WO1998031809A1

WO1998031809A1 - Human cc chemokine slc

Info

Publication number: WO1998031809A1
Application number: PCT/JP1998/000154
Authority: WO
Inventors: Morio Nagira; Toshio Imai; Osamu Yoshie
Original assignee: Shionogi & Co., Ltd.
Priority date: 1997-01-20
Filing date: 1998-01-19
Publication date: 1998-07-23
Also published as: AU5496798A

Abstract

A novel human CC chemokine SLC; its variants; proteins which are fragments of the same; and polynucleotide molecules encoding the same. The above proteins, the above polynucleotide molecules encoding these proteins, antibodies against these proteins, and agonists or antagonists to biological effects due to the bonding of the SLC proteins to receptors specific thereto provide useful means for the treatment and diagnosis of diseases such as inflammation, immunity, infection and cancer.

Description

Specifi cations C C-type chemokines S L C Technical field

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

A variety of extrinsic or endogenous tissue injuries, invasions, or antigen exposures that are caused by physical, chemical, or biological mechanisms can induce strong inflammatory and immune responses. These reactions are important host defense responses, but can also sometimes cause acute or chronic illness. When 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. 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 α 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). As human CXC chemokines, IL-8, β-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. As human CC chemokines, MIP-1α, ΜΙΡ-1β, 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. In addition, some 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. 丄 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. Mutants of the human CC-type chemokines having substantially the same function or activity, or functioning as antagonists of the human CC-type chemokines, or A protein which is a fragment; a pharmaceutical composition containing the protein of the present invention; an antibody against the protein of the present invention, preferably an antibody which is a monoclonal antibody; a hybridoma cell producing the monoclonal antibody of the present invention; A polynucleotide molecule encoding the protein, preferably a nucleotide sequence from A at position 128 to A at position 46 of SEQ ID NO: 1, preferably from A to position 460 of position 59 of SEQ ID NO: 1 A polynucleotide molecule of the present invention containing the full length or a part of the nucleotide sequence up to A, or a variant thereof due to base substitution, base addition or allelic mutation; A polynucleotide or oligonucleotide molecule having a sequence complementary to the full length or part of the nucleotide sequence of A molecule that is a mutant due to base addition or allelic mutation and that inhibits the activity or function of the protein of the present invention; a vector containing the polynucleotide molecule of the present invention, preferably an expression vector or a therapeutic vector A transformant obtained by introducing the expression vector of the present invention into a host cell; culturing the transformant of the present invention, and recovering the produced protein from the culture; A pharmaceutical composition containing the polynucleotide molecule of the present invention or a mutant thereof; a pharmaceutical composition containing the therapeutic vector of the present invention; a protein of the present invention and its specific receptor CCR7. 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

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. 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.

As a result of functional analysis of the SLC of the present invention, it was determined that the specific receptor was 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. In addition, 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). Induced receptor (Ralf Burgs tah Ier, et al. Biochem. Biophys. Res. Com. 215: 737-743, 1995; Hi toshi Masegawa, et al. J. Virol. 68: 5326 — 5329, 1994), and it has been suggested that EBV and HHV-6, 7 are involved in diseases caused by them. Therefore, it is presumed that the ligand of the present invention, SLC, regulates the life cycle of EBV and HHV-6, 7, and is involved in diseases caused by these viruses.

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. Is 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.

As used herein, the term "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.

In one embodiment, 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. And a human CC-type chemokine mutant that can function as an antagonist to the human CC-type chemokine. Preferably, 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. Preferably, a mutant in which the amino acid at amino acid residue 82 of SEQ ID NO: 1 is not a Tyr residue.

As used herein, the term "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. A protein that functions as

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. A protein or a protein that functions as an antagonist of the CC-type chemokine.

In another aspect, the present invention relates to a polynucleotide encoding the CC-type chemokine of the present invention and a mutant of the protein.

As used herein, the term "polynucleotide molecule" can also include non-natural molecules including DNA, RNA, or S-lig. In the case of 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.

Specifically, 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.

Furthermore, 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. As a result, 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

The term “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 molecule which is a mutant due to base addition or allelic mutation and which inhibits the activity or function of the protein according to claim 1.

Particularly, 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.

In another aspect, 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. As 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.

In a further aspect, the present invention relates to 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.

In order to make 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.

Further, 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. In another aspect, 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. And measuring the direct binding and / or reactivity to the target receptor. 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. Hereinafter, the process for preparing the protein of the present invention will be described. In this specification, unless otherwise indicated, unless otherwise indicated, 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.

I. Preparation of the protein of the present invention

1 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

(1) Search for SLC cDNA partial sequence from EST library

It is part of the 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.

(2) Determination of nucleotide sequence of full-length cDNA of SLC

Next, using these primers, for example, rapid amplification of cDNA ends (RACE method) from mRNA of human fetal lung tissue (Frohman et al., Proc. Natl. Acad. Sci. USA 85: 8998-9002, 1988) Determine the nucleotide sequence over the entire length of the cDNA. That is, 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). That is, first, 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). Next, 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. Next, 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. By superimposing the base sequences of the two types of cDNA clones determined in this way using a region common to both, the base sequence corresponding to the full-length cDNA is determined.

(3) Preparation of full-length SLC cDNA

Based on the base sequence of the obtained SLC full-length cDNA, 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).

(4) Expression of recombinant SLC

Next, 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. In prokaryotic microorganisms such as Escherichia coli, It can be expressed under the control of a strong promoter (eg, T7 promoter) as a fusion protein in which the derived signal sequence (eg, signal peptide OMPa) and the mature SLC protein are fused. In 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 α prebub sequence) and a mature SLC protein are fused. In animal cells, 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-1α promoter), and is used as an effective selection marker (eg, dihydrofolate reductase). At the same time, 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.

Alternatively, 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).

Expression of the 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.

II. Preparation of antibodies against the protein of the present invention

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.

Alternatively, 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. For example, a monoclonal antibody of the SLC protein can be obtained by the following steps.

(a) Immunization of mice with SLC protein,

(b) removal of spleen and separation of spleen cells from immunized mice,

(c) Fusing by the method described by Kohler et al. in the presence of a fusion promoter (eg, polyethylene glycol) between the isolated spleen cells and mouse myeloma cells.

(d) culturing the obtained hybridoma cells in a selective medium in which unfused myeloma cells do not grow,

(e) Selection of hybridoma cells producing the desired antibody by methods such as enzyme-linked immunosorbent assay (ELISA) and western blotting, and cloning by limiting dilution, etc.

(f) Culture the hybridoma cells producing the SLC monoclonal antibody and harvest the monoclonal antibody.

[0 0]

III. Detection of mRNA and protein of SLC protein

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). For example, mRNA can be detected by Northern blot analysis using an antisense RNA or cDNA as a probe. Also, 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.

IV. Immunoassay for SLC protein

For example, 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) After appropriately changing the concentration of the unlabeled antigen, the labeled antigen bound to the antibody and the labeled antigen not bound to the antibody are separated by an appropriate method. Measure the amount of radioactivity, enzymatic activity or fluorescence intensity of the labeled antigen bound to the antibody As the amount of unlabeled antigen increases, the amount of labeled antigen bound to the antibody decreases This relationship is graphed to obtain a standard curve. In addition, one of the two types of monoclonal antibodies that recognize different epitopes on the SLC protein is immobilized, and the other is any one of the methods described above. Labeled, you detect quantify the amount of SLC bound to the solid phase antibody in the label antibody, the so-called sandwich method is also possible.

Next, 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. When fitted to a standard curve, 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.

V. Measurement of chemokine activity of SLC protein

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. Example

The present invention is further described by the following examples.

Example 1

Isolation of SLC cDNA and determination of its structure

(1) Search for partial sequence of SLC cDNA from EST library.

A part of 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). These data are cDNAs from the human fetal lung (W17274), human fetal heart (W84422, W84375, W67885, W67812) and colorectal cancer (T25128) cDNA libraries, respectively, and are 535 bp, 455 bp, 400 bp, 482 bp, 196 bp, 164 bp, respectively, April 29, 1996, June 27, 1996, June 27, 1996, October 15, 1996, October 15, 1996, August 24, 1995 , Are registered with GenBank.

(2) Isolation of SLC cDPJA

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). First, 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-8-5 'RACE 5' -CCTTCTTGCATCTTGGGTTCAGGCTTC-3 '(SEQ ID NO: 2)

NCC-8-3 'RACE 5'-GAAGCCTGAACCCAAGATGCAAGAAGG-3' (SEQ ID NO: 3) Next, mRNA was extracted from human fetal lung tissue using a QuickPrep Micro mRNA purification kit (Pharmacia). CDNA was synthesized from this mRNA using the Marathon cDNA Amplification Kit (Clontech). That is, 5 μi of an aqueous solution containing human fetal lung mRNA 1 Atg and Marathon cDNA synthesis primer 10 pmole at 70 ° C Heat for 2 minutes, cool on ice, add dATP, dCTP, dGTP, dTTP (1 mM each) and MMLV (Moloney Murine Leukemia Virus) reverse transcriptase (100 units), and add 50 mM Tris-HCI (pH 8. 3) A single-stranded cDNA synthesis reaction was performed at 42 ° C for 1 hour in 10 I of a reaction mixture of 6 mM MgCI ₂ and 75 mM KCI. After the reaction, the 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 _{厶, 5 mM MgC 1 2, 0.15} mM β -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.

Next, 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 μl of distilled water. Add 20 pmole of Marathon cDNA adapter and T4 DNA ligase (1 unit) to 5 μl 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. for about 20 hours, adapters were bound to both ends of the double-stranded cDNA. After the reaction, heat at 70 ° C for 2 minutes to inactivate the ligase, dilute 250-fold with 10 mM Tricine-K0H (pH 9.2), 0.1 mM EDTA, heat at 94 ° C for 2 minutes, and remove the adapter. The bound double-stranded cDNA was denatured.

Next, a RACE reaction was performed. First, in the 5 'RACE reaction, 5A of the denatured cDNA was added to dATP, dCTP, dGTP, dTTP (0.2 mM each), TAKARA LA Taq (2,5 units), and TaqStart antibody (0.55 μ. g) Add 10 pmole of API primer that binds to a part of the adapter and 10 pmole of NCC-8-5 'RACE primer (SEQ ID NO: 2), and in lx TAKARA LA Taq buffer reaction mixture at 50 ° C, 94 ° C. Immediately after pretreatment for 1 minute, 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. After the reaction, each PCR product was separated by 2¾ 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. , After performing an ethanol precipitation operation, each was dissolved in 10 μl of distilled water. 5 μI of 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 (Ε · col i) XLI-Blue MRF '

(Stratagene) was transformed to obtain colonies.

(3) Identification of positive clones and nucleotide sequencing

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. The presence of a highly hydrophobic signal sequence-like sequence on the N-terminal side, which is a characteristic feature, suggests that this protein is a novel chemokine.

(4) Analysis of deduced amino acid sequence of SLC

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.

Amino acid sequence similarity analysis was performed using the ClustalW program. Figure 2 shows the results. Amino acids conserved in all CC-type chemokines, including proteins deduced from 0RF of the obtained cDNA base sequence, are indicated by asterisks, and amino acids conserved in most CC-type chemokines are indicated by solid circles. Indicated by In addition, the degree of homology between the protein deduced from 0RF of the obtained cDNA base sequence and other CC-type chemokines was determined for mature proteins after signal peptide cleavage. ! ! Indicated on the right side of the display. That is, the amino acid sequence of the mature secreted protein belongs to the CC-type chemokine. For example, MIP-1β (Lipes et a, Proc. Nat, Acad. Sc, USA 85: 9704-9708, 1988) and 33¾, MIP- 1a (Obaru et al., J. Biochem. 99: 885-894, 1986) and 31%, LD78-β (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. 159: 249-255, 1989; Yoshimura et al., FEBS Lett. 244: 487-493, 1989) and 31¾, MCP-2 (Van Damme et al., J. Exp. Med. 176: 59-65, 1992) ) And 31¾, CP-3

(Opdenakker et al., Biochem.Biophys.Res.Commun.191: 535-542, 1993) and 30¾, CP-4 (Uguccioni et al., J. Exp.Med.183: 2379-2384, 1996) and 28 %, RANTES

(Schal I et al., J. Immunol. 141: 1018-1025, 1988) and 27¾, 1-309 (Miler et al., J. Immunol. 143: 2907-2916,〗 989) and 24¾, TARC (Imai et al., J. Biol. Chem. 271: 21514-21521, 1996) and 21% homology. It was also found that four cysteines stored in all CC chemokines were also stored in SLC. Therefore, the obtained amino acid sequence is considered to be that of a novel human CC-type chemokine.

Example 2

Expression analysis of SLC mRNA by Northern blot analysis 2 μg of poly (A) + RNA isolated from various human tissues was subjected to agarose gel electrophoresis and was already transferred to a nylon membrane (multiple tissue plot) Was purchased from Clonetech and probed with SLC 5'-RACE cDNA labeled with ³² P using a multiprime DNA labeling system (Pharmacia) as a probe. A Zession reaction was performed. The hybridization solution was 50% formamide, 5XSSC, 50m Tris.CI pH7.5, 5x Denhard's solution, 0.5¾SDS. And 100 μg / ml of salmon sperm DNA added at 42 ° C. Time hybridisation. After washing the membrane under the conditions of 0. h SSC and 0. U SDS buffer at 65 ° C, the membrane was exposed to an X-ray film (Kodak), developed, and analyzed. Figure 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.

Example 3

Preparation of SLC protein

(1) Expression of SLC protein

The SLC protein was produced in insect cells by introducing cDNA encoding the SLC protein. First, 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) to prepare 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. In this culture supernatant, a protein of about 15 kD, which was not present in the culture supernatant infected with wild-type baculovirus, was detected at a high concentration. It is shown in Test Example 2 below that the protein of about 15 kD is a mature processed SLC protein, but the increase in molecular weight is presumed to be due to some modification.

(2) Purification of SLC protein

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 ρΗ6.5, 1ΜNaC 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.

(Nacalai Tesque, Inc.), washed with solution C (0.05 FA), and purified by reverse phase chromatography using daradiene with solution D (acetonitrile, 0.05 ， TFA). SLC protein is acetate nitrile 28¾! Was identified as a single peak at the position, and almost a single band was observed by silver staining after SDS-PAGE, and SLC proteins with a purity of 95% or more were successfully obtained. The purified SLC protein was lyophilized, dissolved in endotoxin-free distilled water (Fuso Pharmaceutical Co., Ltd.), and the chemotaxis activity was measured. Test example 1

Cell migration activity of purified SLC protein

Using the culture supernatant containing the recombinant SLC obtained in Example 3, cell migration activity was examined. As the cells, 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),〗! ¾ BSA), and placed under a chemoaxis chamber (Chemotaxis chamber, Neuro Probe) of 48-well. In addition to the wells, ⁴ × 10 ⁵ TUT cell line HUT78 cells suspended in the above culture solution were added to the upper wells. Separation of top and bottom wells was performed using a polyvinylpyrrolidone-free polycarbonate membrane (5 μm pore size, manufactured by Neuro Probe) coated with a type IV collagen solution (5 μg / ml aqueous solution) for 2 hours at room temperature. . After culturing at 37 ° C for 2 hours, the membrane was removed, the upper side was washed with PBS, and fixed and stained. The migrated cells were counted under a microscope at 800 × magnification for 5 fields randomly selected per well. Figure 6 shows the results. As shown in the graph of FIG. 6, the SLC-purified protein showed very strong migration activity on HUT78 and HUT102 cells, but no such migration activity was detected in the lysis buffer. Test example 2

Determination of N-terminus of SLC

To determine the N-terminus of mature secreted SLC The 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. In the 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. Test example 3

Identification of specific receptors for SLC

To determine the specific receptor for SLC, the responsivity of SLC to various receptors was examined by increasing the intracellular calcium concentration induced by the binding of SLC to the receptor. 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.

These cells (5 × 10 ⁶⁾ were loaded in a culture solution (RPMI-1640, 20 mM Hepes (pH 7.4), 1% FCS) at 2 mM calcium indica overnight. After washing twice with HBSS containing 1% FCS, the volume was adjusted to 2 ml with the same buffer. The fluorescence derived from Fura-2 was temporarily measured with a fluorescence intensity meter (Perkin-Elmer). Figure 7 shows the results. As shown in FIG. 7, a strong transient increase in intracellular calcium concentration was observed when the SLC protein was added to the CCR7 transfectant {η}. On the other hand, no reaction was observed when 10 nM SLC protein was added to the parent strain L1.2 or its CCR 1 to CCR 6 transfectants, and the intracellular calcium concentration increased when と ηΜ was added to each of the appropriate ligands. was detected. This indicated that CCR7 was a specific receptor for SLC.

Next, the cell migration activity of SLC against CCR7 transfectants was examined. As in Test Example 1, the test solution was diluted with a culture solution (RPM-1640, 20 mM Hepes (pH 7.4), 1¾ BSA), added to the lower well of a transwell chamber (Costar), and added to the upper well. the addition of a murine pre-B-cell line L1.2 or CCR7 transflector Ek Tan Bok of 5Xl0 ⁶ suspended above培溶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. 7, the purified SLC protein showed a migration activity against the CCR7 transfectant, but did not show a migration activity against its parent strain L1.2. This indicates that CCR7 is essential for the migration of CCR7 transfectants to SLC, and that CCR7 is a functional receptor for SLC. Test example 4

SLC promotes HIV virus growth

Peripheral blood mononuclear cells (PBMCs) collected from healthy adult volunteers are cultured for 2 days in a culture solution (RPN 1640, 10¾FCS) 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. At this time, 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.

ΙΟΟμΙ of 50mM Tris-HCI, pH8.3, 150m KCI, lOmM MgCI 0.1¾ Nonidet P-40, 10mM Jichiosurei torr, 5 mg / nil poly (rA), 5mg / ml ( dt), 2. 18. [3 H] 10 µL of the culture supernatant was added to the reaction solution containing dTTP, and the mixture was reacted at 37 ° C for 3 hours, and then cooled with ice to stop the reaction. The reaction with base star Seruha, DEAE-filter mat is adsorbed on (Fuar Macia Co.), 5¾ Na ₂ HP0 ₄ and washed with H ₂ 0, LKB base Isseki play Bok Shin Chireshiyonsu Bae click Bok Rosukopi ( Radioactivity was measured by a method manufactured by Pharmacia.

Fig. 8 shows the results. FIG. 8 shows that SLC significantly promotes viral replication of HIV at concentrations of 160-4000 ng / ml. The virus strain used, NL432, is a T cell finger. Although SF 162 is tropotrophic and is macrotrophic for monocytes, SLC promotes virus growth for both strains. Industrial applicability

Chemokines, which induce leukocyte migration and infiltration into tissues, are essential substances for inflammatory and immune responses in vivo. Currently, 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.

Since SLC exhibits chemotactic activity on lymphocytes such as T cells, it is easily expected that SLC will be involved in acute or chronic inflammatory and immune reactions involving lymphocytes. Therefore, 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. In addition, since 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. 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.

That is, 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. To make things possible. Further, 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) provided by the present invention may be a suitable one. Depending on the method, 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. It can be administered directly into the body. As a result, it is possible to enhance or suppress the production of SLC proteins in the target tissues or cells, thereby enabling acute or chronic diseases, cancers, viruses and other diseases caused by various inflammatory or immune reactions. It is useful for treatment of infectious diseases caused by microorganisms and diseases associated with abnormal SLC gene structure and expression.

Further, the 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.

In addition, it was shown that SLC promotes virus growth on both T cell-tropic viruses and macrophage monocyte-trophic virus strains and acts irrespective of virus cell tropism. Although 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). The fact that 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

Array length: 864

Sequence type: Nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: cDNA to mRNA

Origin

Organism name: Human

Array features

Characteristic symbol: CDS

Location: 59..460

How the features were determined: S

C TTG CAG CTG CCC ACC TCA CCC TCA GCT CTG GCC TCT TAC TCA 43

CCC TCT ACC ACA GAC ATG GCT CAG TCA CTG GCT CTG AGC CTC CTT 88

Met Ala Gin Ser Leu Ala Leu Ser Leu Leu

1 5 10

ATC CTG GTT CTG GCC TTT GGC ATC CCC AGG ACC CAA GGC AGT GAT 133 I I e Leu Va I Leu Ala Phe Gly lie Pro Arg Thr Gin Gly Ser Asp

15 20 25

GGA GGG GCT CAG GAC TGT TGC CTC AAG TAC AGC CAA AGG AAG ATT 178 Gly Gly Ala Gin Asp Cys Cys Leu Lys Tyr Ser Gin Arg Lys l ie

30 35 40

CCC GCC AAG GTT GTC CGC AGC TAC CGG AAG CAG GAA CCA AGC TTA 223 Pro Ala Lys Val Val Arg Ser Tyr Arg Lys Gin Glu Pro Ser Leu

45 50 55

GGC TGC TCC ATC CCA GCT ATC CTG TTC TTG CCC CGC AAG CGC TCT 268 Gly Cys Ser lie Pro Ala lie Leu Phe Leu Pro Arg Lys Arg Ser

60 65 70 2 ν α ^ · 涎 ¾: ¾

^ m: one

瞵伞: om

98 WW VVVVVVVV33 1013110103 VVVVlVVOi

028 3VV133V1V1 丄丄 V3 丄 V1VVV 1311331131 0DV3333V3V 13VD333133 D3V1VD133V 09 DD13D3V00V 31V31VV3VD 0VV0113333 1313V00V30 V303V3DVDV 91V33V303V 00 033131DVDV 339V3DVDD3 VDV3VDV331 D3V303DV39 V3V333D10D V3130D1VDD 0 ^ 9 13D3V13i30 V03311V331 01V3333V03 VV 丄丄丄 33_m 10111313V3 VOOOilDOOO 089 13D1V3300V DODVOOVODi D333DDDV31 331V13D0V0 DVV3VV391V 3VV333VVD1

029 309VV0il33 0VV03V3133 0V3DV3333V OVDDiOOOOV 3D133DV33V OiDVODOOVI sX "i OJd

09f V33 D3D VVV 133

οει 9ZI 0 1

"Review" ID J9S 3- <niO "4 丄 8JV" i SAQ 3 "ΊJ9S 3 s A | D

8 30V DVD V3i 333 3V3 丄. V 3DV 0VV 3D1 ODD VVV 331 333 DVV V33

911 Oil 901

3 "Rs sA J8S B I v ΛIg 3JV dsy sAi 3JV 5λ3 3" 13

£ 0 VVV DVV 333 13V 3VV 331 309 DDD DOV 3V9 DVV D3V 001 ODD 9VD

001 S6 06

B | V OJd sA "i u | 3 OJd J3S OJd Jij 丄 sA, dsy Π31 S! H u | 3 law nan

8 ε 033 V33 VVV DV3 V33 031 V33 V3V 3VV 3V9 913 iVO 9V3 91V 913

98 08 9Z

"ID u | 3 ΙΒΛ 丄 na, Π | 3 sAi o" d dsv BIV sX3 na n | 3 B | VU | 0 ειε 0V3 0V3 3 丄 3 33 丄 313 9 9 3VV V33 3V3 V09 丄 3 丄 13 DVD V09 9V0

^ SI00 / 86dUT / XDJ Array

CCTTCTTGCA TCTTGGGTTC AGGCTTC 27 SEQ ID NO: 3

Array length: 25

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Other nucleic acid Synthetic DNA

Array

GAAGCCTGAA CCCAAGATGC AAGAAGG 27

Claims

The scope of the claims

1. A protein that is a ligand for CCR7, a human CC-type chemokine or a mutant thereof, or a fragment thereof, which is mainly constitutively expressed in secondary lymphoid tissues by Northern blot analysis.

2. A human CC-type chemokine having the amino acid sequence of amino acid residues 24 to 134 of SEQ ID NO: 1, or one or several amino acid residues substituted, deleted, inserted, or added to this sequence. A mutant of said human CC-type chemokine having a sequence containing at least one selected and having a function or activity substantially the same as the function or activity of said human CC-type chemokine, or a fragment thereof. The evening protein according to claim 1.

3. Select from substitution, deletion, insertion and addition of one or several amino acid residues to the sequence of human CC-type chemokine having amino acid residues 24 to 134 of SEQ ID NO: 1. The protein according to claim 1, which is a mutant of the human CC-type chemokine or a fragment thereof, which has a sequence containing at least one of the above-mentioned human CC-type chemokines and functions as an antagonist of the human CC-type chemokine.

4. A human CC-type chemokine having the amino acid sequence of amino acid residues 1 to 134 of SEQ ID NO: 1, or a substitution, deletion, insertion or addition of one or several amino acid residues to this sequence A variant of said human CC-type chemokine, or a fragment thereof, having a sequence or sequence comprising at least one of the above-mentioned human CC-type chemokines and having a function or activity substantially the same as that of said human CC-type chemokine. Even protein described in Item 1.

5. It is selected from substitution, deletion, insertion or addition of one or several amino acid residues to the sequence of human CC-type chemokine having the amino acid residues 1 to 134 of SEQ ID NO: 1. 2. The protein according to claim 1, which is a mutant of the human CC-type chemokine having a sequence containing at least one and functions as an antagonist of the human CC-type chemokine, or a fragment thereof.

6. A pharmaceutical composition containing the protein according to any one of claims 1 to 5.

7. An antibody against the protein according to any one of claims 1 to 5.

8. The antibody according to claim 7, wherein the antibody is a monoclonal antibody.

9. A hybridoma cell producing the monoclonal antibody according to claim 8.

10. A polynucleotide molecule encoding the protein according to any one of claims 1 to 5.

11. The polynucleotide molecule according to claim 10, comprising the full length or a part of the nucleotide sequence from A at position 128 to A at position 46 of SEQ ID NO: 1, or a base substitution or base addition thereof. Or a mutant due to an allelic mutation.

12. The polynucleotide molecule according to claim 10, comprising the full length or a part of the nucleotide sequence from A at position 59 to A at position 46 of SEQ ID NO: 1, or a base substitution, base addition or Mutant due to allelic mutation.

1 3. Polynucleotide or oligonucleotide molecule having a sequence complementary to the full length or a part of the nucleotide sequence from C at position 1 to Α at position 864 of column number 1, or base substitution, base addition or allele thereof A molecule that is a mutant due to a mutation and inhibits the activity or function of the protein according to claim 1.

14. A vector containing the polynucleotide molecule according to any one of claims 10 to 13.

15. The vector according to claim 14, which is an expression vector or a therapeutic vector.

1 6. Claim 1 5 transformants ₀ obtained by introducing into host cells an expression vector according to

17. The protein according to any one of claims 1 to 5, wherein the transformant according to claim 15 is cultured, and the produced protein is recovered from the culture. How to make.

18. A pharmaceutical composition comprising the polynucleotide molecule according to any one of claims 10 to 13 or a variant thereof.

19. A pharmaceutical composition comprising the therapeutic vector according to claim 15.

20. An agonist for the biological action induced by the binding of the protein according to any one of claims 1 to 5 to its specific receptor, inverted ago A method for screening a second or antagonist, comprising adding a sample presumed to contain the agonist, inverse agonist or antagonist to a binding reaction between the protein and its specific receptor CCR7. Measuring the inhibition of the binding, or reacting directly with the specific receptor CCR7 of the protein to determine the binding and / or reactivity to the receptor,

21. Biological action elicited by the binding of the protein according to any of claims 1 to 5 to its specific receptor CCR 7, which can be obtained by the screening method according to claim 20. Agonist, inverse agonist, or antagonist.