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WO1996034891A1 - Chimiokine beta-8, chimiokine beta-1 et proteine 4 inflammatoire des macrophages d'origine humaine - Google Patents

Chimiokine beta-8, chimiokine beta-1 et proteine 4 inflammatoire des macrophages d'origine humaine Download PDF

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WO1996034891A1
WO1996034891A1 PCT/US1995/009058 US9509058W WO9634891A1 WO 1996034891 A1 WO1996034891 A1 WO 1996034891A1 US 9509058 W US9509058 W US 9509058W WO 9634891 A1 WO9634891 A1 WO 9634891A1
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polypeptide
polynucleotide
dna
cellε
seq
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PCT/US1995/009058
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English (en)
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Craig A. Rosen
Steven M. Ruben
Haodong Li
Mark D. Adams
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Human Genome Sciences, Inc.
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Priority to MX9708537A priority Critical patent/MX9708537A/es
Priority to AU31346/95A priority patent/AU3134695A/en
Priority to JP8533269A priority patent/JPH11505417A/ja
Priority to EP95927260A priority patent/EP0871672A4/fr
Publication of WO1996034891A1 publication Critical patent/WO1996034891A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • Thi ⁇ invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, a ⁇ well a ⁇ the production of such polynucleotides and polypeptide ⁇ . More particularly, the polypeptides of the present invention have been putat vely identified a ⁇ human Chemokine Beta-8 (Ck/3-8) , macrophage inflammatory protem-4 (MIP-4) and Chemokine Beta-l (Ck/3-1) . The invention also relates to inhibiting the action of such polypeptides.
  • Chemokine al ⁇ o referred to a ⁇ intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are 8-10 kd in size. In general, chemokme ⁇ exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bond ⁇ . Based on the arrangement of the first two cysteine residues, r chemok ⁇ ne ⁇ have been cla ⁇ ified into two ⁇ ubfamilie ⁇ , alpha and beta. In the alpha ⁇ ubfamily, the fir ⁇ t two cysteine ⁇ are separated by one amino acid and hence are referred to a ⁇ the "C-X-C" ⁇ ubfamily. In the beta ⁇ ubfamily, the two cy ⁇ teine ⁇ are in an adjacent po ⁇ ition and are, therefore, referred to as the "C-C" subfamily. Thus far, at least eight different members of this family have been identified in humans.
  • the intercrine cytokines exhibit a wide variety of functions. A hallmark feature i ⁇ their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, ba ⁇ ophil ⁇ and fibroblast ⁇ . Many chemokines have proinflammatory activity and are involved in multiple ⁇ tep ⁇ during an inflammatory reaction.
  • the ⁇ e activitie ⁇ include ⁇ timulation of histamine release, lyso ⁇ omal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cell ⁇ , enhanced binding of complement protein ⁇ , induced expression of granulocyte adhesion molecules and complement receptor ⁇ , and respiratory burst.
  • MIP-1 macrophage inflammatory protein 1
  • PF-4 platelet factor-4
  • IL-8 Interleukin-8
  • chemokines have been implicated in a number of physiological and disease condition ⁇ , including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological di ⁇ order ⁇ ⁇ uch a ⁇ allergy, asthma and arthritis.
  • MIP-1 wa ⁇ originally identified a ⁇ an endotoxin- induced proinflammatory cytokine produced from macrophage ⁇ .
  • Subsequent studie ⁇ have ⁇ hown that MIP-l i ⁇ composed of two different, but related, protein ⁇ MlP-l ⁇ and MIP-l/3.
  • Both MlP-l ⁇ and MIP-10 are chemo-attractant ⁇ for macrophage ⁇ , monocyte ⁇ and T lymphocytes.
  • biochemical purification and sub ⁇ equent ⁇ equence analy ⁇ is of a multi- potent stem cell inhibitor (SCI) revealed that SCI is identical to MIP-l ⁇ .
  • MIP-13 can counteract the ability of MlP-l ⁇ to ⁇ uppre ⁇ s hematopoietic stem cell proliferation. This finding leads to the hypothesis that the primary physiological role of MIP-l is to regulate hematopoie ⁇ i ⁇ in bone marrow, and that the propo ⁇ ed inflammatory function i ⁇ ⁇ econdary.
  • MIP-l ⁇ a ⁇ a ⁇ tem cell inhibitor relate ⁇ to its ability to block the cell cycle at the Gl/S interphase. Furthermore, the inhibitory effect of MlP-l ⁇ seems to be restricted to immature progenitor cell ⁇ and it i ⁇ actually ⁇ timulatory to late progenitors in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) .
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • MIP-l proteins can be detected in early wound inflammation cell ⁇ and have been ⁇ hown to induce production of IL-1 and IL-6 from wound fibrobla ⁇ t cell ⁇ .
  • purified native MIP-l (comprising MIP-l, MIP-lo. and MIP-13 polypeptides) causes acute inflammation when injected either subcutaneou ⁇ ly into the footpads of mice or intracisternally into the cerebro ⁇ pinal fluid of rabbit ⁇ (Wolpe and Cerami, 1989, FASEB J. 3:2565-73).
  • MIP-l has been recovered during the early inflammatory pha ⁇ e ⁇ of wound healing in an experimental mouse model employing sterile wound chambers (Fahey, et al., 1990, Cytokine, 2:92) .
  • PCT application WO 92/05198 filed by Chiron Corporation, discloses a DNA molecule which is active as a template for producing mammalian macrophage inflammatory proteins (MIPs) in yeast.
  • MIP-lo- and MIP-l/3 are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. MlP-l ⁇ has been expre ⁇ sed in yeast cells and purified to homogeneity. Structural analysis confirmed that MlP-la has a very ⁇ imilar secondary and tertiary structure to PF-4 and IL-8 with which it shares limited sequence homology. It has also been demonstrated that MIP-l ⁇ is active in vivo to protect mouse stem cells from subsequent in vitro killing by tritiated thymidine.
  • MIP-l ⁇ was also ⁇ hown to enhance the proliferation of more committed progenitor granulocyte macrophage colony-forming cells in response to granulocyte macrophage colony- stimulating factor (Clemens, J.M. , et al., Cytokine, 4:76-82 (1992) ) .
  • the polypeptide ⁇ of the pre ⁇ ent invention, CkjS-1, originally referred to a ⁇ MIP-17 in the parent patent application, is a new member of the ⁇ chemokine family based on amino sequence homology.
  • novel mature polypeptides which are human Ck0-8, human MIP-4 and human Ck/3-1 as well a ⁇ biologically active and diagno ⁇ tically or therapeutically useful fragments, analogs and derivative ⁇ thereof.
  • nucleic acid molecules encoding such polypeptide ⁇ , including mRNA ⁇ , DNA ⁇ , cDNA ⁇ , genomic DNA a ⁇ well a ⁇ biologically active and diagno ⁇ tically or therapeutically useful fragments, analogs and derivatives thereof.
  • a proce ⁇ for producing ⁇ uch polypeptide ⁇ by recombinant technique ⁇ which compri ⁇ e ⁇ culturing recombinant prokaryotic and/or eukaryotic ho ⁇ t cell ⁇ , containing nucleic acid ⁇ equence ⁇ , under condition ⁇ promoting expression of said proteins and sub ⁇ equent recovery of ⁇ aid protein ⁇ .
  • a proces ⁇ for utilizing such polypeptides, or polynucleotides encoding ⁇ uch polypeptide ⁇ for therapeutic purpo ⁇ e ⁇ for example, to protect bone marrow ⁇ tern cell ⁇ from chemotherapeutic agents during chemotherapy, to remove leukemic cells, to stimulate an immune response, to regulate hematopoie ⁇ i ⁇ and lymphocyte trafficking, to treat p ⁇ oria ⁇ is, solid tumors, to enhance ho ⁇ t defenses against resi ⁇ tant chronic and acute infection, and to ⁇ timulate wound healing.
  • antibodie ⁇ again ⁇ t such polypeptide ⁇ there are provided antibodie ⁇ again ⁇ t such polypeptide ⁇ .
  • antagonist ⁇ to ⁇ uch polypeptide ⁇ which may be used to inhibit the action of such polypeptides, for example, to inhibit production of IL-1 and TNF- ⁇ , to treat aplastic anemia, myelodysplastic syndrome, asthma and arthritis.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the Ck/3-8, Ck3-1 and MIP-4 nucleic acid sequence ⁇ .
  • diagno ⁇ tic a ⁇ ay ⁇ for detecting di ⁇ ea ⁇ e ⁇ related to the underexpre ⁇ ion and overexpre ⁇ sion of the polypeptide ⁇ and for detecting mutation ⁇ in the nucleic acid ⁇ equence ⁇ encoding ⁇ uch polypeptide ⁇ .
  • a proce ⁇ for utilizing such polypeptides, or polynucleotide ⁇ encoding such polypeptides, as research reagents for in vitro purpose ⁇ related to ⁇ cientific research, synthesi ⁇ of DNA and manufacture of DNA vector ⁇ , for the purpo ⁇ e of developing therapeutics and diagnostics for the treatment of human disease.
  • FIG. l display ⁇ the cDNA ⁇ equence encoding Ck/3-8 and the corre ⁇ ponding deduced amino acid ⁇ equence.
  • the initial 21 amino acid ⁇ represents the putative leader sequence. All the signal sequences were as determined by N-terminal peptide sequencing of the baculovirus expre ⁇ ed protein.
  • FIG. 2 displays the cDNA sequence encoding Ck3-1 and the corresponding deduced amino acid sequence.
  • the initial 19 amino acids represent the leader sequence.
  • FIG. 3 displays the cDNA sequence encoding MIP-4 and the corresponding deduced amino acid sequence.
  • the initial 20 amino acids represent the leader sequence.
  • FIG. 4 illustrate ⁇ the amino acid homology between Ck/3-8 (top) and human MlP-l ⁇ (bottom) .
  • the four cy ⁇ teine ⁇ characteri ⁇ tic of all chemokine ⁇ are ⁇ hown.
  • FIG. 5 displays two amino acid sequence ⁇ wherein, the top sequence i ⁇ the human MIP-4 amino acid ⁇ equence and the bottom sequence i ⁇ human MIP-l ⁇ (Human Ton ⁇ illar lymphocyte LD78 Beta protein precursor) .
  • FIG. 6 illustrates the amino acid sequence alignment between Ck/3-1 (top) and human MIP-l ⁇ (bottom) .
  • FIG. 8 is a photograph of a SDS-PAGE gel after expression and purification of Ck / 3-1 in a baculovirus expre ⁇ ion ⁇ y ⁇ tem.
  • FIG. 9 i a photograph of an SDS-PAGE gel after expre ⁇ ion and a three-step purification of Ck/3-8 in a baculovirus expres ⁇ ion sy ⁇ tem.
  • FIG. 10 The chemoacttractant activity of Ck/3-8 wa ⁇ determined with chemotaxi ⁇ a ⁇ ay ⁇ using a 48-well microchamber device (Neuro Probe, Inc.). The experimental procedure wa ⁇ a ⁇ de ⁇ cribed in the manufacturer ⁇ manual. For each concentration of Ck/3-8 te ⁇ ted, migration in 5 high-power fields wa ⁇ examined. The results presented represent the average values obtained from two independent experiments. The chemoacttractant activity on THP-l (A) cell ⁇ and human PBMCs (B) is shown.
  • FIG. 11 Change in intracellular calcium concentration in response to Ck/3-8 wa ⁇ determined using a Hitachi F-2000 fluorescence ⁇ pectrophotometer. Bacterial expressed Ck/3-8 was added to Indo-1 loaded THP-l cells to a final concentration of 50 nM and the intracellular level of calcium concentration wa ⁇ monitored.
  • FIG. 12 The monocyte cell line THP-l wa ⁇ treated for 16 hour ⁇ with LPS (0.1-10 ng/ml) or Ck / 3-8 (to 50 ng/ml). Tissue culture supernatants were subjected to ELISA analysis to quantify the secretion of TNF- ⁇ .
  • FIG. 13 Human peripheral blood monocytes purified by elutriation were treated for 16 hours with increasing amounts of Ck/3-8 (produced by baculovirus) . Tissue culture supernatant ⁇ were ⁇ ubjected to ELISA analy ⁇ i ⁇ to quantify the ⁇ ecretion of TNF- ⁇ , IL-6, IL-1, GM-CSF, and granulocyte- colony ⁇ timulating factor (G-CSF) .
  • FIG. 14 Human peripheral blood monocytes purified by elutriation were treated for 16 hours with increasing amounts of Ck/3-8 (produced by baculovirus) . Tissue culture supernatant ⁇ were ⁇ ubjected to ELISA analy ⁇ i ⁇ to quantify the ⁇ ecretion of TNF- ⁇ , IL-6, IL-1, GM-CSF, and granulocyte- colony ⁇ timulating factor (G-CSF) .
  • G-CSF granulocyte- colony ⁇ timulating
  • the data ⁇ hown repre ⁇ ent ⁇ the average obtained from two independent experiment ⁇ (each performed in duplicate) .
  • Colonie ⁇ were counted 14 day ⁇ after plating.
  • FIG. 15 illu ⁇ trate ⁇ the effect of Ck/3-8 and Ck/3-1 on mou ⁇ e bone marrow colony formation by HPP-CFC (A) and LPP-CFC (B) .
  • FIG. 16 illu ⁇ trate ⁇ the effect of baculoviru ⁇ -expressed Ck / 3-1 and Ck/3-8 on M-CFS and SCF-stimulated colony formation of freshly isolated bone marrow cell ⁇ .
  • FIG. 17 illu ⁇ trate ⁇ the effect of Ck/3-8 and Ck/3-l on IL- 3 and SCF- ⁇ timulated proliferation and differentiation of the linpopulation of bone marrow cell ⁇ .
  • FIG. 18 Effect of Ck/3-8 and Ck/3-1 on the generation of GR-l and Mac-1 ( ⁇ urface marker ⁇ ) po ⁇ itive population of cells from lin" population of bone marrow cells.
  • lin " cells were incubated in growth medium supplemented with IL-3 (5 ng/ml) and SCF (100 ng/ml) alone (a) and Ck/3-8 (50 ng/ml) (b) or Ck/3-l (50 ng/ml) .
  • Cells were then stained with Monoclonal antibodies against myeloid differentiation GR.l, Mac-1, Sca- 1, and CD45R surface antigens and analyzed by FACScan. Data is presented as percentage of positive cell ⁇ in both large (A) and ⁇ mall (B) cell population ⁇ .
  • FIG. 19 illustrates that the presence of Ck/3-8 (+) inhibits bone marrow cell colony formation in respon ⁇ e to IL- 3, M-CSF and GM-CSF.
  • FIG. 20 Do ⁇ e response of Ck/3-8 inhibits bone marrow cell colony formation.
  • Cells were isolated and treated as in Figure 19. The treated cells were plated at a density of 1,000 cells/dish in agar-ba ⁇ ed colony formation assays in the presence of IL-3, GM-CSF or M-CSF (5 ng/ml) with or without Ck / 3-8 at 1, 10, 50 and 100 ng/ml.
  • the data i ⁇ pre ⁇ ented a ⁇ colony formation a ⁇ a percentage of the number of colonies formed with the specific factor alone.
  • FIG. 21 Induction of apoptosis by Ck/3-8 and Ck/3-1 in the pre ⁇ ence or ab ⁇ ence of hematopoietic growth factor ⁇ . Mou ⁇ e bone marrow cell ⁇ were flu ⁇ hed from both the femur and tibia, ⁇ eparated on a ficol density gradient and monocytes removed by plastic adherence.
  • the resulting population of cells were then incubated overnight in an MEM-based medium supplemented with IL-3 (5 ng/ml) , GM-CSF (5 ng/ml) , M-CSF (10 ng/ml) and G-CSF (10 ng/ml) with or without the addition of Ck/3-8 (50 ng/ml) or Ck/3-1 (250 ng/ml) .
  • cells were cultured in medium alone, with or without Ck/3-8 and Ck/3- 1.
  • cell ⁇ were harvested and proces ⁇ ed for apopto ⁇ i ⁇ using the boehringer mannheim cell death ELISA kit. Data i ⁇ shown a ⁇ percentage increase above background with the background considered a ⁇ the amount of apopto ⁇ i ⁇ occurring in the culture ⁇ incubated in the presence of each of the growth factor ⁇ .
  • FIG. 22 Expre ⁇ ion of RNA encoding Ck/3-8 in human monocytes.
  • Total RNA from fresh elutriated monocytes wa ⁇ i ⁇ olated and treated with 100 U/ml hu rIFN-g. 100 ng/ml LPS, or both.
  • RNA (8 ⁇ g) from each treatment wa ⁇ ⁇ eparated electrophoretically on a 1.2% agaro ⁇ e gel and tran ⁇ ferred to a nylon membrane.
  • Ck/3-8 mRNA wa ⁇ quantified by probing with 3 P-labeled cDNA and the band ⁇ on the re ⁇ ulting autoradiograph were quantified den ⁇ itometrically.
  • nucleic acid ⁇ (polvnucleotide ⁇ ) which encode for the mature polypeptide ⁇ having the deduced amino acid sequence of Figures l, 2 and 3 (SEQ ID No. 2, 4 and 6, respectively) or for the mature Ck/3-8 polypeptide encoded by the cDNA of the clone(s) depo ⁇ ited a ⁇ ATCC Depo ⁇ it No. 75676 on February 9, 1994, and for the mature MIP-4 polypeptide encoded by the cDNA of the clone depo ⁇ ited a ⁇ ATCC Depo ⁇ it No. 75675 on February 9, 1994 and for the mature Ck/3-1 polypeptide encoded by the cDNA of the clone depo ⁇ ited a ⁇ ATCC Depo ⁇ it No. 75572, depo ⁇ ited on October 13, 1993.
  • Polynucleotide ⁇ encoding polypeptide ⁇ of the present invention are structurally related to the pro-inflammatory supergene "intercrine" which i ⁇ in the cytokine or chemokine family. Both Ck/3-8 and MIP-4 are MIP-l homologue ⁇ and are more homologou ⁇ to MIP-l ⁇ than to MIP-1/3.
  • the polynucleotide encoding for Ck/3-8 was derived from an aortic endothelium cDNA library and contains an open reading frame encoding a polypeptide of 120 amino acid residue ⁇ , which exhibit ⁇ significant homology to a number of chemokine ⁇ . The top match i ⁇ to the human macrophage inflammatory protein 1 alpha, ⁇ howing 36% identity and 66% ⁇ imilarity (figure 4) .
  • the four cysteine re ⁇ idue ⁇ occurring in all chemokine ⁇ in a characteri ⁇ tic motif are con ⁇ erved in both clone( ⁇ ).
  • the polynucleotide encoding from Ck/3-1 contains and open reading frame encoding a polypeptide of 93 amino acids of which the first 19 are a leader sequence such that the mature polypeptide contains 74 amino acid residue ⁇ .
  • Ck/3-1 exhibit ⁇ ⁇ ignificant homology to human macrophage inflammatory protein with 48% identity and 72% ⁇ imilarity over a ⁇ tretch of 80 amino acid ⁇ . Further, the four cy ⁇ teine residue ⁇ compri ⁇ ing a characteri ⁇ tic motif are con ⁇ erved.
  • the polynucleotide ⁇ of the pre ⁇ ent invention may be in the form of RNA or in the form of DNA, which DNA include ⁇ cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single- ⁇ tranded, and if ⁇ ingle ⁇ tranded may be the coding ⁇ trand or non-coding (anti- ⁇ ense) strand.
  • the coding sequence which encodes the mature polypeptides may be identical to the coding sequence shown in Figures l, 2 and 3 (SEQ ID No.
  • the polynucleotides which encode for the mature polypeptides of Figures 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or for the mature polypeptides encoded by the deposited cDNA(s) may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide ⁇ and additional coding sequence ⁇ uch a ⁇ a leader or ⁇ ecretory sequence or a proprotein sequence; the coding sequence for the mature polypeptides (and optionally additional coding sequence) and non-coding sequence, such a ⁇ intron ⁇ or non- coding ⁇ equence 5' and/or 3' of the coding ⁇ equence for the mature polypeptide ⁇ .
  • polynucleotide encoding a polypeptide encompa ⁇ e ⁇ a polynucleotide which includes only coding sequence for the polypeptide a ⁇ well a ⁇ a polynucleotide which include ⁇ additional coding and/or non-coding ⁇ equence.
  • the pre ⁇ ent invention further relate ⁇ to variant ⁇ of the hereinabove de ⁇ cribed polynucleotide ⁇ which encode for fragment ⁇ , analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figures l, 2 and 3 (SEQ ID No. 2, 4 and 6) or the polypeptide ⁇ encoded by the cDNA of the depo ⁇ ited clone( ⁇ ) .
  • the variants of the polynucleotide ⁇ may be a naturally occurring allelic variant of the polynucleotide ⁇ or a non-naturally occurring variant of the polynucleotide ⁇ .
  • the pre ⁇ ent invention include ⁇ polynucleotide ⁇ encoding the ⁇ ame mature polypeptide ⁇ a ⁇ ⁇ hown in Figure ⁇ 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or the same mature polypeptides encoded by the cDNA of the deposited clone(s) a ⁇ well a ⁇ variant ⁇ of ⁇ uch polynucleotide ⁇ which variant ⁇ encode for a fragment, derivative or analog of the polypeptide ⁇ of Figure ⁇ 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or the polypeptide ⁇ encoded by the cDNA of the depo ⁇ ited clone( ⁇ ).
  • nucleotide variant ⁇ include deletion variants, substitution variant ⁇ and addition or insertion variants.
  • the polynucleotide may have a coding sequence which i ⁇ a naturally occurring allelic variant of the coding ⁇ equence ⁇ hown in Figure ⁇ 1, 2 and 3 (SEQ ID No. 1, 3 and 5) or of the coding ⁇ equence of the depo ⁇ ited clone( ⁇ ) .
  • an allelic variant i ⁇ an alternate form of a polynucleotide sequence which may have a sub ⁇ titution, deletion or addition of one or more nucleotide ⁇ , which doe ⁇ not ⁇ ub ⁇ tantially alter the function of the encoded polypeptide.
  • the pre ⁇ ent invention al ⁇ o include ⁇ polynucleotide ⁇ , wherein the coding sequence for the mature polypeptide ⁇ may be fu ⁇ ed in the ⁇ ame reading frame to a polynucleotide ⁇ equence which aid ⁇ in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions a ⁇ a ⁇ ecretory ⁇ equence for controlling tran ⁇ port of a polypeptide from the cell.
  • the polypeptide having a leader ⁇ equence i ⁇ a preprotein and may have the leader ⁇ equence cleaved by the ho ⁇ t cell to form the mature form of the polypeptide.
  • the polynucleotide ⁇ may al ⁇ o encode for a proprotein which i ⁇ the mature protein plu ⁇ additional 5' amino acid re ⁇ idue ⁇ .
  • a mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
  • the polynucleotide ⁇ of the pre ⁇ ent invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence) .
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention.
  • the marker sequence may be a hexa- hi ⁇ tidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag correspond ⁇ to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequence ⁇ if there is at least 50% and preferably 70% identity between the sequence ⁇ .
  • the pre ⁇ ent invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotide ⁇ .
  • stringent condition ⁇ mean ⁇ hybridization will occur only if there is at least 95% and preferably at lea ⁇ t 97% identity between the ⁇ equence ⁇ .
  • polypeptide ⁇ which hybridize to the hereinabove de ⁇ cribed polynucleotide ⁇ in a preferred embodiment encode polypeptide ⁇ which retain ⁇ ub ⁇ tantially the same biological function or activity a ⁇ the mature polypeptide ⁇ encoded by the cDNA of Figure 1, 2 and 3 (SEQ ID No. l, 3 and 5) or the depo ⁇ ited cDNA ⁇ .
  • the polynucleotides may be polynucleotide ⁇ which ha ⁇ at lea ⁇ t 20 ba ⁇ e ⁇ , preferably 30 ba ⁇ e ⁇ , and more preferably at lea ⁇ t 50 ba ⁇ e ⁇ which hybridize to a polynucleotide of the pre ⁇ ent invention and which ha ⁇ an identity thereto, a ⁇ hereinabove de ⁇ cribed, and which does not retain activity.
  • Such polynucleotide ⁇ may be employed a ⁇ probe ⁇ for the polynucleotide ⁇ of SEQ ID NOS:l, 3 and 5 for example, for recovery of the polynucleotide or a ⁇ a diagno ⁇ tic probe or a ⁇ a PCR primer.
  • the depo ⁇ it( ⁇ ) referred to herein will be maintained under the term ⁇ of the Budape ⁇ t Treaty on the International Recognition of the Depo ⁇ it of Micro-organism ⁇ for purposes of Patent Procedure.
  • These deposit ⁇ are provided merely as convenience to those of skill in the art and are not an admis ⁇ ion that a depo ⁇ it i ⁇ required under 35 U.S.C. ⁇ 112.
  • the ⁇ equence of the polynucleotide ⁇ contained in the depo ⁇ ited material ⁇ , a ⁇ well a ⁇ the amino acid ⁇ equence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with description of sequence ⁇ herein.
  • a license may be required to make, u ⁇ e or ⁇ ell the depo ⁇ ited material ⁇ , and no ⁇ uch license i ⁇ hereby granted.
  • the present invention further relates to Ck/3-8, MIP-4 and Ck/3-1 polypeptides which have the deduced amino acid sequence of Figures l, 2 and 3 (SEQ ID No. 2, 4 and 6) or which have the amino acid sequence encoded by the deposited cDNAs, as well a ⁇ fragment ⁇ , analog ⁇ and derivatives of such polypeptides.
  • fragment when referring to the polypeptides of Figure ⁇ 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or that encoded by the depo ⁇ ited cDNA, mean ⁇ a polypeptide which retain ⁇ e ⁇ entially the ⁇ ame biological function or activity a ⁇ ⁇ uch polypeptide.
  • an analog include ⁇ a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • polypeptides of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptides of Figures 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are sub ⁇ tituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such sub ⁇ tituted amino acid re ⁇ idue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid re ⁇ idue ⁇ include ⁇ a substituent group, or (iii) one in which the mature polypeptide ⁇ are fu ⁇ ed with another compound, ⁇ uch a ⁇ a compound to increa ⁇ e the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acid ⁇ are fu ⁇ ed to the mature polypeptide ⁇ , such as a leader or secretory sequence or a sequence which is employed for purification
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • gene or "cistron” mean ⁇ the segment of DNA involved in producing a polypeptide chain; it include ⁇ regions preceding and following the coding region (leader and trailer) as well a ⁇ intervening ⁇ equence ⁇ (intron ⁇ ) between individual coding segments (exon ⁇ ) .
  • i ⁇ olated mean ⁇ that the material i ⁇ removed from it ⁇ original environment (e.g., the natural environment if it i ⁇ naturally occurring) .
  • a naturally- occurring polynucleotide ⁇ or polypeptide ⁇ present in a living animal i ⁇ not i ⁇ olated, but the ⁇ ame polynucleotide ⁇ or DNA or polypeptide ⁇ , ⁇ eparated from ⁇ ome or all of the coexi ⁇ ting material ⁇ in the natural ⁇ y ⁇ tem, i ⁇ i ⁇ olated.
  • Such polynucleotide ⁇ could be part of a vector and/or ⁇ uch polynucleotide ⁇ or polypeptide ⁇ could be part of a composition, and still be isolated in that such vector or composition i ⁇ not part of it ⁇ natural environment.
  • the pre ⁇ ent invention al ⁇ o relate ⁇ to vector ⁇ which include polynucleotide ⁇ of the pre ⁇ ent invention, ho ⁇ t cell ⁇ which are genetically engineered with vectors of the invention and the production of polypeptide ⁇ of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expres ⁇ ion vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified a ⁇ appropriate for activating promoter ⁇ , selecting transformant ⁇ or amplifying the Ck/3-8, MIP-4 and Ck/3-1 gene ⁇ .
  • the culture conditions, such a ⁇ temperature, pH and the like, are those previously used with the host cell selected for expres ⁇ ion, and will be apparent to the ordinarily ⁇ killed arti ⁇ an.
  • the polynucleotide ⁇ of the pre ⁇ ent invention may be employed for producing polypeptide ⁇ by recombinant technique ⁇ . Thu ⁇ , for example, the polynucleotide sequence may be included in any one of a variety of expres ⁇ ion vehicle ⁇ , in particular vector ⁇ or pla ⁇ mid ⁇ for expre ⁇ ing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and ⁇ ynthetic DNA ⁇ equence ⁇ , e.g., derivative ⁇ of SV40; bacterial pla ⁇ mid ⁇ ; phage DNA; yea ⁇ t plasmids; vectors derived from combinations of pla ⁇ mid ⁇ and phage DNA, viral DNA ⁇ uch a ⁇ vaccinia, adenoviru ⁇ , fowl pox viru ⁇ , and p ⁇ eudorabie ⁇ .
  • any other pla ⁇ mid or vector may be used as long they are replicable and viable in the ho ⁇ t.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence i ⁇ inserted into an appropriate restriction endonuclease ⁇ ite( ⁇ ) by procedure ⁇ known in the art.
  • procedure ⁇ known in the art.
  • the DNA sequence in the expres ⁇ ion vector i ⁇ operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoter ⁇ known to control expres ⁇ ion of gene ⁇ in prokaryotic or eukaryotic cell ⁇ or their viruses.
  • the expression vector also contain ⁇ a ribosome binding ⁇ ite for tran ⁇ lation initiation and a tran ⁇ cription terminator.
  • the vector may also include appropriate sequence ⁇ for amplifying expre ⁇ ion.
  • the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such a ⁇ dihydrofolate reducta ⁇ e or neomycin re ⁇ i ⁇ tance for eukaryotic cell culture, or ⁇ uch a ⁇ tetracycline or ampicillin re ⁇ i ⁇ tance in E. coli.
  • the vector containing the appropriate DNA ⁇ equence a ⁇ hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the ho ⁇ t to expre ⁇ the protein.
  • a ⁇ representative examples of appropriate ho ⁇ t ⁇ there may be mentioned: bacterial cell ⁇ , ⁇ uch a ⁇ E. coli, Streptomyce ⁇ . Salmonella Tvphimurium: fungal cell ⁇ , ⁇ uch a ⁇ yea ⁇ t; in ⁇ ect cell ⁇ ⁇ uch a ⁇ Dro ⁇ ophila S2 and Sf9; adenoviru ⁇ e ⁇ ; animal cell ⁇ ⁇ uch a ⁇ CHO, COS or Bowe ⁇ melanoma; plant cell ⁇ , etc.
  • the selection of an appropriate host is deemed to be within the scope of tho ⁇ e ⁇ killed in the art from the teaching ⁇ herein.
  • the pre ⁇ ent invention al ⁇ o include ⁇ recombinant con ⁇ truct ⁇ compri ⁇ ing one or more of the ⁇ equence ⁇ a ⁇ broadly de ⁇ cribed above.
  • the con ⁇ truct ⁇ compri ⁇ e a vector, ⁇ uch a ⁇ a pla ⁇ mid or viral vector, into which a ⁇ equence of the invention ha ⁇ been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vector ⁇ and promoters are known to those of skill in the art, and are commercially available.
  • the following vector ⁇ are provided by way of example.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen) , pb ⁇ , pDIO, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; pTRC99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transfera ⁇ e) vector ⁇ or other vector ⁇ with ⁇ electable marker ⁇ .
  • Two appropriate vector ⁇ are PKK232-8 and PCM7.
  • Particular named bacterial promoter ⁇ include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kina ⁇ e, early and late SV40, LTR ⁇ from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary ⁇ kill in the art.
  • the pre ⁇ ent invention relate ⁇ to ho ⁇ t cell ⁇ containing the above-de ⁇ cribed con ⁇ truct.
  • the ho ⁇ t cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, ⁇ uch a ⁇ a yea ⁇ t cell, or the ho ⁇ t cell can be a prokaryotic cell, ⁇ uch a ⁇ a bacterial cell.
  • Introduction of the con ⁇ truct into the host cell can be effected by calcium phosphate tran ⁇ fection, DEAE- Dextran mediated tran ⁇ fection, or electroporation (Davi ⁇ , L., Dibner, M. , Battey, I., Ba ⁇ ic Method ⁇ in Molecular Biology, (1986) ) .
  • the con ⁇ tructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthe ⁇ izer ⁇ .
  • Mature protein ⁇ can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoter ⁇ .
  • Cell-free translation sy ⁇ tem ⁇ can also be employed to produce such proteins using RNAs derived from the DNA construct ⁇ of the pre ⁇ ent invention.
  • Appropriate cloning and expre ⁇ ion vector ⁇ for use with prokaryotic and eukaryotic host ⁇ are de ⁇ cribed by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. , (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting element ⁇ of DNA, usually about from 10 to 300 bp that act on a promoter to increase it ⁇ tran ⁇ cription.
  • Example ⁇ including the SV40 enhancer on the late ⁇ ide of the replication origin bp 100 to 270, a cytomegaloviru ⁇ early promoter enhancer, the polyoma enhancer on the late ⁇ ide of the replication origin, and adenoviru ⁇ enhancer ⁇ .
  • recombinant expre ⁇ ion vector ⁇ will include origin ⁇ of replication and ⁇ electable marker ⁇ permitting transformation of the ho ⁇ t cell, e.g., the ampicillin re ⁇ i ⁇ tance gene of E. coli and S. cerevi ⁇ iae TRPl gene, and a promoter derived from a highly-expre ⁇ ed gene to direct tran ⁇ cription of a down ⁇ tream ⁇ tructural ⁇ equence.
  • promoter ⁇ can be derived from operon ⁇ encoding glycolytic enzyme ⁇ such a ⁇ 3-pho ⁇ phoglycerate kinase (PGK) , ⁇ -factor, acid phosphata ⁇ e, or heat ⁇ hock protein ⁇ , among other ⁇ .
  • PGK 3-pho ⁇ phoglycerate kinase
  • the heterologou ⁇ ⁇ equence can encode a fu ⁇ ion protein including an N-terminal identification peptide imparting de ⁇ ired characteri ⁇ tic ⁇ , e.g., ⁇ tabilization or ⁇ implified purification of expre ⁇ sed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a de ⁇ ired protein together with ⁇ uitable tran ⁇ lation initiation and termination ⁇ ignal ⁇ in operable reading pha ⁇ e with a functional promoter.
  • the vector will compri ⁇ e one or more phenotypic ⁇ electable marker ⁇ and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus ⁇ ubtili ⁇ . Salmonella tvphimurium and variou ⁇ species within the genera Pseudomona ⁇ , Streptomyce ⁇ , and Staphylococcu ⁇ , although other ⁇ may also be employed a ⁇ a matter of choice.
  • useful expres ⁇ ion vector ⁇ for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available pla ⁇ mid ⁇ compri ⁇ ing genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wl, USA) .
  • pBR322 "backbone" ⁇ ections are combined with an appropriate promoter and the ⁇ tructural ⁇ equence to be expre ⁇ ed.
  • Cell ⁇ are typically harve ⁇ ted by centrifugation, di ⁇ rupted by phy ⁇ ical or chemical mean ⁇ , and the re ⁇ ulting crude extract retained for further purification.
  • Microbial cell ⁇ employed in expre ⁇ ion of protein ⁇ can be di ⁇ rupted by any convenient method, including freeze-thaw cycling, ⁇ onica ion, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.
  • mammalian cell culture sy ⁇ tem ⁇ can also be employed to express recombinant protein.
  • mammalian expres ⁇ ion system ⁇ include the COS-7 line ⁇ of monkey kidney fibrobla ⁇ t ⁇ , de ⁇ cribed by Gluzman, Cell, 23:175 (1981) , and other cell line ⁇ capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a ⁇ uitable promoter and enhancer, and also any nece ⁇ sary ribosome binding sites, polyadenylation site, ⁇ plice donor and acceptor ⁇ ite ⁇ , tran ⁇ criptional termination sequences, and 5' flanking nontranscribed ⁇ equence ⁇ .
  • DNA ⁇ equence ⁇ derived from the SV40 ⁇ plice, and polyadenylation ⁇ ite ⁇ may be u ⁇ ed to provide the required nontran ⁇ cribed genetic element ⁇ .
  • Ck3-8, MIP-4 and Ck/3-1 are recovered and purified from recombinant cell culture ⁇ by method ⁇ including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography.
  • Protein refolding step ⁇ can be u ⁇ ed, as neces ⁇ ary, in completing configuration of the mature protein.
  • HPLC high performance liquid chromatography
  • the polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic ho ⁇ t (for example, by bacterial, yea ⁇ t, higher plant, insect and mammalian cell ⁇ in culture) .
  • a prokaryotic or eukaryotic ho ⁇ t for example, by bacterial, yea ⁇ t, higher plant, insect and mammalian cell ⁇ in culture
  • the polypeptide ⁇ of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated.
  • Polypeptide ⁇ of the invention may also include an initial methionine amino acid residue.
  • the polypeptides of the present invention may be employed in a variety of immunoregulatory and inflammatory functions and also in a number of disease conditions.
  • Ck/3-8, MIP-4 and Ck/3-1 are in the chemokine family and therefore they are chemoattractants for leukocyte ⁇ ( ⁇ uch a ⁇ monocyte ⁇ , neutrophil ⁇ , T ly phocyte ⁇ , eosinophils, basophil ⁇ , etc.).
  • Ck/3-8 i ⁇ hown to play an important role in the regulation of the immune response and inflammation.
  • lipopolysaccharide induces the expres ⁇ ion of Ck/3-8 from human monocyte ⁇ . Accordingly, in re ⁇ pon ⁇ e to the pre ⁇ ence of an endotoxin, Ck/3-8 i ⁇ expre ⁇ sed from monocytes and, therefore, administration of Ck/3-8 may be employed to regulate the immune response of a ho ⁇ t.
  • Ck/3-8, MIP-4 and Ck/3-1 can be employed to facilitate wound healing by controlling infiltration of target immune cell ⁇ to the wound area.
  • the polypeptide ⁇ of the pre ⁇ ent invention can enhance ho ⁇ t defenses against chronic infections, e.g., mycobacterial, via their attraction and activation of microbicidal leukocytes.
  • polypeptides of the present invention may be employed in anti-tumor therapy since there is evidence that chemokine expres ⁇ ing cell ⁇ injected into tumor ⁇ have cau ⁇ ed regre ⁇ ion of the tumor, for example, in the treatment of Karposi sarcoma.
  • Ck/3-8, MIP-4 and Ck/3-1 stimulate the invasion and activation of host defense (tumoricidal) cells, e.g., cytotoxic T-cells and macrophage ⁇ via their chemotactic activity, and in thi ⁇ way may also be employed to treat ⁇ olid tumor ⁇ .
  • the polypeptide ⁇ may also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy.
  • Figures 14 and 15 illustrate that Ck/3-8 and Ck/3-1 inhibit colony formation of low proliferative potential colony forming cell ⁇ , and that Ck/3-8 i ⁇ a potent and specific inhibitor of LPP-CFC colony growth.
  • Figure 16 illu ⁇ trate ⁇ that Ck/3-1 ⁇ pecifically inhibit ⁇ M-CSF- ⁇ timulated colony formation, while Ck/3-8 doe ⁇ not.
  • a ⁇ al ⁇ o shown, both Ck/3-8 and Ck3-l significantly inhibit growth or differentiation of bone marrow cells .
  • Thi ⁇ antiproliferative effect allows a greater exposure to chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.
  • the inhibitory effect of the Ck/3-1 and Ck/3-8 polypeptides on the subpopulation of committed progenitor cell ⁇ may be employed therapeutically to inhibit proliferation of leukemic cell ⁇ .
  • a ⁇ ⁇ hown in Figure 20 a do ⁇ e re ⁇ pon ⁇ e of Ck / 3-8 i ⁇ ⁇ hown to inhibit colony formation.
  • Thi ⁇ inhibition could be due to a ⁇ pecific blockage of the differentiative signal mediated by these factors or to a cytotoxic effect on the progenitor cells.
  • polypeptides Another employment of the polypeptides is the inhibition of T-cell proliferation via inhibition of IL-2 bio ⁇ ynthe ⁇ i ⁇ , for example, in auto-immune disease ⁇ and lymphocytic leukemia.
  • Ck/3-8, MIP-4 and Ck/3-1 may al ⁇ o be employed for inhibiting epidermal keratinocyte proliferation for p ⁇ oriasi ⁇ (keratinocyte hyper-proliferation) ⁇ ince Langerhan ⁇ cell ⁇ in skin have been found to produce MlP-l ⁇ .
  • Ck/3-8, MIP-4 and Ck/3-1 may be employed to prevent scarring during wound healing both via the recruitment of debris-cleaning and connective tissue-promoting inflammatory cells and by control of excessive TGF/3-mediated fibrosi ⁇ .
  • the ⁇ e polypeptide ⁇ may be employed to treat ⁇ troke, thrombocyto ⁇ i ⁇ , pulmonary emboli and myeloproliferative di ⁇ order ⁇ , ⁇ ince Ck/3-8, MIP-4 and Ck/3-1 increa ⁇ e va ⁇ cular permeability.
  • Ck/3-8 may al ⁇ o be employed to treat leukemia and abnormally proliferating cell ⁇ , for example tumor cell ⁇ , by inducing apopto ⁇ i ⁇ .
  • Ck/3-8 induce ⁇ apopto ⁇ is in a population of hematopoietic progenitor cell ⁇ a ⁇ ⁇ hown in Figure 21.
  • polypeptide ⁇ of the present invention may be employed as research reagents for in vitro purposes related to scientific research, synthe ⁇ i ⁇ of DNA and manufacture of DNA vector ⁇ , and for the purpose of developing therapeutics and diagnostic ⁇ for the treatment of human disease.
  • Ck/3-l and Ck/3-8 may be employed for the expansion of immature hematopoietic progenitor cells, for example, granulocytes, macrophage ⁇ or monocyte ⁇ , by temporarily preventing their differentiation.
  • the ⁇ e bone marrow cell ⁇ may be cultured in vi tro .
  • Fragment ⁇ of the full length Ck/3-8, MIP-4 or Ck/3-1 gene ⁇ may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or ⁇ imilar biological activity.
  • the probe ⁇ Preferably, however, the probe ⁇ have at lea ⁇ t 30 ba ⁇ e ⁇ and may contain, for example, 50 or more base ⁇ .
  • the probe may al ⁇ o be u ⁇ ed to identify a cDNA clone corre ⁇ ponding to a full length tran ⁇ cript and a genomic clone or clone ⁇ that contain the complete genes including regulatory and promotor regions, exons, and intron ⁇ .
  • a ⁇ creen compri ⁇ e ⁇ i ⁇ olating the coding region of the gene ⁇ by using the known DNA sequence to synthesize an oligonucleotide probe.
  • Labeled oligonucleotide ⁇ having a ⁇ equence complementary to that of the gene ⁇ of the pre ⁇ ent invention are u ⁇ ed to ⁇ creen a library of human cDNA, genomic DNA or mRNA to determine which member ⁇ of the library the probe hybridize ⁇ to.
  • This invention i ⁇ al ⁇ o related to the use of the Ck/3-8, MIP-4 and Ck ⁇ -1 gene as part of a diagnostic as ⁇ ay for detecting di ⁇ ea ⁇ e ⁇ or susceptibility to di ⁇ ea ⁇ e ⁇ related to the presence of mutations in the nucleic acid sequence ⁇ .
  • di ⁇ ea ⁇ e ⁇ are related to under-expression of the chemokine polypeptide ⁇ .
  • Nucleic acid ⁇ for diagno ⁇ i ⁇ may be obtained from a patient' ⁇ cell ⁇ , ⁇ uch a ⁇ from blood, urine, ⁇ aliva, tissue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al. , Nature, 324:163-166 (1986)) prior to analysi ⁇ .
  • RNA or cDNA may also be used for the ⁇ ame purpo ⁇ e.
  • PCR primers complementary to the nucleic acid encoding Ck/3-8, MIP-4 and Ck/3-1 can be used to identify and analyze Ck/3-8, MIP-4 and Ck/3-1 mutations. For example, deletions and insertion ⁇ can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled Ck/3-8, MIP-4 and Ck/3-1 RNA or alternatively, radiolabeled Ck/3-8, MIP-4 and Ck/3-1 antisense DNA sequence ⁇ . Perfectly matched ⁇ equence ⁇ can be di ⁇ tingui ⁇ hed from mi ⁇ matched duplexe ⁇ by RNa ⁇ e A digestion or by differences in melting temperatures.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertion ⁇ can be vi ⁇ ualized by high resolution gel electrophoresis. DNA fragments of different ⁇ equence ⁇ may be di ⁇ tingui ⁇ hed on denaturing formamide gradient gels in which the mobilitie ⁇ of different DNA fragment ⁇ are retarded in the gel at different po ⁇ itions according to their specific melting or partial melting temperature ⁇ ( ⁇ ee, e.g., Myer ⁇ et al., Science, 230:1242 (1985)).
  • Sequence changes at specific locations may also be revealed by nuclease protection assay ⁇ , ⁇ uch a ⁇ RNa ⁇ e and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)).
  • the detection of a ⁇ pecific DNA ⁇ equence may be achieved by method ⁇ ⁇ uch as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of re ⁇ triction enzyme ⁇ , (e.g., Re ⁇ triction Fragment Length Polymorphi ⁇ ms (RFLP) ) and Southern blotting of genomic DNA.
  • re ⁇ triction enzyme ⁇ e.g., Re ⁇ triction Fragment Length Polymorphi ⁇ ms (RFLP)
  • RFLP Re ⁇ triction Fragment Length Polymorphi ⁇ ms
  • mutation ⁇ can al ⁇ o be detected by in situ analy ⁇ i ⁇ .
  • the present invention al ⁇ o relate ⁇ to a diagno ⁇ tic assay for detecting altered level ⁇ of Ck/3-8, MIP-4 and Ck/3-1 protein in variou ⁇ ti ⁇ ue ⁇ ⁇ ince an over-expre ⁇ ion of the protein ⁇ compared to normal control ti ⁇ ue samples may detect the presence of a disease or susceptibility to a disease, for example, a tumor.
  • a ⁇ ay ⁇ used to detect levels of Ck/3-8, MIP-4 and Ck/3-1 protein in a sample derived from a ho ⁇ t are well-known to tho ⁇ e of ⁇ kill in the art and include radioimmunoa ⁇ ay ⁇ , competitive-binding assay ⁇ , Western Blot analysis, ELISA as ⁇ ay ⁇ and "sandwich” as ⁇ ay.
  • An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initially comprise ⁇ preparing an antibody ⁇ pecific to the Ck/3-8, MIP-4 and Ck/3-l antigen ⁇ , preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody.
  • a detectable reagent such as a ⁇ radioactivity, fluore ⁇ cence or, in thi ⁇ example, a hor ⁇ eradi ⁇ h peroxida ⁇ e enzyme.
  • a ⁇ araple i ⁇ removed from a ho ⁇ t and incubated on a ⁇ olid ⁇ upport, e.g. a_ poly ⁇ tyrene di ⁇ h, that bind ⁇ the protein ⁇ in the ⁇ ample. Any free protein binding ⁇ ite ⁇ on the di ⁇ h are then covered by incubating with a non-specific protein like BSA.
  • the monoclonal antibody is incubated in the di ⁇ h during which time the monoclonal antibodie ⁇ attach to any Ck/3-8, MIP-4 and Ck/3-1 protein ⁇ attached to the poly ⁇ tyrene di ⁇ h. All unbound monoclonal antibody is washed out with buffer.
  • the reporter antibody linked to horseradi ⁇ h peroxida ⁇ e i ⁇ now placed in the di ⁇ h re ⁇ ulting in binding of the reporter antibody to any monoclonal antibody bound to Ck/3-8, MIP-4 and Ck/3-1. Unattached reporter antibody i ⁇ then wa ⁇ hed out.
  • Peroxida ⁇ e ⁇ ub ⁇ trate ⁇ are then added to the di ⁇ h and the amount of color developed in a given time period i ⁇ a mea ⁇ urement of the amount of Ck/3-8, MIP-4 and Ck/3-1 protein pre ⁇ ent in a given volume of patient ⁇ ample when compared again ⁇ t a ⁇ tandard curve.
  • a competition a ⁇ say may be employed wherein antibodie ⁇ specific to Ck/3-8, MIP-4 and Ck/3-1 are attached to a ⁇ olid support and labeled Ck/3-8, MIP-4 and Ck/3-1 and a ⁇ ample derived from the host are pa ⁇ ed over the ⁇ olid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of protein in the ⁇ ample.
  • MIP-4 and Ck/3-1 i ⁇ passed over a ⁇ olid ⁇ upport and bind ⁇ to antibody attached to a ⁇ olid ⁇ upport.
  • a ⁇ econd antibody i ⁇ then bound to the Ck/3-8, MIP-4 and Ck/3-1.
  • a third antibody which i ⁇ labeled and ⁇ pecific to the ⁇ econd antibody i ⁇ then pa ⁇ ed over the ⁇ olid support and binds to the second antibody and an amount can then be quantified.
  • This invention provides a method for identification of the receptors for the chemokine polypeptide ⁇ .
  • the gene encoding the receptor can be identified by numerou ⁇ method ⁇ known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in I mun., 1(2), Chapter 5, (1991)) .
  • expres ⁇ ion cloning is employed wherein polyadenylated RNA is prepared from a cell respon ⁇ ive to the polypeptide ⁇ , and a cDNA library created from thi ⁇ RNA i ⁇ divided into pool ⁇ and u ⁇ ed to transfect COS cell ⁇ or other cell ⁇ that are not re ⁇ pon ⁇ ive to the polypeptide ⁇ .
  • Tran ⁇ fected cell ⁇ which are grown on gla ⁇ s slide ⁇ are exposed to the labeled polypeptides.
  • the polypeptides can be labeled by a variety of mean ⁇ including iodination or inclusion of a recognition ⁇ ite for a ⁇ ite- ⁇ pecific protein kina ⁇ e.
  • the ⁇ lide ⁇ are ⁇ ubjected to autoradiographic analysis. Positive pool ⁇ are identified and ⁇ ub-pool ⁇ are prepared and retran ⁇ fected u ⁇ ing an iterative sub-pooling and re ⁇ creening proce ⁇ s, eventually yielding a ⁇ ingle clones that encode ⁇ the putative receptor.
  • the labeled polypeptide ⁇ can be photoaffinity linked with cell membrane or extract preparation ⁇ that express the receptor molecule. Cros ⁇ -linked material i ⁇ re ⁇ olved by PAGE analy ⁇ i ⁇ and expo ⁇ ed to X-ray film.
  • the labeled complex containing the receptor ⁇ of the polypeptide ⁇ can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be u ⁇ ed to de ⁇ ign a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptor ⁇ .
  • This invention provides a method of screening compound ⁇ to identify agonist ⁇ and antagoni ⁇ t ⁇ to the chemokine polypeptide ⁇ of the pre ⁇ ent invention.
  • An agoni ⁇ t i ⁇ a compound which ha ⁇ ⁇ imilar biological function ⁇ of the polypeptide ⁇ , while antagoni ⁇ t ⁇ block ⁇ uch function ⁇ .
  • Chemotaxi ⁇ may be a ⁇ ayed by placing cell ⁇ , which are chemoattracted by either of the polypeptide ⁇ of the pre ⁇ ent invention, on top of a filter with pore ⁇ of ⁇ ufficient diameter to admit the cell ⁇ (about 5 ⁇ m) .
  • Solution ⁇ of potential agoni ⁇ t ⁇ are placed in the bottom of the chamber with an appropriate control medium in the upper compartment, and thus a concentration gradient of the agonist is measured by counting cells that migrate into or through the porou ⁇ membrane over time.
  • the chemokine polypeptides of the present invention are placed in the bottom chamber and the potential antagonist is added to determine if chemotaxis of the cell ⁇ i ⁇ prevented.
  • a mammalian cell or membrane preparation expressing the receptors of the polypeptide ⁇ would be incubated with a labeled chemokine polypeptide, eg. radioactivity, in the pre ⁇ ence of the compound.
  • chemokine polypeptide eg. radioactivity
  • the ability of the compound to block thi ⁇ interaction could then be mea ⁇ ured.
  • the chemokines would be absent and the ability of the agoni ⁇ t itself to interact with the receptor could be measured.
  • Examples of potential Ck/3-8, MIP-4 and Ck/3-1 antagonists include antibodies, or in some cases, oligonucleotides, which bind to the polypeptides.
  • Another example of a potential antagonist i ⁇ a negative dominant mutant of the polypeptides. Negative dominant mutants are polypeptides which bind to the receptor of the wild-type polypeptide, but fail to retain biological activity.
  • Antisen ⁇ e con ⁇ truct ⁇ prepared u ⁇ ing anti ⁇ en ⁇ e technology are al ⁇ o potential antagoni ⁇ ts.
  • Antisen ⁇ e technology can be u ⁇ ed to control gene expre ⁇ ion through triple-helix formation or anti ⁇ en ⁇ e DNA or RNA, both of which method ⁇ are ba ⁇ ed on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide ⁇ equence which encodes for the mature polypeptide ⁇ of the pre ⁇ ent invention, i ⁇ u ⁇ ed to de ⁇ ign an anti ⁇ ense RNA oligonucleotide of from about 10 to 40 base pair ⁇ in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple- helix, see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988) ; and Dervan et al., Science, 251: 1360 (1991) ) , thereby preventing tran ⁇ cription and the production of the chemokine polypeptide ⁇ .
  • the anti ⁇ en ⁇ e RNA oligonucleotide hybridize ⁇ to the mRNA in vivo and block ⁇ tran ⁇ lation of the mRNA molecule into the polypeptide ⁇ (anti ⁇ en ⁇ e - Okano, J. Neurochem.
  • oligonucleotide ⁇ de ⁇ cribed above can also be delivered to cells such that the anti ⁇ en ⁇ e RNA or DNA may be expre ⁇ ed in vivo to inhibit production of the chemokine polypeptide ⁇ .
  • chemokine antagoni ⁇ t i ⁇ a peptide derivative of the polypeptide ⁇ which are naturally or synthetically modified analog ⁇ of the polypeptides that have lost biological function yet still recognize and bind to the receptors of the polypeptides to thereby effectively block the receptors.
  • peptide derivatives include, but are not limited to, ⁇ mall peptides or peptide-like molecules.
  • the antagoni ⁇ t ⁇ may be employed to treat di ⁇ order ⁇ which are either MlP-induced or enhanced, for example, auto-immune and chronic inflammatory and infective di ⁇ ea ⁇ e ⁇ .
  • di ⁇ order ⁇ which are either MlP-induced or enhanced, for example, auto-immune and chronic inflammatory and infective di ⁇ ea ⁇ e ⁇ .
  • Example ⁇ of auto-immune di ⁇ ea ⁇ e ⁇ include multiple ⁇ clero ⁇ i ⁇ , and insulin- dependent diabete ⁇ .
  • the antagoni ⁇ t ⁇ may also be employed to treat infectious disea ⁇ e ⁇ including silicosi ⁇ , ⁇ arcoido ⁇ is, idiopathic pulmonary fibrosi ⁇ by preventing the recruitment and activation of mononuclear phagocyte ⁇ . They may al ⁇ o be employed to treat idiopathic hyper-eo ⁇ inophilic ⁇ yndrome by preventing eo ⁇ inophil production and migration. Endotoxic shock may also be treated by the antagonist ⁇ by preventing the migration of macrophage ⁇ and their production of the chemokine polypeptide ⁇ of the pre ⁇ ent invention.
  • the antagoni ⁇ t ⁇ may al ⁇ o be employed for treating atherosclero ⁇ i ⁇ , by preventing monocyte infiltration in the artery wall.
  • the antagoni ⁇ t ⁇ may al ⁇ o be employed to treat hi ⁇ tamine- mediated allergic reactions and immunological disorder ⁇ including late pha ⁇ e allergic reactions, chronic urticaria, and atopic dermatitis by inhibiting chemokine-induced mast cell and ba ⁇ ophil degranulation and relea ⁇ e of hi ⁇ tamine.
  • IgE-mediated allergic reaction ⁇ ⁇ uch a ⁇ allergic a ⁇ thma, rhiniti ⁇ , and eczema may al ⁇ o be treated.
  • the antagoni ⁇ t ⁇ may also be employed to treat chronic and acute inflammation by preventing the attraction of monocytes to a wound area. They may al ⁇ o be employed to regulate normal pulmonary macrophage populations, since chronic and acute inflammatory pulmonary disea ⁇ e ⁇ are a ⁇ ociated with ⁇ equestration of mononuclear phagocytes in the lung.
  • Antagonist ⁇ may al ⁇ o be employed to treat rheumatoid arthriti ⁇ by preventing the attraction of monocyte ⁇ into ⁇ ynovial fluid in the joints of patient ⁇ .
  • Monocyte influx and activation play ⁇ a ⁇ ignificant role in the pathogenesi ⁇ of both degenerative and inflammatory arthropathie ⁇ .
  • the antagoni ⁇ t ⁇ may be employed to interfere with the deleterious cascades attributed primarily to IL-l and TNF, which prevents the biosynthesis of other inflammatory cytokines. In this way, the antagoni ⁇ t ⁇ may be employed to prevent inflammation.
  • the antagoni ⁇ t ⁇ may al ⁇ o be employed to inhibit prostaglandin-independent fever induced by chemokine ⁇ .
  • the antagonists may also be employed to treat ca ⁇ e ⁇ of bone marrow failure, for example, apla ⁇ tic anemia and myelody ⁇ pla ⁇ tic ⁇ yndrome.
  • the antagoni ⁇ t ⁇ may al ⁇ o be employed to treat a ⁇ thma and allergy by preventing eo ⁇ inophil accumulation in the lung.
  • the antagonist ⁇ may al ⁇ o be employed to treat ⁇ ubepithelial ba ⁇ ement membrane fibro ⁇ i ⁇ which i ⁇ a prominent feature of the a ⁇ thmatic lung.
  • the antagoni ⁇ t ⁇ may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
  • the chemokine polypeptides and agonists and antagonist ⁇ may be employed in combination with a ⁇ uitable pharmaceutical carrier.
  • a ⁇ uitable pharmaceutical carrier Such compo ⁇ ition ⁇ compri ⁇ e a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient.
  • a carrier include ⁇ but i ⁇ not limited to ⁇ aline, buffered ⁇ aline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredient ⁇ of the pharmaceutical compo ⁇ ition ⁇ of the invention.
  • a ⁇ ociated with ⁇ uch container( ⁇ ) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical ⁇ or biological product ⁇ , which notice reflect ⁇ approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides and agonist ⁇ and antagonists may be employed in conjunction with other therapeutic compound ⁇ .
  • the pharmaceutical compo ⁇ ition ⁇ may be admini ⁇ tered in a convenient manner ⁇ uch a ⁇ by the topical, intravenous, intraperitoneal, intramuscular, intratumor, subcutaneous, intranasal or intradermal routes.
  • the pharmaceutical compositions are administered in an amount which i ⁇ effective for treating and/or prophylaxi ⁇ of the ⁇ pecific indication.
  • the polypeptide ⁇ will be admini ⁇ tered in an amount of at lea ⁇ t about 10 ⁇ g/kg body weight and in most case ⁇ they will be admini ⁇ tered in an amount not in excess of about 8 mg/Kg body weight per day.
  • the dosage is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administratic-n, ⁇ ymptom ⁇ , etc.
  • the chemokine polypeptide ⁇ , and agoni ⁇ t ⁇ or antagoni ⁇ t ⁇ which are polypeptide ⁇ may be employed in accordance with the present invention by expres ⁇ ion of ⁇ uch polypeptide ⁇ in vivo, which i ⁇ often referred to a ⁇ "gene therapy.”
  • cell ⁇ from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cell ⁇ then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expres ⁇ ion of a polypeptide in vivo by, for example, procedure ⁇ known in the art.
  • procedure ⁇ known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expres ⁇ ion vehicle for engineering cell ⁇ may be other than a retroviru ⁇ , for example, an adenoviru ⁇ which may be u ⁇ ed to engineer cell ⁇ in vivo after combination with a ⁇ uitable delivery vehicle.
  • the retroviral pla ⁇ mid vector ⁇ may be derived from retroviru ⁇ e ⁇ which include, but are not limited to, Moloney Murine Sarcoma Viru ⁇ , Moloney Murine Leukemia Viru ⁇ , spleen necrosi ⁇ viru ⁇ , Rou ⁇ Sarcoma Viru ⁇ and Harvey Sarcoma Viru ⁇ .
  • HSV herpes simplex virus
  • tk thymidine kinase
  • the vector ⁇ include one or more ⁇ uitable promoter ⁇ which include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegaloviru ⁇ (CMV) promoter de ⁇ cribed in Miller, et al., Biotechni ⁇ ue ⁇ , Vol. 7, No. 9, 980-990 (1989) , or any other promoter (e.g., cellular promoter ⁇ ⁇ uch a ⁇ eukaryotic cellular promoters including, but not limited to, the hi ⁇ tone, pol III, and /3-actin promoters) .
  • a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the nucleic acid sequence encoding the polypeptide of the present invention i ⁇ under the control of a ⁇ uitable promoter which include ⁇ , but i ⁇ not limited to, viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs, the /3-actin promoter, and the native promoter which controls the gene encoding the polypeptide.
  • a ⁇ uitable promoter include ⁇ , but i ⁇ not limited to, viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs, the /3-actin promoter, and the native promoter which controls the gene encoding the polypeptide.
  • the retroviral plasmid vector i ⁇ employed to tran ⁇ duce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317 and GP+aml2.
  • the vector may transduce the packaging cells through any means known in the art. Such mean ⁇ include, but are not limited to, electroporation, the u ⁇ e of liposomes, and CaP0 4 precipitation.
  • the producer cell line generate ⁇ infectiou ⁇ retroviral vector particle ⁇ which include the nucleic acid sequence (s) encoding the polypeptide ⁇ .
  • retroviral vector particle ⁇ then may be employed, to tran ⁇ duce eukaryotic cell ⁇ , either in vi tro or in vivo .
  • the transduced eukaryotic cell ⁇ will expre ⁇ the nucleic acid ⁇ equence ( ⁇ ) encoding the polypeptide.
  • Eukaryotic cell ⁇ which may be tran ⁇ duced include but are not limited to, fibrobla ⁇ t ⁇ and endothelial cell ⁇ .
  • the ⁇ equence ⁇ of the pre ⁇ ent invention are al ⁇ o valuable for chromo ⁇ ome identification.
  • the ⁇ equence i ⁇ specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • there i ⁇ a current need for identifying particular ⁇ ite ⁇ on the chromo ⁇ ome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphism ⁇ ) are presently available for marking chromosomal location.
  • the mapping of DNA to chromosome ⁇ according to the present invention i ⁇ an important fir ⁇ t ⁇ tep in correlating tho ⁇ e sequences with gene ⁇ as ⁇ ociated with di ⁇ ea ⁇ e.
  • ⁇ equence ⁇ can be mapped to chromo ⁇ omes by preparing PCR primer ⁇ (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosome ⁇ . Only those hybrid ⁇ containing the human gene corre ⁇ ponding to the primer will yield an amplified fragment.
  • mapping of ⁇ omatic cell hybrid ⁇ i ⁇ a rapid procedure for a ⁇ igning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panel ⁇ of fragment ⁇ from ⁇ pecific chromo ⁇ ome ⁇ or pool ⁇ of large genomic clone ⁇ in an analogou ⁇ manner.
  • Other mapping ⁇ trategie ⁇ that can ⁇ imilarly be u ⁇ ed to map to it ⁇ chromosome include in si tu hybridization, prescreening with labeled flow- ⁇ orted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of cDNA clones to a metaphase chromo ⁇ omal ⁇ pread can be u ⁇ ed to provide a preci ⁇ e chromo ⁇ omal location in one ⁇ tep.
  • Thi ⁇ technique can be used with cDNA as short a ⁇ 500 or 600 ba ⁇ e ⁇ .
  • a cDNA preci ⁇ ely localized to a chromo ⁇ omal region a ⁇ ociated with the di ⁇ ease could be one of between 50 and 500 potential causative genes. (This assumes 1 megaba ⁇ e mapping resolution and one gene per 20 kb) .
  • the polypeptide ⁇ , their fragment ⁇ or other derivatives, or analogs thereof, or cells expres ⁇ ing them can be u ⁇ ed a ⁇ an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodie ⁇ .
  • the pre ⁇ ent invention al ⁇ o include ⁇ chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of an Fab expre ⁇ ion library. Variou ⁇ procedure ⁇ known in the art may be u ⁇ ed for the production of ⁇ uch antibodie ⁇ and fragment ⁇ .
  • Antibodie ⁇ generated again ⁇ t the polypeptide ⁇ corre ⁇ ponding to a ⁇ equence of the pre ⁇ ent invention or it ⁇ in vivo receptor can be obtained by direct injection of the polypeptide ⁇ into an animal or by admini ⁇ tering the polypeptides to an animal, preferably a nonhuman. The antibody ⁇ o obtained will then bind the polypeptide ⁇ it ⁇ elf . In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptide ⁇ . Such antibodie ⁇ can then be u ⁇ ed to isolate the polypeptides from tis ⁇ ue expre ⁇ ing that polypeptide.
  • any technique which provide ⁇ antibodie ⁇ produced by continuous cell line culture ⁇ can be used.
  • Examples include the hybridoma technique (Kohler and Mil ⁇ tein, 1975, Nature, 256:495-497) , the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodie ⁇ and Cancer Therapy, Alan R. Li ⁇ , Inc., pp. 77-96) .
  • Technique ⁇ de ⁇ cribed for the production of ⁇ ingle chain antibodie ⁇ (U.S. Patent 4,946,778) can be adapted to produce ⁇ ingle chain antibodie ⁇ to immunogenic polypeptide ⁇ product ⁇ of thi ⁇ invention.
  • Al ⁇ o, transgenic mice may be used to expre ⁇ humanized antibodie ⁇ to immunogenic polypeptide product ⁇ of thi ⁇ invention.
  • “Plasmid ⁇ ” are de ⁇ ignated by a lower ca ⁇ e p preceded and/or followed by capital letter ⁇ and/or number ⁇ .
  • the ⁇ tarting plasmids herein are either commercially available, publicly available on an unrestricted basi ⁇ , or can be con ⁇ tructed from available pla ⁇ mid ⁇ in accord with published procedures.
  • equivalent plasmid ⁇ to tho ⁇ e de ⁇ cribed are known in the art and will be apparent to the ordinarily ⁇ killed arti ⁇ an.
  • “Dige ⁇ tion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction condition ⁇ , cofactors and other requirements were used a ⁇ would be known to the ordinarily skilled artisan.
  • analytical purpo ⁇ e ⁇ typically 1 ⁇ g of pla ⁇ mid or DNA fragment i ⁇ u ⁇ ed with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume.
  • buffer ⁇ and ⁇ ub ⁇ trate amount ⁇ for particular re ⁇ triction enzyme ⁇ are specified by the manufacturer. Incubation times of about 1 hour at 37'C are ordinarily used, but may vary in accordance with the supplier' ⁇ instructions. After dige ⁇ tion the reaction i ⁇ electrophore ⁇ ed directly on a polyacrylamide gel to i ⁇ olate the de ⁇ ired fragment.
  • Oligonucleotides refer ⁇ to either a ⁇ ingle ⁇ tranded polydeoxynucleotide or two complementary polydeoxynucleotide ⁇ trand ⁇ which may be chemically ⁇ ynthe ⁇ ized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a pho ⁇ phate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that ⁇ ha ⁇ not been depho ⁇ phorylated.
  • Ligaation refer ⁇ to the proce ⁇ of forming pho ⁇ phodie ⁇ ter bond ⁇ between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unle ⁇ otherwi ⁇ e provided, ligation may be accompli ⁇ hed using known buffer ⁇ and conditions with 10 units to T4 DNA ligase ("liga ⁇ e") per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragment ⁇ to be ligated.
  • liga ⁇ e T4 DNA ligase
  • the DNA ⁇ equence encoding Ck/3-8, ATCC # 75676, wa ⁇ initially amplified u ⁇ ing PCR oligonucleotide primer ⁇ corre ⁇ ponding to the 5' and 3' end ⁇ equences of the processed Ck/3-8 protein (minus the ⁇ ignal peptide sequence) and the vector sequence ⁇ 3' to the Ck/3-8 gene. Additional nucleotide ⁇ corre ⁇ ponding to Bam HI and Xbal were added to the 5' and 3' ⁇ equence ⁇ re ⁇ pectively.
  • the 5' oligonucleotide primer ha ⁇ the sequence 5' TCAGGATCCGTCACAAAAGATGCAGA 3' (SEQ ID No.
  • re ⁇ triction enzyme sites correspond to the restriction enzyme sites on the bacterial expres ⁇ ion vector PQE-9 (Qiagen, Inc., Chatsworth, CA) .
  • PQE-9 encodes antibiotic resistance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribo ⁇ ome binding ⁇ ite (RBS) , a 6- Hi ⁇ tag and re ⁇ triction enzyme ⁇ ite ⁇ .
  • pQE-9 i ⁇ then dige ⁇ ted with BamHI and Xbal.
  • the amplified ⁇ equence ⁇ are ligated into PQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS.
  • the ligation mixture i ⁇ then u ⁇ ed to tran ⁇ form E. coli ⁇ train M15/rep4 available from Qiagen.
  • M15/rep4 contain ⁇ multiple copies of the plasmid pREP4, which expresses the lad repressor and also confer ⁇ kanamycin re ⁇ i ⁇ tance (Kan r ) .
  • Tran ⁇ formant ⁇ are identified by their ability to grow on LB plate ⁇ and ampicillin/kanamycin re ⁇ i ⁇ tant colonie ⁇ are ⁇ elected.
  • Pla ⁇ mid DNA i ⁇ isolated and confirmed by restriction analysi ⁇ . Clone ⁇ containing the de ⁇ ired constructs were grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
  • the O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cell ⁇ are grown to an optical den ⁇ ity 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG I ⁇ opropyl-B-D-thiogalacto pyrano ⁇ ide
  • IPTG induce ⁇ by inactivating the lad repre ⁇ sor, clearing the P/O leading to increa ⁇ ed gene expre ⁇ ion.
  • Cell ⁇ are grown an extra 3 to 4 hour ⁇ . Cell ⁇ are then harvested by centrifugation.
  • the cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HC1.
  • Ck/3-8 (5% pure) is eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpo ⁇ e of renaturation adju ⁇ ted to 3 molar guanidine HC1, lOOmM ⁇ odium pho ⁇ phate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in thi ⁇ ⁇ olution for 12 hour ⁇ the protein i ⁇ dialyzed to 10 mmolar sodium pho ⁇ phate.
  • the 5' oligonucleotide primer ha ⁇ the ⁇ equence 5' TC ⁇ GGATCC.TGTGCACAAGTTGGTACC 3' (SEQ ID No.
  • the restriction enzyme site ⁇ correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc., Chatsworth, CA) .
  • pQE-9 encode ⁇ antibiotic re ⁇ istance (Amp 1 ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribo ⁇ ome binding ⁇ ite (RBS) , a 6- Hi ⁇ tag and re ⁇ triction enzyme ⁇ ite ⁇ .
  • pQE-9 wa ⁇ then dige ⁇ ted with BamHI and Xbal and the amplified ⁇ equence ⁇ were ligated into pQE-9 and inserted in frame with the sequence encoding for the histidine tag and the RBS.
  • the ligation mixture wa ⁇ then u ⁇ ed to tran ⁇ form E. coli strain available from Qiagen.
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expres ⁇ e ⁇ the lad repres ⁇ or and al ⁇ o confer ⁇ kanamycin re ⁇ istance (Kan r ) .
  • Transformant ⁇ are identified by their ability to grow on LB plates and ampicillin/kanamycin resi ⁇ tant colonie ⁇ were ⁇ elected. Pla ⁇ mid DNA wa ⁇ isolated and confirmed by restriction analysi ⁇ . Clone ⁇ containing the de ⁇ ired con ⁇ truct ⁇ were grown overnight (O/N) in liquid culture in LB media ⁇ upplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
  • the cell ⁇ were grown to an optical den ⁇ ity 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG I ⁇ opropyl-B-D-thiogalacto pyrano ⁇ ide
  • IPTG induce ⁇ by inactivating the lad repres ⁇ or, clearing the P/0 leading to increa ⁇ ed gene expre ⁇ ion.
  • Cell ⁇ were grown an extra 3 to 4 hour ⁇ . Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HC1.
  • MIP-4 wa ⁇ purified from thi ⁇ ⁇ olution by chromatography on a Nickel-Chelate column under condition ⁇ that allow for tight binding by protein ⁇ containing the 6-Hi ⁇ tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)).
  • the 5' oligonucleotide primer ha ⁇ the ⁇ equence 5' GCCCGCGGATCCTCCTCACGGGGACCTTAC 3' (SEQ ID No.
  • PQE-9 encode ⁇ antibiotic resistance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/0) , a ribosome binding ⁇ ite (RBS) , a 6-Hi ⁇ tag and restriction enzyme ⁇ ite ⁇ .
  • pQE-9 was then digested with BamHI and Xbal and the amplified sequence ⁇ were ligated into PQE-9 and were in ⁇ erted in frame with the ⁇ equence encoding for the histidine tag and the RBS.
  • the ligation mixture wa ⁇ then used to transform E. coli strain available from Qiagen under the trademark M15/rep 4.
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expres ⁇ es the lad repressor and also confers kanamycin resi ⁇ tance (Kan r ) .
  • Tran ⁇ formant ⁇ are identified by their ability to grow on LB plate ⁇ and ampicillin/kanamycin re ⁇ i ⁇ tant colonie ⁇ were ⁇ elected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containingthe desired construct ⁇ were grown overnight (O/N) in liquid culture in LB media ⁇ upplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
  • the cells were grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG Isopropyl-B-D-thiogalacto pyrano ⁇ ide
  • IPTG induce ⁇ by inactivating the lad repre ⁇ or, clearing the P/O leading to increased gene expres ⁇ ion.
  • Cell ⁇ were grown an extra 3 to 4 hour ⁇ . Cell ⁇ were then harvested by centrifugation. The cell pellet was ⁇ olubilized in the chaotropic agent 6 Molar Guanidine HC1.
  • Ck/3-1 wa ⁇ purified from thi ⁇ ⁇ olution by chromatography on a Nickel- Chelate column under condition ⁇ that allow for tight binding by protein ⁇ containing the 6-Hi ⁇ tag (Hochuli, E. et al., J. Chromatocraphv 411:177-184 (1984)).
  • Ck/3-1 (95% pure) was eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpo ⁇ e of renaturation adjusted to 3 molar guanidine HC1, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this ⁇ olution for 12 hour ⁇ the protein wa ⁇ dialyzed to 10 mmolar ⁇ odium pho ⁇ phate.
  • the expre ⁇ sion of plasmid, CMV-Ck/3-8 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resi ⁇ tance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
  • the HA tag corre ⁇ pond to an epitope derived from the influenza hemagglutinin protein a ⁇ previou ⁇ ly de ⁇ cribed (I. Wil ⁇ on, et al., Cell, 37:767 (1984)).
  • the infu ⁇ ion of HA tag to the target protein allow ⁇ easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the PCR product 14 contains complementary sequence ⁇ to an Xba I ⁇ ite, tran ⁇ lation ⁇ top codon, HA tag and the la ⁇ t 20 nucleotide ⁇ of the Ck/3-8 coding ⁇ equence (not including the ⁇ top codon) . Therefore, the PCR product contain ⁇ a Hindlll ⁇ ite, Ck/3-8 coding ⁇ equence followed by HA tag fu ⁇ ed in frame, a tran ⁇ lation termination ⁇ top codon next to the HA tag, and an Xbal ⁇ ite.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp are dige ⁇ ted with Hindlll and Xbal re ⁇ triction enzyme and ligated.
  • the ligation mixture i ⁇ transformed into E. coli ⁇ train SURE (Stratagene Cloning Sy ⁇ tem ⁇ , La Jolla, CA) the tran ⁇ formed culture i ⁇ plated on ampicillin media plate ⁇ and re ⁇ i ⁇ tant colonie ⁇ are ⁇ elected. Pla ⁇ mid DNA i ⁇ isolated from transformant ⁇ and examined by re ⁇ triction analy ⁇ i ⁇ for the pre ⁇ ence of the correct fragment.
  • COS cell ⁇ are tran ⁇ fected with the expre ⁇ ion vector by DEAE-DEXTRAN method (J. Sambrook, E. Frit ⁇ ch, T.
  • CMV-MIP-4 HA i ⁇ derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin re ⁇ i ⁇ tance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation ⁇ ite.
  • a DNA fragment encoding the entire MIP-4 precur ⁇ or and a HA tag fu ⁇ ed in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wil ⁇ on, et al., Cell, 37:767 (1984)) .
  • the infu ⁇ ion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognize ⁇ the HA epitope.
  • the DNA sequence encoding MIP-4 is constructed by PCR using two primer ⁇ : the 5' primer 5' GGAAAGC-TTATGAAGGGCCTTGCAGCTGCC 3' (SEQ ID No. 15) contain ⁇ a Hindlll ⁇ ite followed by 20 nucleotides of MIP-4 coding sequence starting from the initiation codon; the 3' sequence 5' CGCTCTAGATC-AABCGTAGTCTGCJGACGTCCT ⁇
  • the PCR product contains complementary sequences to an Xba I site, translation stop codon, HA tag and the last 19 nucleotide ⁇ of the MIP-4 coding sequence (not including the stop codon) . Therefore, the PCR product contains a Hindlll site, MIP-4 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xbal site.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp are digested with Hindlll and Xbal restriction enzyme and ligated. The ligation mixture is transformed into E.
  • CMV-Ck/3-1 HA The expression of plasmid, CMV-Ck/3-1 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resi ⁇ tance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
  • the HA tag corre ⁇ pond to an epitope derived from the influenza hemagglutinin protein a ⁇ previou ⁇ ly de ⁇ cribed (I. Wil ⁇ on, et al., Cell, 37:767 (1984)).
  • the infu ⁇ ion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the PCR product contains complementary sequences to an Xba I site, translation stop codon, HA tag and the last 19 nucleotides of the Ck/3-1 coding sequence (not including the stop codon) . Therefore, the PCR product contains a Hindlll site, Ck/3-1 coding sequence followed by an HA tag fused in frame, a tran ⁇ lation termination ⁇ top codon next to the HA tag, and an Xbal ⁇ ite.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp were dige ⁇ ted with Hindlll and Xbal re ⁇ triction enzyme and ligated. The ligation mixture wa ⁇ tran ⁇ formed into E.
  • Northern blot analysi ⁇ wa ⁇ carried out to examine the level ⁇ of expre ⁇ sion of Ck/3-8 in human tis ⁇ ue ⁇ .
  • Total cellular RNA samples were isolated with RNAzolTM B sy ⁇ tem (Biotecx Laboratorie ⁇ , Inc., Hou ⁇ ton, TX 77033) .
  • the labeling reaction is done according to the Stratagene Prime-It kit with 50ng DNA fragment.
  • the labeled DNA i ⁇ purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. Boulder, CO) .
  • the filter i ⁇ then hybridized with radioactive labeled full length Ck/3-8 gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65 'C. After wa ⁇ h twice at room temperature and twice at 60'C with 0.5 x SSC, 0.1% SDS, the filter i ⁇ then expo ⁇ ed at -70 'C overnight with an intensifying ⁇ creen.
  • Northern blot analy ⁇ i ⁇ wa ⁇ carried out to examine the levels of expres ⁇ ion of MIP-4 in human cell ⁇ .
  • Total cellular RNA ⁇ ample ⁇ were isolated with RNAzolTM B sy ⁇ tem (Biotecx Laboratorie ⁇ , Inc., Hou ⁇ ton, TX) .
  • About lOug of total RNA i ⁇ olated from each human ti ⁇ ue ⁇ pecified was separated on 1% agarose gel and blotted onto a nylon filter (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Pre ⁇ , (1989)) .
  • the labeling reaction wa ⁇ done according to the Stratagene Prime-It kit with 50ng DNA fragment.
  • the labeled DNA wa ⁇ purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc., Boulder, CO) .
  • the filter wa ⁇ then hybridized with radioactive labeled full length MIP-4 gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65 ° C. After wash twice at room temperature and twice at 60 'C with 0.5 x SSC, 0.1% SDS, the filter was then exposed at -70 * C overnight with an inten ⁇ ifying ⁇ creen.
  • Northern blot analy ⁇ i ⁇ wa ⁇ carried out to examine the level ⁇ of expre ⁇ ion of Ck/3-1 in human tissue ⁇ .
  • Total cellular RNA samples were isolated with RNAzolTM B system (Biotecx Laboratories, Inc. Houston, TX) .
  • the labeling reaction wa ⁇ done according to the Stratagene Prime-It kit with 50ng DNA fragment.
  • the labeled DNA wa ⁇ purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc., Boulder, CO).
  • the filter wa ⁇ then hybridized with radioactive labeled full length Ck/3- 1 gene at 1,000,000 cpm/ml in 0.5 M NaP0 4# pH 7.4 and 7% SDS overnight at 65"C. After wa ⁇ h twice at room temperature and twice at 60'C with 0.5 x SSC, 0.1% SDS, the filter wa ⁇ then expo ⁇ ed at -70 * C overnight with an inten ⁇ ifying ⁇ creen.
  • SF9 cell ⁇ were infected with a recombinant baculoviru ⁇ de ⁇ igned to expre ⁇ the Ck/3-8 cDNA.
  • Cell ⁇ were infected at an MOI of 2 and cultured at 28°C for 72-96 hour ⁇ .
  • Cellular debri ⁇ from the infected culture wa ⁇ removed by low ⁇ peed centrifugation.
  • Protea ⁇ e inhibitor cocktail wa ⁇ added to the supernatant at a final concentration of 20 ⁇ g/ml Pefabloc SC, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml E-64 and 1 mM EDTA.
  • Ck/3-8 in the supernatant wa ⁇ monitored by loading 20-30 ⁇ l of ⁇ upernatant only 15% SDS-PAGE gels.
  • Ck/3-8 wa ⁇ detected a ⁇ a vi ⁇ ible 9 Kd band, corresponding to an expres ⁇ ion level of ⁇ everal mg per liter.
  • Ck/3-8 wa ⁇ further purified through a three- ⁇ tep purification procedure: Heparin binding affinity chromatography.
  • Example wa ⁇ then applied to a heparin binding column (HEl poro ⁇ 20, Bio- Perceptive Sy ⁇ tem Inc.) .
  • Ck/3-08 wa ⁇ eluted at approximately 300 mM NaCl in a linear gradient of 50 to 500 mM NaCl in 50 mM HEPES/MES/NaOAc at pH 6; Cation exchange chromatography.
  • Ck/3-8 wa ⁇ eluted at 250 mM NaCl in a linear gradient of 25 to 300 mM NaCl in 50 mM HEPES/MES/NaOAc at pH 6; Size exclusion chromatography.
  • Ck/3-8 was further purified by applying to a size exclusion column (HW50, TOSO HAAS, 1.4 x 45 cm) .
  • the purified Ck/3-8 was also tested for endotoxin/LPS contamination.
  • the LPS content wa ⁇ less than 0.1 ng/ml according to LAL assay ⁇ (BioWhittaker) .
  • Example 11 Effect of baculovirus-expressed Ck/3-l and Ck/3-8 on M-CSF and SCF-stimulated colony formation of freshly isolated bone marrow cells.
  • a low density population of mou ⁇ e bone marrow cell ⁇ were incubated in a treated ti ⁇ ue culture di ⁇ h for one hour at 37°C to remove monocytes, macrophages, and other cells that adhere to the pla ⁇ tic ⁇ urface.
  • the non-adherent population of cell ⁇ were then plated (10,000 cell ⁇ /dish) in agar containing growth medium in the pre ⁇ ence or ab ⁇ ence of the factor ⁇ ⁇ hown in Figure 16.
  • Culture ⁇ were incubated for 10 day ⁇ at 37°C (88% N 2 , 5% C0 2 , and 7% 0 2 ) and colonies were scored under an inverted microscope.
  • Example 12 Effect of Ck/3-8 and Ck ⁇ 3-1 on IL-3 and SCF stimulated proliferation and differentiation of lin-population of bone marrow cells.
  • the re ⁇ ulting population of cells (Lin ' cells) were plated (5 x 10 4 cells/ml) in the presence or absence of the indicated chemokine (50 ng/ml) in a growth medium supplemented with IL-3 (5 ng/ml) plus SCF (100 ng/ml) .
  • cell ⁇ were harvested and as ⁇ ayed for the HPP-CFC, and immature progenitor ⁇ .
  • cell ⁇ were analyzed for the expre ⁇ ion of certain differentiation antigen ⁇ by FACScan. Colony data are expre ⁇ ed a ⁇ mean number of colonie ⁇ +/- SD) and were obtained from a ⁇ say ⁇ performed in ⁇ ix dishes for each population of cell ⁇ ( Figure 17) .
  • Example 13 Ck/3-8 inhibit ⁇ colony formation in re ⁇ ponse to IL-3. M-CSF. and GM-CSF.
  • Mouse bone marrow cells were flushed from both the femur and tibia, separated on a ficol density gradient and monocyte ⁇ removed by pla ⁇ tic adherence. The re ⁇ ulting population of cell ⁇ were incubated overnight in an MEM-based medium ⁇ upplemented with IL-3 (5 ng/ml) , GM-CSF (5 ng/ml) , M- CSF (10 ng/ml) and G-CSF (10 ng/ml) .
  • Fibroblast ⁇ are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the ti ⁇ ue are placed on a wet ⁇ urface of a tissue culture flask, approximately ten pieces are placed in each fla ⁇ k. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the fla ⁇ k is inverted and the chunks of ti ⁇ ue remain fixed to the bottom of the fla ⁇ k and fre ⁇ h media (e.g., Ham' ⁇ F12 media, with 10% FBS, penicillin and ⁇ treptomycin, i ⁇ added. Thi ⁇ i ⁇ then incubated at 37°C for approximately one week.
  • fre ⁇ h media e.g., Ham' ⁇ F12 media, with 10% FBS, penicillin and ⁇ treptomycin, i ⁇ added.
  • a monolayer of fibroblast ⁇ emerge.
  • the monolayer i ⁇ tryp ⁇ inized and scaled into larger flasks.
  • pMV-7 (Kirsch eier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeat ⁇ of the Moloney murine sarcoma viru ⁇ , i ⁇ dige ⁇ ted with EcoRI and Hindlll and subsequently treated with calf intestinal phosphata ⁇ e.
  • the cDNA encoding a polypeptide of the present invention is amplified u ⁇ ing PCR primer ⁇ which corre ⁇ pond to the 5' and 3' end ⁇ equence ⁇ re ⁇ pectively.
  • the 5' primer containing an EcoRI ⁇ ite and the 3' primer having contain ⁇ a Hindlll ⁇ ite.
  • Equal quantitie ⁇ of the Moloney murine ⁇ arcoma viru ⁇ linear backbone and the EcoRI and HimdIII fragment are added together, in the pre ⁇ ence of T4 DNA liga ⁇ e.
  • the resulting mixture is maintained under condition ⁇ appropriate for ligation of the two fragment ⁇ .
  • the ligation mixture i ⁇ used to transform bacteria HB101, which are then plated onto agar- containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
  • the amphotropic pA317 or GP+aml2 packaging cell ⁇ are grown in ti ⁇ ue culture to confluent den ⁇ ity in Dulbecco' ⁇ Modified Eagles Medium (DMEM) with 10% calf ⁇ eru (CS) , penicillin and streptomycin.
  • DMEM Dulbecco' ⁇ Modified Eagles Medium
  • CS calf ⁇ eru
  • penicillin and streptomycin The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector.
  • the packaging cell ⁇ now produce infectiou ⁇ viral particle ⁇ containing the gene (the packaging cell ⁇ are now referred to a ⁇ producer cells) .
  • the engineered fibroblast ⁇ are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
  • the fibroblast ⁇ now produce the protein product.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • Gin Arg lie Met A ⁇ p Tyr Tyr Glu Thr A ⁇ n Ser Gin Cy ⁇ Ser
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:7: TCAGGATCCG TCACAAAAGA TGCAGA 26
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:9: TCAGGATCCT GTGCACAAGT TGGTACC 27
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:10: CGCTCTAGAG TAAAACGACG GCCAGT 26
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:11: GCCCGCGGAT CCTCCTCACG GGGACCTTAC 30 (2 ) INFORMATION FOR SEQ ID NO : 12 :
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:12: GCCTGCTCTA GATCAAAGCA GGGAAGCTCC AG 32
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:13: GGAAAGCTTA TGAAGGTCTC CGTGGCT 27
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:17: GGAAAGCTTA TGAAGATTCC GTGGCTGC 28

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Abstract

L'invention porte sur la chimiokine bêta-8, la chimiokine bêta-1, la protéine 4 inflammatoire des macrophages, et de l'ADN (ARN) d'origine humaine codant pour ces polypeptides de chimiokine ainsi que sur une procédure permettant de fabriquer ces polypeptides par des techniques de recombinaison. L'invention se rapporte également à des procédés permettant d'utiliser ces polypeptides de chimiokine pour le traitement de la leucémie, des tumeurs, des infections chroniques, des maladies auto-immunes, des pathologies fibreuses, de la cicatrisation des plaies et du psoriasis. Elle concerne également des antagonistes agissant contre ces polypeptides de chimiokine et leur emploi en tant qu'agents thérapeutiques permettant de traiter la polyarthrite rhumatoïde, les maladies inflammatoires et infectieuses auto-immunes, chroniques et aigües, les réactions allergiques, la fièvre non dépendante de la prostaglandine et l'insuffisance médullaire. L'invention décrit également des méthodes de dosage diagnostique permettant de détecter des maladies se rapportant à des mutations dans les séquences d'acide nucléique et de déceler des concentrations de polypeptides modifiées.
PCT/US1995/009058 1995-05-05 1995-06-23 Chimiokine beta-8, chimiokine beta-1 et proteine 4 inflammatoire des macrophages d'origine humaine WO1996034891A1 (fr)

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MX9708537A MX9708537A (es) 1995-05-05 1995-06-23 Quimiocina beta-8 quimiocina beta-1 y proteina-4 inflamatoria de los macrofagos, humanas.
AU31346/95A AU3134695A (en) 1995-05-05 1995-06-23 Human chemokine beta-8, chemokine beta-1 and macrophage infl ammatory protein-4
JP8533269A JPH11505417A (ja) 1995-05-05 1995-06-23 ヒトケモカインベータ−８、ケモカインベータ−１、およびマクロファージ炎症性タンパク質−４
EP95927260A EP0871672A4 (fr) 1995-05-05 1995-06-23 Chimiokine beta-8, chimiokine beta-1 et proteine 4 inflammatoire des macrophages d'origine humaine

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GENE, Volume 151, issued 1994, POWER et al., "Cloning of a Full-Length cDNA Encoding the Neutrophil-Activating Peptide ENA-78 from Human Platelets", pages 333-334. *
See also references of EP0871672A4 *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 269, Number 41, issued 14 October 1994, CHANG et al., "Cloning and Characterization of the Human Neutrophil-Activating Peptide (ENA-78) Gene", pages 25277-25282. *

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JP2003102486A (ja) 2003-04-08
KR19990008335A (ko) 1999-01-25
JPH11505417A (ja) 1999-05-21
AU3134695A (en) 1996-11-21
EP0871672A4 (fr) 1999-05-12
CN1515672A (zh) 2004-07-28
CA2220123A1 (fr) 1996-11-07
MX9708537A (es) 1998-02-28
EP0871672A1 (fr) 1998-10-21
CN1186501A (zh) 1998-07-01
CN1125082C (zh) 2003-10-22

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