Genome Sciences, Inc. Human G-protein chemokine receptor HSATU68
US6184358B1 (en) 1996-09-10 2001-02-06 Millennium Pharmaceuticals, Inc. IP-10/Mig receptor designated CXCR3, antibodies, nucleic acids, and methods of use therefor
US7407655B2 (en) 1996-09-10 2008-08-05 Millennium Pharmaceuticals, Inc. Method of inhibiting leukocytes with human CXC chemokine receptor 3 antibody
US6576445B1 (en) 1996-09-12 2003-06-10 Human Genome Sciences, Inc. Chemokine α-4
WO1999013082A1 (fr) * 1997-09-12 1999-03-18 Incyte Pharmaceuticals, Inc. Chimiokine cxc
WO1999033990A1 (fr) * 1997-12-30 1999-07-08 Chiron Corporation CHEMOKINE HUMAINE CXC (Tim-1)
EP1066310A4 (fr) * 1998-02-10 2002-11-27 Millennium Pharm Inc Proteines et molecules d'acides nucleiques de neokine et leur utilisation
US6573095B1 (en) 1998-04-29 2003-06-03 Genesis Research & Development Corporation Limited Polynucleotides isolated from skin cells
EP1676861A3 (fr) * 1998-10-13 2006-07-19 Arena Pharmaceuticals, Inc. Récepteurs non-endogènes de la protéine G humaine ayant une activité constitutive
US8440391B2 (en) 1998-10-13 2013-05-14 Arena Phamaceuticals, Inc. Constitutively activated human G protein coupled receptors
US6936248B1 (en) 1999-03-11 2005-08-30 Millennium Pharmaceuticals, Inc. Methods of inhibiting GPR-9-6 function
US6884574B2 (en) 1999-03-11 2005-04-26 Millennium Pharmaceuticals, Inc. Methods of identifying agents which inhibit GPR-9-6
US7381412B2 (en) 1999-03-11 2008-06-03 Millennium Pharmaceuticals, Inc. Anti-TECK antibodies and methods of use therefor
US7485301B2 (en) 1999-03-11 2009-02-03 Millennium Pharmaceuticals, Inc. Methods of treating inflammatory disease using antibodies which bind GPR-9-6
US6689570B2 (en) 1999-03-11 2004-02-10 Millennium Pharmaceuticals, Inc. Methods of identifying agents which bind GPR-9-6
US6329159B1 (en) 1999-03-11 2001-12-11 Millennium Pharmaceuticals, Inc. Anti-GPR-9-6 antibodies and methods of identifying agents which modulate GPR-9-6 function
US6919176B2 (en) 2001-05-07 2005-07-19 Amgen Inc. Polypeptides and nucleic acids associated with cancer
WO2003065045A3 (fr) * 2002-02-01 2004-02-19 Bayer Healthcare Ag Diagnostic et traitement de maladies associees au recepteur gpr65

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