WO1998031810A2 - Chemokines de mammiferes, recepteurs, reactifs, utilisations - Google Patents

Chemokines de mammiferes, recepteurs, reactifs, utilisations Download PDF

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WO1998031810A2
WO1998031810A2 PCT/US1998/000218 US9800218W WO9831810A2 WO 1998031810 A2 WO1998031810 A2 WO 1998031810A2 US 9800218 W US9800218 W US 9800218W WO 9831810 A2 WO9831810 A2 WO 9831810A2
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WO1998031810A3 (fr
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Constance F. Huffine
Devora L. Rossi
Myriam Capone
Joseph A. Hedrick
Alain Vicari
Daniel M. Gorman
Albert Zlotnik
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Schering Corporation
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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
    • C07K14/705—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • MAMMALIAN CHEMOKINES RECEPTORS: REAGENTS:
  • 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.)
  • 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.,
  • GPCR G-protein coupled receptor
  • GPLR G-protein coupled receptors
  • 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
  • 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)
  • the present invention is based, in part, upon the discovery of new genes encoding various chemokines, e.g., those designated CKDLR20.1, which encode a CXC chemokine; or 7 transmembrane receptors, e.g., those designated 69A08, which are exemplified by a mouse clone; and HSD12, which are exemplified by a human clone.
  • Each of the 7 transmembrane receptors is probably a G-protein coupled (or linked) receptors (GPCR or GPLR), though a ligand for each has not yet been identified.
  • 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 a CKDLR20.1; a 69A08; or an HSD12; 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 mouse or human; it comprises a sequence of SEQ ID NO: 2, 4, 6 or 8; 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 or 8; 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.
  • binding compositions e.g., of producing an antigen:antibody complex, comprising contacting: a
  • the method is one wherein: the complex is purified from other chemokine or chemokine receptor; the complex is purified from other antibody; the contacting is with a sample comprising a CKDLR20.1 chemokine antigen; the contacting is with a sample comprising either 69A08 or HSD12 receptor antigen; the contacting allows quantitative detection of the antigen; the contacting is with a sample comprising the antibody; or the contacting allows quantitative detection of the antibody.
  • 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; comprises a sequence of SEQ ID NO: 1, 3, 5 or 7; or comprises a deoxyribonucleic acid nucleotide.
  • the invention also provides a kit with such nucleic acids. With nucleic acids are provided fusion proteins, comprising: a sequence of SEQ ID NO: 2, 4, 6 or 8; and /or sequence of another chemokine or 7 transmembrane receptor, as appropriate. Also, provided is a cell comprising a recombinant nucleic acid, as described, and methods of producing a polypeptide comprising expressing the nucleic acid in an expression system.
  • a ligand:receptor complex comprising contacting: a protein made by expression of a CKDLR20.1 nucleic acid with a G protein coupled receptor; a protein or peptide made by expression of a 69A08 nucleic acid with a chemokine or ligand; or a protein or peptide made by expression of an HSD12 nucleic acid with a chemokine or ligand; thereby allowing the complex to form.
  • the complex results in a Ca++ flux; the G protein coupled receptor is on a cell; the complex results in a physiological change in a cell expressing the receptor or protein; the 69A08 or HSD12 protein is on a cell; the contacting is with a sample comprising a chemical antagonist to block production of the complex; or the contacting allows quantitative detection of ligand.
  • the invention further provides 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 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
  • Chemokines are secreted by activated leukocytes and act as a chemoattractant for a variety of cells which are involved in inflammation. Besides chemoattractant properties, 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. Thus, 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 CKDLR20.1.
  • the invention provides two genes encoding novel G-protein coupled receptors, designated 69A08 and HSD12. 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. Alternatively, the ligands may be other molecules, including molecules such as epinephrine, serotonin, or glucagon.
  • chemokines or receptors should be important for mediating various aspects of cellular, organ, tissue, or organismal physiology or development.
  • Mouse CKDLR20.1 chemokine nucleotide and amino acid sequences are shown in SEQ ID NO: 1 and 2.
  • 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. The gene was first found while screening a- rag lung library with the human MIP-3a probe (complete cDNA). A ELRCLC motif can be seen at residues 2-7 of SEQ ID NO: 2.
  • the CKDLR20.1 gene encodes a novel protein exhibiting structure and motifs characteristic of a chemokine.
  • the protein exhibits an ELR motif just upstream of the CXC sequence, implicating the chemokine in pro-inflammatory immune responses.
  • the mRNA expression appears highly restricted to lung, and is induced in infection by the parasite Nippostongylus brasiliensis.
  • Nucleotide and amino acid sequences of a novel GPCR, from a mouse, designated 69A08, are provided in SEQ ID NO: 3, 4, 5 and 6.
  • the nucleotide sequence of SEQ ID NO: 3 was first isolated from pre-T cells, and part of the sequence was derived by PCR. The corresponding amino acid sequence is also provided. (SEQ ID NO: 4). Subsequent sequencing suggests that nucleotides 158, 159, and 276 are absent, resulting in a region of frameshift, as indicated in the revised sequences provided in SEQ ID NO: 5 and 6.
  • GPCR from human, designated HSD12, are shown in SEQ ID NO: 7 and 8. Generic descriptions of physical properties of polypeptides, nucleic acids, and antibodies, where directed to one embodiment clearly are generally applicable to other chemokines or receptors described herein.
  • 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.
  • CKDLR20.1 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 species counterpart, e.g., which exhibits similar sequence, and is more homologous in natural encoding sequence than other genes from that species, particularly primate species.
  • 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.
  • Homologous proteins would be found in mammalian species other than mouse, e.g., rats, dogs, cats, and primates. Non-mammalian species should also possess structurally or functionally related genes and proteins. Similar concepts apply to GPCR embodiments 69A08 and HSD12, in the context of a receptor.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. (1983) Chapter One in Time Warps, String Edits, and Macromolecules: The Theory and Practice of Sequence Comparison Addison- Wesley,
  • 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 or 8, 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 warm blooded animals, e.g., mammals and birds.
  • descriptions are generally meant to encompass the various chemokine or receptor proteins, not limited to the mouse or human embodiments specifically discussed.
  • Chemokine or receptor mutagenesis can be conducted by making amino acid insertions or deletions. Substitutions, deletions, insertions, or combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed 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).
  • PCR polymerase chain reaction
  • 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 b-sheet or a-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.
  • 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 or receptor 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 haptens.
  • 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., phosphotyrosine, 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.
  • cytokines are homodimeric entities, and a repeat construct may have various advantages, including lessened susceptibility to proteolytic cleavage. Moreover, 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. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins.
  • Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a 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.
  • 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-
  • 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 amino acid sequences shown in SEQ ID NO: 2, 4, 6 or 8, 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 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., rodents, lagomorphs, carnivores, artiodactyla, perissodactyla, and 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.
  • 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 species counterparts, e.g., rat and other mammalian species or allelic or polymorphic variants.
  • 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.
  • 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. They also 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. Further, 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 Immuno chemistry, 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 Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press; Goding (1986)
  • 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.
  • Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See, Huse, et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281; and Ward, et al. (1989) Nature 341:544-546.
  • polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • 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.
  • 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 other species, e.g., warm blooded animals, such as birds and mammals. Cross hybridization will allow isolation of ligand from other 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. See, e.g., SEQ ID NO: 1, 3, 5 and 7.
  • synthetic oligonucleotides will be useful in selecting correct clones from a library. Complementary sequences will also be used as probes or primers.
  • the third peptide should be particularly useful, e.g., coupled with anchored vector or poly-A complementary PCR techniques or with complementary DNA of other peptides.
  • 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 Tables 1 through 3.
  • 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 nucle
  • 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 other species, including primates. Various chemokine proteins should be homologous and are encompassed herein, as would be receptors. However, even proteins that have a more distant evolutionary relationship to the ligands or receptors can readily be isolated under appropriate conditions using these sequences if they are sufficiently homologous. Primate chemokines or receptors are of particular interest.
  • This invention further covers recombinant DNA molecules and fragments having a DNA sequence identical to or highly homologous to the isolated DNAs set forth herein.
  • the sequences will often be operably linked to DNA segments which control transcription, translation, and DNA 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. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al.
  • homologous nucleic acid sequences when compared, exhibit significant similarity, or identity.
  • the standards for homology in nucleic acids are either measures for homology generally used in the art by sequence comparison or based upon hybridization conditions. The hybridization conditions are described in greater detail below.
  • 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 Tables 1 through 3.
  • 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, Kanehisa (1984) Nuc. Acids Res. 12:203-213.
  • the length of homology comparison may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, 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 (1968) T. Mol. Biol.
  • Corresponding chemokines or receptors from other mammalian species can be cloned and isolated by cross-species hybridization of closely related species.
  • sequences from a data base may be recognized as having similarity. Homology may be relatively 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.
  • This DNA 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 Rodriguez, 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
  • Lower eukaryotes e.g., yeasts and Dictyostelium, may be transformed with chemokine or receptor sequence containing nucleic acids.
  • 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.
  • 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-phosphogly cerate 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) Ceil 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
  • chemokines and receptors have been characterized, fragments or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) Solid Phase Peptide Synthesis. Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis, Springer- Verlag, New York; and Bodanszky (1984) The Principles of Peptide -Synthesis. Springer- Verlag, New York.
  • an azide process for example, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p-nitrophenyl ester, N- hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative-reductive process, or a dicyclohexylcarbodiimide (DCCD)/ additive process 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.
  • 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.
  • 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 supernatants of cells producing the desired proteins as a result of DNA techniques, see below.
  • 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 value.
  • 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. Moreover, 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.,
  • 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 and methods for detecting the presence of ligand, antibodies, or receptors 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 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.
  • 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.
  • an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate.
  • 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. However, other techniques may also be employed, such as using biotin modified nucleotides for introduction into a polynucleotide.
  • the biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like.
  • antibodies may be employed which can recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes.
  • the antibodies in -turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • probes to the novel anti-sense RNA may be carried out in conventional techniques such as nucleic acid hybridization, plus and minus screening, recombinational probing, hybrid released translation (HRT), and hybrid arrested translation (HART). This also includes amplification techniques such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • 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
  • the CKDLR20.1 was isolated from a cDNA library made from the lung from a RAG-1 "knockout" mouse. See, Mombaerts, et al. (1992) Ceil 68:869-877. A cDNA probe which comprises the entire coding portion of human MIP-3a (see Gish, et al., U.S.S.N. 08/675,814) was used as a probe. This identified a gene designated CKDLR20.1, which is characterized in SEQ ID NO: 1. 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 1 (met) to about 18 (thr), so the mature form should begin with gin and run about 142 amino acids. Additional processing may occur in a physiological system.
  • the message is upregulated in Nippostrongylus brasilensis parasite infected lung tissue, and its mRNA expression appears fairly restricted to lung tissue. But such may suggest a role in other mucosal boundaries, e.g., skin or gut.
  • the mouse 69A08 clone was derived from mouse pre-T cells.
  • the nucleotide sequence is provided in SEQ ID NO: 3 and 5, encoding a polypeptide of about 359 amino acids.
  • the closest related genes are orphan G-protein coupled receptors. These include the chemokine receptors, and protease, e.g., thrombin, receptors. Structural motifs suggest that the receptor contains motifs characteristic of the chemokine receptor family, and of the protease receptor family.
  • transmembrane segments based upon hydrophobicity plots and comparisons with other similar GPCRs, should be about as follows: TM1 from 57 (ala) to 74 (leu); TM2 from 93 (leu) to 109 (ala); TM3 from 122 (ala) to 147 (leu); TM4 from 167 (leu) to 189 (his), but with the hydrophobic region reaching as far as 205 (met); TM5 from 222 (ala) to 248 (ala); TM6 from 256 (ala) to 281 (his); and TM7 from 293 (leu) to 318 (val). See, e.g., Loetscher, et al. (1996) T.
  • a DRY motif is found, e.g., near residue 149.
  • 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 TM1 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.
  • the human HSD12 clone was derived from a cDNA library made from human monocytes or dendritic cells. Individual cDNA clones are sequenced using standard methods, and the sequence was identified and further characterized. The nucleotide sequence is provided in SEQ ID NO: 7, encoding a polypeptide of about 371 amino acids. The natural message appears to be about 2.8 kB, which contains a poly-A tail. The message contains an Alu repeat in the region of about 2450-2825.
  • transmembrane segments based upon hydrophobicity plots and comparisons with other similar GPCRs, should be about as follows: TM1 from 34 (ile) to 50 (ala); TM2 from 67 (val) to 83 (thr); TM3 from 112 (ile) to 128 (ser); TM4 from 147 (arg) to 165 (his); TM5 from 193 (ala) to 209 (thr); TM6 from 238. (val) to 254 (leu); and TM7 from 282 (val) to 298 (ile).
  • 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 cytoplasmic, or intracellular, segments should then run Cl between TM1 and TM2; C2 between TM3 and TM4, C3 between TM5 and TM6, and C4 the carboxy terminus from the end of TM7. Additional processing may occur in a physiological system.
  • the C3 segment which is usually the longest of the cytoplasmic segments, and which probably provides specificity for binding of signaling components, e.g., the G proteins.
  • 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 Tables 2 or 3. 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
  • the antibodies provided will be useful as immuno affinity reagents, as detection reagents, for immunohistochemistry, and as potential therapeutic reagents, either as agonist or antagonist reagents. They will often be in sterile formulations.
  • V Assays for chemotactic activity of chemokines
  • 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. See, de Wet, et al. (1987) Mol.
  • a positive control e.g., recombinant murine MIP- 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) L Immunol. 154:3654-3666.
  • lymphocytes After stimulation with various chemokines, lymphocytes often exhibit a measurable intracellular Ca2+ flux. MlP-la, e.g., is capable of inducing immediate transients of calcium mobilization. Typically, the levels of chemokine used in these assays will be comparable to those used for the chemotaxis assays (1/1000 dilution of conditioned supernatants).
  • 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.
  • receptors 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.
  • Various ligand sources are tested to find a source of ligand which results in a G- protein coupled response.
  • 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.
  • RNA blot and hybridization are performed according to the standard methods in Maniatis, et al. (1982) Molecular Cloning: A
  • RNA loaded in each lane is reprobed with a control cDNA, e.g., 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. However, in certain cases, 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. Alternatively, in situ nucleic acid hybridization may also be used in such analysis.
  • the CKDLR20.1 was isolated from a RAG-1 "knockout" mouse.
  • Several cell lines were tested for expression using Northern blot technology, and were found negative. These cell lines included bone marrow stroma (3D1), mast cells (MC9), ab CD4- CD8- hybridoma (A3.2), T cell clone (HT-2), fibroblast (L cell), pro-T hybridoma, pre-T hybridoma, B cell (A20-2J) and CD3- CD4- CD8- (BW) cells.
  • the expression level was high in lung, with weak signals in fetal lung and heart, and no detectable signal in fetal liver, thymus, activated spleen, lymph node, brain, or kidney.
  • the expression pattern might suggest that the pro-inflammatory chemokine may be involved various aspects of the lung physiology, e.g., the initiation or maintenance of an asthmatic condition. It may play a role in pneumonia, or in various occupational lung conditions, e.g., black lung, farmer's lung, silicosis, asbestosis, or various hypersensitivity lung conditions. See, e.g., Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; and Thorn, et al. Harrison's Principles of Internal Medicine, McGraw-Hill,
  • the ELR motif may also suggest a role in angiogenesis, which may suggest that antagonists, or possibly agonists in other situations, may be useful in treating lung or other tumors, e.g., of various mucosal surfaces such as the gut or skin. It may also be useful in treatment of lung neoplastic conditions, e.g., lung cancers.
  • Samples for mouse mRNA isolation may include, e.g.: resting mouse fibroblastic L cell line (C200); Braf:ER (Braf fusion to estrogen receptor) transfected cells, control (C201); T cells, THl polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IFN-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-g; T201); T cells, highly THl polarized (see Openshaw, et al.
  • IL-10 K.O. spleen see Kuhn, et al. (1991) Cell 75:263-274; X201); total adult spleen, normal (O201); total spleen, rag-1 (O207); IL-10 K.O. Peyer's patches (O202); total Peyer's patches, normal (O210); IL-10 K.O. mesenteric lymph nodes (X203); total mesenteric lymph nodes, normal (0211); IL- 10 K.O. colon (X203); total colon, normal (0212); NOD mouse pancreas (see Makino, et al.
  • the 69A08 gene was identified from a cDNA library made from thymus pre-T cells. Hybridization analysis detected a positive mRNA signal in activated T cells, Thl and Th2 cell libraries, macrophages, and tissue prepared from mice infected with Nippostrongylus brasiliensis. This suggests a role of the molecule in the immune response, e.g., inflammation or vascular biology.
  • the molecule, or its antagonist, should be useful in various inflammatory disease states or conditions, e.g., in the lung or elsewhere, including skin and gut. C.
  • HSD12 Southern Analysis 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 human mRNA isolation include, e.g.: U937 premonocytic line, resting (MlOO); elutriated monocytes, activated with LPS, IFNg, anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes, activated with LPS, IFNg, IL-10 for 1, 2, 6, 12, 24 h pooled (M103); elutriated monocytes, activated LPS for 1 h (M108); elutriated monocytes, activated LPS for 6 h (M109); DC 70% CDla+, from CD34+ GM-CSF, TNFa 12 days, resting (D101); DC 70% CDla+, from CD34+
  • peripheral blood mononuclear cells peripheral blood mononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells), resting (T100); peripheral blood mononuclear cells, activated with anti-CD3 for 2, 6, 12 h pooled (T101); T cell, THl clone HY06, resting (T107); T cell, THl clone HY06, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, THl clone HY06, anergic treated with specific peptide for 2, 6, 12 h pooled (T109); T cell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935, activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (Till); T cell, TH2 clone HY935, resting (T110); T cell, TH2 clo
  • the HSD12 gene was identified from a cDNA library made from activated dendritic cells. It has also been detected by hybridization in monocytes and dendritic cells, with lower signals detected in Thl cells and NK cells. Dendritic cells derived from CD34+ cells seem to express more than those which are monocyte derived. The expression levels seem lower in either resting or anergic cell libraries. In the NK cells, the activated cells had higher expression levels than resting. The expression in dendritic cells suggests a role in immune function, e.g., where dendritic cells are important. Thus includes antigen presentation, and initiation of an immune response. Thus, agonists or antagonists of the receptor should be useful in such immune functions.
  • Screening for receptor /ligand 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. Then plate COS cells at 2-3. x 10 ⁇ cells per chamber in 1.5 ml of growth media. Incubate overnight at 37° C.
  • a positive control is prepared, e.g., of huIL-10- FLAG cDNA at 1 and 1/200 dilution, and a negative mock. Rinse cells with serum free DME. Add the DNA solution and incubate 5 hr at 37° C. Remove the medium and add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and wash once with DME. Add 1.5 ml growth medium and incubate overnight.
  • HBSS HBSS
  • PFA paraformaldehyde
  • glucose glucose
  • the slides may be stored at -80° C after all liquid is removed.
  • 0.5 ml incubations are performed as follows. Add HBSS/saponin(0.1%) with 32 ml/ml of IM NaN3 for 20 min. Cells are then washed with HBSS/saponin IX. Add antibody complex to cells and incubate for 30 min. Wash cells twice with HBSS/saponin. Add second antibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, and incubate for 30 min.
  • second antibody e.g., Vector anti-mouse antibody
  • ELISA solution e.g., Vector Elite ABC horseradish peroxidase solution, and preincubate for 30 min.
  • Use e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solution and incubate for 30 min. Wash cells twice with HBSS, second wash for 2 min, which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5 to 10 min.
  • DAB Vector diaminobenzoic acid
  • 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. All references cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Many modification an variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of the equivalents to which such claims are entitled.
  • SEQ ID NO: 1 is a rodent CKDLR20.1 nucleotide sequence
  • SEQ ID NO: 2 is a rodent CKDLR20.1 amino acid sequence.
  • SEQ ID NO: 3 is a rodent 69A08 nucleotide sequence
  • SEQ ID NO: 4 is a rodent 69A08 amino acid sequence
  • SEQ ID NO: 5 is a revised rodent 69A08 nucleotide sequence
  • SEQ ID NO: 6 is a revised rodent 69A08 amino acid sequence.
  • S SEEQQ I IDD N NOO:: 7 7 is a primate HSD12 nucleotide sequence.
  • SEQ ID NO: 8 is a primate HSD12 amino acid sequence.
  • CTGCTGCTGG GGTGGGTCCC CACGAAGCTG GTACCTGCCC TCTATGGGCT TGTGGTGGCT 1350 GTGGGGCTGC CTGCCAATGG GCTGGCGCTG TGGGTGCTGG CCACAAGGGT GCCACGCCTG 1410
  • TAGGCTGGGC TATGTGAGAG TCCAGAGGCA GAAAGGAGTT ATGAGGTCAC TAGCTAGAGG 2550
  • GGAGGCCGCG CAGGGCCGTG GGGCTGAGCC
  • ACGCTCTCGT TTTGTCAGGC AGCTATGCAG 2422
  • TTGCTCTTCC TTGTTTTTGT TTTGTTTTTG TTTTTGTTTTTT TAATATTTAT TTTTTTAGAG 2482
  • CATCCCAGCA CTTTGGGAGG CCGAGACGGG AGGATCAGTT GAGGTCAGGA GTTTGAGACC 2782
  • MOLECULE TYPE protein

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Abstract

L'invention concerne des chémokines et sept récepteurs transmembranaires extraits de mammifères, ainsi que des réactifs associés, notamment des protéines purifiées, des anticorps spécifiques et des acides nucléiques codant pour lesdites chémokines ou lesdits récepteurs. Elle concerne également des méthodes permettant d'utiliser lesdits réactifs et des trousses diagnostiques.
PCT/US1998/000218 1997-01-21 1998-01-20 Chemokines de mammiferes, recepteurs, reactifs, utilisations WO1998031810A2 (fr)

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AU60170/98A AU6017098A (en) 1997-01-21 1998-01-20 Mammalian chemokines; receptors; reagents; uses

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PCT/US1998/000218 WO1998031810A2 (fr) 1997-01-21 1998-01-20 Chemokines de mammiferes, recepteurs, reactifs, utilisations

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WO2000031258A2 (fr) * 1998-11-20 2000-06-02 Arena Pharmaceuticals, Inc. Recepteurs humains couples a la proteine g orphan
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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7176283B1 (en) 1996-10-30 2007-02-13 The Regents Of The University Of California Protease-activated receptor 3 and uses thereof
WO1999043809A3 (fr) * 1998-02-27 1999-10-14 Univ California Recepteur 4 active par la protease et ses utilisations
WO1999043809A2 (fr) * 1998-02-27 1999-09-02 The Regents Of The University Of California Recepteur 4 active par la protease et ses utilisations
US7816492B2 (en) 1998-11-20 2010-10-19 Arena Pharmaceuticals, Inc. Human G protein-coupled receptors
EP1780280A1 (fr) * 1998-11-20 2007-05-02 Arena Pharmaceuticals, Inc. Récepteurs humains orphelins couplés à une protéine G
US8198049B2 (en) 1998-11-20 2012-06-12 Arena Pharmaceuticals, Inc. Nucleic acids encoding RUP3 and methods of using same
US8097452B2 (en) 1998-11-20 2012-01-17 Arena Pharmaceuticals, Inc. Human orphan G protein-coupled receptors
EP2295573A1 (fr) * 1998-11-20 2011-03-16 Arena Pharmaceuticals, Inc. Récepteurs humains orphelins couplés à une protéine g
EP1580271A2 (fr) * 1998-11-20 2005-09-28 Arena Pharmaceuticals, Inc. Recepteurs humains couples à la protéine G sans ligands connus
EP1584683A1 (fr) * 1998-11-20 2005-10-12 Arena Pharmaceuticals, Inc. Récepteurs humaines orphelins couplés à une protéine g
EP2295574A1 (fr) * 1998-11-20 2011-03-16 Arena Pharmaceuticals, Inc. Récepteurs humains orphelins couplés à une protéine g
EP1580271A3 (fr) * 1998-11-20 2006-01-18 Arena Pharmaceuticals, Inc. Recepteurs humains couples à la protéine G sans ligands connus
US7108991B2 (en) 1998-11-20 2006-09-19 Arena Pharmaceuticals, Inc. Human orphan G protein-coupled receptors
WO2000031258A3 (fr) * 1998-11-20 2000-10-05 Arena Pharm Inc Recepteurs humains couples a la proteine g orphan
USRE42190E1 (en) 1998-11-20 2011-03-01 Arena Pharmaceuticals, Inc. Method of identifying a compound for inhibiting or stimulating human G protein-coupled receptors
EP1849866A2 (fr) * 1998-11-20 2007-10-31 Arena Pharmaceuticals, Inc. Récepteurs humains orphelins couplés à une protéine g
EP1849866A3 (fr) * 1998-11-20 2007-11-07 Arena Pharmaceuticals, Inc. Récepteurs humains orphelins couplés à une protéine g
US7893235B2 (en) 1998-11-20 2011-02-22 Arena Pharmaceuticals, Inc. Nucleic acids encoding the GPCR, RUP3, and methods of use thereof
WO2000031258A2 (fr) * 1998-11-20 2000-06-02 Arena Pharmaceuticals, Inc. Recepteurs humains couples a la proteine g orphan
WO2001012663A2 (fr) * 1999-08-19 2001-02-22 Curagen Corporation Facteurs de regulation hematopoietique et procedes d'utilisation
AU783994B2 (en) * 1999-08-19 2006-01-12 Curagen Corporation Novel hematopoietic regulatory factors and methods of use thereof
JP2003509015A (ja) * 1999-08-19 2003-03-11 キュラゲン コーポレイション 新規な造血調節因子およびその使用方法
WO2001012663A3 (fr) * 1999-08-19 2001-10-25 Curagen Corp Facteurs de regulation hematopoietique et procedes d'utilisation
US7341851B2 (en) 2002-08-19 2008-03-11 Ares Trading, S.A. IL-8 like protein
WO2004016654A1 (fr) * 2002-08-19 2004-02-26 Ares Trading S.A. Proteine du type il-8

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AU6017098A (en) 1998-08-07

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