EP0951549A1 - Chemokines mammaliennes - Google Patents

Chemokines mammaliennes

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Publication number
EP0951549A1
EP0951549A1 EP97944422A EP97944422A EP0951549A1 EP 0951549 A1 EP0951549 A1 EP 0951549A1 EP 97944422 A EP97944422 A EP 97944422A EP 97944422 A EP97944422 A EP 97944422A EP 0951549 A1 EP0951549 A1 EP 0951549A1
Authority
EP
European Patent Office
Prior art keywords
chemokine
mctap3
mpf4
chrl9kine
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP97944422A
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German (de)
English (en)
Inventor
Joseph A. Hedrick
Albert Zlotnik
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Merck Sharp and Dohme Corp
Original Assignee
Schering Corp
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Filing date
Publication date
Application filed by Schering Corp filed Critical Schering Corp
Publication of EP0951549A1 publication Critical patent/EP0951549A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • the present invention contemplates compositions related to proteins which function in controlling development, differentiation, trafficking, and physiology of mammalian cells, e.g., cells of a mammalian immune system.
  • mammalian cells e.g., cells of a mammalian immune system.
  • it provides proteins which regulate or evidence development, differentiation, and function of various cell types, including hematopoietic cells.
  • 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 Hematolocrv (2d ed.) Lippincott, Philadelphia, PA; Jandl (1987) Blood: Textbook of Hematolocrv. Little, Brown and Co., Boston, MA.; and Paul (ed. ) (1993) Fundamental Immunology (3d ed. ) Raven Press, N.Y.
  • Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to support the proliferation, growth, and differentiation of the pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. These interactions between the cellular components are necessary for a healthy immune response. These different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents .
  • the chemokines are a large and diverse superfamily of proteins. The superfamily is subdivided into two classical branches, based upon whether the first two cysteines in the chemokine motif are adjacent (termed the "C-C” branch), or spaced by an intervening residue (“C-X- C") .
  • a more recently identified branch of chemokines lacks two cysteines in the corresponding motif, and is represented by the chemokines known as lymphotactins .
  • Another recently identified branch has three intervening residues between the two cysteines, e.g., CX3C chemokines. See, e.g., Schall and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy & Immunol. 109:97-109.
  • the present invention reveals the existence of previously unknown chemokine-motif containing molecules which are hereby designated mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or ChrlSkine chemokine. Based on sequence analysis of the chemokine protein sequences described below, it is apparent that mpf4, mCTAP3 , and Chrl9kine belong to the CXC chemokine family, and m6Ckine and h6Ckine belong to the CC chemokine family.
  • the present invention provides a composition of matter selected from a composition comprising an antigen binding site from an antibody which specifically binds to mpf4, mCTAP3, m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine; an expression vector encoding mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine or fragment thereof; a substantially pure protein which is specifically recognized by the respective antigen binding site; and a substantially pure mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine or peptide thereof, or fusion protein comprising a 30 amino acid fragment of mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine sequence.
  • the antigen binding site may be: specifically immunoreactive with a mature protein selected from the group consisting of the polypeptides of SEQ ID NO: 2, 4, 6, 8, or 10; raised against a purified or recombinantly produced mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine; in a monoclonal antibody, Fab, or F(ab)2; or in a labeled antibody.
  • the antigen binding site is detected in a biological sample by a method of: contacting a binding agent having an affinity for the mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine protein with the biological sample; incubating the binding agent with the biological sample to form a binding agent:mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine protein complex; and detecting the complex.
  • the biological sample is human or mouse, and the binding agent is an antibody.
  • kits embodiment possessing a compound, described above, with either instructional material for the use of the compound; or a compartment into which the compound is segregated.
  • a nucleic acid embodiment of the invention includes an expression vector encoding a mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine protein, wherein the protein specifically binds an antibody generated against an immunogen selected from the mature polypeptide portions of SEQ ID NO: 2, 4, 6, 8, or 10, more particularly a natural protein.
  • the vector may: encode a mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine polypeptide with complete sequence identity to a naturally occurring mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine protein; encode a mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein comprising sequence selected from the polypeptides of SEQ ID NO: 2, 4, 6, 8, or 10, and particularly a natural protein; or comprise sequence selected from the nucleic acids of SEQ ID NO: 1, 3, 5, or 7.
  • the vector is capable of selectively hybridizing to a nucleic acid encoding a mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine protein, e.g., a mature protein coding segment of SEQ ID NO: 1, 3, 5, or 7.
  • the isolated nucleic acid is detected in a biological sample by a method of : contacting a biological sample with a nucleic acid probe capable of selectively hybridizing to the nucleic acid; incubating the nucleic acid probe with the biological sample to form a hybrid of the nucleic acid probe with complementary nucleic acid sequences present in the biological sample; and determining the extent of hybridization of the nucleic acid probe to the complementary nucleic acid sequences.
  • the nucleic acid probe is capable of hybridizing to a nucleic acid encoding a protein consisting of the polypeptides of SEQ ID NO: 2, 4, 6, 8, or 10.
  • the isolated mpf4 , mCTAP3 , m ⁇ Ckine is from a mouse; the isolated h6Ckine is from a human; consists of a polypeptide comprising sequence from SEQ ID NO: 2, 4, 6, 8, or 10; recombinantly produced, or a naturally occurring protein.
  • fusion proteins comprising a sequence of SEQ ID NO: 2, 4, 6, 8, or 10 and/or a sequence of another cytokine or chemokine.
  • the present invention also embraces a cell transfected with the nucleic acid encoding a mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine, e.g., where the nucleic acid has SEQ ID NO: 1, 3, 5, or 7.
  • the cell may be either prokaryote or eukaryote.
  • the invention also provides a method of modulating physiology or development of a cell by contacting the cell with a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine, or an antagonist, e.g., neutralizing antibody, of the chemokine.
  • the physiology is attraction of a cell which possesses a natural receptor for the chemokine.
  • the present invention provides mouse and human DNA sequences encoding mammalian proteins which exhibit structural properties or motifs characteristic of a cytokine or chemokine.
  • chemokine family see, e.g., Lodi, et al . (1994) Science 263:1762- 1767; Gronenborn and Clore (1991) Protein Engineering 4:263-269; Miller and Kranger (1992) Proc. Nat ' 1 Acad. Sci. USA 89:2950-2954; Matsushima and Oppenheim (1989) Cvtokine 1:2-13; Stoeckle and Baker (1990) New Bioi. 2:313-323; Oppenheim, et al . (1991) Ann. Rev. Immunol. 9:617-648; Schall (1991) Cvtokine 3:165-183; and The Cytokine Handbook Academic Press, NY.
  • novel cytokines described herein are designated mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine.
  • the descriptions below are directed, for exemplary purposes, to mammalian embodiments, e.g., human and mouse, but are likewise applicable to related embodiments from other, e.g., natural, sources. These sources should include various vertebrates, typically warm blooded animals, e.g., birds and mammals, particularly domestic animals, and primates.
  • the nucleic acid and amino acid sequences of the novel Mouse mpf4 chemokine are provided in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the mpf4 coding sequence begins at base 141 and ends at base 458, the CXC motif is at amino acid residues 5-7. Absence of an ELR motif immediately preceding the CXC suggests likely anti-inflammatory properties. A signal sequence is indicated, but based upon related genes, variable forms may be produced by different cell types.
  • the nucleic acid and amino acid sequences of the novel mouse CTAP3 (mCTAP3) are provided in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the CXC motif corresponds to amino acid residues numbered 10-12.
  • a predicted signal sequence is indicated, but may actually be of longer or shorter length depending, in part, upon the cell expressing the protein.
  • the ELR motif immediately preceeding the CXC motif suggests function in inflammatory mediator trafficking.
  • the nucleic acid and amino acid sequences of the novel mouse 6Ckine (m6Ckine) chemokine are provided in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the predicted coding region begins at about position 71 and ends at position 469.
  • a predicted signal sequence is indicated and the CC motif is at residues numbered 7-8.
  • m6Ckine has a total of 6 conserved cysteine residues in the mature peptide, suggesting a new subclass of CC chemokines exhibiting extended carboxy terminal sequence.
  • the nucleic acid sequence and the corresponding predicted amino acid sequence of the novel human 6Ckine are provided in SEQ ID NO: 7 and SEQ ID NO: 8, -y- respectively .
  • Predicted coding sequence runs from position 6 through position 407 .
  • the CC motif corresponds to residues numbered 8-9 ; predicted signal sequence is indicated .
  • h6Ckine has 6 conserved cysteine residues in the mature peptide, similar to m6Ckine , and exhibits carboxy terminal extension .
  • Table 1 depicts the alignment of porcine 6Ckine (p ⁇ Ckine) ( SEQ ID NO : 9 ) , mouse and human 6Ckine amino acid sequences .
  • Chrl9kine Partial amino acid sequence of human Chrl9kine (CXC chemokine; SEQ ID NO: 10) .
  • the CXC motif is underlined.
  • the absense of the ELR motif suggests anti-inflammatory properties .
  • Chrl9kine was found by search and careful analysis of a public EST database. See, e.g., gnl
  • chemokine proteins of this invention are defined in part by their physicochemical and biological properties .
  • the biological properties of the chemokines described herein, e.g., mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine are defined, in part, by their amino acid sequence, and mature size. They also should share at least some biological properties with other similar chemokines.
  • sequence variations may be tolerated, e.g., conservative substitutions or positions remote from the critical residues for receptor interaction or important tertiary structure features, without altering significantly the biological activity of the molecule.
  • chemokines are present in specific tissue types, e.g., lymphoid tissues, and the interaction of the protein with a receptor will be important for mediating various aspects of cellular physiology or development .
  • tissue types e.g., lymphoid tissues
  • the cellular types which express message encoding mpf4, mCTAP3, m ⁇ Ckine, h6Ckine, or Chrl9kine suggest that signals important in cell differentiation and development are mediated by them. See, e.g., Gilbert (1991) Developmental Biology (3d ed. ) Sinauer Associates, Sunderland, MA; Browder, et al. (1991) Developmental Biology (3d ed.
  • mpf4 exhibits approximately 71% amino acid identity to rat platelet factor 4 (pf4) , which functions as a marker for megakaryocyte differentiation. See, e.g., Doi, et al. (1987) Mol. Cell. Bioi. 7:898-904; and GenBank Accession number M15254 (1987) . Identity comparisons of mCTAP3 sequence, see, e.g., Marra, et al .
  • mCTAP3 is likely to be the murine counterpart of the human protein.
  • m6Ckine was also found by search and careful analysis of the public EST database with sequences exhibiting chemokine structure in the SWISS PROT database.
  • the nucleotide sequence of Table 3 and SEQ ID NO 5 is found on three murine ESTs, see, e.g., gnl
  • h6Ckine was found in the similar manner.
  • the nucleotide sequence of Table 4 and SEQ ID NO: 6 can be found, e.g., on gnl
  • Chrl9kine was found by search and careful analysis of a public EST database. See, e.g., gnl
  • IP-10 and Mig are known to have angiostatic properties. See, e.g., Keane, et al. (1997) J. Immunol. 159:1437-1443; and Farber (1997) J. Leukoc . Bioi. 61:246- 257. Because of this angiostatic effect, Mig and IP-10 prevent tumor related angiogenesis as well as regulate the rate of wound healing and scar tissue formation. Since 6Ckine shares a receptor with IP-10 and Mig, ⁇ Ckine may also play a role in preventing tumor formation, tumor metastasis, and/or regulating wound healing.
  • ⁇ Ckine is a T cell chemoattractant, especially for activated T cells, it may serve to enhance many T-cell mediated anti-tumor effects. This may occur with 6Ckine alone, or in combination with IP-10, Mig, lymphotactin, see, e.g., Kelner, et al . (1994) Science 266:1395-1399, and/or MIP3 ⁇ , see, e.g., U.S.S.N.
  • ⁇ Ckine may mediate rapid lymphocyte adhesion.
  • ⁇ Ckine may be used to direct activated T cells to an antigen, e.g., an infectious agent.
  • binding composition refers to molecules that bind with specificity and selectivity to a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine, respectively, e.g., in an antibody-antigen interaction.
  • other compounds e.g., receptor proteins, may also specifically and/or selectively associate with mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokines to the exclusion of other molecules.
  • the association will be in a natural physiologically relevant protein- protein interaction, either covalent or non-covalent, and may include members of a multiprotein complex, including carrier compounds or dimerization partners.
  • the molecule may be a polymer, or chemical reagent. No implication as to whether a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine is either the ligand or the receptor of a ligand-receptor interaction is necessarily represented, other than whether the interaction exhibits 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 the receptor, see, e.g., Goodman, et al.
  • binding agent:mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein complex refers to a complex of a binding agent and a mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein that is formed by specific binding of the binding agent to the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein.
  • Specific binding of the binding agent means that the binding agent has a specific binding site, e.g., antigen binding site, that recognizes a site on the mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein.
  • a specific binding site e.g., antigen binding site
  • antibodies raised to a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein and recognizing an epitope on the mpf4, CTAP3 , or ⁇ Ckine chemokine protein are capable of forming a binding agent :mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein complex by specific binding.
  • a binding agent:mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine protein complex allows the measurement of mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine protein in a mixture of other proteins and biologies.
  • the term "antibody: pf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein complex” refers to an embodiment in which the binding agent, e.g., is the antigen binding portion from an antibody.
  • the antibody may be monoclonal, polyclonal, or a binding fragment of an antibody, e.g., an Fab or F(ab)2 fragment.
  • the antibody will preferably be a polyclonal antibody for cross-reactivity testing purposes.
  • 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 and/or phylogenetic relationship, or based upon hybridization conditions. Hybridization conditions are described in greater detail below.
  • nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other biologic components which naturally accompany a native sequence, e.g., proteins 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 usually contain homogeneous nucleic acid molecules, but will, in some embodiments, contain nucleic acids with minor sequence heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
  • the term "mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein” shall encompass, when used in a protein context, a protein having amino acid sequences, particularly from the chemokine motif portions, shown in SEQ ID NO: 2, 4, 6, 8, or 10, or a significant fragment unique to and/or characteristic of such a protein, preferably a natural embodiment.
  • the invention also embraces a polypeptide which exhibits similar structure to mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine, e.g., which interacts with mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine specific binding components.
  • binding components typically bind to a mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine 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.
  • polypeptide or "protein” as used herein includes a significant fragment or segment of chemokine motif portion of a mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine, 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., 35, 40, 45, 50, 60, 70, 80, etc.
  • the segments may have amino and carboxy termini, with appropriate lengths, e.g., starting at residue 1, 2, 3, etc., and ending at residue 134, 133, 132, etc.
  • the invention encompasses
  • 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 mutants.
  • products made by transforming cells with any non-naturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any 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.
  • 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.
  • Solubility is reflected by sedimentation measured in Svedberg units, which are a measure of the sedimentation velocity of a molecule under particular conditions. The determination of the sedimentation velocity was classically performed in an analytical ultracentrifuge, but is typically now performed in a standard ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d ed.) W.H. Freeman & Co., San Francisco, CA; and Cantor and Schimmel (1980) Biophysical Chemistry parts 1-3, W.H. Freeman & Co., San Francisco, CA.
  • a sample containing a putatively soluble polypeptide is spun in a standard full sized ultracentrifuge at about 50K rpm for about 10 -IS - minutes, and soluble molecules will remain in the supernatant.
  • a soluble particle or polypeptide will typically be less than about 3OS, 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. 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.
  • 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 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. Usually the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5.
  • a detergent will be added, typically a mild non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS (3- [3- cholamidopropyl- dimethylammonio] -1-propane sulfonate) , or a low enough concentration as to avoid significant disruption of structural or physiological properties of the protein.
  • CHS cholesteryl hemisuccinate
  • CHAPS 3- [3- cholamidopropyl- dimethylammonio] -1-propane sulfonate
  • Substantially pure in a protein context typically means that the protein is isolated from other contaminating proteins, nucleic acids, and other biologicals derived from the original source organism. Purity, or “isolation” may be assayed by standard methods, and will ordinarily be at least about 50% pure, more ordinarily at least about 60% pure, generally at least about 70% pure, more generally at least about 80% pure, often at least about 85% pure, more often 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. Similar concepts apply, e.g., to antibodies or nucleic acids.
  • “Substantial similarity" 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 56%, more generally at least 59%, ordinarily at least 62%, more ordinarily at least 65%, often at least 68%, more often at least 71%, typically at least 74%, more typically at least 77%, usually at least 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 similarity exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence derived from SEQ ID NO: 1, 3, 5, or 7.
  • selective hybridization will occur when there is at least about 55% similarity 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 similarity 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, e.g., 150, 200, etc.
  • "Stringent conditions” in referring to homology or substantial similarity in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters, typically those controlled in hybridization reactions . The combination of parameters is more important than the measure of any single parameter.
  • a nucleic acid probe which binds to a target nucleic acid under stringent conditions is specific for said target nucleic acid.
  • Such a probe is typically more than 11 nucleotides in length, and is sufficiently identical or complementary to a target nucleic acid over the region specified by the sequence of the probe to bind the target under stringent hybridization conditions.
  • mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines from other mammalian species can be cloned and isolated by cross-species hybridization of closely related species. See, e.g., below. Similarity may be relatively low between distantly related species, and thus hybridization of relatively closely related species is advisable. Alternatively, preparation of an antibody preparation which exhibits less species specificity may be useful in expression cloning approaches .
  • the specified antibodies bind to a particular protein and do not significantly bind other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised to the mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein immunogen with the amino acid sequence depicted in SEQ ID NO: 2, 4, 6, 8, or 10 can be selected to obtain antibodies specifically immunoreactive with mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine proteins and not with other proteins .
  • the antibodies may be species specific, e.g., also recognizing polymorphic and splicing or developmental variants.
  • Nucleic Acids mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine is exemplary of structurally and functionally related proteins . These soluble chemokine proteins will serve to transmit signals between different cell types.
  • the preferred embodiments, as disclosed, will be useful in standard procedures to isolate genes from different individuals or other species, e.g., warm blooded animals, such as birds and mammals.
  • Cross hybridization will allow isolation of related genes encoding proteins from individuals, strains, or species.
  • a number of different approaches are available to successfully isolate a suitable nucleic acid clone based upon the information provided herein. Southern blot hybridization studies can qualitatively determine the presence of homologous genes in human, monkey, rat, mouse, dog, cat, cow, and rabbit genomes under specific hybridization conditions .
  • Complementary sequences will also be used as probes or primers . Based upon identification of the likely amino terminus, other peptides should be particularly useful, e.g., coupled with anchored vector or poly-A complementary PCR techniques or with complementary DNA of other peptides .
  • Techniques for nucleic acid manipulation of genes encoding mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins, such as subcloning nucleic acid sequences encoding polypeptides into expression vectors, labeling probes, DNA hybridization, and the like are described generally in Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual (2nd ed. ) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, which is incorporated herein by reference. This manual is hereinafter referred to as “Sambrook, et al . "
  • DNA sequences encoding mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine proteins are various methods.
  • DNA is isolated from a genomic or cDNA library using labeled oligonucleotide probes having sequences identical or complementary to the sequences disclosed herein. Full-length probes may be used, or oligonucleotide probes may be generated by comparison of the sequences disclosed.
  • probes can be used directly in hybridization assays to isolate DNA encoding mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins, or probes can be designed for use in amplification techniques such as PCR, for the isolation of DNA encoding mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins.
  • Reverse translation computer programs can also provide alternative nucleic acid sequences which encode the same proteins .
  • cDNA is prepared from cells which expresses a mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine protein.
  • cDNA is prepared from the mRNA and ligated into a recombinant vector.
  • the vector is transfected into a recombinant host for propagation, screening, and cloning. Methods for making and screening cDNA libraries are well known. See Gubler and Hoffman (1983) Gene 25:263-269 and Sambrook, et al .
  • the DNA can be extracted from tissue and either mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb.
  • the fragments are then separated by gradient centrifugation and cloned in bacteriophage lambda vectors . These vectors and phage are packaged in vitro, as described in Sambrook, et al . Recombinant phage are analyzed by plaque hybridization as described in Benton and Davis (1977) Science 196:180-182. Colony hybridization is carried out as generally described in e.g., Grunstein, et al. (1975) Proc. Nati. Acad. Sci. USA. 72:3961-3965.
  • DNA encoding a mpf4 , mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein can be identified in either cDNA or genomic libraries by its ability to hybridize with the nucleic acid probes described herein, e.g., in colony or plaque hybridization assays .
  • the corresponding DNA regions are isolated by standard methods familiar to those of skill in the art. See, e.g., Sambrook, et al.
  • Various methods of amplifying target sequences can also be used to prepare DNA encoding mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins .
  • Polymerase chain reaction (PCR) technology is used to amplify such nucleic acid sequences directly from mRNA, from cDNA, and from genomic libraries or cDNA libraries.
  • the isolated sequences encoding mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins may also be used as templates for PCR amplification.
  • oligonucleotide primers complementary to two 5' regions in the DNA region to be amplified are synthesized. The polymerase chain reaction is then carried out using the two primers. See Innis, et al. (eds.) (1990) PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA. Primers can be selected to amplify the entire regions encoding a full-length mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein or to amplify smaller DNA segments as desired.
  • oligonucleotide probes can be prepared from sequence obtained using standard techniques . These probes can then be used to isolate DNA's encoding mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins. Oligonucleotides for use as probes are usually chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Carruthers (1983) Tetrahedron Lett . 22 (20) : 1859-1862, or using an automated synthesizer, as described in Needham-VanDevanter, et al . (1984) Nucleic Acids Res.
  • oligonucleotides Purification of oligonucleotides is performed e.g., by native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom. 255:137-149.
  • the sequence of the synthetic oligonucleotide can be verified using, e.g., the chemical degradation method of Maxam, A.M. and Gilbert, W. in Grossman, L. and Moldave (eds.) (1980) Methods in Enzymology 65:499-560 Academic Press, New York.
  • nucleic acid encoding a mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein was identified.
  • the nucleotide sequence and corresponding open reading frame are provided in SEQ ID NO : 1 , 3 , 5 , or 7.
  • mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokines exhibit limited similarity to portions of chemokines. See, e.g., Matsushima and Oppenheim (1989) Cytokine 1:2-13; Oppenheim, et al . (1991) Ann . Rev. Immunol . 9:617-648; Schall (1991) Cvtokine 3:165-183; and Gronenborn and Clore (1991) Protein Engineering 4:263- 269. Other features of comparison are apparent between the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine and chemokine families.
  • ⁇ -sheet and ⁇ -helix residues can be determined using, e.g., RASMOL program, see Sayle and Milner-White (1995) TIBS 20:374-376; or Gronenberg, et al . (1991) Protein Engineering 4:263-269; and other structural features are defined in Lodi, et al . (1994) Science 263:1762-1767.
  • the 6Ckine embodiments provided herein exhibit peculiar carboxy termini for the chemokine class of molecules .
  • the sequence of h6Ckine extending beyond residue Asp68 to Prolll therein may be peculiar to, and may represent a heretofore unrecognized subgroup of the CC chemokines .
  • This invention provides isolated DNA or fragments to encode a mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein.
  • this invention provides isolated or recombinant DNA which encodes a protein or polypeptide which is capable of hybridizing under appropriate conditions, e.g., high stringency, with the DNA sequences described herein.
  • Said biologically active protein or polypeptide can be an intact ligand, or fragment, and have an amino acid sequence as disclosed in SEQ ID NO: 2, 4, 6, 8, or 10, particularly natural embodiments.
  • Preferred embodiments will be full length natural sequences, from isolates, e.g., about 11,000 to 12,500 daltons in size when unglycosylated, or fragments of at least about 6,000 daltons, more preferably at least about 8,000 daltons. In glycosylated form, the protein may exceed 12,500 daltons.
  • the 6Ckine embodiments exhibit a correspondingly larger size. Further, this invention contemplates the use of isolated or recombinant DNA, or fragments thereof, which encode proteins which are homologous to a mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or
  • Chrl9kine chemokine protein or which were isolated using cDNA encoding a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein 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. Also embraced are methods for making expression vectors with these sequences, or for making, e.g., expressing and purifying, protein products .
  • DNAs which encode a mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine or fragments thereof can be - 2-? - obtained by chemical synthesis, screening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples . Methods for doing so, or making expression vectors are described herein.
  • DNAs can be expressed in a wide variety of host cells for the synthesis of a full-length protein or fragments which can in turn, e.g., be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified molecules; and for structure/function studies.
  • Each mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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 antigen e.g., mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine, or portions thereof, may be expressed as fusions with other proteins or possessing an epitope tag.
  • Expression vectors are typically self-replicating DNA or RNA constructs containing the desired antigen gene or its fragments, usually operably linked to appropriate genetic control elements that are recognized in a suitable host cell .
  • 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 from the host cell.
  • the vectors of this invention contain DNAs which encode a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine, or a fragment thereof, typically encoding, e.g., a biologically active polypeptide, or protein.
  • 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 a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein 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.
  • vectors that cause integration of a mpf4 , mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine gene or its fragments into the host DNA by recombination, or to integrate a promoter which controls expression of an endogenous gene.
  • Vectors contemplate plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host.
  • Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector, but many other forms of vectors which serve an equivalent function are suitable for use herein. See, e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors : A Laboratory Manual Elsevier, N.Y.; and Rodriguez, et al . (eds.) (1988) Vectors : A Survey of Molecular Cloning Vectors and Their Uses Buttersworth, Boston, MA. 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.
  • a representative vector for amplifying DNA is pBR322 or its derivatives .
  • Vectors that can be used to express mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines or mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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) .
  • Lower eukaryotes e.g., yeasts and Dictyostelium, may be transformed with mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine sequence containing vectors .
  • the most common lower eukaryotic host is the baker's yeast, Saccharomyces cerevisiae. It will be used generically to represent lower eukaryotes although a number of other strains and species are also available.
  • Yeast vectors typically consist of a replication origin (unless of the - ⁇ za - 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
  • 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 typically the preferred host cells for expression of the functionally active mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine protein.
  • many higher eukaryotic tissue culture cell lines may be used, e.g., insect baculovirus expression systems, whether from an invertebrate or vertebrate source.
  • mammalian cells are preferred to achieve proper processing, both cotranslationally and posttranslationally. Transformation or transfection and propagation of such cells is routine.
  • 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 (e.g., if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also may contain a selection gene or amplification gene.
  • Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, e.g., from such sources as from adenovirus, SV40, parvoviruses , vaccinia virus, or cytomegalovirus .
  • suitable expression vectors include pCDNAl; pCD, see Okayama, et al . (1985) Mol. Cell Bioi. 5:1136-1142; pMClneo Poly-A, see Thomas, et al . (1987) Cell 51:503- 512; and a baculovirus vector such as pAC 373 or pAC 610.
  • mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines need not be glycosylated to elicit biological responses.
  • the usual pattern will be that provided naturally by the expression system.
  • the pattern will be modifiable by exposing the polypeptide, e.g., in unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system.
  • the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes. It is further understood that over glycosylation may be detrimental to mpf4 , mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine biological activity, and that one of skill may perform routine testing to optimize the degree of glycosylation which confers optimal biological activity.
  • a mpf4, mCTAP3, m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine, 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.
  • a phosphatidyl inositol cleaving enzyme e.g., phosphatidyl inositol phospholipase-C.
  • mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokines 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 Peotide Synthesis Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis Springer-Verlag, New York, NY; and Bodanszky (1984) The Principles of Peptide Synthesis Springer-Verlag, New York, NY.
  • an azide process for example, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p-nitrophenyl ester, N- hydroxysuccinimide ester, or cyanomethyl ester) , a carbodiimidazole process, an oxidative-reductive process, *or a dicyclohexylcarbodiimide (DCCD) /additive process
  • Solid phase and solution phase syntheses are both applicable to the foregoing processes. See also chemical ligation, e.g., Dawson, et al . (1994) Science 266:776-779, a method of linking long synthetic peptides by a peptide bond.
  • the prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, precipitation, electrophoresis and various forms of chromatography, and the like.
  • the mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokines of this invention can be obtained in varying degrees of purity depending upon its desired use. Purification can be accomplished by use of known protein purification techniques or by the use of the antibodies or binding partners herein described, e.g., in immunoabsorbant affinity chromatography.
  • This immunoabsorbant affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate source cells, lysates of other cells expressing the ligand, or lysates or supernatants of cells producing the mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokines as a result of recombinant DNA •techniques, see below. Multiple cell lines may be screened for one which expresses a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine at a high level compared with other cells.
  • Natural mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine '-chemokines can be isolated from natural sources, or by -2.9- expression from a transformed cell using an appropriate expression vector. Purification of the expressed protein is achieved by standard procedures, or may be combined with engineered means for effective purification at high efficiency from cell lysates or supernatants . Epitope or other tags, e.g. , FLAG or Hisg segments, can be used for such purification features.
  • Antibodies can be raised to various mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines, including individual, polymorphic, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in their recombinant forms. Additionally, antibodies can be raised to mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines in either their active forms or in their inactive forms . Anti-idiotypic antibodies may also be used. The antibodies may exhibit various binding specificities for species, individual or polymorphic variants A. Antibody Production
  • a number of immunogens may be used to produce antibodies specifically reactive with mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins.
  • Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
  • Naturally occurring protein may also be used either in pure or impure form.
  • Synthetic peptides, made using the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein sequences described herein, may also used as an immunogen for the production of antibodies to mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokines.
  • Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described herein, and purified as described. Naturally folded or denatured material can be used, as appropriate, for producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein. Methods of producing polyclonal antibodies are known to those of skill in the art. Typically, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized with the mixture.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein of interest.
  • titer of reactivity to the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein of interest When appropriately high titers of antibody to the immunogen are obtained, usually after repeated immunizations, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired. See, e.g. , Harlow and Lane; or Coligan. Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art.
  • spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein (1976) Eur . J. Immunol . 6:511-519, incorporated herein by reference) .
  • Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses , or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.
  • DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according, e.g., to the general protocol outlined by Huse, et al . (1989) Science 246:1275-1281.
  • Antibodies, including binding fragments and single chain versions, against predetermined fragments of mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines can be raised by immunization of animals with conjugates of the fragments with carrier proteins as described above.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody.
  • These antibodies can be screened for binding to normal or defective mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines, or screened for agonistic or antagonistic activity, e.g., mediated through a receptor.
  • 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.
  • 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) Monoclonal Antibodies : Principles and Practice (2d ed.
  • 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.
  • recombinant immunoglobulins may be produced. See, Cabilly, U.S. Patent No. 4,816,567; and Queen, et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.
  • the antibodies of this invention are useful for affinity chromatography in isolating mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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 or supernatant may be passed through the column, the column washed, followed by increasing concentrations of a mild denaturant, whereby purified mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein will be released.
  • antibody binding to the chemokine may be capable of neutralizing receptor binding, and may serve as a receptor antagonist. They may also be useful as Western blot detection reagents, or ELISA reagents.
  • 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.
  • Chrl9kine chemokines may be used for the identification of cell populations expressing mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines. By assaying the expression products of cells expressing mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines it is possible to diagnose disease, e.g., immune-compromised conditions.
  • Antibodies raised against each mpf4 , mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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 .
  • Immunoassays for measurement of mpf4 , mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine proteins can be performed by a variety of methods known to those skilled in the art.
  • immunoassays to measure the protein can be either competitive or noncompetitive binding assays.
  • the sample to be analyzed competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface.
  • the capture agent is an antibody specifically reactive with mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins produced as described above.
  • the concentration of labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample .
  • the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein present in the sample competes with labeled protein for binding to a specific binding agent, for example, an antibody specifically reactive with the mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein.
  • the binding agent may be bound to a solid surface to effect separation of bound labeled protein from the unbound labeled protein.
  • the competitive binding assay may be conducted in liquid phase and a variety of techniques known in the art may be used to separate the bound labeled protein from the unbound labeled protein. Following separation, the amount of bound labeled protein is determined. The amount of protein present in the sample is inversely proportional to the amount of labeled protein binding.
  • a homogeneous immunoassay may be performed in which a separation step is not needed.
  • the label on the protein is altered by the binding of the protein to its specific binding agent. This alteration in the labeled protein results in a decrease or increase in the signal emitted by label, so that measurement of the label at the end of the immunoassay allows for detection or quantitation of the protein.
  • mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins may also be determined by a variety of noncompetitive immunoassay methods. For example, a two- site, solid phase sandwich immunoassay may be used.
  • a binding agent for the protein for example an antibody
  • a second protein binding agent which may also be an -ZSr- antibody, and which binds the protein at a different site, is labeled. After binding at both sites on the protein has occurred, the unbound labeled binding agent is removed and the amount of labeled binding agent bound to the solid phase is measured. The amount of labeled binding agent bound is directly proportional to the amount of protein in the sample.
  • Western blot analysis can be used to determine the presence of mpf4 , mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins in a sample.
  • Electrophoresis is carried out, for example, on a tissue sample suspected of containing the protein. Following electrophoresis to separate the proteins, and transfer of the proteins to a suitable solid support, e.g., a nitrocellulose filter, the solid support is incubated with an antibody reactive with the protein. This antibody may be labeled, or alternatively may be detected by subsequent incubation with a second labeled antibody that binds the primary antibody.
  • the immunoassay formats described above employ labeled assay components .
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. A wide variety of labels and methods may be used. Traditionally, a radioactive label incorporating ⁇ H,
  • Non-radioactive labels include ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation.
  • U.S. Patent No. 4,391,904 which is incorporated herein by reference.
  • Antibodies reactive with a particular protein can also be measured by a variety of immunoassay methods .
  • immunoassay methods For a review of immunological and immunoassay procedures applicable to the measurement o antibodies by immunoassay techniques, see Stites and Terr (eds.) Basic and Clinical Immunology (7th ed.) supra; Maggio (ed.)
  • immunoassays to measure antisera reactive with mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins can be either competitive or noncompetitive binding assays .
  • the sample analyte competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface.
  • the capture agent is a purified recombinant mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein produced as described above.
  • Noncompetitive assays include sandwich assays, in which the sample analyte is bound between two analyte- specific binding reagents. One of the binding agents is used as a capture agent and is bound to a solid surface. The second binding agent is labeled and is used to measure or detect the resultant complex by visual or instrument means. A number of combinations of capture agent and labeled binding agent can be used. A variety of different immunoassay formats, separation techniques, and labels can be also be used similar to those described above for the measurement of mpf4, mCTAP3, m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins.
  • Mouse nucleotide and amino acid sequences are provided in SEQ ID NO: 1 to 6, and human nucleotide and amino acid sequences for h ⁇ Ckine are provided in SEQ ID NO: 7 and 8.
  • 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 polyclonal and monoclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY, which are incorporated herein by reference.
  • a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine receptor can be useful as a specific binding reagent, and advantage can be taken of its specificity of binding, for, e.g., purification of a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine ligand.
  • the specific binding composition can be used for screening an expression library made from a cell line which expresses a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine.
  • Many methods for screening are available, e.g., 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 produce appropriate oligonucleotides to screen a library.
  • the genetic code can be used to select appropriate oligonucleotides useful as probes for screening.
  • synthetic oligonucleotides will be useful in selecting desired clones from a library, including natural allelic an polymorphic variants.
  • the peptide sequences allow preparation of peptides to generate antibodies to recognize such segments, and allow preparation of oligonucleotides which encode such sequences .
  • the sequence also allows for synthetic preparation, e.g., see Dawson, et al. (1994) Science
  • mCTAP3 m6Ckine, h ⁇ Ckine, or Chrl9kine chemokines
  • the gene will normally possess an N-terminal signal sequence, which is removed upon processing and secretion.
  • the exact processing point may be vary in different cell types, and forms of different lengths are often detected.
  • Prediction of the signal cleavage point can be performed, e.g., using the methods of Nielsen, et al . (1997) Protein Eng. 10:1-8.
  • Analysis of the structural features in comparison with the most closely related reported sequences has revealed similarities with other cytokines, particularly the class of proteins known as CC and CXC chemokines .
  • the longer carboxy terminus of the ⁇ Ckine embodiments suggest a distinct subfamily of CC chemokines. This subfamily may exhibit additional biological or structural features.
  • This invention also encompasses proteins or peptides having substantial amino acid sequence similarity with an amino acid sequence of a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine. Natural variants include individual, polymorphic, allelic, strain, or species variants.
  • Amino acid sequence similarity, 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 include natural polymorphic, allelic, and interspecies variations in each respective protein sequence.
  • Typical homologous proteins or peptides will have from 50-100% similarity (if gaps can be introduced) , to 75-100% similarity (if conservative substitutions are included) with the amino acid sequence of the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine. Similarity measures will be at least about 50%, generally at least 60%, more generally 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) J. Mol. Bioi. 48:443-453; Sankoff, et al .
  • nucleic acids encoding mpf4, mCTAP3 , m ⁇ Ckine, or h6Ckine chemokine proteins will normally hybridize to the nucleic acid of SEQ ID NO: 1, 3, 5, or 7 under stringent hybridization conditions.
  • stringent conditions are selected to be about 10° C lower than the thermal melting point (Tm) for the probe sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • stringent conditions will be those in which the salt concentration is about 0.2 molar at pH 7 and the temperature is at least about 50° C.
  • a preferred embodiment will include nucleic acids which will bind to disclosed sequences in 50% formamide and 200 mM NaCI at 42° C.
  • An isolated mpf4 , mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and short inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine antigens, their derivatives, or - ⁇ -D ⁇ proteins having highly similar physiological, immunogenic, or antigenic activity.
  • Modified sequences can be used to produce mutant antigens or to enhance expression. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms.
  • Such mutant mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine derivatives include predetermined or site-specific mutations of the respective protein or its fragments .
  • “Mutant mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine” encompasses a polypeptide otherwise falling within the homology definition of the human mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine as set forth above, but having an amino acid sequence which differs from that of a mpf4 , mCTAP3 , m6Ckine, h ⁇ Ckine, or
  • Chrl9kine chemokine as found in nature, whether by way of deletion, substitution, or insertion.
  • site specific mutant mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine generally includes proteins having significant similarity with a protein having a sequence of SEQ ID NO: 2, 4, 6, 8, or 10, e.g., natural embodiments, and as sharing various biological activities, e.g., antigenic or immunogenic, with those sequences, and in preferred embodiments contain most or all of the disclosed sequence. This applies also to polymorphic variants from different individuals.
  • mutants need not be site specific, mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine mutagenesis can be conducted by making amino acid insertions or deletions. Substitutions, deletions, - - insertions, or any combinations may be generated to arrive at a final construct. Insertions include amino- or carboxyl- terminal fusions, e.g. epitope tags. Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity.
  • the mutations in the DNA normally should not place coding sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins .
  • the present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins .
  • a heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner.
  • the fusion product of an immunoglobulin with a mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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 concept applies to heterologous nucleic acid sequences .
  • new constructs may be made from combining similar functional domains from other proteins.
  • protein-binding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, e.g., Cunningham, et al . (1989) Science 243:1330-1336; and O'Dowd, et al . (1988) J. Bioi. Chem. 263:15985-15992.
  • new chimeric polypeptides exhibiting new combinations of specificities will result from the functional linkage of protein-binding specificities and other functional domains.
  • Binding Agent mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or
  • the immunoassay uses a polyclonal antiserum which was raised to a protein of SEQ ID NO: 2, 4, 6, 8, or 10. This antiserum is selected to have low crossreactivity against other chemokines and any such crossreactivity is removed by immunoabsorbtion prior to use in the immunoassay.
  • the protein of SEQ ID NO: 2, 4, 6, 8, or 10 is isolated as described herein.
  • recombinant protein may be produced in a mammalian cell line.
  • An inbred strain of mice such as balb/c is immunized with the protein of SEQ ID NO: 2, 4, 6, 8, or 10, using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and Lane, supra) .
  • a synthetic peptide, preferably near full length, derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
  • Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 ⁇ or greater are selected and tested for their cross reactivity against C, CC, CX3C, and CXC chemokines, using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573.
  • two chemokines are used in this determination in conjunction with human mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine.
  • Immunoassays in the competitive binding format can be used for the crossreactivity determinations .
  • a protein of SEQ ID NO: 2, 4, 6, 8, or 10 can be ⁇ ⁇ 3- immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the protein of SEQ ID NO: 2, 4, 6, 8, or 10 can be ⁇ ⁇ 3- immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the protein of SEQ ID NO: 2, 4, 6, 8, or 10 can be ⁇ ⁇ 3- immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding
  • the percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorbtion with the above-listed proteins.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein to the immunogen protein
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than twice the amount of the protein, e.g., of SEQ ID NO: 2, 4, 6, 8, or 10 that is required, then the second protein is said to specifically bind to an antibody generated to the immunogen.
  • each of mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine proteins are members of respective families of homologous proteins that comprise two or more genes.
  • the term refers not only to the amino acid sequences disclosed herein, but also to other proteins that are polymorphic, allelic, or non-allelic variants.
  • mpf4, mCTAP3, m6Ckine, h ⁇ Ckine, or Chrl9kine includes nonnatural mutations introduced by deliberate mutation using conventional recombinant technology such as single site mutation, or by excising short sections of DNA encoding mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins, or by substituting new amino acids, or adding new amino acids . Such minor alterations should substantially maintain the immunoidentity of the original molecule and/or its biological activity.
  • these alterations include proteins that are specifically immunoreactive with a designated naturally occurring mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine protein, for example, the mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine protein shown in SEQ ID NO: 2, 4, 6, 8, or 10.
  • the biological properties of the altered proteins can be determined by expressing the protein in an appropriate cell line and measuring, e.g., a chemotactic effect.
  • Particular protein modifications considered minor would include conservative substitution of amino acids with similar chemical properties, as described above for the mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine as a whole.
  • the blocking of physiological response to mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine may result from the inhibition of binding of the protein to its receptor, e.g., through competitive inhibition.
  • in vitro assays of the present invention will often use isolated protein, membranes from cells expressing a recombinant membrane associated mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine, soluble fragments comprising receptor binding segments of these proteins, or fragments attached to solid phase substrates.
  • binding segment mutations and modifications or protein mutations and modifications, e.g., protein analogs.
  • This invention also contemplates -HS-- the use of competitive drug screening assays, e.g., where neutralizing antibodies to antigen or receptor fragments compete with a test compound for binding to the protein. In this manner, the antibodies can be used to detect the presence of a polypeptide which shares one or more antigenic binding sites of the protein and can also be used to occupy binding sites on the protein that might otherwise interact with a receptor.
  • “Derivatives" of mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine antigens include amino acid sequence mutants , glycosylation variants , and covalent or aggregate conjugates with other chemical moieties. Covalent derivatives can be prepared by linkage of functionalities to groups which are found in mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine 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, 0-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. Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties, including ribosyl groups or cross-linking reagents.
  • phosphorylated amino acid residues e.g., phosphotyrosine, phosphoserine, or phosphothreonine
  • a major group of derivatives are covalent conjugates of the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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 protein derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues.
  • Fusion polypeptides between mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine and other homologous or heterologous proteins are also provided.
  • Many growth factors and cytokines are homodimeric entities, and a repeat construct may have various advantages, including lessened susceptibility to proteolytic degradation.
  • many receptors require dimerization to transduce a signal, and various dimeric proteins or domain repeats can be desirable.
  • Heterologous polypeptides may be fusions between different surface markers, resulting in, e.g., a hybrid protein exhibiting receptor binding specificity.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins .
  • Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a protein, e.g., a receptor-binding segment, so that the presence or location of the fused protein may be easily determined.
  • a reporter polypeptide e.g., luciferase
  • a segment or domain of a protein e.g., a receptor-binding segment
  • Other gene fusion partners include bacterial ⁇ -galactosidase, trpE, Protein A, ⁇ -lactamase, alpha amylase, alcohol dehydrogenase, 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, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups.
  • the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands.
  • This invention also contemplates the use of derivatives of mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine other than variations in amino acid sequence or glycosylation.
  • Such derivatives may involve covalent or aggregative association with chemical moieties.
  • 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 mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine 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-mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine antibodies or its receptor.
  • a solid support such as cyanogen bromide-activated SEPHAROSE
  • the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine can also be labeled with a detectable group, e.g., radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.
  • a detectable group e.g., radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.
  • Purification of mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokines may be effected by immobilized antibodies or receptor.
  • Isolated mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine genes will allow transformation of cells lacking expression of corresponding mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine, e.g., either species types or cells which lack corresponding proteins 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 mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine receptor proteins. Subcellular fragments, e.g., cytoplasts or membrane fragments, can be isolated and used.
  • 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 .
  • mpf4, mCTAP3, m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine nucleotides e.g., mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine DNA or RNA, may be used as a component in a forensic assay.
  • nucleotide sequences provided may be labeled using, e.g., 32p or biotin and used to probe standard restriction fragment polymorphism blots, providing a measurable character to aid in distinguishing between individuals .
  • probes may be used in well-known forensic techniques such as genetic fingerprinting.
  • nucleotide probes made from mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine sequences may be used in in si tu assays to detect chromosomal abnormalities .
  • rearrangements in the human chromosome encoding a mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine gene may be detected via well-known in situ techniques, using mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine probes in conjunction with other known chromosome markers .
  • Antibodies and other binding agents directed towards mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or ' Chrl9kine chemokine proteins or nucleic acids may be used to purify the corresponding mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine molecule .
  • antibody purification of mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine components is both possible and practicable.
  • Antibodies and other binding agents may also be used in a diagnostic fashion to determine whether mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine components are present in a tissue sample or cell population using well-known techniques described herein.
  • the ability to attach a binding agent to a mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine provides a means to diagnose disorders associated with mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine misregulation.
  • Antibodies and other mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine binding agents may also be useful as histological markers.
  • mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine expression is limited to specific tissue types.
  • a probe such as an antibody or nucleic acid
  • This invention also provides reagents with significant therapeutic value.
  • the mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine (naturally occurring or recombinant) , fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity to a mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine, are useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions. 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 mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine is a target for an agonist or antagonist of the protein.
  • the proteins likely play a role in regulation or development of neuronal or - - hematopoietic cells, e.g., lymphoid cells, which affect immunological responses .
  • an antagonist for h ⁇ Ckine may have potential for T cell mediated autoimmunity, e.g., joint inflammation, organ/tissue inflammation.
  • T cell mediated autoimmunity e.g., joint inflammation, organ/tissue inflammation.
  • secondary lymphoid organs e.g., lymph nodes, tonsils
  • h ⁇ Ckine may be a preliminary molecule involved in the initial inflammatory response .
  • Other abnormal developmental conditions are known in cell types shown to possess mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine mRNA by northern blot analysis. See Berkow (ed.
  • chemokines have also been implicated in viral replication mechanisms. See, e.g., Cohen (1996) Science 272:809-810; Feng, et al . (1996) Science 272:872- 877; and Cocchi, et al . (1995) Science 270:1811-1816.
  • the mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine may be useful in a similar context.
  • the stalk structure may be very important in presentation of the ligand domain, and other chemokines may be advantageously substituted for the chemokine domain in this molecule.
  • Recombinant mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine or mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine antibodies can be purified and then administered to a patient.
  • 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 .
  • Drug screening using antibodies or receptor or fragments thereof can identify compounds having binding affinity to mpf4, mCTAP3, m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine, 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 protein. Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of a mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine.
  • This invention further contemplates the therapeutic use of antibodies to mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine as antagonists.
  • This approach should be particularly useful with other mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman, et al . (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press; and (1990) Remington ' s Pharmaceutical Sciences (17th ed.
  • Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co., Rahway, NJ. 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.
  • mpf4, mCTAP3, m6Ckine, h ⁇ Ckine, or Chrl9kine chemokines, fragments thereof, and 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 any conventional dosage formulation. While it is possible for the active ingredient to be administered alone, it is 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.
  • Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8th ed. ) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17th ed. ) Mack Publishing Co., Easton, PA; Avis, et al .
  • the therapy of this invention may be combined with or used in association with other therapeutic agents .
  • kits and assay methods which are capable of screening compounds for binding activity to the proteins .
  • automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. See, e.g., Fodor, et al . (1991) Science 251:767-773, and other descriptions of chemical diversity libraries, which describe means for testing of binding affinity by a plurality of compounds.
  • Suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine as provided by this invention.
  • antagonists can normally be found once the protein has been structurally defined. Testing of potential protein analogs is now possible upon the development of highly automated assay methods using a purified receptor. In particular, new agonists and antagonists will be discovered by using screening techniques described herein.
  • This invention is particularly useful for screening compounds by using recombinant protein in a variety of drug screening techniques.
  • the advantages of using a recombinant protein in screening for specific ligands include: (a) improved renewable source of the mpf4, mCTAP3, m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine from a specific source; (b) potentially greater number of ligands per cell giving better signal to noise ratio in assays; and (c) species variant specificity (theoretically giving greater biological and disease specificity) .
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing a mpf4 , mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine receptor, e.g., CXCR3 for ⁇ Ckine.
  • Cells may be isolated which express a receptor in isolation from any others. Such cells, either in viable or fixed form, can be used for standard ligand/receptor binding assays. See also, Parce, et al. (1989) Science 246:243-247; and Owicki, et al . (1990) Proc. Nat'l Acad. Sci.
  • any one of numerous techniques can be used to separate bound from free ligand to assess the degree of ligand binding.
  • This separation step could typically involve a procedure such as adhesion to filters followed by washing, adhesion to plastic followed by washing, or centrifugation of the cell membranes .
  • Viable cells could also be used to screen for the effects of drugs on mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine mediated functions, e.g., second messenger levels, i.e., Ca ++ ; cell proliferation; inositol phosphate pool changes; and others.
  • Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system. Calcium sensitive dyes will be useful for detecting Ca ++ levels, with a fluorimeter or a fluorescence cell sorting apparatus .
  • the 6Ckine-CXCR3 ligand receptor pair can also be used as a positive control to identify other CXCR3 ligands.
  • CXCR3 can be attached to a solid support, e.g., a chip, a bead in an affinity column, a microtiter plate well . Unknown samples containing potential ligands are incubated with the receptor. ⁇ Ckine is treated similarly in a separate sample. Binding affinities are measured as described above and compared to 6Ckine. Similarly, cells expressing CXCR3 , which respond biologically to ligand binding, can be assayed and responses compared to 6Ckine induced activity.
  • Another method utilizes membranes from transformed eukaryotic or prokaryotic host cells as the source of a mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine. These cells are stably transformed with DNA vectors directing the expression of a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine, e.g., an engineered membrane bound form. Essentially, the membranes would be prepared from the cells and used in a receptor/ligand binding assay such as the competitive assay set forth above .
  • Still another approach is to use solubilized, unpurified or solubilized, purified mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine from transformed eukaryotic or prokaryotic host cells .
  • This allows for a "molecular" binding assay with the advantages of increased specificity, the ability to automate, and high drug test throughput.
  • Another technique for drug screening involves an approach which provides high throughput screening for compounds having suitable binding affinity to a mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine antibody and is described in detail in Geysen, European Patent Application 84/03564, published on September 13, 1984.
  • a solid substrate e.g., plastic pins or some other appropriate surface
  • Rational drug design may also be based upon structural studies of the molecular shapes of the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine and other effectors or analogs. See, e.g., Methods in
  • 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.
  • a purified mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine can be coated directly onto plates for use in the aforementioned drug screening techniques .
  • non-neutralizing antibodies to these ligands can be used as capture antibodies to immobilize the respective ligand on the solid phase.
  • mpf4, mCTAP3, m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine proteins, fragments thereof, peptides, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine or a mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine receptor.
  • the kit will have a compartment containing either a defined mpf4, mCTAP3, m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine peptide or gene segment or a reagent which recognizes one or the other, e.g., receptor fragments or antibodies.
  • a kit for determining the binding affinity of a test compound to a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine would typically comprise a test compound; a labeled compound, e.g.
  • mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine can be evaluated in suitable biological assays, as are well known in the art, to determine whether they act as agonists or antagonists to the receptor.
  • suitable biological assays as are well known in the art, to determine whether they act as agonists or antagonists to the receptor.
  • the availability of recombinant mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine polypeptides also provide well defined standards for calibrating such assays.
  • a preferred kit for determining the concentration of, for example, a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine in a sample would typically comprise a labeled compound, e.g., receptor or antibody, having known binding affinity for the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine, a source of mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine (naturally occurring or recombinant) , and a means for separating the bound from free labeled compound, for example, a solid phase for immobilizing the mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine.
  • a labeled compound e.g., receptor or antibody
  • Antibodies including antigen binding fragments, specific for the mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine or ligand fragments are useful in diagnostic applications to detect the presence of elevated levels of mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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 in serum, or the like.
  • Diagnostic assays may be homogeneous (without a separation step between free reagent and antigen-mpf4 , mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine complex) or heterogeneous (with a separation step) .
  • Various commercial assays exist, such as radioimmunoassay (RIA) , enzyme-linked immunosorbent assay (ELISA) , enzyme immunoassay (EIA) , enzyme- multiplied immunoassay technique (EMIT) , substrate- labeled fluorescent immunoassay (SLFIA) , and the like.
  • unlabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the antibody to a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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,
  • Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against a mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine, as such may be diagnostic of various abnormal states. For example, overproduction of mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or
  • Chrl9kine chemokine may result in production of various immunological or other medical reactions which may be diagnostic of abnormal physiological states, e.g., in cell growth, activation, or differentiation.
  • 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 receptor, or labeled mpf4, mCTAP3, m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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 protein, test compound, mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine, or antibodies thereto can be labeled either directly or indirectly.
  • Possibilities for direct labeling include label groups: radiolabels such as 125 f enzymes (U.S. Pat. No.
  • the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine can be immobilized on various matrices followed by washing. Suitable matrices include plastic such as an ELISA plate, filters, and beads. Methods of immobilizing the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling, and biotin- avidin.
  • the last step in this approach involves the precipitation of ligand/receptor or ligand/antibody complex by any of several methods including those utilizing, e.g., an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate.
  • suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678.
  • Another diagnostic aspect of this invention involves use of oligonucleotide or polynucleotide sequences taken from the sequence of a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine .
  • These sequences can be used as probes for detecting levels of the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine message in samples from natural sources, or patients suspected of having an abnormal condition, e.g., cancer or developmental problem.
  • the preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature.
  • an oligonucleotide probe should have at least about 14 nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes may be up to several kilobases .
  • Various labels may be employed, most commonly radionuclides, particularly 32p_ 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, fluorophores, 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 using many 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 these and 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. Qualitative or quantitative expression of each chemokine may be evaluated by standard methods at the protein or mRNA levels .
  • a binding partner of a specific interaction methods exist for isolating the counter- partner. See, Gearing, et al . (1989) EMBO J. 8:3667- -fit- 3676.
  • means to label a mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine without interfering with the binding to its receptor can be determined.
  • an affinity label or epitope tag can be fused to either the amino- or carboxyl- terminus of the ligand.
  • An expression library can be screened for specific binding of the mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine, e.g., by cell sorting, or other screening to detect subpopulations which express such a binding component. See, e.g., Ho, et al. (1993) Proc. Nat ' 1 Acad. Sci. USA 90:11267-11271. Alternatively, a panning method may be used. See, e.g., Seed and Aruffo (1987) Proc. Nat ' 1 Acad. Sci. USA 84:3365-3369.
  • a two-hybrid selection system may also be applied making appropriate constructs with the available chemokine sequences. See, e.g., Fields and Song (1989) Nature 340:245-246. Standard Ca ++ flux methods can also be utilized. See, e.g., Coligan, et al . (eds.) (1992 and periodic supplements) Current Protocols in Immunology Greene/Wiley, New York, NY.
  • Protein cross-linking techniques with label can be applied to isolate binding partners of a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine. This would allow identification of proteins which specifically interact with a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or
  • Chrl9kine chemokine e.g., in a ligand-receptor like manner.
  • the chemokine family binds to receptors of the seven transmembrane receptor family, and the receptor for the mpf4, mCTAP3 , m ⁇ Ckine, h6Ckine, or Chrl9kine chemokine is likely to exhibit a similar structure.
  • the receptor will be found by expression in a system which is capable of expressing such a membrane protein in a form capable of exhibiting ligand binding capability.
  • 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,” Methods in Enzymology vol. 182, and other volumes in this series; and manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, NJ, or Bio-Rad, Richmond, CA. Combination with recombinant techniques allow fusion to appropriate segments (epitope tags) , e.g.
  • Standard immunological techniques are described, e.g., in Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Methods in Enzymology volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.
  • Assays for neural cell biological activities are described, e.g., in Wouterlood (ed. 1995) Neuroscience Protocols modules 10, Elsevier; Methods in Neurosciences Academic Press; and Neuromethods Humana Press, Totowa, NJ.
  • Methodology of developmental systems is described, e.g., in Meisami (ed.) Handbook of Human Growth and Developmental Biology CRC Press; and Chrispeels (ed.) Molecular Techniques and Approaches in Developmental Biology Interscience.
  • telomeres A clone encoding the mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine is isolated from a natural source by many different possible methods. Given the sequences provided herein, PCR primers or hybridization probes are selected and/or constructed to isolate either genomic DNA segments or cDNA reverse transcripts. Appropriate cell sources include listed tissues, e.g., brain libraries. Tissue distribution below also suggests source tissues. Genetic and polymorphic or allelic variants are isolated by screening a population of individuals . PCR based detection is performed by standard methods, preferably using primers from opposite ends of the coding sequence, but flanking segments might be selected for specific purposes.
  • hybridization probes are selected. Particular AT or GC contents of probes are selected depending upon the expected homology and mismatching expected. Appropriate stringency conditions are selected to balance an appropriate positive signal to background ratio. Successive washing steps are used to collect clones of greater homology.
  • Further clones are isolated using an antibody based selection procedure. Standard expression cloning methods are applied including, e.g., FACS staining of membrane associated expression product.
  • the antibodies are used to identify clones producing a recognized protein.
  • antibodies are used to purify a mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine chemokine, with protein sequencing and standard means to isolate a gene encoding that protein.
  • Genomic sequence based methods will also allow for identification of sequences naturally available, or otherwise, which exhibit homology to the provided sequences .
  • Similar methods are used as above to isolate an appropriate primate chemokine gene from another primate. Similar source materials as indicated above are used to isolate natural genes, including genetic, polymorphic, allelic, or strain variants. Other species variants are also isolated using similar methods. Alternatively, gene databases may be searched for the appropriate motifs .
  • rodent source is selected as above, e.g., rat, hamster, etc. Similar methods are utilized to isolate a species variant, though the level of similarity will typically be lower for rodent chemokine as compared to a human to other primate sequence.
  • the coding sequence is inserted into an appropriate expression vector.
  • This may be in a vector specifically selected for a prokaryote, yeast, insect, or higher vertebrate, e.g., mammalian expression system. Standard methods are applied to produce the gene product, preferably as a soluble secreted molecule, but will, in certain instances, also be made as an intracellular protein. Intracellular proteins typically require cell lysis to recover the protein, and insoluble inclusion bodies are a common starting material for further purification.
  • recombinant production means are used, although natural forms may be purified from appropriate sources .
  • the protein product is purified by standard methods of protein purification, in certain cases, e.g., coupled with immunoaffinity methods. Immunoaffinity methods are used either as a purification step, as described above, or as a detection assay to determine the separation properties of the protein.
  • the protein is secreted into the medium, and the soluble product is purified from the medium in a soluble form.
  • inclusion bodies from prokaryotic expression systems are a useful source of material.
  • the insoluble protein is solubilized from the inclusion bodies and refolded using standard methods .
  • Purification methods are developed as described above.
  • the product of the purification method described above is characterized to determine many structural features. Standard physical methods are applied, e.g., amino acid analysis and protein sequencing.
  • the resulting protein is subjected to CD spectroscopy and other spectroscopic methods, e.g., NMR, ESR, mass spectroscopy, etc.
  • the product is characterized to determine its molecular form and size, e.g., using, gel chromatography and similar techniques . Understanding of the chromatographic properties will lead to more gentle or efficient purification methods.
  • Prediction of glycosylation sites may be made, e.g., as reported in Hansen, et al . (1995) Biochem. J. 308:801- 813.
  • DNA for expression or protein produced, e.g., as above, animals are immunized to produce antibodies.
  • Polyclonal antiserum is raised, in some cases, using non- purified antigen, though the resulting serum will exhibit higher background levels.
  • the antigen is purified using standard protein purification techniques, including, e.g., affinity chromatography using polyclonal serum indicated above. Presence of specific antibodies is detected using defined synthetic peptide fragments .
  • Polyclonal serum is raised against a purified antigen, purified as indicated above, or using, e.g., a plurality of, synthetic peptides.
  • a series of overlapping synthetic peptides which encompass all of the full length sequence, if presented to an animal, will produce serum recognizing most linear epitopes on the protein.
  • Such an antiserum is used to affinity purify protein, which is, in turn, used to introduce intact full length protein into another animal to produce another antiserum preparation.
  • Similar techniques are used to generate induce monoclonal antibodies to either unpurified antigen, or, preferably, purified antigen.
  • the antiserum or antibodies may recognize native protein, or may recognize denatured antigen.
  • Cellular and tissue distribution Distribution of the protein or gene products are determined, e.g. , using immunohistochemistry with an antibody reagent, as produced above, or by screening for nucleic acids encoding the chemokine. Hybridization or PCR methods are used to detect DNA, cDNA, or message content. Histochemistry allows determination of the specific cell types within a tissue which express higher or lower levels of message or DNA. Antibody techniques are useful to quantitate protein in a biological sample, including a liquid or tissue sample. Immunoassays are developed to quantitate protein. Also, FACS analysis may be used to evaluate expression in a cell population.
  • the pro-migratory activities of mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine are assessed in microchemotaxis assays. See, e.g., Bacon, et al . (1988) Br. J. Pharmacol . 95:966-974.
  • Chemokines may also be assayed for activity in hemopoietic assays as described, e.g., by H. Broxmeyer. They may be assayed for angiogenic activities as described, e.g., by R. Streiter.
  • IX Biological activities, direct and indirect
  • a robust and sensitive assay is selected as described above, e.g., on immune cells, neuronal cells, or stem cells .
  • Chemokine is added to the assay in increasing doses to see if a dose response is detected.
  • a proliferation assay cells are plated out in plates. Appropriate culture medium is provided, and chemokine is added to the cells in varying amounts. Growth is monitored over a period of time which will detect either a direct effect on the cells, or an indirect effect of the chemokine.
  • an activation assay or attraction assay is used.
  • An appropriate cell type is selected, e.g., hematopoietic cells, myeloid (macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid (T cell, B cell, or NK cells) , neural cells (neurons, neuroglia, oligodendrocytes, astrocytes, etc.), or stem cells, e.g., progenitor cells which differentiate to other cell types, e.g., gut crypt cells and undifferentiated cell types.
  • IX Structure activity relationship Information on the criticality of particular residues is determined using standard procedures and analysis.
  • Standard mutagenesis analysis is performed, e.g., by generating many different variants at determined positions, e.g., at the positions identified above, and evaluating biological activities of the variants . This may be performed to the extent of determining positions which modify activity, or to focus on specific positions to determine the residues which can be substituted to either retain, block, or modulate biological activity.
  • analysis of natural variants can indicate what positions tolerate natural mutations . This may result from populational analysis of variation among individuals, or across strains or species. Samples from selected individuals are analysed, e.g., by PCR analysis and sequencing. This allows evaluation of population polymorphisms .
  • Agonists or antagonists are screened for ability to induce or block biological activity.
  • the candidate compounds e.g., sequence variants of natural mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine, are assayed for their biological activities.
  • compounds are screened, alone or in combinations, to determine effects on biological activity.
  • a mpf4, mCTAP3 , m6Ckine, h6Ckine, or Chrl9kine chemokine can be used as a specific binding reagent to identify its binding partner, by taking advantage of its specificity of binding, much like an antibody would be used.
  • a binding reagent is either labeled as described above, e.g., fluorescence or otherwise, or immobilized to a substrate for panning methods.
  • the typical chemokine receptor is a seven transmembrane receptor.
  • the binding composition e.g., chemokine
  • chemokine is used to screen an expression library made from a cell line which expresses a binding partner, i.e. receptor.
  • Standard staining techniques are used to detect or sort intracellular or surface expressed receptor, or surface expressing transformed cells are screened by panning. Screening of intracellular expression is performed by various staining or immunofluorescence procedures. See also McMahan, et al . (1991) EMBO J. 10:2821-2832.
  • CXC chemokine receptor a receptor for m6Ckine was found to be a CXC chemokine receptor, designated CXCR3. This receptor is shared with IP-10 and Mig, chemokines which possess potent angiostatic and antitumor properties. See, e.g., Keane, et al. (1997) J. Immunol. 159:1437-1443; and Farber (1997) J. Leukoc. Bioi. 61:246-257.
  • a positive control is prepared, e.g., of human mpf4, mCTAP3 , m6Ckine, h ⁇ Ckine, or Chrl9kine chemokine 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.
  • the slides may be stored at -80 * C after all liquid is removed.
  • 0.5 ml incubations are performed as follows. Add HBSS/saponin (0.1%) with 32 ⁇ l/ml of 1 M NaN3 for 20 min. Cells are then washed with HBSS/saponin IX. Add chemokine or chemokine/antibody complex to cells and incubate for 30 min. Wash cells twice with HBSS/saponin. If appropriate, add first antibody for 30 min. Add second antibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, and incubate for 30 min.
  • second antibody e.g., Vector anti-mouse antibody
  • ELISA solution e.g., Vector Elite ABC horseradish peroxidase solution
  • 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.
  • chemokine reagents are used to affinity purify or sort out cells expressing a receptor. See, e.g., Sambrook, et al . or Ausubel, et al . Another strategy is to screen for a membrane bound receptor by panning.
  • the receptor cDNA is constructed as described above.
  • the ligand can be immobilized and used to immobilize expressing cells . Immobilization may be achieved by use of appropriate antibodies which recognize, e.g., a FLAG sequence of a chemokine fusion construct, or by use of antibodies raised against the first antibodies. Recursive cycles of selection and amplification lead to enrichment of appropriate clones and eventual isolation of receptor expressing clones .
  • Phage expression libraries can be screened by chemokine. Appropriate label techniques, e.g., anti-FLAG antibodies, will allow specific labeling of appropriate clones.
  • the antibody described above is used to identify expression of mpf4, mCTAP3 , m ⁇ Ckine, h ⁇ Ckine, or Chrl9kine in various tissues .
  • Methods for immunohistochemical staining are described, e.g., in - 7 - Sheehan, et al . (eds.) (1987) Theory and Practice of
  • SEQ ID NO 1 is mpf4 mouse nucleic acid sequence
  • SEQ ID NO 2 is mpf4 mouse amino acid sequence
  • SEQ ID NO 3 is mCTAP3 mouse nucleic acid sequence .
  • SEQ ID NO 4 is mCTAP3 mouse amino acid sequence
  • SEQ ID NO 5 is m ⁇ Ckine mouse nucleic acid sequence .
  • SEQ ID NO 6 is m ⁇ Ckine mouse amino acid sequence
  • SEQ ID NO 7 is h ⁇ Ckine human nucleic acid sequence.
  • S SEEQQ I IDD N NOO: 8 8 is h ⁇ Ckine human amino acid sequence.
  • SEQ ID NO 9 is ⁇ Ckine porcine amino acid sequence.
  • SEQ ID NO 1 10 is Chrl9kine human amino acid sequence.
  • Lys Asn Gly Arg Lys lie Cys Leu Asp Arg Gin Ala Pro Leu Tyr Lys 45 50 55
  • Lys Val lie Lys Lys lie Leu Glu Ser 60 65 (2) INFORMATION FOR SEQ ID NO: 3:
  • Asp Pro Tyr lie Glu Leu Arg Cys Arg Cys Thr Asn Thr lie Ser Gly 5 10 15 ATC CCA TTC AAT TCT ATC TCC CTT GTG AAT GTG TAC AGG CCA GGA GTT 301
  • MOLECULE TYPE protein
  • ATCCCCCACG AACTTCAAGC TGGGTGGTTC ACGGTCCAAC TCACAGGCAA AGAGGGAGCT 559 AGAAAACAGA CTCAGGAGCC CAAAGCAGCC ACCTCATGCT GGCCTCCGTC CACACCCTTG 619
  • TCTTCCTTGT AAATATGATT TATACCTAAC TGAATAAAAA GCTGTTCTGT CTTCCCACCC 797 AAAAAAAAAA AAAAAAA 814

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Abstract

L'invention porte sur de nouvelles chémokines CC et CXC issues de la souris et de l'homme, sur leurs réactifs apparentés comprenant des protéines purifiées, des anticorps spécifiques et des acides nucléiques codant ces chémokines. L'invention porte également sur des procédés de fabrication et d'utilisation de ces réactifs et de kits de diagnostic.
EP97944422A 1996-10-02 1997-10-02 Chemokines mammaliennes Withdrawn EP0951549A1 (fr)

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JP2001523946A (ja) 2001-11-27
AU4592397A (en) 1998-04-24
WO1998014581A1 (fr) 1998-04-09
CA2267092A1 (fr) 1998-04-09

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