JP2002504823A - Human L105 protein and polynucleotide encoding the same - Google Patents

Abstract

  (57) [Summary] Disclosed is the chemokine, human L105.

Description

DETAILED DESCRIPTION OF THE INVENTION Human L105 Protein and Polynucleotide Encoding the Same The present invention relates to a 60 / XXXXXX filed on Jun. 11, 1997 (provisional application filed from 08/87704, which is not a provisional application). ) Is a continuation-in-part application, which is hereby incorporated by reference. FIELD OF THE INVENTION The present invention provides chemokines called human L105 proteins and polynucleotides encoding them. BACKGROUND OF THE INVENTION Chemokines are a subclass of cytokines that direct the migration or chemotaxis of specific cell populations toward or away from higher concentrations of chemokines. Many chemokines that cause migration of the major blood cell population have been identified. These factors are useful for migrating a cell population to a desired area or from an undesirable area. Therefore, it is desirable to identify new chemokines and the polynucleotides that encode them. Applicants' U.S. Patent No. 5,707,829 and International Patent Application WO 97/07198 disclose a novel murine chemokine identified as "L105". Murine L105 has been shown to have chemotactic / chemoattractant activity against several cell types, including thymic lymphocytes, renal glomerular stromal cells, and lymphocytes localized to lymph nodes. . Because of these beneficial activities, it would be desirable to identify a human homolog of murine L105. SUMMARY OF THE INVENTION In one embodiment of the invention, the invention provides a polynucleotide comprising: (a) a polynucleotide comprising the nucleotide sequence from nucleotide 80 to nucleotide 481 of SEQ ID NO: 1; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 149 to nucleotide 481 of SEQ ID NO: 1. (C) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO: 1 encoding a protein having chemokine activity; (d) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 (E) a polynucleotide encoding a protein comprising the amino acid sequence from amino acid 14 to amino acid 134 of SEQ ID NO: 2; (f) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having chemokine activity Nucleotides; (g) SEQ ID NO: (H) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 from nucleotide 85 to nucleotide 519; (i) a nucleotide comprising the nucleotide sequence of SEQ ID NO: 3 from nucleotide 124 to nucleotide 519 A polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO: 3 encoding a protein having chemokine activity; (k) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 4; (L) a polynucleotide encoding a protein comprising the amino acid sequence from amino acid 14 to amino acid 145 of SEQ ID NO: 4; (m) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 having chemokine activity (N) A polynucleotide which is an allelic variant of SEQ ID NO: 3; and (o) as in (a), (b), (d), (e), (h), (i), (k) or (l) above. A composition comprising an isolated polynucleotide selected from the group consisting of a polynucleotide capable of hybridizing to any one of the indicated polynucleotides. Preferably, such a polynucleotide comprises a polynucleotide comprising the nucleotide sequence from nucleotide 80 to nucleotide 481 of SEQ ID NO: 1; a polynucleotide comprising the nucleotide sequence from nucleotide 149 to nucleotide 481 of SEQ ID NO: 1; accession number ATCC 98429 The nucleotide sequence of the full-length protein coding sequence of clone huL1053 deposited as accession number; or the mature protein sequence of clone huL1053 deposited as accession number ATCC 98429. In another preferred embodiment, such a polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone huL1053 deposited under accession number ATCC 98429. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having chemokine activity (preferably, the fragment comprises 8 of SEQ ID NO: 2, more preferably A polynucleotide that encodes a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having chemokine activity (comprising 20 amino acids, most preferably 30 consecutive amino acids); Amino acid sequence up to amino acid 71). Preferably, such a polynucleotide comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 from nucleotide 85 to nucleotide 519; a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 from nucleotide 124 to nucleotide 519; accession number ATCC 98431 The nucleotide sequence of the full-length protein coding sequence of clone huL1057 deposited as No .; or the mature protein sequence of clone huL1057 deposited as accession number ATCC 98431. In another preferred embodiment, such a polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone huL1057 deposited under accession number ATCC 98431. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 having chemokine activity (preferably, the fragment comprises eight of SEQ ID NO: 4, more preferably A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 having chemokine activity (comprising 20 amino acids, most preferably containing 30 consecutive amino acids); Amino acid sequence up to amino acid 76). In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The present invention also provides host cells, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Further, a method for producing a protein, comprising: (a) growing a culture of a host cell transformed with such a polynucleotide composition in a suitable medium; and (b) purifying the protein from the culture. There is also provided a method comprising: Other embodiments provide an isolated gene corresponding to the cDNA sequence of SEQ ID NO: 1, and an isolated gene corresponding to the cDNA sequence of SEQ ID NO: 3. An organism having enhanced, reduced, or modified expression of a gene corresponding to a polynucleotide sequence disclosed herein is provided by the invention. Also disclosed are compositions comprising a protein having chemokine activity, in a preferred embodiment, the protein comprises: (a) the amino acid sequence of SEQ ID NO: 2; (b) the amino acid sequence from amino acid 14 to amino acid 134 of SEQ ID NO: 2 (C) a fragment of the amino acid sequence of SEQ ID NO: 2 having chemokine activity; (d) an amino acid sequence of SEQ ID NO: 4; (e) an amino acid sequence from amino acid 14 to amino acid 145 of SEQ ID NO: 4; A) a fragment of the amino acid sequence of SEQ ID NO: 4 having chemokine activity; and an amino acid sequence selected from the group consisting of: and substantially free of other mammalian proteins. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence from amino acid 14 to amino acid 134 of SEQ ID NO: 2. In a further preferred embodiment, the invention relates to a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having chemokine activity (preferably the fragment comprises 8, more preferably 20, and most preferably 20 A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having chemokine activity (including the amino acid sequence from amino acid 62 to amino acid 71 of SEQ ID NO: 2) having chemokine activity. provide. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 4, or the amino acid sequence from amino acid 124 to amino acid 134 of SEQ ID NO: 4, or the amino acid sequence of amino acid 14 to amino acid 145 of SEQ ID NO: 4. In a further preferred embodiment, the invention relates to a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 having chemokine activity (preferably said fragment comprises 8, more preferably 20 and most preferably 20 A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 having chemokine activity (the fragment includes the amino acid sequence from amino acid 67 to amino acid 76 of SEQ ID NO: 4) having chemokine activity. provide. Such compositions may further comprise a pharmaceutically acceptable carrier. Compositions comprising antibodies specifically reactive with such proteins are also provided by the present invention. Also provided is a method for preventing, treating or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier. DETAILED DESCRIPTION A human L105 sequence was found in the Genbank EST database by searching using the murine L105 sequence (polynucleotide is shown in SEQ ID NO: 5, protein is shown in SEQ ID NO: 6). The search revealed two possible related human ESTs, AA027314 (SEQ ID NO: 7) and W17274 (SEQ ID NO: 8), which were partially overlapping. To isolate the full length human L105 clone, the sequence (X = biotin) (SEQ ID NO: 9) was designed. The probe was used to screen an adult heart cDNA library. Screening revealed full-length clones and identified them as "huL1053" and "huL1057". The polynucleotide and amino acid sequence of huL1053 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The coding sequence of huL1053 contains nucleotides 80-481, which encodes a full-length protein of 134 amino acids. The mature protein starts at amino acid 14 (SEQ ID NO: 2). The polynucleotide and amino acid sequence of huL1057 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The coding sequence of huL1053 contains nucleotides 85-519, which encodes a full-length protein of 145 amino acids. The mature protein starts at amino acid 14 (SEQ ID NO: 4). A comparison of huL1053 and huL1057 reveals that huL1053 is such that huL1053 includes an insertion region. The insertion region is found at nucleotides 454-486 of SEQ ID NO: 3. The huL1053 and huL1057 cDNAs were deposited with the American Type Culture Collection (10801 United States Boulevard, Nanassas, Virginia 20110-2209 USA) on May 9, 1997 in accordance with the provisions of the Budapest Treaty, and have accession numbers ATCC 98429 and ATCC 98429, respectively. ATCC 98431. All restrictions on public use of the deposited material are set forth in 37 CFR. F. R. Except for the provisions of § 1.808 (b), this will be terminated by the grant of a patent, and the deposit period will be 37 C.E. F. R. § 1.806. Comparison of the human L105 sequence (SEQ ID NO: 2 and SEQ ID NO: 4) with the murine L105 sequence (SEQ ID NO: 6) confirms that these proteins are human homologs of the murine chemokine. As a result, human L105 protein shares the chemokine activity of murine L105. A fragment of the protein of the present invention that can exhibit biological activity is also included in the present invention. Protein fragments may be linear or, for example, HUSaragovi, et al., Bio / Technology 10,773-778 (1992) and RSM Dowell, et al., J. Amer. Chem. Soc. 114,9245-9253. (1992) (both references are deemed as described herein by reference) and may be cyclized using known methods. Such fragments may be fused to the carrier molecule along with the immunoglobulin for many purposes, including increasing the number of valencies at the protein binding site. For example, a protein fragment may be fused to the Fc portion of an immunoglobulin via a "linker". For a bivalent form of the protein, such a fusion can be made to the Fc portion of an IgG molecule. Such fusions may be obtained using other immunoglobulin isotypes. For example, a protein-IgM fusion will yield a ten-valent form of a protein of the invention. The invention also provides the full length and mature forms of the disclosed proteins. The full length form of such a protein has been identified in the sequence listing by translation of the nucleotide sequence of the disclosed clone. Mature forms of such proteins may be obtained by expressing the disclosed full-length polynucleotides (preferably those deposited with the ATCC) in appropriate mammalian cells or other host cells. The sequence of the mature form of the protein may be determined from the amino acid sequence of the full-length form. The present invention also provides a gene corresponding to the cDNA sequence disclosed herein. A “corresponding gene” is a region of the genome that is transcribed to produce mRNA from which cDNA is derived and whose flanking regions (coding sequences, 5 ′ and 3 ′) are required for regulated expression of such gene. 'Including untranslated regions) or spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. Corresponding genes can be isolated using the sequence information disclosed herein. Corresponding genes can be isolated using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence information to identify and / or amplify genes in a suitable genomic library or other source of genomic material. An "isolated gene" is a gene that has been separated from flanking coding sequences, if present, present in the genome of the organism from which the gene was isolated. Organisms having enhanced, reduced, or altered expression of a gene corresponding to the polynucleotide sequences disclosed herein are provided. Desirable changes in gene expression are achieved by using antisense polynucleotides or by using ribozymes that bind and / or cleave mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15). Lavarosky et al., 1997, Biochem. Mol. Med. 62 (1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1. -39; all of which are hereby incorporated by reference). A transgenic animal having a multicopy gene corresponding to a polynucleotide disclosed herein, preferably obtained by transforming cells with a genetic construct that is stably maintained in transformed cells and their progeny. A transgenic animal is provided. Also provided are transgenic animals having altered genetic control regions that increase or decrease gene expression levels or alter the temporal or spatial pattern of gene expression (see European Patent No. 0 649 464 B1; All of which are considered to be described herein by reference). In addition, an organism is provided wherein the gene corresponding to the polynucleotide sequence disclosed herein has been incompletely or completely inactivated, either by insertion of an external sequence or by deletion of all or part of the corresponding gene. By insertion, preferably by insertion after incorrect excision of the transposable element (Plasterk, 1992, Bioessays 14 (9): 629-633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90 (16): 7431-7435; Clark et al., 1994, Proc. Natl. Acad. Sci. USA 91 (2): 719-722; all of which are hereby incorporated by reference. ) Or by homologous recombination, preferably by homologous recombination detected by a positive / negative genetic selection method (Mansour et al., 1988, Nature 336: 348-352; US. Patent Nos. 5,464,764; 5,487,992; 5,627,059; 5,616,491; and 5,679,523; all of which are deemed to be described herein by reference), incomplete or complete gene inactivation can be performed. Those organisms with altered gene expression are preferably eukaryotes, more preferably mammals. Such organisms are useful for developing non-human models for studying diseases associated with the corresponding gene, and for developing assay systems for identifying molecules that interact with protein products from the corresponding gene. The proteins and protein fragments of the invention have an amino acid sequence that is at least 25% (more preferably at least 50%, most preferably at least 75%) of the amino acid sequence of the disclosed protein and at least 60% of the disclosed protein. Of proteins having a sequence identity (more preferably at least 75% identity, most preferably at least 90% or 95% identity). Sequence identity is determined by comparing the amino acid sequences of the proteins when the sequences are aligned to maximize overlap and identity while minimizing sequence gaps. It has at least 75% sequence identity (more preferably at least 85% identity, most preferably at least 95% identity) to any of the disclosed protein segments, preferably 8 or more ( Proteins or protein fragments containing segments comprising more preferably 20 or more, most preferably 30 or more) contiguous amino acids are also encompassed by the present invention. Species homologs of the disclosed polynucleotides and proteins are also provided by the invention. As used herein, the term "species homolog" refers to a protein or polynucleotide for a species that is different from the source of the protein or polynucleotide, but that has significant sequence similarity as determined by those skilled in the art. Preferably, the polynucleotide species homolog has at least 60% (more preferably at least 75%, most preferably at least 90%) sequence identity to the particular polynucleotide, and the protein species homolog is Have at least 30% (more preferably at least 45%, most preferably at least 60%) sequence identity to any of the proteins. Here, sequence identity is determined by comparing the nucleotide sequence of a polynucleotide or the amino acid sequence of a protein juxtaposed such that overlap and identity are maximized and sequence gaps are minimized. Species homologs may be isolated and identified by generating appropriate probes or primers from the sequences provided herein, and then screening for an appropriate nucleic acid source from the desired species. Preferably, the species homolog is isolated from a mammalian species. Most preferably, the species homologs are, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirus, Micros, busin musculus, Rattus norvegicus, Cricetulus, griseus, Felis catus, Mustela vison, Canis famil iaris, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa and Eq uus caballus, isolated from specific mammals, The genetic map has been created to identify the association of gene sequence order between the genomic organization of a gene in one species and the genomic organization of related genes in another species ( O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al., 1993, Nature Genetics 3: 103-112; Johansson et al., 1995, Genomics 25: 682-690. ; Lyons et al., 1997, Nature Genetics 15: 47-56; O'B rien et al., 1997, Trends in Genetics 13 (10): 393-399; Carver and Stubbs, 1997, Genome Research 7: 1123-1137, all of which are deemed to be incorporated herein by reference. )). The invention also encompasses allelic variants of the disclosed polynucleotides, i.e., naturally occurring alternative forms of isolated polynucleotides that encode proteins identical, homologous, or related to the protein encoded by the disclosed polynucleotides. I do. Preferably, the allelic variant has at least 60% (more preferably at least 75%, most preferably at least 90%) identity to the particular polynucleotide, wherein sequence identity is an overlap and identity Is determined by comparing the nucleotide sequences of the aligned polynucleotides such that sex is maximized and sequence gaps are minimized. Allelic variants may be isolated and identified by generating appropriate probes or primers from the sequences described herein and screening for an appropriate source of nucleic acid from an individual of the appropriate species. The present invention also encompasses polynucleotides having sequences complementary to the sequences of the polynucleotides disclosed herein. The invention also encompasses polynucleotides that are capable of hybridizing to the polynucleotides described herein under reduced stringency conditions, more preferably under stringent conditions, and most preferably under very stringent conditions. I do. The following table shows examples of strictness conditions. The very strict conditions are, for example, at least as strict as the conditions A to F, and the strict conditions are, for example, at least as strict as the conditions GL, and the reduced conditions are, for example, For example, it is at least as strict as the conditions M to R. ^‡ : The hybrid length is the expected length for the hybridizing region (s) of the hybridizing polynucleotide. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be the length of the hybridizing polynucleotide. When polynucleotides of known sequence hybridize, the length of the hybrid can be determined by juxtaposing the polynucleotide sequences and identifying the region (s) of optimal sequence complementarity. ^† : SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ and 1.25 mM EDTA, pH 7.4) in hybridization and wash buffer with SSC (1 × SSC is 0.15 M NaCl and 15 mM citrate) Sodium). After completion of the hybridization, washing is performed for 15 minutes. * T _B -T _R : The hybridization temperature for hybrids that are expected to be less than 50 base pairs in length should be 5-10 ° C. below the melting temperature (T _m ) of the hybrid. T _m is determined by the following equation. For hybrids less than 18 base pairs in length, T _m (° C.) = 2 (number of A + T bases) +4 (number of G + C bases). For hybrids 18 to 49 base pairs in length, T _m (° C.) = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) − (600 / N), where N is the hybrid And the sodium ion concentration in the hybridization buffer ([Na ⁺ ] for 0.1X SSC = 0.165M). Further examples of stringency conditions for polynucleotide hybridization are described in Sambrook, J., EF. Fritsch, and T.W. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, FM. Ausubel et al., Eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, which are deemed to be incorporated herein by reference. Preferably, each such hybridizing polynucleotide has a length of at least 25% (more preferably at least 50%, most preferably at least 75%) of the polynucleotide of the invention to be hybridized, Has at least 60% sequence identity (more preferably, at least 75% identity, most preferably, at least 90% or 95% identity) to the polynucleotide of the invention to be hybridized. Sequence identity is determined by comparing the amino acid sequences of the proteins when the sequences are aligned to maximize overlap and identity while minimizing sequence gaps. The isolated polynucleotide encoding the protein of the present invention can be prepared as described in Kaufman et al., Nucleic Acids Res. The protein may be produced recombinantly by operably linking it to an expression control sequence such as the pMT2 or pED expression vector disclosed in 19, 4485-4490 (1991). Many suitable expression control sequences are known in the art. Many suitable expression control sequences are known in the art. General methods for recombinant protein expression are also known and are disclosed in Kaufman, Methods in Enzymology 185, 537-566 (1990) is a typical example. "Operably linked," as defined herein, means that the isolated polynucleotide of the present invention and the expression control sequence are placed in a vector or cell and transformed (transfected) with the ligated polynucleotide / expression control sequence. It is meant to be expressed by a host cell. Many types of cells can act as suitable host cells for expressing the proteins of the present invention. Mammalian cells include, for example, monkey COS cells, Chinese hamster ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primates Includes cell lines, normal diploid cells, cell lines obtained from in vitro culture of primary tissues, primary explants, HeLa cells, mouse cells, BHK, HL-60, U937HaK or Jurkat cells. Alternatively, the protein can be produced in lower eukaryotic cells such as yeast or prokaryotic cells such as bacteria. Potentially suitable yeast strains include Saccharomyces ce revisiae, Schizosaccharomyces pombe, Kluyveromyces strain, Candida, or yeast strains capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or bacterial strains capable of expressing heterologous proteins. If the protein is produced in yeast or bacteria, it may be necessary to modify the protein produced therein, for example, by phosphorylation or glycosylation at appropriate sites to obtain a functional protein. Such covalent linkages may be made using known chemical or enzymatic methods. The isolated polynucleotides of the present invention may be operably linked to appropriate control sequences in one or more insect expression vectors and the protein obtained by using an insect expression system. Materials and methods for baculovirus / insect cell expression systems, for example In vitrogen, San Diego, California, are commercially available in kit form from USA (MaxBac ^R kit), such methods are well known in the art Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (11987), which is hereby incorporated by reference. As used herein, insect cells capable of expressing a polynucleotide of the present invention have been "transformed." The protein of the present invention may be produced by culturing the transformed host cell under culture conditions suitable for expressing the recombinant protein. The resulting expressed protein may then be purified from such culture (ie, medium or cell extract) using known purification processes, such as gel filtration and ion exchange chromatography. Purification of the protein may also be accomplished by affinity columns containing an agent that will bind to the protein; one or more column steps with an affinity column such as Concanavalin A-agarose, Heparin-Toyopearl ^R or Cibacrom blue 3GA Sepharose ^R ; One or more steps using hydrophobic interaction chromatography with a resin such as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the protein of the invention may be expressed in an easily purified form. For example, it may be expressed as a fusion protein such as a fusion protein with maltose binding protein (MBP), glutathione-S-tonsferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from NowEnglan BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively. The protein can also be tagged with an epitope and then purified by using a specific antibody directed against such epitope. One such epitope ("flag") is commercially available from Kodak (New Haeven, CT). Finally, the protein is further purified using one or more reverse phase high quality liquid chromatography (RP-PLC) steps using a hydrophobic RP-HPLC medium, such as silica gel with pendant methyl or other aliphatic groups. be able to. Some or all of the above purification steps may be used in various combinations to obtain a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in the present invention as "isolated protein". The protein of the present invention is expressed as a product of a transgenic animal, for example, as a component of milk of a transgenic cow, goat, pig, or sheep characterized by somatic cells or germ cells containing a nucleotide sequence encoding the protein. You may. The protein may be produced by known conventional chemical synthesis. Methods for constructing the protein of the present invention by synthetic means are known to those skilled in the art. Synthetically constructed protein sequences may have common biological properties, including protein activity, by sharing primary, secondary or tertiary structure and / or conformational properties. Thus, they may be used as biological activities or immunological substitutes for naturally occurring purified proteins in immunological processes for screening therapeutic compounds and generating antibodies. The proteins described herein are characterized by an amino acid sequence similar to the amino acid sequence of the purified protein, but may be modified therein, either naturally or by derivatization. For example, modifications of the peptide or DNA sequence can be made by those skilled in the art using known methods. Modifications of interest in the protein sequence include changes, substitutions, exchanges, insertions or deletions of selected amino acid residues in the coding sequence. For example, one or more cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Methods for such alterations, substitutions, exchanges, insertions or deletions are well known to those skilled in the art (see, for example, US Pat. No. 4,158,584). Preferably, such changes, substitutions, exchanges, insertions or deletions retain the desired activity of the protein. Other fragments and derivatives of the protein sequence that are expected to retain protein activity in whole or in part, and thus may be useful in screening or other immunological methods, can also be made by those skilled in the art from the disclosure herein. . Such modifications are believed to be encompassed by the present invention. The protein of the present invention may be used for chemotaxis to mammalian cells including, for example, lymphocytes (eg, thymic lymphocytes, monocytes, neutrophils and T cells), mast cells, eosinophils and / or endothelial cells. It may have chemokinetic activity (eg, acts as a chemokine). The murine L105 protein has been shown to have chemotaxis for thymic lymphocytes, renal glomerular stromal cells and lymphocytes localized to lymph nodes. The human L105 protein shares these activities while having other chemotactic and chemokinetic activities. The chemotactic and chemokinetic properties can be used to recruit or attract a desired cell population to a desired site of action. Chemotaxis or chemokinetic proteins are particularly advantageous in treating wounds and other trauma to tissues as well as treating local infections. For example, attracting lymphocytes, monocytes or neutrophils to a tumor or site of infection may result in an improved immune response against the tumor or infectious agent. The chemokinetic properties of the proteins of the invention can also be used to block chemotaxis, for example, to block inflammation (eg, by including large amounts of protein in the blood to induce "chemotaxis paralysis"). (Ie, no gradient exists for the cell, and therefore no movement occurs)). A protein or peptide has chemotaxis if the protein or peptide can directly or indirectly stimulate the orientation or movement of the overlying cell population. Preferably, the protein or peptide is capable of directly stimulating the directed movement of a cell. Whether a particular protein has chemotactic activity on a cell population can be readily determined by using such a protein or peptide in a known assay for cell chemotaxis. (1) chemoattractant or chemotaxis in a chemoattractant or chemotaxis assay (preferably an assay in which the mature protein of the corresponding species has activity), or (2) a factor-dependent cell proliferation assay (preferably, Indicates a biological activity in an assay when the mature protein of the corresponding species has an activity, or (3) an activity to induce lymphokine production or effector function in an immune cell function assay. The protein has "chemokine activity". Examples of effector functions include, but are not limited to, tumor killing activity, granule release, adhesion molecule expression, and the like. Activity may be monitored using assays known in the art. Chemoattraction or chemotaxis can also be measured in vivo by injecting the protein at a specific site and performing histological tests on cell types that migrate to the injection site. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for chemotactic activity (assays to identify proteins that induce or interfere with chemotaxis) induce migration across cell membranes. Assays that measure the ability of a protein to induce adhesion of one cell population to another cell population as well as the ability of the protein to adhere to another cell population. Assays suitable for migration and attachment are described in Current Protocols in Immunology, Ed by JE. Coligan, AM. Kruisbeek, DH. Margulies, EM. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95: 1370-1376, 1995; Lind et al. APMIS 103: 140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152: 5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994. Including, but not limited to, the proteins of the present invention, which may be from any source, including recombinant and non-recombinant methods, (But not limited to these) may be used in pharmaceutical compositions by mixing with a pharmaceutically acceptable carrier, such as a diluent, filler, salts, buffers, The term "pharmaceutically acceptable" may include stabilizers, solubilizing agents, and other materials well known in the art. A non-toxic substance that does not interfere with the efficacy of the biological activity, the characteristics of the carrier will depend on the route of administration, and the pharmaceutical compositions of the present invention may be M-CSF, GM-CSF, TNF, IL-1, IL- 2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14 , IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and cytokines such as erythropoietin, lymphokines, or other hematopoietic factors. The pharmaceutical composition may further comprise other agents that enhance protein activity or supplement its activity or utility in therapy. It may be included to exert a synergistic effect with the protein of the present invention or to minimize side effects, and conversely, specific cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors, or anti-inflammatory agents May contain a protein of the invention to minimize the side effects of cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors, or anti-inflammatory agents. A protein of the invention may be active in multimers (eg, heterodimers or homodimers) or in complexes with itself or other proteins. Consequently, the pharmaceutical composition of the present invention may contain such a multimeric or complexed form of the protein of the present invention. The pharmaceutical composition of the present invention may be in the form of a complex of the protein of the present invention and a protein or peptide antigen. Protein and / or peptide antigens will deliver a stimulatory signal to B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) after presentation of the antigen by MHC proteins. MHC and structurally related proteins, including those encoded by the host cell's class I and class II MHC genes, will serve to present peptide antigens to T lymphocytes. The antigen component can be supplied as a purified MHC-peptide complex alone or with a costimulatory molecule that can directly signal T cells. Alternatively, antibodies that can bind to surface immunoglobulins on B cells and TCRs and other molecules on T cells can be mixed with the pharmaceutical compositions of the present invention. The pharmaceutical composition of the present invention may be in the form of a liposome, in which the protein of the present invention further comprises another pharmaceutically acceptable carrier, an amphipathic agent such as a lipid (in the form of an aggregate such as micelles in an aqueous solution). , Present as an insoluble monolayer, liquid crystal or lamellar layer). Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatide, lysolecithin, phospholipids, saponins, bile acids, and the like. The preparation of such liposome formulations is within the level of ordinary skill in the art and is described, for example, in US Pat. Nos. 4,235,871, 4501728, 4837028, and 4737323, all of which are hereby incorporated by reference. It's like it was done. As used herein, the term "therapeutically effective amount" refers to the amount of each active ingredient of a pharmaceutical composition or method that is sufficient to show significant patient benefit, e.g., amelioration of symptoms of such symptoms, healing, increased rate of healing. Means the total amount. When used for an individual active ingredient administered alone, the term refers only to that ingredient. When used in a combination of active ingredients, it refers to the total amount of active ingredients that produces a therapeutic effect regardless of sequential or simultaneous administration. In practicing the therapeutic methods of the invention, a therapeutically effective amount of a protein of the invention is administered to a mammal having the condition to be treated. According to the method of the invention, the protein of the invention may be administered alone or with other therapeutic agents, such as cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, the proteins of the invention may be administered simultaneously or sequentially with cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors. If administered sequentially, the attending physician will determine the appropriate order of administration for combinations of the proteins of the present invention with cytokines, lymphokines, other hematopoietic, thrombolytic or antithrombotic factors. The administration of the protein of the present invention in the pharmaceutical composition of the present invention or used in the practice of the present invention can be performed by various conventional methods, for example, oral feeding, inhalation, or intradermal, subcutaneous, or intravenous injection. Intravenous injection into a patient is preferred. When a therapeutically effective amount of a protein of the invention is administered orally, the protein of the invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may further contain a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain from about 5 to 95% of a protein of the invention, preferably from about 25 to 90%. When administered in liquid form, water, petroleum, oils of animal or vegetable origin, such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. Liquid form pharmaceutical compositions may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains about 0.5 to 90% by weight of a protein of the present invention, preferably about 1 to 50%. When a therapeutically effective amount of a protein of the present invention is administered by intravenous, intradermal or subcutaneous injection, the protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution having appropriate pH, isotonicity, and stability is within the skill of the art. Preferred pharmaceutical compositions for intravenous, intradermal, or subcutaneous injection include, in addition to the protein of the present invention, sodium chloride for injection, Ringer's solution, dextrose for injection, dextrose and sodium chloride for injection, and Ringer's solution containing lactic acid for injection, Or it should contain other carriers known in the art. The pharmaceutical composition of the present invention may contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. The amount of the protein of the present invention in the pharmaceutical composition of the present invention depends on the nature and severity of the condition to be treated and the nature of the treatment that the patient has already received. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each patient. First, the attending physician administers a low dose of the protein of the invention and observes the patient's response. Higher doses of the protein of the invention may be administered until the optimal therapeutic effect is obtained, with no further increase in the dose at the time the optimal therapeutic effect is obtained. Various pharmaceutical compositions for carrying out the method of the present invention comprise about 0.01 μg to about 100 mg of the protein of the present invention per kg of body weight (preferably, about 0.1 μg to about 10 mg per kg of body weight, more preferably It should contain from 0.1 μg to about 1 mg of the protein of the invention.The duration of intravenous treatment with the compositions of the invention will vary depending on the severity of the disease to be treated and the condition and response of each patient. It is believed that each application period of the protein of the present invention will be in the range of 12 to 24 hours for continuous intravenous administration, and ultimately the attending physician will administer treatment by intravenous administration using the pharmaceutical composition of the present invention. Animals are immunized with the protein of the present invention to obtain polyclonal and monoclonal antibodies that specifically react with the protein of the present invention. The whole protein or a fragment thereof may be used as an immunogen to obtain such antibodies.The peptide immunogen may further include a cysteine residue at the carboxy terminus, such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art and include, for example, RPM Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); JLKrstenansky, et.al., FEBS Lett. .211, 10 (1987) Monoclonal antibodies that bind to the protein of the present invention can be useful diagnostic agents for immunodetection of the protein of the present invention Neutralizing antibodies that bind to the protein of the present invention Can be useful as a therapeutic agent for a symptom related to the protein of the present invention, and further useful in the treatment of certain tumors in the treatment of some forms of cancer in which abnormal expression of the protein of the present invention is involved. In the case of cancer cells or white blood cells, neutralizing monoclonal antibodies against the protein of the present invention may be useful for detecting and preventing metastatic spread of cancer cells mediated by the protein of the present invention. Such polynucleotides can be introduced into cells in vivo or ex vivo and expressed in a mammalian subject. Polynucleotides of the invention can be expressed by other known methods for introducing nucleic acids into cells or organisms. Nucleotides may be administered (including, but not limited to, in a viral vector, or in the form of naked DNA, etc.). It may produce a desired effect on a cell or a desired activity in such a cell. The treated cells can then be introduced in vivo for therapeutic purposes. The patents and publications cited herein are considered to be entirely incorporated herein by reference.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/04 C07K 14/47 43/00 111 14/475 C07K 14/47 14/52 14/475 16 / 24 14/52 C12P 21/02 C 16/24 C12N 15/00 ZNAA C12N 5/10 5/00 B C12P 21/02 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM ), AL, M, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU , ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW

Claims (1)

[Claims]   1. (A) nucleotides from nucleotide 80 to nucleotide 481 of SEQ ID NO: 1 A polynucleotide comprising a nucleotide sequence;   (B) the nucleotide sequence from nucleotide 149 to nucleotide 481 of SEQ ID NO: 1 A polynucleotide comprising a reotide sequence;   (C) the nucleotide of SEQ ID NO: 1, which encodes a protein having chemokine activity A polynucleotide comprising a fragment of the sequence;   (D) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 ;   (E) the amino acid sequence from amino acid 14 to amino acid 134 of SEQ ID NO: 2 A polynucleotide encoding a protein comprising;   (F) a fragment of the amino acid sequence of SEQ ID NO: 2 having chemokine activity A polynucleotide encoding a protein comprising;   (G) a polynucleotide that is an allelic variant of SEQ ID NO: 1;   (H) nucleotides from nucleotide 85 to nucleotide 519 of SEQ ID NO: 3 A polynucleotide comprising an otide sequence;   (I) the nucleotide sequence from nucleotide 124 to nucleotide 519 of SEQ ID NO: 3 A polynucleotide comprising a reotide sequence;   (J) nucleotide of SEQ ID NO: 3, which encodes a protein having chemokine activity A polynucleotide comprising a fragment of the sequence;   (K) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 4 ;   (L) the amino acid sequence from amino acid 14 to amino acid 145 of SEQ ID NO: 4 A polynucleotide encoding a protein comprising;   (M) a fragment of the amino acid sequence of SEQ ID NO: 4 having chemokine activity A polynucleotide encoding a protein comprising;   (N) a polynucleotide that is an allelic variant of SEQ ID NO: 3; and   (O) (a), (b), (d), (e), (h), (i), (k) or Can hybridize with any one of the polynucleotides shown in (l) Polynucleotide A composition comprising an isolated polynucleotide selected from the group consisting of:   2. 2. The polynucleotide of claim 1 operably linked to an expression control sequence.   3. A host cell transformed with the polynucleotide of claim 2.   4. 4. The host cell of claim 3, which is a mammalian cell.   5. (A) growing the culture of the host cell of claim 3 in a suitable medium;   (B) Purifying the protein from the culture And a method for producing a protein.   6. A protein produced by the method of claim 5.   7. (A) the amino acid sequence of SEQ ID NO: 2;   (B) an amino acid sequence from amino acid 14 to amino acid 134 of SEQ ID NO: 2;   (C) a fragment of the amino acid sequence of SEQ ID NO: 2 having chemokine activity;   (D) the amino acid sequence of SEQ ID NO: 4;   (E) an amino acid sequence from amino acid 14 to amino acid 145 of SEQ ID NO: 4; and   (F) a fragment of the amino acid sequence of SEQ ID NO: 4 having chemokine activity A composition comprising a protein comprising an amino acid sequence selected from the group consisting of: A composition wherein the white is substantially free of other mammalian proteins.   8. The composition of claim 7, further comprising a pharmaceutically acceptable carrier.   9. A composition comprising an antibody that specifically reacts with the protein of claim 7.   10. Administering to a mammalian subject a therapeutically effective amount of the composition of claim 8. , Methods for the prevention, treatment or amelioration of a medical condition.   11. The polynucleotide is a nucleotide from nucleotide 80 of SEQ ID NO: 1; 2. The composition of claim 1, which comprises the nucleotide sequence up to 481.   12. The polynucleotide is a nucleoside from nucleotide 149 of SEQ ID NO: 1 2. The composition of claim 1, which comprises the nucleotide sequence up to Tide 481.   13. The polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 2 2. The composition of claim 1, wherein said composition comprises a polynucleotide.   14． The polynucleotide comprises amino acid 14 to amino acid 134 of SEQ ID NO: 2. Includes polynucleotides encoding proteins containing amino acid sequences up to The composition of claim 1.   15. The polynucleotide is a nucleotide from nucleotide 85 of SEQ ID NO: 3 The composition of claim 1, comprising a nucleotide sequence up to 519.   16. The polynucleotide is a nucleoside from nucleotide 124 of SEQ ID NO: 3 The composition of claim 1 which comprises a nucleotide sequence up to 519.   17． The polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 4 2. The composition of claim 1, wherein said composition comprises a polynucleotide.   18. The polynucleotide comprises amino acids 14 to 145 of SEQ ID NO: 4. Includes polynucleotides encoding proteins containing amino acid sequences up to The composition of claim 1.   19. The composition of claim 7, wherein said protein comprises the amino acid sequence of SEQ ID NO: 2. object.   20. The protein is an amino acid having amino acids 14 to 134 of SEQ ID NO: 2 The composition of claim 7, which comprises an acid sequence.   21. The composition of claim 7, wherein said protein comprises the amino acid sequence of SEQ ID NO: 4. object.   22. The protein is an amino acid consisting of amino acids 14 to 145 of SEQ ID NO: 4. The composition of claim 7, which comprises an acid sequence.   23. An isolated gene corresponding to SEQ ID NO: 1.   24. An isolated gene corresponding to SEQ ID NO: 3.