WO1998031809A1 - Slc humaine de chemokine cc - Google Patents

Slc humaine de chemokine cc Download PDF

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Publication number
WO1998031809A1
WO1998031809A1 PCT/JP1998/000154 JP9800154W WO9831809A1 WO 1998031809 A1 WO1998031809 A1 WO 1998031809A1 JP 9800154 W JP9800154 W JP 9800154W WO 9831809 A1 WO9831809 A1 WO 9831809A1
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protein
human
sequence
slc
amino acid
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PCT/JP1998/000154
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English (en)
Japanese (ja)
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Morio Nagira
Toshio Imai
Osamu Yoshie
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Shionogi & Co., Ltd.
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Priority to AU54967/98A priority Critical patent/AU5496798A/en
Publication of WO1998031809A1 publication Critical patent/WO1998031809A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel human CC-type chemokine SL, a polynucleotide encoding the protein, a method for producing the protein, a pharmaceutical composition containing the protein or a polynucleotide encoding the protein, and a monoclonal antibody comprising the protein. Screening antibodies and hybridomas capable of producing the antibodies, and further screening agonists against agonism against biological effects induced by the binding of the proteins to their specific receptors. About the method. Background art
  • extrinsic or endogenous tissue injuries, invasions, or antigen exposures can induce strong inflammatory and immune responses. These reactions are important host defense responses, but can also sometimes cause acute or chronic illness.
  • a cause that induces an inflammatory or immune response is added to a tissue, first, inflammatory cells such as neutrophils, granulocytes, lymphocytes or macrophages or immunocompetent cells adsorb to vascular endothelial cells, extravascular And accumulation in invaded or damaged tissues or in the presence of antigens.
  • chemokines As a substance that induces such a series of cell migration reactions, there is a group of chemotactic sites, so-called chemokines.
  • Chemokines are a group of site forces that induce a chemotactic reaction (chemotactic reaction), and are closely related structurally to each other due to similarities in amino acid sequences. To date, more than 30 chemokines have been reported in humans. Chemokines are largely ⁇ or CXC (two cysteines separated by one amino acid) and i8 or CC from the arrangement of the first two of the four conserved cysteine residues in common. Type (two cysteines are next to each other).
  • CXC chemokines As human CXC chemokines, IL-8, ⁇ -TG, PF-4, MGSA / GRO, ENA-78, NAP-2, GCP-K GCP-2, IP-10, SDF-1 / PBSF, MIG, NA which is known.
  • cxc-type chemokines mainly induce neutrophil activation and migration.
  • human CC chemokines MIP-1 ⁇ , ⁇ -1 ⁇ , RANTES, MCP-K MCP-2, MCP-3, U-309, eotaxin and the like are known in humans.
  • CC-type chemokines mainly induce monocyte / macrophage activation and migration.
  • CC-type chemokines are known to exhibit activation and migration induction on T cells, basophils, and eosinophils (Oppenheim et al, Annu. Rev. Immunol. 9: 617- 648, 1991; Baggiol ini et a I., Adv. Immunol. 55: 97-179, 1994; Ben—Baruch et al., J. Biol. Chem. 270: 11703-11706, 1995; M. Baggiol ini et al. , Annu. Rev. Immunol. 15: 675-705, 1997; B. ⁇ Rollins, Blood 90: 909-928, 1997). Summary of the Invention
  • the present invention relates to a newly discovered human CC-type chemokine that is a ligand of CCR7 or a mutant thereof, which is found to be mainly constitutively expressed in secondary lymphoid tissues by a single-blot analysis.
  • Proteins that are fragments thereof preferably a human CC type chemokai having the amino acid residues 24 to 134 of SEQ ID NO: 1 or the amino acid residues 1 to 134 of SEQ ID NO: 1 Or a sequence containing at least one selected from substitution, deletion, insertion, and addition of one or several amino acid residues in this sequence and the function or activity of the human CC-type chemokine.
  • Screening agonist, inverse agonist or antagonist for biological action induced by binding A method in which a sample presumed to contain the agonist, inverse agonist or antagonist is added to a binding reaction between the protein and its specific receptor CCR7, and the inhibition of the binding is measured. Or a method comprising directly reacting with the protein specific receptor CCR7 to measure the binding and / or reactivity to the receptor; the present invention, which can be obtained by the screening method of the present invention.
  • Arginist, inverse agonist or antagonist for biological action induced by the binding of the protein to its specific receptor CCR7 BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows the nucleotide sequence and deduced amino acid sequence of human SLC cDNA.
  • FIG. 2 is a view showing the results of comparing the amino acid sequences of the SLC protein of the present invention and 14 known human CC-type chemokines. The numbers on the right show the amino acid identity with the SLC protein.
  • FIG. 3 is a photograph instead of a drawing showing the results of Northern blot analysis of SLC mRNA expression in various human tissues.
  • FIG. 4 is a diagram showing a purity assay of purified SLC protein using SDS-PAGE.
  • FIG. 5 is a graph showing induction of a chemotaxis reaction in human T cell line HUT78 cells and HUT102 cells by purified SLC protein.
  • Figure 6 shows the reactivity of purified SLC protein to various receptors It is a graph shown by the change of the waterfall.
  • FIG. 7 is a graph showing induction of a chemotaxis reaction in a mouse L1.2 cell line stably expressing human CCR 7 by purified SLC protein.
  • FIG. 8 is a graph showing promotion of HIV virus growth by SLC. Protein. Disclosure of the invention
  • CC-type chemokine In order to find a new CC-type chemokine, the present inventors, based on the amino acid sequence of various human CC-type chemokines, used a part of the nucleic acid sequence database GenBank published by PJCBI of the U.S.A. An Expressed Sequence Tag (EST) database composed of partial sequences was searched using TBLASTN search software. As a result, we found the presence of the sequence of a DNA fragment that is thought to encode a new CC-type chemokine-like protein, and isolated two types of cDNA clones that complement the full-length cDNA from human fetal lung tissue tnRNA. Then, the nucleotide sequence over the entire length was determined.
  • EST Expressed Sequence Tag
  • This gene is constitutively expressed mainly in secondary lymphoid tissues such as lymph nodes, small intestine, cecum, and spleen, and the protein produced using genetic engineering techniques is transferred to human T cells. On the other hand, they have shown that they have cell migration activity, and thus completed the present invention.
  • the new human CC-type chemokine was named SLC (Secondary Lymphoid tissue Chemokne).
  • SLC is predicted to be a protein consisting of 134 amino acids based on the open reading frame (0RF) predicted from the nucleotide sequence of cDNA. In mature proteins, glycine at position 23 and serine at position 24 are considered. The signal sequence is cleaved between the two, and it is presumed to be a basic protein consisting of 111 amino acids and having a molecular weight of about 12.2 kDa. Mature SLC shows significant homology to known CC chemokines, especially all four cysteines conserved in CC chemokines. However, the homology with existing CC-type chemokines is about 33% even for the highest MIP-13.
  • CCR7 described in Mark Birkenbach et al., J. Virol. 67: 2209-2220, 1993. This CCR 7 has been reported to be specifically expressed in lymphoid cell lines. Therefore, [Supra, Vicki and Schweickart et al., GENOMICS 23: 643-650, 1994], it is presumed that the ligand SLC of the present invention has a function of migrating lymphocytes.
  • CCR7 (described as EBI1 in the above-cited reference) is caused by infection with Epstein-Barr virus (EBV) and Human Herpesvirus 6 or 7 (HHV-6, 7).
  • the present invention provides a human CC-type chemokine that is a ligand of CCR7, a mutant thereof, or a fragment thereof, which is confirmed to be mainly constitutively expressed in secondary lymphoid tissues by Northern blot analysis.
  • a protein Preferably, the protein is a human CC-type chemokine having amino acid residues of at least 99 or a mutant thereof, or a fragment thereof.
  • secondary lymphoid tissue refers to a primary lymphoid tissue, such as bone marrow or thymus, which controls the production and differentiation of progenitor cells from which immunocompetent cells are derived, and includes lymph nodes, small intestine, Lymphoid tissues such as the cecum and spleen and tissues rich in lymphocytes, which means tissues that actually play an immune response.
  • the present invention relates to a human CC-type chemokine (SLC) having an amino acid sequence of amino acid residues 24 to 134 of SEQ ID NO: 1 or one or several amino acid residues of this sequence.
  • the human CC having a sequence containing at least one selected from substitution, deletion, insertion, and addition, and having a function or activity substantially the same as the function or activity of the human CC chemokine.
  • a human CC-type chemokine mutant that can function as an antagonist to the human CC-type chemokine.
  • the mutant is a variant in which the amino acid at amino acid residue 82 of SEQ ID NO: 1 is not a Tyr residue.
  • the present invention also relates to a human CC type chemokine (SLC precursor) having the amino acid sequence of amino acid residues 1 to 134 of SEQ ID NO: 1, or one or several amino acids added to this sequence.
  • a function comprising a sequence containing at least one selected from substitution, deletion, insertion and addition of a non-acid residue, and having substantially the same function or activity as that of the human CC-type chemokine; or
  • the present invention relates to a mutant of the human CC-type chemokine having activity, or a mutant of the human CC-type chemokine capable of functioning as an antagonist to the human CC-type chemokine.
  • a mutant in which the amino acid at amino acid residue 82 of SEQ ID NO: 1 is not a Tyr residue.
  • CC type chemokine variant refers to a protein having an amino acid sequence of the original protein having a sequence containing amino acid or amino acid sequence substitution, deletion, insertion, or addition, and / or chemical or biochemical A modified protein capable of containing a natural or unnatural amino acid, a protein whose function or activity is substantially the same as the CC-type chemokine, or an antagonist of the CC-type chemokine.
  • the present invention also includes a protein which is a fragment of the CC-type chemokine of the present invention or a mutant thereof.
  • the length of this fragment is, for example, 5 to 100 amino acid residues, or 20 to 80 amino acid residues, and each function or activity is substantially the same as that of the CC-type chemokine of the present invention.
  • the present invention relates to a polynucleotide encoding the CC-type chemokine of the present invention and a mutant of the protein.
  • polynucleotide molecule can also include non-natural molecules including DNA, RNA, or S-lig.
  • DNA it can be cDNA, genomic DNA or synthetic DNA. Both DNA and RNA may be double-stranded or single-stranded. In the case of a single strand, the coding strand or non-coding strand can be used.
  • it includes the nucleotide sequence from A at position 128 to A at position 46 of SEQ ID NO: 1, or the nucleotide sequence from A at position 590 to A at position 450 in SEQ ID NO: 1. It relates to a polynucleotide molecule.
  • the present invention provides variants of these polynucleotide molecules by base substitution, base addition or allelic mutation.
  • a variant by base substitution or base addition refers to the nucleotide sequence set forth in SEQ ID NO: 1.
  • the same amino acid sequence as the amino acid sequence of amino acids 1 to 134 shown in SEQ ID NO: 1 or the amino acid sequence of amino acids 24 to 134 shown in SEQ ID NO: 1 A variant that can encode the same protein as
  • mutant due to allelic mutation means a naturally occurring base mutation based on individual or ethnic differences, and the amino acid sequence to be encoded may be changed.
  • the present invention further provides a polynucleotide or oligonucleotide molecule having a sequence complementary to the entire length or a part of the nucleotide sequence from C at position 1 to A at position 864 of SEQ ID NO: 1, or a base substitution thereof, 2.
  • a sequence complementary to the 5 'non-coding portion is preferred, and more preferably a sequence complementary to the transcription initiation site, translation initiation site, 5' untranslated region, boundary region between exon and intron or 5 'CAP region. It is desirable that Preferred lengths are from about 10 base pairs (bp) to about 60 bp.
  • the present invention relates to a vector containing the polynucleotide molecule of the present invention.
  • the vectors of the present invention include vectors having various uses, such as expression vectors, cloning vectors, and therapeutic vectors.
  • the expression vector can be used for mass production of the protein of the present invention. Details of the expression vector are shown in the following section.
  • Therapeutic vector is used for the method of administering the polynucleotide molecule of the present invention and introducing it into cells.
  • the method using a viral vector and other methods (Nikkei Science, April 1994, pp. 20-45; Monthly Pharmaceutical Affairs, 36 (1) 23-48 (1 994); Experimental Medicine Special Edition, 12 (15), (1 994)). And any of these cited references (etc.) can be applied.
  • a method using a viral vector for example, the DNA or RNA of the present invention is incorporated into a viral vector such as a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a box virus, a polio virus, or a simbis virus.
  • the method introduced in is mentioned. Among them, a method using a retrovirus, an adenovirus, an adeno-associated virus, a vaccinia virus and the like is particularly preferable. Other methods include administration of plasmid directly into the muscle (DNA method), ribosome method, lipofectin method, microinjection method, calcium phosphate method, and electroporation method. DNA vaccine method and ribosome method are preferred.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a protein of the invention or a variant thereof or a polynucleotide molecule encoding them or a therapeutic vector containing it.
  • the pharmaceutical composition of the present invention includes, for example, anti-inflammatory agents, immune response regulators, anti-infective agents, anti-cancer agents, prophylactic agents or diagnostic agents for diseases related to inflammation and / or immunity.
  • the polynucleotide molecule of the present invention act as a medicine, an in vivo method of direct introduction into the body, and a method of introducing a gene into the cell outside the body by using a certain cell from a human and returning the cell to the body There is a way.
  • the dose of the protein or polynucleotide molecule of the present invention can be appropriately adjusted depending on the age, body weight, etc. of the patient, but usually 0.001 mg to 100 mg of the protein of the present invention, It is preferably administered once every few months. Since the protein of the present invention is an active substance in a living body, acute toxicity may not be a problem in the amount in which the activity of the protein occurs, that is, in the amount of the pharmaceutical composition containing the protein of the present invention. Easily guessed.
  • the present invention relates to an antibody against the protein of the present invention or a mutant thereof, particularly a monoclonal antibody, and a hybridoma cell producing the monoclonal antibody.
  • the present invention provides an agonist for the biological effects induced by the binding of the protein of the present invention to its specific receptor CCR7 by providing a relationship between the protein of the present invention and its specific receptor.
  • a method of searching for and evaluating a substance acting as an inverse agonist or antagonist That is, a sample presumed to contain the agonist, inverse agonist or antagonist is added to the binding reaction between the protein of the present invention and its specific receptor CCR7 to measure the inhibition of the binding, or to determine the specificity of the protein.
  • the present invention also relates to an agonist, inverted agonist or antagonist against a biological effect induced by binding of the protein of the present invention to its specific receptor CCR7, which can be obtained by the screening method of the present invention. You. Preferred embodiments of the invention
  • the present invention relates to a human CC-type chemokine whose constitutive expression is mainly observed in secondary lymphoid tissues and in the thymus by Northern blot analysis.
  • a gene recombination technique known in the art a technique for producing a recombinant protein in animal cells, insect cells, yeast and Escherichia coli, a method for separating and purifying expressed proteins, and an assay. Methods and immunological techniques can be employed.
  • DNA fragment containing DNA encoding the SLC protein of the present invention illustrates a method for sequencing a DNA fragment containing DNA encoding the SLC protein of the present invention.
  • the sequence of this DNA fragment can be obtained from cDNA derived from, for example, human lymph nodes, small intestine, cecum, thymus, spleen, or fetal lung tissue. Primers are required for cloning cDMA that encodes
  • GenBank a nucleic acid sequence database released by NCBI of the United States.It uses an Expressed Sequence Tag (EST) database consisting of cDNA partial sequences, and provides TBLASTN search software based on various human CC-type chemokine amino acid sequences. A search is performed to obtain a partial cDNA sequence that has significant homology to CC-type chemokines, but is thought to encode a protein different from known chemokines. Based on the obtained cDNA partial sequence, a primer pair for polymerase chain reaction (PCR) is synthesized.
  • PCR polymerase chain reaction
  • RACE method rapid amplification of cDNA ends
  • mRNA of human fetal lung tissue Frohman et al., Proc. Natl. Acad. Sci. USA 85: 8998-9002, 1988
  • Poly (A) + RNA was extracted from human fetal lung tissue using a Quickprep Micro mRNA purification kit (Pharmacia), and a marathon cDNA amplification kit (Ciontech) was extracted from this poly (A) + RNA. Perform 5 'RACE. And 3' RACE using).
  • cDNA is amplified from the upstream 5 'primer to the downstream 3' end to the 3 'end of the cDNA, and the resulting cDNA fragment is converted into an appropriate nucleotide sequence determination vector, for example, pGEM-T ( Promega) or pBluescript (Stratagene).
  • an appropriate nucleotide sequence determination vector for example, pGEM-T ( Promega) or pBluescript (Stratagene).
  • the recombinant plasmid was recovered, and the nucleotide sequence of the cloned cDNA fragment was determined by, for example, the Sanger method (Sanger et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467, 1977). decide.
  • cDNA is similarly amplified from the downstream 3 'primer to the upstream 5' end to the 5 'end of cDMA.
  • the obtained cDNA fragment is inserted into an appropriate nucleotide sequence determination vector in the same manner as described above, the recombinant plasmid is recovered, and the nucleotide sequence of the cloned cDNA is determined.
  • the base sequence corresponding to the full-length cDNA is determined.
  • amplification was performed by the polymerase chain reaction (PCR) using two types of primers synthesized based on the base sequences at both ends of the region encoding the SLC protein. Insert the DNA into an appropriate nucleotide sequence determination vector (described above), recover the recombinant vector, and determine the nucleotide sequence of the cloned cDNA (described above).
  • PCR polymerase chain reaction
  • the obtained cDNA encoding the SLC protein is inserted into an appropriate expression vector to provide an expression vector for expressing the SLC protein.
  • Appropriate expression vectors include, for example, pRSET, pGEMEX.pKK233-2 for bacteria, pYES2 for yeast, pVU393 for insect cells, pFAST-Bac, pEF-B0S for animal cells, pSRa, pDR2, and the like.
  • the expression vector is introduced into a suitable host cell, for example, a bacterium, yeast, insect cell, or animal cell, to produce a transformant.
  • prokaryotic microorganisms such as Escherichia coli
  • a strong promoter eg, T7 promoter
  • yeast it can be expressed as a fusion protein in which a signal sequence derived from a natural precursor of a yeast secretory protein (eg, a pheromone ⁇ prebub sequence) and a mature SLC protein are fused.
  • the gene for the precursor protein of the SLC protein which already contains a signal sequence, is inserted downstream of a strong promoter (eg, EF-1 ⁇ promoter), and is used as an effective selection marker (eg, dihydrofolate reductase).
  • a strong promoter eg, EF-1 ⁇ promoter
  • the cells can be introduced into animal cells (eg, CH0 dhfr- cells), and cells can be selected based on their resistance to the drug (in this case, metrexrexate), thereby establishing a highly expressing cell line. It can also be expressed by incorporating the gene for the SLC protein precursor containing the signal sequence into a virus or retrovirus and infecting animal cells with the recombinant virus. By culturing these transformants, SLC proteins can be produced and secreted.
  • the mature SLC protein can be prepared using the reported method (Clark-Lewis et al., Biochemistry 30: 3128-) using, for example, a solid phase method, paying attention to the presence of three disulfide bonds. 3135, 1991).
  • the obtained protein can be purified by a method known to those skilled in the art, for example, by a combination of affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, and hydrophobic chromatography. (Imai et al., J. Biol. Chem. 271: 21514-21521, 1996).
  • mutant of the SLC protein of the present invention can be performed by a gene recombination technique (.Sambrook et al., Molecular Cloning: AI aboraroy manual, 2nd edn.New York, Cold Spring Harbor Laboratory) well known to those skilled in the art. This can be done using the introduced DNA.
  • Antibodies to the SLC protein of the present invention include, for example, a synthetic peptide synthesized by an ordinary peptide synthesizer based on a part of the deduced SLC amino acid sequence, and a bacterium, yeast, or insect transformed with a vector that expresses SLC. Purify SLC proteins produced by cells, animal cells, etc. by ordinary protein chemical methods and immunize them As a source, immunize animals such as mice, rats, eight-stars, and egrets to produce antibodies (polyclonal antibodies) derived from their sera.
  • lymphocytes are removed from the spleen or lymph nodes of immunized mouse rats, fused with myeloma cells, and subjected to Kohler and Milstein's method t Nature, 256, 495-497 (1975). )] Or a modified method of Ueda et al. [Proc. Natl. Acad. Sci. USA, 79: 4386-4390, 1982)] to produce a hybridoma and prepare a monoclonal clone from the hybridoma.
  • Antibodies can be produced.
  • a monoclonal antibody of the SLC protein can be obtained by the following steps.
  • the presence of the SLC mRNA and protein of the present invention can be carried out by using a detection method for ordinary specific mRNA and protein (Sambrook et al., Molecular Cloning: AI aboraroy manual, 2nd edn. New York, Cold Spring Harbor Laboratory 1989; Harlow and Lane, Antibodies: A laboratory manual, New York, Cold Spring Harbor Laboratory 1988).
  • mRNA can be detected by Northern blot analysis using an antisense RNA or cDNA as a probe.
  • mRNA is converted to CDNA with reverse transcriptase. It can also be detected by polymerase chain reaction (PCR) using appropriate primer combinations.
  • Protein can be detected by immunoprecipitation using an antibody specific to the SLC protein, Western blot, or the like.
  • a fixed amount of SLC labeled with a radioisotope, an enzyme such as peroxidase or alkaline phosphatase, or a fluorescent dye is added to a known concentration of unlabeled SL (: and anti-SLC polyclonal antibody or monoclonal antibody derived from serum)
  • a known concentration of unlabeled SL : and anti-SLC polyclonal antibody or monoclonal antibody derived from serum
  • a sample containing an unknown amount of antigen in place of the unlabeled antigen of known degree is added to the above reaction system, and the radioactivity, enzyme activity, or fluorescence intensity obtained after the reaction is determined.
  • the amount of antigen, ie, SLC protein in a sample can be determined. Quantification of SLC proteins could provide a new way to monitor inflammatory and immune responses.
  • the chemokine activity of the SLC protein of the present invention can be determined, for example, by placing SLC on one side of a culture vessel partitioned by a filter having a certain diameter of pores in a test tube and placing target cells on the other side. After a certain period of time, the number of cells that have moved through the pores of the filter to the side where SLC is present can be compared with the number of random migration. In vivo, it can also be demonstrated by administering purified SLC protein to animals and detecting cell invasion and aggregation by histological methods.
  • GenBank a nucleic acid sequence database released by NCBI in the United States, based on the Expressed Sequence Tag (EST) database composed of partial sequences derived from various cDNAs based on the amino acid sequences of various human CC-type chemokines.
  • EST data (GenBank Accession No .: W17274, 84422.), which was searched using TBLASTN search software and is thought to encode a chemokine protein that has significant homology to CC-type chemokines but is different from known chemokines. W84375, 67885, W67812, T25128).
  • the SLC cDNA was isolated by the RACE method using SLC-specific primers (Frohman et ai., Proc. Natl. Acad. Sci. USA 85: 8998-9002, 1988).
  • primers for RACE, NCC-8-5′RACE primer and NCC-8R-3 ′ RACE primer were synthesized based on the sequence of GenBank EST data W17274.
  • the sequences of the fJCC-8-5 'RACE primer and NCC-8R-3' RACE primer are as follows:
  • NCC-8-3 'RACE 5'-GAAGCCTGAACCCAAGATGCAAGAAGG-3' SEQ ID NO: 3
  • CDNA was synthesized from this mRNA using the Marathon cDNA Amplification Kit (Clontech).
  • reaction solution was cooled with water, and dATP, dCTP, dGTP, dTTP (0.2 mM each), E. coli DMA polymerase I (24 units), E. coli DNA ligase (4.8 units), and E . col i RNase H to (1 Yuni' g) was added, 100 mM KC and 10 mM sulfuric Anmoniu ⁇ , 5 mM MgC 1 2, 0.15 mM ⁇ -NAD, 20 mM Tris-HCI (pH7.5), 0.05 mg / ml A double-stranded cDNA synthesis reaction was performed for 1 hour and a half at 16 in 80 I of a serum albumin reaction solution.
  • T4 DNA polymerase (10 units) was added to the reaction solution, and the mixture was reacted at 16 ° C for 45 minutes to blunt the cDNA. After the reaction, phenol extraction and ethanol precipitation were performed, and the DNA was dissolved in 10 ⁇ l of distilled water. Add 20 pmole of Marathon cDNA adapter and T4 DNA ligase (1 unit) to 5 ⁇ l of the solution, and add 50 mM Tris-HCI (pH 7.8), 10 mM MgCI or 1 mM DTT, 1 mM ATP, 5 mM (w / v) In a reaction solution of polyethylene glycol (MW 8,000) in 10 I at 16 ° C.
  • PCR was carried out for 30 cycles at 94 ° C, 30 seconds; 60 ° C, 30 seconds; 68 ° C, 4 minutes.
  • the 3 ′ RACE reaction was performed under the same reaction conditions as above, except that the NCC-8-3 ′ RACE primer (SEQ ID NO: 3) was used instead of the NCC-8-5 ′ RACE primer.
  • each PCR product was separated by 23 ⁇ 4 low-melting point agarose gel electrophoresis, and the main 5 'RACE fragment (about 540 bp) and the main 3' RACE fragment (about 350 bp) were recovered by ethanol extraction.
  • each was dissolved in 10 ⁇ l of distilled water.
  • each DMA aqueous solution was mixed with 1.0 I of the vector pCR-lI (Stratagene), reacted with T4 DMA ligase at 16 ° C for about 20 hours, and ligated.
  • a replacement plasmid was prepared. Escherichia coli ( ⁇ ⁇ col i) XLI-Blue MRF '
  • Plasmid DNA was extracted from several of the colonies obtained in the above step, and the nucleotide sequences at the 5 'and 3' ends of the cDNA were examined using SP6 motor and primer and T7 promoter and primer. All had almost the same nucleotide sequence as EST W1727. Therefore, one clone obtained from each of the 5 'RACE reaction and the 3' RACE reaction was selected one by one (hereinafter referred to as 5'-RACE cDNA and 3'-RACE cDNA), and the entire nucleotide sequence of them was determined by Sanger et al. Natl. Acad. Sci. USA 74: 5463-5467, 977 977).
  • the full-length SLC cDNA was determined from the two partially overlapping cDNA sequences thus obtained. As a result, it was found that there was a nucleotide sequence encoding a protein consisting of 134 amino acid residues including methionine defined by the translation initiation codon ATG which appears first.
  • the amino acid sequence of this protein does not correspond to known chemokines, but has significant homology and contains the four conserved cysteine residues that are structural features of chemokines.
  • FIG. 1 shows the determined nucleotide sequence of the full-length cDNA and the amino acid sequence of the longest open reading frame (0RF) starting from the predicted start codon.
  • This gene has 0RF consisting of 134 amino acids, and has about 20 strongly hydrophobic amino acid sequences at its N-terminus, which are presumed to be signal peptides characteristic of secreted proteins.
  • the molecular weight of the protein consisting of 134 amino acids was 14,629.
  • the calculated cleavage site for the signal peptide was estimated to be between glycine at position 23 and serine at position 24.
  • the putative mature protein of 111 amino acids after signal peptide cleavage is presumed to be a secreted protein, and The molecular weight was 12, 23, and the isoelectric point was 10.72.
  • MIP-1 ⁇ Lipes et a, Proc. Nat, Acad. Sc, USA 85: 9704-9708, 1988
  • MIP- 1a Olet al., J. Biochem. 99: 885-894, 1986
  • 31% LD78- ⁇ (Nakao et al., Mol. Cel I. Biol. 10: 3646- 3658, 1990) '' And 3, eotaxin (Kitaura et al., J. Biol. Chem. 271: 7725-7730, 1996) and 3, MCP-1 (Furutan i et a I., Biochem. Biophys. Res. Commun.
  • FIG. 3 shows the expression of SLC mRNA in various human tissues. The results in FIG. 3 revealed that SLC mRNA was strongly expressed in immune system tissues, particularly in lymph nodes, secondary lymph tissues such as the small intestine, cecum, and spleen, and in the thymus.
  • the SLC protein was produced in insect cells by introducing cDNA encoding the SLC protein.
  • the SLC 5'-RACE cDNA (described above), which encodes the full-length SLC protein, is digested with the restriction enzymes BamHI and Xbal, and the vector pFAST-Bac (Gibco-BRL Inc.) is used to recombine with baculovirus in E. coli.
  • the vector pFAST-Bac-SLC was prepared by inserting the vector between BamHI and Xbal site. This vector was introduced into Escherichia coli (E.
  • coli) DmOBac manufactured by G'ibco-BRL
  • G'ibco-BRL G'ibco-BRL
  • a recombinant baculovirus DNA expressing the SLC protein.
  • This recombinant baculovirus DMA was introduced into insect cells Sf9 using CellFectin Reagent (manufactured by Gibco-BRL), and a recombinant baculovirus was obtained from the culture supernatant.
  • the recombinant baculovirus obtained in this manner was infected into High Five, an insect cell with high expression efficiency, and two days after the culture, the culture supernatant was recovered.
  • the culture supernatant of the insect cells infected with the recombinant baculovirus is sterilized by filtration through a 0.22 nm filter, and the ion exchange column HiTrap. SP cation exchange column (Pharmacia), washed with buffer A (50 mM HES pH 6.5), and eluted the protein with a gradient using buffer B (50 mM ES ⁇ 6.5, 1 ⁇ NaC I). The fraction containing SLC was identified using SDS-PAGE, and a protein of 15 kD was found in the eluted fraction of 0.4-0.5M NaCI. This fraction is collected on a Cosmosil reverse phase column.
  • Example 3 Using the culture supernatant containing the recombinant SLC obtained in Example 3, cell migration activity was examined.
  • human T cell lines HUT78 and HU02 imai et al., J. Biol. Chem. 271: 21514-21521, 1996) were used.
  • the test solution is diluted with a culture solution (RPM 1640, 20 mM Hepes (pH 7.4), ⁇ ! 3 ⁇ 4 BSA), and placed under a chemoaxis chamber (Chemotaxis chamber, Neuro Probe) of 48-well.
  • 4 ⁇ 10 5 TUT cell line HUT78 cells suspended in the above culture solution were added to the upper wells.
  • N-terminal amino acid sequence of purified SLC protein was determined using an amino acid sequencer (Shimadzu), and Ser-Asp-Gly-Gly-Ala-Gin-Asp-XX was used. -Leu-Lys-Tyr.
  • amino acid sequence deduced from the base sequence shown in FIG. 1 the amino acid sequence between the dalysine residue at position 23 and the serine residue at position 24, which is the cleavage site of the deduced signal sequence, is shown.
  • the signal peptide was truncated and agreed with the fJ-terminal amino acid sequence expected when a mature secreted SLC protein consisting of 111 amino acids was obtained.
  • Cells include mouse pre-B cell line L1.2 as a control and CC chemokine receptor of mouse pre-B cell line L1.2, which stably expresses seven types of receptors, human chemoforce-in receptor (CCR) 1 to CCR 7, respectively. Transfectants were used.
  • test Example 1 the test solution was diluted with a culture solution (RPM-1640, 20 mM Hepes (pH 7.4), 13 ⁇ 4 BSA), added to the lower well of a transwell chamber (Costar), and added to the upper well.
  • a murine pre-B-cell line L1.2 or CCR7 transflector Ek Tan Bok of 5Xl0 6 suspended above ⁇ solution After culturing at 37 ° C for 4 hours, the number of cells that had migrated to the lower well was counted using a cell sorter (Becton Dickinson).
  • Figure 7 shows the results. As shown in FIG.
  • PBMCs Peripheral blood mononuclear cells collected from healthy adult volunteers are cultured for 2 days in a culture solution (RPN 1640, 103 ⁇ 4FCS) containing mutadaltinin (PHA) and IL-2 at 20 U / ml. Then, human immunodeficiency virus (HIV) strain, NL432 or SF162 was added to these cells at a moi of about 0.1 and incubated at 37 ° C for 2 hours to infect the cells. After washing the infected cells, the cells were suspended in a culture solution containing IL-2 and 20 U / ml (RPN 1640, 10 UFCS) so that the cell flow rate became 1 ⁇ 10 6 / ml.
  • a culture solution RPN 1640, 103 ⁇ 4FCS
  • the culture was maintained for 7 days with or without SLC at the indicated concentration of 32-4000 ng / ml, and the amount of HIV production was determined by the HIV reverse transcriptase activity in the culture supernatant.
  • Reverse transcriptase activity was measured as follows.
  • FIG. 8 shows the results.
  • SLC significantly promotes viral replication of HIV at concentrations of 160-4000 ng / ml.
  • the virus strain used, NL432 is a T cell finger.
  • SF 162 is tropotrophic and is macrotrophic for monocytes, SLC promotes virus growth for both strains.
  • Chemokines which induce leukocyte migration and infiltration into tissues, are essential substances for inflammatory and immune responses in vivo.
  • CXC type and CC type are mainly known as chemokines, and there are multiple types of each type, ranging from producing tissues, producing cells, types of stimuli to induce production, induction of production to production cessation They exhibit different properties with respect to reaction time, types of target cells that induce migration, and the presence of specific receptors.
  • the SLC of the present invention structurally belongs to the group of CC-type chemokines, and is structurally expressed mainly in lymphoid tissues such as lymph nodes, small intestine, cecum, thymus, and spleen and in tissues rich in lymphocytes, and T cells It has the property of showing chemotactic activity for lymphocytes such as.
  • the SLC of the present invention provides a new means for elucidating its function, understanding the inflammatory and immune reactions involving lymphocytes, and inducing or suppressing such phenomena.
  • the SLC of the present invention is constitutively expressed mainly in secondary lymphoid tissues, for example, lymphatic tissues such as lymph nodes, small intestine, cecum and spleen, and thymus, the SLC is normal.
  • lymphocytes In living organisms, it controls the pharmacokinetics of lymphocytes, that is, circulatory circulation, which controls homing in lymphoid tissues, and is involved in the migration and establishment of lymphocytes in lymphoid tissues, maturation and differentiation, antigen recognition, survival, proliferation, etc. It is expected that Therefore, the SLC of the present invention, by elucidating its function, is useful for understanding the migration and establishment of lymphocytes in various lymphoid tissues, differentiation and maturation, antigen recognition, regulation of cell growth and survival, etc. It provides a useful tool for regulating such phenomena.
  • the SLC protein or a mutant thereof provided by the present invention regulates a physiological or pathological biological reaction involving SLC by enhancing or suppressing the action of SLC in vivo.
  • the polynucleotide molecule non-naturally occurring polynucleotide molecule including DNA, RNA or S-lignin, double-stranded or single-stranded, including DNA, RNA or S-ligigo
  • it may be directly administered to a living body as a polynucleotide, introduced into an appropriate vector and introduced into cells outside the body, and the cells (transformed cells) may be returned to the body, or introduced into an appropriate vector.
  • nucleotide sequence of SLC provided by the present invention, a polynucleotide molecule (non-natural molecule including DNA, RNA or S-oligo) which encodes SLC in full length or in part, and a specific antibody against SLC are those of SLC. It is useful for detecting and analyzing gene mutations, and is also useful for specifically detecting and quantifying SLC gene expression (mRNA) and protein expression. As a result, it provides new means for diagnosis and investigation of the causes of inflammatory diseases, blood system diseases, immune system diseases, infectious diseases, cancers, etc. involving the SLC gene or SLC protein. It is expected to provide a new means of treatment.
  • SLC promotes virus growth on both T cell-tropic viruses and macrophage monocyte-trophic virus strains and acts irrespective of virus cell tropism.
  • virus particles are detected in the blood of HIV immediately after infection, the amount of virus in the blood drops sharply for a long time until the onset of HIV. During this time, it is known that HIV is not at rest but is continuously growing in the lymph nodes (persistent infection).
  • SLC expressed in lymph nodes promotes the viral propagation of HIV in vitro indicates that HIV may be involved in the sustained growth of HIV in lymph nodes in vivo.
  • Inhibitors that inhibit the binding of SLC to its receptor CCR7 may be new anti-HIV drugs. Sequence listing SEQ ID NO: 1
  • Sequence type nucleic acid

Abstract

Nouvelle SLC humaine de chémokine CC; ses variants; ses protéines fragments; et molécules polynucléotidiques codant ladite substance. Les protéines considérées et les molécules polynucléotidiques qui les codent, les anticorps dirigés contre ces protéines, ainsi que les agonistes ou les antagonistes par rapport aux effets biologiques dus à la liaison des protéines SLC sur des récepteurs spécifiques sont utiles pour le traitement et le diagnostic de maladies comme l'inflammation, l'immunité, l'infection et le cancer.
PCT/JP1998/000154 1997-01-20 1998-01-19 Slc humaine de chemokine cc WO1998031809A1 (fr)

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Cited By (5)

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WO2000009151A1 (fr) * 1998-08-17 2000-02-24 Schering Corporation Regulation de l'activite de cellules dendritiques
WO2000038706A3 (fr) * 1998-12-31 2000-11-23 Chiron Corp Methodes de traitement du cancer et de mediation de la chimiotaxie des cellules dendritiques
US6153441A (en) * 1998-02-17 2000-11-28 Smithkline Beecham Corporation Methods of screening for agonists and antagonists for human CCR7 receptor and CKβ-9 ligand and interaction thereof
WO2004104574A3 (fr) * 2003-05-23 2005-02-03 Bayer Healthcare Ag Agents diagnostiques et therapeutiques destines a des maladies associees au recepteur de chimiokine c-c de type 7 (ccr7)
WO2005083440A2 (fr) * 2004-02-19 2005-09-09 Yale University Identification de biomarqueurs proteiques du cancer par des techniques proteomiques

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WO1996006169A1 (fr) * 1994-08-23 1996-02-29 Human Genome Sciences, Inc. Chemokine beta 9 humaine
WO1996025497A1 (fr) * 1995-02-17 1996-08-22 Incyte Pharmaceuticals, Inc. Nouvelles chemokines exprimees dans le pancreas

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1996006169A1 (fr) * 1994-08-23 1996-02-29 Human Genome Sciences, Inc. Chemokine beta 9 humaine
WO1996025497A1 (fr) * 1995-02-17 1996-08-22 Incyte Pharmaceuticals, Inc. Nouvelles chemokines exprimees dans le pancreas

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6153441A (en) * 1998-02-17 2000-11-28 Smithkline Beecham Corporation Methods of screening for agonists and antagonists for human CCR7 receptor and CKβ-9 ligand and interaction thereof
WO2000009151A1 (fr) * 1998-08-17 2000-02-24 Schering Corporation Regulation de l'activite de cellules dendritiques
WO2000038706A3 (fr) * 1998-12-31 2000-11-23 Chiron Corp Methodes de traitement du cancer et de mediation de la chimiotaxie des cellules dendritiques
WO2004104574A3 (fr) * 2003-05-23 2005-02-03 Bayer Healthcare Ag Agents diagnostiques et therapeutiques destines a des maladies associees au recepteur de chimiokine c-c de type 7 (ccr7)
WO2005083440A2 (fr) * 2004-02-19 2005-09-09 Yale University Identification de biomarqueurs proteiques du cancer par des techniques proteomiques
WO2005083440A3 (fr) * 2004-02-19 2006-03-16 Univ Yale Identification de biomarqueurs proteiques du cancer par des techniques proteomiques
AU2005217375B2 (en) * 2004-02-19 2011-05-19 Yale University Identification of cancer protein biomarkers using proteomic techniques
AU2005217375C1 (en) * 2004-02-19 2012-06-07 Yale University Identification of cancer protein biomarkers using proteomic techniques
US8975379B2 (en) 2004-02-19 2015-03-10 Yale University Identification of cancer protein biomarkers using proteomic techniques
US9470688B2 (en) 2004-02-19 2016-10-18 Yale University Identification of cancer protein biomarkers using proteomic techniques
US10168334B2 (en) 2004-02-19 2019-01-01 Yale University Identification of cancer protein biomarkers using proteomic techniques

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