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WO2006009309A1 - Facteur de proteine de liaison a la galectine-9 - Google Patents

Facteur de proteine de liaison a la galectine-9 Download PDF

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Publication number
WO2006009309A1
WO2006009309A1 PCT/JP2005/013792 JP2005013792W WO2006009309A1 WO 2006009309 A1 WO2006009309 A1 WO 2006009309A1 JP 2005013792 W JP2005013792 W JP 2005013792W WO 2006009309 A1 WO2006009309 A1 WO 2006009309A1
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Prior art keywords
galectin
protein
group
polypeptide
amino acid
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PCT/JP2005/013792
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English (en)
Japanese (ja)
Inventor
Mitsuomi Hirashima
Nozomu Nishi
Akira Yamauchi
Yumiko Kashio
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Galpharma Co., Ltd.
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Publication of WO2006009309A1 publication Critical patent/WO2006009309A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the control of the biological activity induced by the interaction such as binding to human galectin 9 and the like.
  • the present invention also relates to a galectin-9-binding protein factor that interacts with human galectin-9.
  • the present invention relates to a technique for controlling the interaction and / or binding between human lectin 9 and galectin 9-binding protein, for example, a technique for inhibiting or promoting, and a technique for using the same (biochemistry, clinical examination, medical care and pharmaceutical application technique). Including). Background art
  • Galectin 9 one of the tandem repeat galectins, was first identified in humans as an autoantigen in patients with Hodgkin's disease (Non-patent Documents 1 and 2), and reciprocal interaction between immune cells. It was imagined that it might play an important role in the action.
  • Mouse galectin 9 was obtained by 5'-RACE PCR using a digitized primer that was designed based on a sequence that was found to be highly conserved in the carbohydrate recognition domain of galectins. It has been cloned from a mouse kidney cDNA library (Non-patent Document 3).
  • Antigen-stimulated human T cells have been found to produce ecalectin, an eosinophil migration factor in vivo and in vitro, and the ecalectin has been previously Although it was structurally different from eosinophil migration factors, it had a binding affinity for a sugar blocker with ⁇ -galactoside structure, which may belong to Galectin family.
  • ekalecti is a variant of galectin 9
  • galectin 9 and ekalectin are the same substance (non-patented Reference 4) has been confirmed.
  • L type galectin 9 (galectin-9 medium isoform or medium type galectin-9: Gal-9L), M type galectin-9 (galectin-9 medium isoform or medium type galectin-9: Gal- 9M) and S type galectin-9 (galectin-9 short isoform or short type galectin-9: Gal-9S) have been reported.
  • Each galectin 9 consists of two sugar chain recognition units 1 (carbohydrate recognition domain: CRD) and a link peptide region connecting them, and L-type galectin 9 has the longest link peptide region.
  • N-terminal domain (NCRD) and C-terminal domain (CCRD) are linked by the linked peptide region
  • Kuching 9 has the shortest link peptide region
  • M-type galectin 9 has a link peptide region having an intermediate length between the two. Is widely known to be found in in vivo tissues and cells. In addition, there has been evidence that genetic polymorphisms exist among the galectin-9 genes cloned from human cells and tissues.
  • Wild-type galectin 9 consists of two sugar recognition sites (CRDs) and a linked region that connects them.
  • Recombinant galectin 9 produced using E. coli as a host has a direct effect on tumor cells (cell-cell adhesion and apoptosis). It has been suggested that cancer-mediated metastasis suppression and regression are induced by the activity through the immune system. It has also been shown that galectin 9 does not act on non-activated lymphocytes and induces apoptosis of activated T cells, particularly CD 4 positive T cells that cause hyperimmune reactions. Furthermore, it has been shown that rheumatism has a strong apoptosis-inducing ability for synovial cells involved in joint deformation.
  • Non-Patent Document 1 Sahin, U. et al., Proc. Natl. Acad. Sci. USA,. 92, 11810-11813 (1995)
  • Non-Patent Document 2 Tureci, 0. et al., J. Biol. Chem., 272 (10), 6416-6422 (1997)
  • Non-Patent Document 3 Wada, J. et al., J. Biol. Chem., 272 (9), 6078-6086 (1997),
  • Non-Patent Document 4 Matsumoto,. Et al., J. Biol. Chem., 273 (27), 16976-16984 (1998) Disclosure of the Invention
  • galectin 9 By utilizing these multifaceted actions of galectin 9, it is expected to treat cancer, refractory autoimmune diseases (including rheumatism), allergic diseases, etc. Elucidate biological activity In addition, elucidation of galectin 9-binding proteins such as galectin 9 receptor, elucidation of their interaction with galectin 9, and development of galectin 9-related technologies including drug development are required. Yes. The present inventors have found that galectin 9 shows cytotoxic activity against tumor cells, but does not show cytotoxic activity against normal cells. Induce a more favorable result by exhibiting a characteristic action on metastatic tumor cells (for example, metastasis-inhibiting activity), or the ability to induce apoptosis against tumor cells.
  • metastatic tumor cells for example, metastasis-inhibiting activity
  • a galectin-9 binding agent characterized by comprising as an active ingredient a substance selected from the group consisting of (1) HSP70, (2) ATP1B3, and (3) HSP90;
  • galectin 9 Interaction between galectin 9 and galectin 9 binding protein, including Or a method of screening compounds or salts thereof that promote or inhibit binding;
  • a substance selected from the group (a) and / or one polypeptide selected from the group (b) is labeled, and by detecting or measuring the label, the galectin 9 and the galectin 9-binding protein
  • the screening method according to any one of the above [2] to [4], wherein the formation of a bond and Z or interaction is detected;
  • One polypeptide selected from group (b) bound to a substance selected from group (a) is a primary antibody against the polypeptide of group (b) or the polypeptide of (b)
  • a primary antibody against another peptide fused with the above By detecting or measuring using a primary antibody against another peptide fused with the above, the interaction between these galectin 9 and galectin 9-binding protein and / or the formation of binding is detected [ 2 ]
  • [8] (a) (1) HSP70, (2) ATP1B3, and (3) a protein or activity containing an amino acid sequence identical or substantially identical to the amino acid sequence of a protein selected from the group consisting of HSP90 Type polypeptide, partial peptide or salt thereof, and (b) amino acid sequence identical or substantially identical to the amino acid sequence of modified galectin 9 modified from galectin 9 or its linked peptide Promoting interaction and Z or binding between galectin 9 and galectin 9-binding protein comprising one polypeptide selected from the group consisting of a polypeptide or a partial peptide thereof or a salt thereof Or a screening kit for inhibiting compounds or salts thereof;
  • a pharmaceutical composition comprising the compound or salt thereof according to [11] above;
  • composition according to the above [12] which is selected from the group consisting of: [14] A bioactivity regulator for galactin 9 characterized by comprising an antibody that recognizes a protein selected from the group consisting of (1) HSP70, (2) ATP1B3, and (3) HSP90;
  • a galectin characterized in that an antibody recognizing a protein selected from the group consisting of (1) HSP70, (2) ATP1B3 and (3) HSP90 is contacted with a cell expressing a protein or polypeptide 9 biological activity control methods;
  • a modified version of galectin 9 or its linked peptide comprising a polypeptide containing the same or substantially the same amino acid sequence as that of galectin 9 or a partial peptide thereof or a salt thereof. It is characterized in that a peptide selected from the group is used as an active ingredient a)
  • HSP70 (2) ATP1B3 and (3) a protein or polypeptide bioactivity control agent containing an amino acid sequence identical or substantially identical to the amino acid sequence of a protein selected from the group consisting of HSP90;
  • HSP70 (1) HSP70, (2) ATP1B3, and (3) a protein or polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence of the protein selected from the group consisting of HSP90
  • a polypeptide containing an amino acid sequence that is identical or substantially identical to the amino acid sequence of galectin 9 or its modified galectin 9 modified in its linked peptide is expressed in the expressing cell.
  • a protein selected from the group consisting of (1) HSP70, (2) ATP1B3 and (3) HSP90 characterized by contacting with a polypeptide selected from the group consisting of peptides or partial peptides or salts thereof
  • Galectin 9-binding protein selected from the group consisting of (1) HSP70, (2) ATP1B3, and (3) HSP90;
  • HSP70 (1) HSP70, (2) ATP1B3 and (3) a protein or active form containing an amino acid sequence identical or substantially identical to the amino acid sequence of a protein selected from the group consisting of HSP90
  • a host cell characterized by: and
  • [21] Provided is a substance that regulates the binding ability of a galectin 9-binding site of a protein selected from the group consisting of (1) HSP70, (2) ATP1B3, and (3) HSP90.
  • a protein selected from the group consisting of (1) HSP70, (2) ATP1B3, and (3) HSP90.
  • Human lectin 9 exhibits cytotoxic activity against tumor cells but induces apoptosis, but does not exhibit normal cell cytotoxic activity and further induces apoptosis, but galectin 9-binding protein is narrowed down. Therefore, it is possible to screen a substance that controls the interaction between galectin 9 and its galectin 9-binding protein and / or the formation of the bond.
  • An antibody against a sugar chain on a cutin 9-binding protein, as well as substances / compounds that control or inhibit or promote the interaction and / or the formation of bonds between galectin-9 and its galectin-9-binding protein Can be used to control the biological activity of galectin-9 9 or galectin 9 binding protein shown using its analogue It is possible to control the biological activity. Therefore, it is possible to develop a technology that uses an antitumor agent (anticancer agent) that does not act on normal cells, shows damaging activity on tumor cells, and induces apoptosis.
  • anticancer agent anticancer agent
  • the present invention makes use of the interaction between galectin 9 and galectin 9-binding protein and the formation of Z or bond, and the biological activity exhibited by human galectin 9, ie, cytotoxicity against malignant tumor cells.
  • Activity apoptosis-inducing activity against malignant tumor cells, anti-tumor activity against malignant tumor cells (anti-cancer activity), apoptosis-inducing activity of activated T cells, especially apoptosis-inducing activity of CD4-positive T cells, immunosuppressive activity
  • the present invention relates to technology for controlling and utilizing anti-inflammatory and anti-allergic effects.
  • the present invention utilizes the relationship between galectin 9 and galectin 9 binding protein to Biological activity of binding protein, ie, apoptosis-inducing activity against malignant tumor cells, apoptosis-inducing activity of activated T cells, immunosuppressive activity
  • anti-cancer activities It relates to technology that controls or uses anti-inflammatory, anti-allergic, and anti-tumor activities (anti-cancer activities).
  • anti-cancer agents anti-cancer agents
  • anti-allergic agents anti-allergic agents
  • immunosuppressive agents immunosuppressive agents
  • autoimmune disease agents anti-inflammatory agents
  • corticosteroid substitutes using galectin 9 and related technologies To do.
  • galectin-9-binding molecules involved in exerting the biological functions of galectin-9 particularly apoptosis-related galectin-9-binding proteins.
  • the search target is not particularly limited, and various biological materials can be used. Typically, cell lines in which cell death is induced by the addition of galectin 9 such as human T cell-derived strain MOLT-4 are used as starting materials. This can be done and is preferred in some cases.
  • the interaction molecular search method various methods known in the art can be used alone or in any combination. For example, in order to identify a protein that interacts with galectin 9 from a scavenger protein, it can be applied by selecting from the following methods, for example. By knowing the interacting proteins, it is possible to know the novel functions of target proteins as well as their regulatory mechanisms, such as those mediated by galectin-9.
  • the immunoprecipitation method adds an antibody specific to the target protein to a variety of protein solutions as samples, and immunoprecipitates the protein interacting with the target protein as an immune complex.
  • This method is effective for examining the presence of proteins that interact with the target protein by any method that is isolated as a product and identified by SDS-PAGE or Western blotting.
  • a specific antibody against the target protein or a specific antibody against a known protein a resin that traps an antibody such as protein A or protein G sepharose can be used, and preferably a protein solution labeled with RI or the like. It is preferable that can be prepared.
  • kits can also be used, such as Affi-Prep 10, Affi-Gel Hz (BIO-RAD), NHS Sepharose HP (Pharmacia).
  • the protein to be measured can be prepared and purified as a fusion protein.
  • the antibody against Tag can be used effectively, and it is convenient to use Escherichia coli, yeast, or mammalian cells. It is also possible to use a cell-free translation system for reticulocytes. It can also be used for co-purification when Si-combined proteins are simply purified by adding an excess of the desired fusion protein to the protein solution.
  • the Western or Far-Western method is a modification of the Western method, which uses a protein such as a protein of interest labeled in place of an antibody or a probe of a known protein to check the binding to the protein transferred to the membrane. By doing this, it is what 'detection' measures. Using the target protein as a probe, it is possible to know the distribution, localization, and molecular weight of the protein to be bound. This method can be applied to screening of expression libraries and used for cDNA cloning.
  • Ligand-blotting is a method for analyzing proteins that bind to a ligand, which is a type of far-western plot. Detection with a streptavidin-conjugated antibody, detection with a specific antibody and a labeled secondary antibody using an unlabeled ligand. It is also possible to use non-RI labels. For example, Soutar, AK & Wade, DP, Protein function. "A practical approach
  • the intermolecular cross-linking method uses a chemical cross-linking agent to cross-link proteins between proteins and separates them by SDS-PAGE and detects them by combining Western plotting and immunoprecipitation methods. This is an effective method for analysis and analysis of interacting proteins or nearby proteins (or domains) and analysis of subunit structures such as receptors.
  • chemical cross-linking agents see PIERCE website
  • any protein in the cDNA pool is expressed in cells, and the target protein or tagged protein is used as a probe to correlate with cells that have been confirmed to interact with each other.
  • a specific cDNA is cloned from the cDNA group used, and analyzed through cloning of receptors, ligands and the like.
  • an expression system using prokaryotic cells such as E. coli
  • an expression system using cultured cells such as mammalian cells
  • an expression system using Xenopus egg cells etc.
  • the expression system known in can be used.
  • ELISA method or a FACS (Fluorescence Activated Cell Sorting) method
  • mRNA is synthesized from specific cDNAs in vitro and microinjected into Xenopus laevis eggs, and the protein expressed by the interaction with the target protein and the resulting cellular response are utilized. It is also possible to select and clone.
  • the two-hyprid system utilizes the phenomenon that the transcriptional activator constructed so that the reporter gene is expressed when the domain structure of the target protein or its protein interacts with an independent protein domain. This is a crawling system that searches for interacting proteins.
  • Commercially available pre-made libraries can be used in this method, but are not limited to this. For example, MATCHMAKER GAL4 cDNA LIBRARY, MATCHMAKER LexA cDNA LIBRARY
  • the phage display method is to repeat the operation of collecting phages that bind to the target protein using a tie library having a random amino acid sequence of 5 to 7 residues on the surface of the phage, and proliferating the phage. This is a technique for searching for a highly specific amino acid sequence.
  • the surface plasmon resonance method is typically a technique developed for the purpose of monitoring the interaction between biomolecules on the sensor chip in real time using the BIAC0RE TM system.
  • BIAC0RE TM when the light is applied to the sensor chip (gold thin film) on the side where biomolecules are not immobilized so as to be totally reflected, a part of the reflected light is observed to have a reduced reflected light intensity. (SPR signal is generated).
  • the angle at which this dark part of light appears depends on the mass on the sensor chip.
  • Sensor chip CM5 Carboxymethyldextrin, which has a lan surface
  • Sensor chip SA Streptavidin immobilized in advance
  • Sensor chip NTA NTA immobilized, chelated with nickel And those capable of immobilizing poly-His fusion proteins.
  • This method is, for example, Setsuko Hashimoto, Bunseki 5, Analysis of biomolecular interactions using surface plasmon resonance phenomenon, P362-368, 1997, Toru Natsume, Biomanual UP series, Protein molecular interaction experiment method, PP211 -230, Yodosha, 1996.
  • Fluorescence polarization uses the phenomenon that when a molecule with a fluorescent label excited by plane-polarized light undergoes a movement such as rotation during the excited state, the emitted fluorescence becomes a plane different from the excitation light. However, since the purity of a molecule is affected by its size, it will maintain its polarization when it becomes a polymer due to complex formation, etc., and it will be depolarized when it is low and highly mobile. So we measured this degree of polarization You can know the interaction.
  • Various fluorescent labels can be used. For example, FS, FITC, FXS, etc. can be used. The measurement can be performed using, for example, FBEAC0N TM.
  • searching for and identifying an apoptosis-related galectin-9-binding protein can be suitably performed using a utility column.
  • Synthetic peptides, fusion proteins, antibodies, etc. can be used as affinity column ligands.
  • affinity column ligands As a typical example of isolation, we will explain the acquisition of galectin-9-binding protein using MOLT-4 cell lysate as a starting material. The MOLT-4 cell lysate is passed through a galectin 9 CT (C-terminal region) column. Collect the galectin 9-binding molecule.
  • the collected cell-derived protein is electrophoresed on a gel, separated according to the molecular weight, and the gel fragment obtained by the separation is analyzed for the amino acid sequence inside the protein to identify the galectin-9-binding protein.
  • Candidate galectin 9-binding proteins can be determined by the above-described method for identifying proteins that interact with galectin 9 from the candidate proteins.
  • galectin 9-binding protein it is a protein derived from human, which is the following: 4F2 heavy chain antigen (177216); ATPase, Na + / K + transporting, alpha 1 polypeptide (21361181); heat shock 90kDa protein 1, beta (20149594); heat shock 90kDa protein 1, alpha; heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) (16507237); heat shock 70kDa protein 8 isoform 2
  • Heat shock 70kDa protein 9B precursor 24234688; Sodium / potassium-transporting ATPase beta-3 chain (1703470 or P54709).
  • the numbers in parentheses () indicate the protein information and the DNA that encodes it by entering the numbers on the NCBI homepage (http: ⁇ www.ncbi.nlm.nih.gov/). Can be obtained. Therefore, the amino acid sequence information and DNA etc.
  • the techniques and methods described in this specification taking galectin 9 as an example can be applied to the protein and the coding gene sequence.
  • Such applicable technologies and methods naturally include antibody production and utilization techniques, including the production and utilization of monoclonal antibodies.
  • the galectin 9-binding protein is a human-derived protein that is: (1) HSP70, (2) ATP1B3 and (3) HSP90 [(1) HSP70 abbreviated genes and proteins Protein name: heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa), source: human, gene name: HSPA5 (or GRP78 / B1P), Protein Accession No. NP 005338, mRNA Accession No.
  • ATP1B3 protein name: Sodium / potassium-transporting ATPase beta-3 chain (Sodium / potassium-dependent ATPase beta-3 subunit), source: human, gene name: ATP1B3, Protein Accession No. NP— 001670, mRNA Accession No. ⁇ — 001679 (3)
  • HSP90 protein name: heat shock 90kDa protein 1, beta, source-'human, gene name: HSPCB (Or GRP94 (HSP90ER homologue)), Protein Ac cession No. NP— 031381, mRNA Accession No.
  • Hsp70 / Hsc70, GRP78 / Bip, mtHsp70, etc. are known.
  • Hsp90 protein families Hsp90 (o ;,
  • the presence of polynucleotide in the biological sample of a galectin-9-binding molecule in a subject's biological sample is tested, and a subject with a higher abundance of polynucleotide compared to those in healthy subjects is galectin-9 sensitive or physiological activity induced by galectin-9 It is possible to provide a diagnostic method characterized in that it is determined that the person can be expected.
  • the abundance of galectin 9-binding molecule protein in the biological sample of the subject was tested, and the abundance of galectin 9-binding molecule protein was higher than that in healthy subjects, and the subject was induced by galectin 9 sensitivity or galectin 9 It is possible to provide a diagnostic method characterized in that it is determined that the person has a desired physiological activity.
  • an oligonucleotide that hybridizes under stringent conditions with a galectin 9-binding molecule polynucleotide or an oligonucleotide that hybridizes under polynucleotide conditions or a galectin 9-binding molecule polynucleotide, a galectin 9-binding molecule polynucleotide
  • a primer set for PCR amplification, an antibody that recognizes a galectin 9-binding molecule, and an antibody that binds to an epitope different from the antibody are also provided.
  • Galectin 9 sensitive or Method for measuring expected degree of physiological activity induced by lectin 9 and measurement kit at least the following elements: (a) plate or membrane on which an antibody recognizing galectin 9-binding molecule is immobilized; and (b) said ( Method or method for measuring or detecting the expectation of galectin 9 sensitivity or physiological activity induced by galectin 9 characterized in that it comprises an antibody that binds to an epitope different from the antibody in a) and is labeled.
  • Kit at least the following steps: (a) a step of contacting a biological sample of a subject with a carrier on which the antibody is immobilized; (b) a step of washing a solid phase carrier that has been brought into contact with the biological sample; A step of contacting the solid phase carrier contacted with the sample with the labeled antibody; (d) a step of measuring the solid phase labeled or free label; (e) in step (d) Using the measured labeling amount as an index of galectin-9-binding molecular weight and comparing it with the result of the control biological sample; and (f) galectin-9-binding molecular protein abundance significantly higher than that of the control biological sample.
  • the method for measuring the degree of galectin-9-related physiological reaction as described above comprising the step of using it as an index indicating the degree of expectation of sensitivity or the physiological activity induced by galectin-9, and at least the following steps: : (A) The process of preparing RNA from the biological sample of the subject; (b) The process of electrophoretic separation of the RNA prepared in step (a); (c) The RNA isolated in process (b) is galectin 9 A step of hybridizing with a labeled nucleotide probe under stringent conditions with the binding molecule polynucleotide; (d) galectin-9 binding to the amount of label hybridized in step (e) And (e) a significantly higher galectin-9-binding molecule polynucleotide expression level as compared to the control biological sample as an indicator of molecular polynucleotide expression level, and galectin-9 sensitive or galectin 9 A method for measuring the degree of galectin-9-related physiological reaction as described above, characterized in that it comprises
  • a step of preparing a strand cDNA (c) a step of PCR amplification using a primer set for PCR amplification of a galactin 9-binding molecule polynucleotide in a cage shape from the cDNA prepared in step (b); (d) (C) electrophoretic separation of the PCR product obtained in step (c); (e) a labeled nucleotide that hybridizes the galectin 9-binding molecule polynucleotide with a polynucleotide under stringent conditions.
  • step (F) a step of hybridizing with the probe; (f) using the amount of label hybridized in step (e) as an indicator of the expression level of the galectin 9-binding molecule polynucleotide, and comparing it with the result of the control biological sample; and (g) normal Galectin 9-binding molecule polynucleotide expression level is significantly higher than that of biological samples, and the degree of expectation of bioactivity induced by galectin 9 or galectin 9
  • a method for measuring the degree of galectin 9-related physiological reaction as described above characterized in that it comprises a step used as an indicator of indication, at least the following steps: (a) subjecting a biological sample of a subject to tissue immobilization treatment (B) a step of using the tissue-immobilized specimen prepared in step (a) as a section; (c) subjecting the sectioned tissue to immunohistochemical staining with an antibody that recognizes a galectin 9-binding molecule; (d) Comparing the degree of immunohisto
  • the method for measuring the degree of galectin-9-related physiological reaction as described above comprising: testing the abundance of a 9-binding molecule polynucleotide and comparing the abundance of the polynucleotide with those of a healthy person The amount of galectin 9-binding molecule protein abundance or galectin 9-binding molecule polynucleotide expression level is quantitatively measured, and the degree of galectin 9-related physiological reaction as described above is measured.
  • a reagent containing the antibody according to any one of the above, which is used in the method for measuring a degree of a physiological reaction related to galectin 9 according to any one of the above, galectin 9 Using an antibody that recognizes the binding molecule, the amount of galectin-9 binding molecule protein in the sample or the expression of galectin-9 binding molecule polynucleotide A method for measuring or diagnosing the degree of expectation of galectin 9 sensitivity or the degree of physiological activity induced by galectin 9 or the degree of galectin 9-related physiological reaction, an antibody that recognizes galectin 9-binding molecule Galectin 9-binding molecule protein amount or galectin 9-binding molecule polynucleotide expression level in a sample sample is measured, and the degree of expectation of galectin 9 sensitivity or physiological activity induced by galectin 9 or galectin 9-related physiology Reagents for measuring or diagnosing the extent of reaction can also be provided.
  • the amount of polynucleotide present in the subject is, for example, 10% or more, preferably 30% or more, more preferably 70% or more, and most preferably compared to that of the control. This is the case when it is 100% or more.
  • a method for producing a microarray As a method for producing a microarray, a method of directly synthesizing oligonucleotides on the surface of a solid-phase carrier (on-chip method) and a method of immobilizing previously prepared oligonucleotides or polynucleotides on the surface of the solid-phase carrier are known. .
  • the microarray used in the present invention can be produced by any of these methods.
  • the on-chip method is a combination of the use of protective groups that are selectively removed by light irradiation, and photolithography and solid-phase synthesis technologies used in semiconductor manufacturing to create a predetermined area of minute Malingera. (Masking technology: for example, Fodor, SPA Science 251: 767, 1991) and the like.
  • an oligonucleotide or polynucleotide prepared in advance is immobilized on the surface of a solid phase carrier
  • an oligonucleotide or polynucleotide into which a functional group is introduced is synthesized and the oligonucleotide or polynucleotide is synthesized on the surface of the solid phase carrier that has been surface-treated.
  • Leotide is spotted and covalently bound (eg Lamture, JB et al. Nucl. Acids Res. 22: 2121-2125, 1994; Guo, Z. et al. Nucl. Acids Res. 22: 5456-5465, 1994 ).
  • Oligonucleotides or polynucleotides are generally covalently bound to a surface-treated solid support via a spacer or a crosslinker.
  • a method is also known in which polyacrylamide gel fragments are aligned on a glass surface and a synthetic oligonucleotide is covalently bound thereto (Yershov, G. et al. Proc. Natl. Acad. Sci. USA 94: 4913). , 1996).
  • a microelectrode array was prepared on a silica microarray, and an agarose permeation layer containing streptavidin was provided on the electrode as a reaction site, and the biotinylated oligonucleotide was charged by positively charging this site.
  • cMA is synthesized using mRNA isolated from a subject's cells as a saddle and amplified by PCR. At that time, the labeled dNTP is incorporated into the labeled cDNA. This labeled cDNA is brought into contact with the macroarray, and the hybridized cDNA is detected by the microarray capture probe (oligonucleotide or polynucleotide). Hybridization can be performed by dispensing a labeled cDNA aqueous solution onto a microarray by dispensing into 96-well or 384-well plastic plates. The amount of spotting can be about 1 to 100 nl.
  • Hybridization is performed at room temperature to 70 ° C for 6 to 20 hours. It is preferable to carry out in an enclosure. After hybridization, wash with a mixed solution of detergent and buffer to remove unreacted labeled cDNA.
  • a mixed solution of detergent and buffer As the surfactant, sodium dodecyl sulfate (SDS) is preferably used.
  • SDS sodium dodecyl sulfate
  • As the buffer solution a citrate buffer solution, a phosphate buffer solution, a borate buffer solution, a Tris buffer solution, a Good buffer solution, and the like can be used, and a citrate buffer solution is preferably used.
  • Galectin 9-binding molecule polynucleotide can also be measured by measuring the expression level of the polynucleotide (specifically, mRNA) using a primer set.
  • the RT-PCT method can be suitably used. It is also preferable to perform quantitative measurements using the competitive PCT method.
  • This primer set can be designed based on a known base sequence and can be prepared through synthesis and purification steps.
  • competitive primers can be designed and synthesized based on the base sequence of the galectin-9 binding molecule gene. For example, the following points can be taken into consideration when designing a primer.
  • the size (number of bases) of the primer is 15- ⁇ ) bases, preferably 15-30 bases, considering that it will satisfy the specific annealing with the vertical DNA. However, when LA (long accurate) PCR is performed, at least 30 bases are effective.
  • the GC content should be about 50% in order to ensure stable binding to the truncated DNA, so that GC-rich or AT-rich is not unevenly distributed in the primer. Since the annealing temperature depends on Tra (melting temperature), in order to obtain highly specific PCR products, primers that are close to each other with a Tm value of S55-65 ° C are selected. It should also be noted that the final concentration of primers used in PCR should be adjusted to about 0.1 to 1 ⁇ .
  • the galectin 9-binding molecule expression gene measurement system apoptosis detection system, anti-tumor detection system, and the reagents, methods, processes, and analysis programs used for it are all based on Toyoaki's technology and it. Included in the system used.
  • the in situ hybridization may include, for example, non-RI in situ hybridization, and may include, for example, a direct method and an indirect method.
  • the direct method uses, for example, a molecule (reporter) that is directly bound to a nucleic acid probe, and the indirect method amplifies a signal using, for example, an antibody against the reporter molecule.
  • Functional groups for example, primary aliphatic amino groups, SH groups, etc.
  • the nucleic acid probe label typically includes digoxigenin (DIG), biotin, fluorescein, etc., but can be used by appropriately selecting from the labels described in the following antibodies. Multiple labeling is also possible.
  • labeled antibodies can also be used.
  • the nucleic acid probe labeling method is appropriately selected from methods known in the art. For example, random prime method, nick 'translation method, DNA amplification by PCR, labeling Z-tiling method, in vitro transcription method, etc. can be mentioned.
  • the treated sample it can be used by appropriately selecting from methods known in the field. For example, dark field microscope, phase contrast microscope, reflection contrast microscope, fluorescent microscope, digital imaging microscope, electron microscope You can also use flow cytometry.
  • a galectin-9-binding molecule protein or a related polypeptide or oligopeptide and a galectin-9-binding molecule-expressing gene can be used as various markers, antitumor markers, carcinogenesis or cancer deterioration markers.
  • It can be used as a measurement reagent set or system, and it can be expected to have excellent functions and effects as a prognosis.
  • the specific criterion is that the abundance of galectin 9-binding molecular protein in the subject is 10% or more compared to that of the control, preferably
  • Galectin 9-binding molecule protein is present in an amount that specifically binds to antibodies that recognize galectin 9-binding molecules or galectin 9-binding molecules It can be measured using an antibody (anti-galectin 9-binding molecule antibody).
  • the antibody may be a polyclonal antibody or a monoclonal antibody, and includes all molecules capable of binding to the galectin 9-binding molecule protein epitope, and Fab, F (ab ′) 2 , Fv fragments, and the like.
  • measurement systems include immunostaining for proteins, protein measurement systems such as immunoelectron microscopy, and expressed gene measurement systems such as in situ hybridization. For tissue extracts, EIA, RIA, FIA, LIA, Western Protein measurement systems such as blotting, Northern blotting, Southern blotting, Dot blot, RNase protection assay, RT-PCR (reverse transcriptasethyl-binding RNA.
  • transcription polymerase chain reaction s Real-Time PCR, combined PCR and other gene expression measurement systems, and protein measurement systems such as EIA, RIA, FIA, LIA and Western blotting for blood, body and fluid Available.
  • an anti-galectin 9-binding molecule antibody is used as an immobilized antibody, and labeled and unlabeled antigens (antigens include galectin 9-binding molecules or fragment peptides thereof).
  • solid-phase anti-galectin 9-binding molecule antibodies and labeled anti-galectin 9-binding molecule antibodies can be used in non-competitive methods, such as sandwich methods, and anti-galectin 9-binding molecule antibodies can be used directly. It is also possible to carry out the labeling or immobilization of the antibody against the anti-galectin 9-binding molecule antibody without immobilization.
  • a combination of a non-enzyme-labeled primary antibody uses a polymer, an enzyme, and a primary antibody (Envisions Formula 1 ); Enhanced polymer one-step staining (EPOS ) A combination of a secondary antibody and a biotin-conjugated avidin complex, ABC
  • treptavidin-biotin complex treptavidin-biotin complex
  • LSAB labeleled streptavidin- biotin
  • CSA catalyzed signal amplification
  • HSP70 (2) because it has a ATP1B3 and (3) active to bind to galectin-9 those selected from the group consisting of HSP90, are useful as gal 9 binding agent and by interacting with g Al9 Since biological activity is induced, it is also useful as a reagent that utilizes this phenomenon.
  • “gene recombination technology” can be used to isolate and sequence a predetermined nucleic acid, produce a recombinant, or obtain a predetermined peptide. Examples of genetic recombination techniques (including recombinant DNA techniques) that can be used herein include those known in the art, such as J. Sambrook, EF Fritsch & T. Maniatis, Molecular Cloning ".
  • homology means each amino acid residue constituting the chain between two chains in a polypeptide sequence (or amino acid sequence) or a polynucleotide sequence (or base sequence). This means the amount (number) of things that can be determined to be the same in the matching relationship between each other or each base, and the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. To do.
  • Homology can be easily calculated. Many methods are known for measuring homology between two polynucleotide or polypeptide sequences, and the term “homology” (also called “identity”) is well known to those skilled in the art.
  • homology also called “identity”
  • homology also called “identity”
  • a preferred method for measuring homology is one designed to obtain the largest fit between the two sequences tested.
  • An example of such a method is one that is assembled as a computer program.
  • Preferred computer programming methods for measuring the homology between two sequences include the GCG program package (Devereux, J.
  • polypeptide may refer to any polypeptide as described below. The basic structure of a polypeptide is well known and is described in numerous references and other publications in the art. In view of this, the term “polypeptide” as used herein refers to any peptide containing two or more amino acids that are linked together by peptide bonds or modified peptide bonds. Or any protein.
  • polypeptide J as used herein is generally referred to in the art as, for example, peptides, oligopeptides or short oligomers, also referred to as peptide oligomers, and proteins.
  • Polypeptides are often commonly referred to as natural amino acids (naturally occurring amino acids: or gene-encoded amino acids), both of which usually mean long chains of known ones
  • Polypeptides may also contain natural amino acids such as many amino acid residues, including terminal amino acid residues, which are translated and processed (or modified) after translation. Not only by the process, but also by the chemical modification techniques well known to those skilled in the art, It will be appreciated that be modified (modified).
  • galectin 9 is typically a natural galectin.
  • Natural type galectin 9 is currently a long type (L type) galectin 9, medium type (M type) galectin 9 and short type.
  • (S-type) Galectin 9 has been reported, but L-type galectin 9 has an N-terminal domain and a C-terminal domain linked by the putative linked peptide region of SEQ ID NO: 4 disclosed in W0 02/37114 A1, M-type galectin 9 has a N-terminal domain and a C-terminal domain linked by the putative link peptide region of SEQ ID NO: 5 of TO 02/37114 Al, and S-type galectin 9 has the sequence of W0 02/37114 A1 It is thought that the N-terminal domain and the C-terminal domain are linked by the putative link peptide region of No. 6. In M-type galactin 9, the W0 is greater than the link peptide region of L-type galectin 9.
  • 02/37114 A1 is different from L-type galectin 9 in that the amino acid residue of SEQ ID NO: 7 is deleted, and in S-type galectin 9, M-type galectin It differs from M-type galectin 9 in that the amino acid residue of SEQ ID NO: 8 of TO 02/37114 A1 is deleted from the linked peptide region of 9, that is, L-type galectin in S-type galectin 9 9 Different from L-type galectin 9 in that the amino acid residue of SEQ ID NO: 9 of W0 02/37114 A1 is deleted from the putative linked peptide region.
  • galectin 9 includes the above-mentioned galectin 9, M-type galectin 9 and S-type galectin 9, other naturally occurring mutants of these galectin 9 families, and further artificial mutations (ie, It may mean that one or more amino acid residues have been deleted, added, modified, inserted, etc., or that include a partial domain or partial peptide fragment thereof.
  • the representative galectin-9 protein of the present invention includes any one of the amino acid sequences of SEQ ID NOs: 1 to 3 (SEQ ID N0: 1 to 3) of W0 02/37114 A1 or substantially equivalent thereto.
  • a polypeptide having an amino acid sequence for example, having at least 5-311, 5-323 or 5-355 contiguous amino acid residues of the amino acid sequence of SEQ ID N0: 1, 2 or 3 And having substantially equivalent biological activity such as equivalent antigenicity, etc., or having these characteristics and any of SEQ ID N0: 1, 2 or 3 in the sequence listing At least 50% homology with any one of these domains, or at least 60% homology, or at least 70% homology, or at least more than 80% homology, or at least 90% Higher homology or less Least 95% homology, or the like having at least 98% homology.
  • the human lectin 9 polypeptide of the present invention includes a sequence of amino acid residues comprising all or part of the amino acid sequence of SEQ ID N0: 1-3 in the sequence listing of W0 02/37114 A1, or the SEQ ID N0: 5 or more, preferably 10 or more, preferably 20 or more, more preferably 30 or more, more preferably 30 or more, more preferably 40 or more, of amino acid sequences of l, 2 and 3 Also preferably 50 or more, more preferably 60 or more, more preferably 70 or more, more preferably 80 or more, still more preferably 90 or more, still more preferably 100 or more, and preferably 110 or more. It has what it has.
  • the human galectin 9-related polypeptide of the present invention may have a part or all of an amino acid sequence selected from the group consisting of SEQ ID N0: 1, 2, and 3 (Met corresponding to the start codon). May be missing). Anything having such a sequence may be included.
  • galectin-9 protein of the present invention galectin-9 disclosed in Japanese Patent Application No. 2004-94401 (filing date: March 29, 2004) or substantially the same activity as that of galectin-9 protein is disclosed.
  • a protein (galectin 9 variant) or a salt thereof in which the link peptide of the protein possessed or a region close thereto is modified is included.
  • the modified galectin 9 is, for example, a protein or a salt thereof in which a linked peptide of a protein having substantially the same activity as that of galectin 9 or a modification thereof, or a salt thereof, a galectin 9 or a substantially equivalent thereof.
  • It consists of an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of the link peptide of the protein having the above activity or in the vicinity thereof, compared with galectin 9 A protein or a salt thereof that has been modified such that its degradation susceptibility to link peptide is modified, a protein having substantially the same activity as galectin 9, and an amino acid sequence of galectin 9 And at least 70% homology, or at least 75% homology, or at least 80% homology, or low A protein or salt thereof having at least 85% homology, or at least 90% homology, or at least 95 ° / 0 homology, or (1) a sugar chain recognition region on the N-terminal side of galectin 9 ( NCRD) or a polypeptide having substantially equivalent activity and (2) a carbohydrate recognition region (CCRD) on the C-terminal side of galectin 9 or a polypeptide having substantially equivalent activity.
  • NCRD N-terminal side of galectin 9
  • CCRD carbohydrate
  • the variant of galectin 9 is, for example, (1) an amino acid sequence shown in SEQ ID NO: 2 shown in the specification of Japanese Patent Application No. 2004-94401 as NCRD of galectin 9 or one in the sequence More amino acids deleted, substituted or added, or at least 70% homology with the amino acid sequence shown in SEQ ID NO: 2 shown in the specification of Japanese Patent Application No.
  • the link region (3) includes at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 9 shown in the specification of Japanese Patent Application No.
  • the link region (3) includes the amino acid sequence shown in SEQ ID NO: 9 shown in the specification of Japanese Patent Application No. 2004-94401, the sequence of ⁇ , RIP, or any two amino acids. And the like substituted with. ,
  • Galectin 9 variant polypeptide includes wild-type galectin 9 native sequence variants, derivatives, analogs (analogs), fragments, chimeric bodies, and variants thereof.
  • the polypeptide is encoded by a recombinantly produced polynucleotide sequence that is intended to be expressed in a host cell and designed to code for a specific galectin-9 variant polypeptide.
  • galectin 9 variant means a polynucleotide encoding a galectin 9 variant (galectin 9 variant polynucleotide) or a molecule derived from a galectin 9 variant polypeptide sequence, such as Galactin 9 variant A polynucleotide or polypeptide variant, variant, analog, chimera, fragment or the like may be included.
  • Nucleic acids encoding the galectin 9 are single-stranded DNA, double-stranded DNA, RNA, and DNA: RNA hive. It may be any of nucleic acid such as lid, synthetic DNA, human genomic DNA, human genomic DNA library, human tissue.cell-derived cDNA, or synthetic DNA.
  • the nucleotide sequence of the nucleic acid encoding galectin 9 can be modified (for example, added, removed, substituted, etc.), and such modified ones can also be included. It may also be a naturally occurring variant.
  • the nucleic acid may encode an L-type, M-type or S-type galectin-9 peptide, a galectin-9 variant or a part thereof, and preferred examples include DNA.
  • the above-mentioned “same base sequence” means, for example, SEQ ID NO: 1 in the sequence listing disclosed in W0 02/37114 A1 under stringent conditions or SEQ ID NO: 1 shown in the specification of Japanese Patent Application No. 2004-94401. 5 or more of the nucleotide sequence encoding the amino acid sequence, preferably 10 or more nucleotide sequences, more preferably 15 or more nucleotide sequences, and even more preferably 20 or more nucleotide sequences. Examples thereof include those that hybridize with a base sequence and encode an amino acid sequence that is substantially equivalent to the galectin 9 or a galectin 9 variant, or a phase capture thereof.
  • the nucleic acid encoding galectin 9 is typically SEQ ID 0: 1 to 3 in the sequence listing disclosed in TO 02/37114 A1 and SEQ ID N0: l shown in the specification of Japanese Patent Application No. 2004-94401. Containing a nucleotide sequence encoding a peptide represented by any one of the above and a part of the continuous amino acid sequence (including those encoding only each characteristic domain), and a coding sequence having a start codon (A codon encoding Met) and a stop codon, and an amino acid sequence having at least 50% homology with the protein encoded by the base sequence and disclosed in SEQ ID NO: W02 / 37114 A1 ID N0: 1 to 3 or SEQ ID N0: disclosed in the specification of Japanese Patent Application No.
  • the primers used in the PCR method are primers complementary to the nucleotide sequence to be amplified in the vertical type.
  • a primer adjacent to the nucleotide sequence is preferably used
  • the primer on the 5 'end side is selected so that it contains at least the initiation codon or can be amplified including the initiation codon, and It is preferable to select a primer that contains at least a stop codon or can be amplified including the stop codon as a primer on the 3 'end. More preferably, an oligonucleotide consisting of 10 or more bases, more preferably an oligonucleotide consisting of 18 to 25 bases.
  • the PCR reaction can be performed by a method known in the art, or a method or modification method substantially similar thereto, for example, R. Saiki, et al.,
  • PCR Protocols a guide to methods and applications, Academic Press, New York (1990)); MJ cPherson, P. Quirke and GR Taylor (Ed.), PCR: a practical approach, IRL Press, Oxford (1991); MA Frohman et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (1988), etc., or can be modified or modified according to a modified method.
  • the PCR method can be performed using a commercially available kit suitable for the PCR method, and can also be performed according to a protocol clarified by the kit manufacturer or the kit vendor.
  • a PCR reaction is carried out, for example, by using a 10 ⁇ reaction buffer containing a cage (for example, DNA synthesized by transforming mRNA into a cage; 1st strand DNA) and a primer designed based on the gene.
  • a cage for example, DNA synthesized by transforming mRNA into a cage; 1st strand DNA
  • a primer designed based on the gene for example, a primer designed based on the gene.
  • dTPs mixed with doxynucleoside triphosphates dATP, dGTP, dCTP, dTTP
  • Taq DNA polymerase and deionized distilled water (included with Taq DNA polymerase).
  • the mixture is subjected to 25-60 cycles under typical PCR cycle conditions using an automated thermal cycler such as the GeneAmp TM PCR System 2700, GeneAmp TM PCR System 9700 Series, Applied Biosystems Japan Ltd. (ABI).
  • an automated thermal cycler such as the GeneAmp TM PCR System 2700, GeneAmp TM PCR System 9700 Series, Applied Biosystems Japan Ltd. (ABI).
  • the number of cycles for amplification can be appropriately set according to the purpose.
  • PCR cycle conditions include, for example, denaturation 90 to 95 ° C 5 to 100 seconds, annealing 40 to 60 ° C 5 to 150 seconds, extension 65 to 75 ° C 30 to 300 seconds, preferably 94 ° denaturation C 15 seconds, annealing 58 ° C 15 seconds, extension 72 ° C 45 seconds cycle, but the reaction temperature and time of annealing can be selected appropriately by experiment, and the reaction of denaturation reaction and extension reaction The time can also be selected appropriately depending on the expected PCR product chain length. Generally, the annealing reaction temperature is preferably changed according to the Tm value of the hybrid of the primer and the vertical DNA.
  • the extension time is usually about 1 minute per lOOObp growth, but a shorter time can be selected in some cases.
  • oligonucleotide J is a relatively short single-stranded or double-stranded polynucleotide, preferably a polydioxynucleotide, Angew. Chem. Int. Ed. Engl. Vol. 28, p. 716-734 (1989) It can be chemically synthesized by a known method such as a phosphophotoester method, a phosphodiester method, a phosphite method, a phosphoramidite method, or a phosphonate method. Usually, it is known that synthesis can be carried out conveniently on a modified solid support. For example, it can be carried out using an automated synthesizer, which is commercially available.
  • the oligonucleotide may contain one or more modified bases, for example, a non-naturally occurring base such as inosine, or a tritylated base, In some cases, it may contain a marked base.
  • a hybridization technique can be used. The hybridization can be performed by the method described in the literature disclosing the “gene recombination technology” or a method or modification method substantially similar thereto. For example, in high predation, a sample containing nucleic acid such as DNA is transferred to a carrier including a membrane such as a nylon filter, and then subjected to transformation treatment, immobilization treatment, washing treatment, etc. as necessary. This is carried out by reacting a substance transcribed on a carrier (for example, a membrane) with a labeled probe DNA fragment modified as necessary in a hybridization buffer.
  • a carrier for example, a membrane
  • the hybridization treatment is usually performed at about 35 to about 80 ° C., more preferably about 50 to about 65 ° C., for about 15 minutes to about 36 hours, more preferably about 1 to about 24 hours.
  • the optimum conditions can be selected and performed.
  • the high pre-diization process is performed at about 55 ° C for about 18 hours.
  • the hybridization buffer can be selected from those commonly used in the field, and for example, Rapid hybridization buffer (Amersham) can be used.
  • Examples of the modification treatment of the transferred carrier include a method of using an alkali-denaturing solution, and it is preferable to treat with a neutralizing solution or a buffer solution after the treatment.
  • the carrier eg, membrane
  • the carrier is usually immobilized at about 40 to about 100 ° (more preferably about 70 to about 90 ° C. for about 15 minutes). It is carried out by baking for about 24 hours, more preferably for about 1 to about 4 hours, but it can be carried out by appropriately selecting preferable conditions. For example, immobilization can be performed by baking a carrier such as a filter at about 80 ° C for about 2 hours.
  • a washing treatment of the transferred carrier (eg, membrane) a washing solution commonly used in the art, for example, 50 mM Tris_HCl buffer containing NaCl, ImM EDTA and 0.1% sodium dodecyl sulfate (SDS), pH 8. This can be done by washing with 0 or the like.
  • the carrier including a membrane such as a nylon filter can be selected from those commonly used in the field.
  • the above-mentioned Al-denaturing solution, neutralizing solution, and buffer solution can be selected from those commonly used in the field.
  • Examples of the alkali-denaturing solution include 0.5M NaOH and 1.5M.
  • Examples of the neutralizing solution include 1.5M NaCl-containing 0.5M Tris-HC1 buffer, pH 8.0, and the like. for example, and the like 2 X SSPE (0. 36M NaCl, 20raM NaH 2 P0 4 and 2 mM EDTA).
  • the transferred carrier for example, a membrane
  • the transferred carrier is preferably prehybridized as necessary to prevent non-specific hybridization reaction.
  • This pre-hybridization treatment can be performed by, for example, pre-hybridization solution [50% formamide, 5 X Denhardt, s solution (0.2% ushi serum albumin, 0.2% polyvinyl pyrrolidone) s 5 X SSPE, 0. 1% SDS, 100 ⁇ g / l heat-denatured salmon sperm DNA], etc., and about 35 to about 50 ° C., preferably about 42 ° C., about 4 to about 24 hours, preferably about 6 to about 8
  • the reaction can be carried out for a time, but those conditions can be determined by those skilled in the art by appropriately repeating the experiment and determining more preferable conditions.
  • the denaturation of the labeled prop DNA fragment used for hybridization is performed, for example, by heating at about 70 to about 100 ° C, preferably about 100 ° C, for about 1 to about 60 minutes, preferably about 5 minutes. It can be carried out .
  • Hybridization is a method known per se, or Although stringent conditions in this specification include, for example, about 15 to about 50 mM, preferably about 19 to about 40 mM, more preferably about 19 to about 20 mM, The temperature is about 35 to about 85 ° C, preferably about 50 to about 70 ° C, more preferably about 60 to about 65 ° C.
  • the carrier such as a filter is thoroughly washed, and the labeled probe other than the labeled probe DNA fragment that has undergone a specific hybridization reaction can be removed before performing the detection process.
  • Washing treatment of the carrier such as a filter can be performed by selecting from those commonly used in the field, for example, 0.1%, containing SDS 0.5 X SSC (0.15M NaCl 15mM It can be carried out by washing with a solution such as kenic acid.
  • the hybridized nucleic acid can typically be detected by one “tradularity”, but can be appropriately selected from methods used in this field for detection.
  • Nucleic acid corresponding to the detected signal band a suitable buffer, for example, SM solution (lOOmM NaCl and 10 mM MgSO 4 containing 50 mM Tris-HCl buffer, pH 7. 5) was suspended in like and then moderately and diluted this suspension, a predetermined
  • the nucleic acid can be isolated and purified, and subjected to further amplification processing.
  • “high homology” refers to, for example, 50% or more, or even 60% or more, depending on the length of the target sequence.
  • the “same-effect base sequence” may be one that hybridizes to, for example, a sequence having a problem sequence under stringent conditions, for example, a sequence of 5 or more consecutive base sequences, preferably Preferably, it hybridizes with 10 or more base sequences, more preferably 15 or more base sequences, more preferably 20 or more base sequences, and encodes an amino acid sequence substantially equivalent to the polypeptide. And what to do. Nucleic acids can also be obtained by chemical synthesis. In that case, the fragments may be chemically synthesized and linked by an enzyme.
  • a target nucleic acid from a nucleic acid sample including a gene library or a cDNA library by a hybridization process can be repeated.
  • Cloned human cDNA libraries such as various human-derived silkworms, are cultured cells (especially, human kidney, brain, pineal gland, pituitary gland, nerve cells, retina, retina).
  • tDNA libraries can be used.
  • a cDNA library used as a cage type a commercially available cDNA library derived from various tissues can be used directly, for example, a cDNA library sold by Stratagene, Invitrogen, Clontech, etc. is used.
  • Typical examples include gene libraries prepared from human avian cells, such as the human PI artificial chromosome genomic library (Human Genome Mapping Resource Center), and human and human cDNA libraries (eg, Clontech). Etc.) can be used.
  • Human genomic DNA libraries or human-derived cDNA libraries constructed from various human tissues or cultured cells can be screened using probes.
  • a commercially available labeling kit such as a random prime DNA labeling kit (Boehringer Mannheim) can be used.
  • Random- priming kit (Pharmacia LKB, Inc., Uppsala) was used a city, a DNA probe [- 32 P] labeled with a dCTP (Amersham Corp.), to obtain a probe having an active radiate Can do.
  • Phage particles, recombinant plasmids, recombinant vectors, etc. that possess a given nucleic acid can be purified and separated by methods commonly used in the art, such as glycerol gradient ultracentrifugation. (Molecular Cloning, a laboratory manual, ed. T. Maniatis, Cold Spring Harbor Laboratory, 2nd ed. 78, 1989), and can be purified by electrophoresis. From phage particles, DNA can be purified and separated by methods commonly used in the field. For example, the obtained phage can be used as a TM solution.
  • subcloning can be performed using Escherichia coli as a host and a plasmid vector.
  • DNA obtained by such subcloning can also be purified and separated by methods such as centrifugation, phenol extraction, and ethanol precipitation in the same manner as described above.
  • nucleic acids including DNA
  • PCR products obtained are usually subjected to 1-2% agarose gel electrophoresis and excised from the gel as a specific band.
  • gene clean kit Bio 101
  • Extract using an extraction kit sold by The extracted DNA is cleaved with an appropriate restriction enzyme, purified as necessary, and further phosphorylated at the 5 'end with T4 polynucleotide kinase as necessary.
  • PCR product cloning include p-Mrect (Clontech), pCR-Script TM SK (+) (Stratag ⁇ ne), GEM-T (Promega), Amp TM (Gibco-BRL), etc.
  • plasmid vectors can be used. In order to transform host cells, for example, phage vectors are used, calcium method, rubidium / calcium method, calcium Z manganese method, TFB high efficacy.
  • the method can be carried out by methods known in the art such as the rate method, FSB frozen combi- tive cell method, rapid colony method, and electroporation method, or substantially similar methods (D. Hanahan, J. Mol. Biol ., 166: 557, 1983, etc.).
  • reverse transcription PCR RACE grapia amplification of cDNA ends
  • RACE should be performed according to the method described in, for example, MA Innis et al. Ed., PCR Protocols (MA Frohman, a guide to methods and applications "), pp. 28 38, Academic Press, New York (1990). Can do.
  • DNA can be cloned as necessary.
  • plasmids phage cosmids, P1 phage, F factor, YAC, etc.
  • Preferred examples include phage-derived vectors, such as Charon 4A, Charon 21A, Lgtl0, Agtl1, ADASHII, AFIXII, ⁇ EMBL3, ⁇ TM
  • the obtained DNA is incorporated into an appropriate vector as described in detail below, for example, a vector such as plasmids pEX, pMAMneo, pKG5, and an appropriate host cell as described in detail below. It can be expressed in E. coli, yeast, CH0 cells, COS cells, etc.
  • the MA fragment can be used as it is or as a DNA fragment to which an appropriate control sequence has been added, or incorporated into an appropriate vector and introduced into an animal to produce a transgenic animal that expresses a predetermined gene. it can. Examples of animals include mammals, such as mice, rats, rabbits, guinea pigs, and rabbits.
  • the DNA fragment can be introduced into a fertilized egg of an animal such as a mouse to create a transgenic animal.
  • Confirmation of a predetermined gene product can be performed using animal cells, such as 293T cells and COS-1 cells, which have been transfected with the foreign gene.
  • a method for introducing a foreign gene into an animal cell such as a mammal a method known in the art or a method substantially similar thereto can be used.
  • calcium phosphate method eg FL Graham et al., Virology, 52: 456, 1973, etc.
  • DEAE-dextran method eg D. Warden et al., J. Gen. Virol., 3: 371, 1968 etc.
  • Elect mouth position method for example, E. Neumann et al., EMBO J, 1: 841, 1982
  • microindi- terion method eg ribosome method, virus infection method, phage particle method, and the like.
  • a plasmid into which a predetermined gene or the like (DNA obtained in the present invention) is incorporated
  • a host cell commonly used in genetic engineering for example, prokaryotic hosts such as bacteria and Bacillus subtilis, yeast, 293T cells, CH0
  • Any plasmid can be used as long as the DNA can be expressed in cells, eukaryotic hosts such as COS cells, and insect cell hosts such as Sf21.
  • prokaryotic hosts such as bacteria and Bacillus subtilis, yeast, 293T cells
  • Any plasmid can be used as long as the DNA can be expressed in cells, eukaryotic hosts such as COS cells, and insect cell hosts such as Sf21.
  • COS cells eukaryotic hosts
  • insect cell hosts such as Sf21.
  • Such sequences can include codons that are suitably modified, for example, to be expressed in the selected host cell, can contain restriction enzyme sites, Control sequences for facilitating the expression of the child, facilitating sequences, linkers, adapters, etc. that help to bind the target gene, antibiotic resistance, etc., control metabolism, It can contain self-trains (including those encoding hybrid proteins and fusion proteins) that are useful for sorting and the like.
  • suitable plasmids such as plasmids that have Escherichia coli as the host include the tritophan fan promoter (trp), the ratatothase promoter (lac), the tryptophan-ratathose promoter (tac), the lipoprotein promoter (lpp), the ⁇ phage P
  • trp tritophan fan promoter
  • lac ratatothase promoter
  • tac tryptophan-ratathose promoter
  • lpp lipoprotein promoter
  • ⁇ phage P For plasmids that use L promoters, etc. as host cells, SV40 rate promoter, MMTV LTR promoter, RSV LTR promoter, CMV promoter, SRa promoter, etc., and for plasmids that use yeast as host, GAL1, GAL10 promoter Etc. can be used.
  • control systems such as CYC1, HIS3, ADH1, PGK, PH05, GAPDH, ADCl, TRPl, URA3, LEU2, ENO, TPl, A0X1 can be used.
  • Monkey An enhancer can be inserted into the vector to promote the transcription of the DNA encoding the desired polypeptide. As such an enhancer, it acts as a promoter and promotes transcription, usually about 10 to 100 bp. The element with the cis action of. Many enhancers are known from mammalian genes such as globin, elastase, anolebumin, fetoprotein, and insulin. Typically, an enhancer obtained from a eukaryotic cell infectious virus can be suitably used.
  • the SV40 enhancer (100-270 bp) in the rate region of the replication origin, the early promoter of cytomegalovirus.
  • examples include enhancers, enhancers in the replication origin rate domain of poliooma, and enhancers of adenoviruses.
  • coli as hosts include, for example, pBR 322 , pUC18, .pUC19, pUC118, pUC119, pSP64, pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGE-3, pGEM-4, pGEM- 3Z, pGEM-4Z, pGEM-5Zf (-), and pBluescript KS TM (Stratagene3 ⁇ 4).
  • Examples of plasmid vectors suitable for expression in E. coli include pAS, pKK223 (Pharmacia), pMC1403, pMC931, pKC30, and pRSET-B (Invitrogen).
  • Examples of plasmids that use animal cells as hosts include SV40 vectors, polio-omas, winolecs vector, vaccinia-winoles vector, retroinores vector, and specifically pcD, pcD-SR. a, CDM8, pCEV4, pME18S, PBC12BI, pSG5 (Stratagene).
  • Examples of plasmids using yeast as a host include Yip type vectors, YEp type vectors, YRp type vectors, YCp type vectors, and the like, for example, pGPD-2.
  • the host cell when the host cell is E. coli, for example, one derived from E.
  • BL21 (DE3) pLysS is derived from ⁇ ⁇ 533, XL, Blue, C600, DH1, DH5, DH11S, DH12S, DH5a, DH10B, HB101, MC1061, JM109, STBL2, and B834.
  • yeast examples thereof include Saccharomyces cerevisiae, Schizosaccharorayces prombe, Pichia pastor is, Kluyveromyces strain, Candida, Trichoderma reesia, and other yeast strains.
  • the host cell is an animal cell, for example, COS-7 cells, COS-1 cells, CV-1 cells, human kidney cell-derived 293 cells, human epidermal cell-derived A431 cells, or human colon-derived cells derived from African green monkey fibroblasts 205 cells, mouse fibroblast-derived C0P cells, M0P cells, W0P cells, Chinese hamster cell-derived CH0 cells, CH0 DHFR— cells, human HeLa cells, mouse cell-derived C127 cells, mouse cell-derived NIH 3T3 cells Mouse L cells, 9BHK, HL60, U937, HaK, Jurkat cells, other transformed cell lines, normal diploid cells, cell lines derived from in vitro primary cultures, etc. .
  • Insect cells include Bombyx tnori nuclear polyhedrosis virus, or derived from it or other suitable vectors, and Spodoptera frugiperda (caterpil lar), Aedes aegypti (mosquito), Aedes albopictus ( mosquito), Drosophila me ⁇ angaster (fruitfly), silkworm larvae or silkworm cultured cells such as BM-N cells (eg, Luckow et al., Bio / Technology, 6, 47-55 (1988) Set low, JK et al. (Eds.), Genetic Engineering, Vol. 8, pp. 277-279, Plenum Publishing, 1986; Maeda et al., Nature, 315, pp.
  • telomeres a restriction enzyme, reverse transcriptase, or DNA fragment known or widely used in the art is modified or converted into a structure suitable for cloning.
  • Enzyme DNA modification ⁇ Degradation enzyme, DNA Polymerer , Terminal nucleotidyl transferase, DNA ligase, etc. can be used.
  • restriction enzymes include RJ Roberts, Nucleic Acids Res., 13: rl65, 1985; S. Linn et al. Ed. Nucleases, p.
  • a transformant transformed with an expression vector containing a nucleic acid encoding a polypeptide (or protein) can be highly expressed by repeated cloning using an appropriate selection marker as necessary.
  • a cell line having stable ability can be obtained. For example, in a transformant using an animal cell as a host cell, when the dhfr gene is used as a selection marker, the strain of the present invention is selected by culturing with gradually increasing sputum concentration and selecting a resistant strain.
  • the transformant of the present invention can be cultured under conditions that allow expression of the nucleic acid encoding the polypeptide of the present invention, and the target product can be produced and accumulated.
  • the transformant can be cultured in a medium commonly used in the art.
  • a liquid medium can be suitably used for a transformant having a prokaryotic cell host such as Escherichia coli or Bacillus subtilis or yeast as a host.
  • the medium contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Examples of carbon sources include dulcose, dextrin, soluble starch, and sucrose.
  • nitrogen sources include ammonium salts, nitrates, corn sheep 'liquor, peptone, casein, meat extract, malt extract, soybean meal,
  • inorganic or organic substances such as potato extracts and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and calcium carbonate.
  • yeast, vitamins, casamino acids, growth promoting factors and the like may be added. If necessary, a drug such as 3-indolylacrylic acid can be added to make the promoter work efficiently.
  • Medium The pH is preferably about 5 to about 8.
  • E. coli is usually cultured at about 15 to about 45 ° C for about 3 to about 75 hours, and if necessary, aeration or agitation can be added.
  • a MEM medium containing about 5 to about 20% fetal bovine serum, a PRMI1640 medium, a DMEM medium, or the like is used as the medium.
  • the pH is preferably from about 6 to about 8.
  • Culture is usually performed at about 30 to about 40 ° C. for about 15 to about 72 hours, and aeration and agitation are added as necessary.
  • the transformant expressing the predetermined gene product can be used as it is, but it can also be used as its cell homogenate, but the predetermined gene product can also be isolated and used.
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, and cultivated by sonication, lysozyme and / or freeze-thawing.
  • a method of obtaining a crude extract by centrifugation or filtration after cell disruption can be used as appropriate.
  • Protein buffers such as urea and guanidine hydrochloride, and surfactants such as Triton X-100 (trade name) and Tween-20 (trade name) may be added to the buffer. If the target product is secreted into the culture solution, after completion of the recharge, isolate the cell or supernatant from the cells using a method known per se, and collect the supernatant.
  • the culture supernatant thus obtained or the target product contained in the extract can be purified by appropriately combining known separation / purification methods, such as ammonium sulfate precipitation.
  • polyacrylamide gel electrophoresis, ligands, etc. are immobilized. It can be purified and processed by chromatography. Examples include gelatin-garose affinity chromatography, heparin-garose chromatography, and the like.
  • the obtained polypeptide (or protein) of the present invention can be modified by a chemical method to modify the amino acid residue contained therein, or a peptidase such as pepsin, chymotrypsin, papain, promeline, It can be modified with enzymes such as endopeptidase and exopeptidase, or partially decomposed into its derivatives.
  • the C-terminus is usually a carboxyl group (-C00H) or force / repoxylate (-C00 "), but the C-terminus is an amide (-C0NH 2 ) or ester (-C00R). or here, as R in the ester, e.g., methyl, E Ji Le, n -.
  • propyl, _ 6 alkyl groups such as isopropyl or n- heptyl, for example, consequent opening pentyl, C 3, such as cyclohexyl - s cycloalkyl groups such as phenyl, alpha-C 6 _ 12 Ariru groups, naphthyl, for example, benzyl, alpha-Na, such as phenyl one C 1-2 alkyl group or alpha-naphthyl-methyl, such as phenethyl: chill - - 2 alkyl C 7 such as -.
  • PIPA Roi Ruo carboxymethyl groups commonly used as ho force oral ester Ararukiru groups are needed use carbonitrile proteins other than the C-terminus of the present invention
  • those in which the carboxyl group is amidated or esterified are also included in the polypeptide of the present invention.
  • the amino group of the N-terminal methionine residue is a protecting group (for example, an alkyl such as a formyl group or acetyl). such as single carbonyl group - 6 is protected with a protecting ⁇ etc.
  • 49 groups, guanidino groups, etc. are protected with appropriate protecting groups (for example, C 6 -acyl groups such as formyl group, acetyl group, etc.) or complex proteins such as so-called glycoproteins to which sugar chains are bound included.
  • protecting groups for example, C 6 -acyl groups such as formyl group, acetyl group, etc.
  • complex proteins such as so-called glycoproteins to which sugar chains are bound included.
  • one or more amino acid substitutions are appropriately performed in the amino acid sequence of a predetermined polypeptide.
  • Corresponding polypeptides into which mutations such as deletion, insertion, transfer or addition have been introduced can be produced.
  • fusion polypeptide fusion protein
  • fusion polypeptides include those fused to a histidine tag, or j3 -galactosidase (3-gal), maltose binding protein (MBP), glutathione-S-transphrase (GST), thioredoxin (TRX) Or those fused to the amino acid sequence of Cre Recombinase.
  • a polypeptide can be tagged with a heterogeneous epitope and can be purified by immunoaffinity-chromatography using an antibody that specifically binds to the epitope.
  • a polyhistidine (poly-His) or polyhistidine-glycine (poly-His-Gly) tag and the epitope tag include, for example, AU5, c-Myc, CruzTag 09, CruzTag 22, CruzTag 41, Glu-Glu, HA, Ha. 11, KT3, FLAG (registered trademark, Sigma-A ⁇ drich), Omni -probe, S-probe, T7, Lex A, V5, VP 16, GAL4, VSV-G (Field et al., Molecular and Cellular Biology, 8: pp. 2159-2165 (1988); Evan et al., Molecular and Cellular Biology, 5: pp.
  • fusion polypeptide can be a detectable protein.
  • the detectable marker may be a biotin Avi Tag based on the biotin / streptavidin system, a fluorescent substance, or the like.
  • the fluorescent substance includes a green fluorescent protein (GFP) derived from a luminescent jellyfish such as Aequorea victorea, and a modified variant (GFP variant) such as EGFP (Enhanced- humanized GFP),. rsGFP (red-shift GFP), yellow fluorescent protein (YFP), green II fluorescent protein (-GFP), cyan fluorescent protein protein (CFP), blue fluorescent protein (BFP), GFP derived from Renilla reniformis, etc.
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • -GFP green II fluorescent protein
  • CFP cyan fluorescent protein protein protein
  • BFP blue fluorescent protein
  • Detection can also be performed using antibodies (including monoclonal antibodies and fragments thereof) that specifically recognize the fusion tag.
  • Fusion polypeptide can be performed using a commercially available kit suitable for it, and can also be performed according to the protocol disclosed by the kit manufacturer or kit distributor.
  • the resulting protein (which may include peptides or polypeptides) can be immobilized by binding it to an appropriate carrier or solid phase using known techniques such as enzyme immunoassay. Solid-phased proteins and solid-phased peptides can be conveniently used for screening of binding materials.
  • the biological activity may include immunological activity, eg, antigenicity.
  • modifications / modifications are deamination, hydroxylation, carboxylation, phosphorylation, sulfation, 05 013792
  • Alkynolation such as tilation, acylation such as acetylation, esterification, amidation, ring-opening, ring-closing, glycosylation, changing the type of sugar chain to a different one, increasing or decreasing the number of sugar chains It may be a lipid bond, substitution to a D-form amino acid residue, or the like.
  • the human peptide or polypeptide (or protein) of the present invention has one or more amino acid residues that differ from the natural ones in terms of identity, and the position of one or more amino acid residues is natural. It may be different from the one.
  • the peptide derived from the human of the present invention has one or more amino acid residues (for example, 1 to 80, preferably 1) unique to human lectin 9 (including L, M and S types) protein or galectin 9 binding protein. -60, more preferably 1-4Q, more preferably 1-20, especially 1-10, etc.) lacking deletion analogs, one or more of the unique amino acid residues (eg 1 to 80, preferably 1-60, more preferably 1-40, more preferably 1-20, in particular 1-10, etc.) substituted with other residues, 1 or more
  • 1 to 80 also includes analogs.
  • the domain structure or sugar-binding ability which is a characteristic of natural human lectin 9 protein or galectin 9-binding protein, is maintained, all the above-mentioned variants are included in the present invention.
  • the peptides or polypeptides of the present invention include those having a primary structure conformation or a part thereof that is substantially equivalent to natural human lectin 9 protein or galactin 9 binding protein. It is also possible that a substance having substantially the same biological activity as that of a natural substance may be included. It can also be one of naturally occurring variants.
  • the human-derived protein (or peptide or polypeptide) of the present invention is, for example, PT / JP2005 / 013792
  • WO 02/37114 Al having an amino acid sequence selected from the group consisting of SEQ ID N0: 1 to 3 in the sequence listing of Al 60%, in some cases higher than 70% homology More preferably 80% or 90 ° /. Those having the above homologous amino acid sequences are mentioned.
  • a part of the human-derived protein of the present invention is a partial peptide of the human-derived protein (that is, a partial peptide of the protein), and the galectin-9 protein of the present invention. And those having substantially the same activity.
  • the partial peptide of the protein of the present invention comprises at least 5 or more, preferably 20 or more, more preferably 50 or more, more preferably 70 or more, more preferably, of the constituent amino acid sequences of the human galectin 9 , Is a peptide having an amino acid sequence of 100 or more, in some cases 200 or more, and preferably a force corresponding to consecutive amino acid residues, or, for example, the TO 02 / Among the amino acid sequences represented by SEQ ID N0: 1 to 3 in the sequence table of 37114 Al, those having homology similar to the above may be mentioned with respect to homology to the corresponding region.
  • substantially equivalent means protein activity, for example, predetermined cytotoxic activity, apoptosis-inducing activity, anti-inflammatory activity, anti-allergic activity, immunosuppressive activity, sugar chain binding activity, physiological activity Means that the biological activity is substantially the same. Furthermore, the meaning of the term may include a case having substantially the same quality activity, and the substantially same quality activity includes binding activity, cytotoxic activity, apoptosis-inducing activity, and the like. Can be mentioned.
  • the substantially homogeneous activity indicates that the activities are qualitatively homogeneous, for example, physiologically, pharmacologically, or biologically homogeneous. For example, activities such as binding activity, cytotoxic activity, apoptosis-inducing activity, etc.
  • amino Acid substitutions, deletions, or insertions often do not cause significant changes in the physiological or chemical properties of the polypeptide, and in some cases give favorable changes. In such cases, the polypeptide with the substitution, deletion or insertion will be considered substantially identical to that without such substitution, deletion or insertion.
  • Substantially identical substitutions of amino acids in the amino acid sequence can be selected from other amino acids of the class to which the amino acids belong.
  • non-polar (hydrophobic) amino acids include alanine, phenenolealanine, leucine, isoguchi isine, parin, proline, tryptophan, methionine, etc.
  • polar (neutral) include glycine, serine, Examples include threonine, cysteine, tyrosin, asparagine, and glutamine.
  • Amino acids with a positive charge include arginine, lysine, and histidine, and amino acids with a negative charge (acidic amino acids).
  • Aspartic acid, glutamic acid and the like include arginine, lysine, and histidine.
  • a protein or a resin for peptide synthesis is used, and appropriately protected amino acids are sequentially bonded to the desired amino acid sequence on the resin by various known condensation methods.
  • various activating reagents known per se are preferably used.
  • strong positive imides such as dicyclohexyl carpositimide can be preferably used.
  • the peptide (or polypeptide) of the present invention When the peptide (or polypeptide) of the present invention is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, or obtained as a salt. In this case, it can be converted into a free form or other salt by a method known per se or a method analogous thereto.
  • the salt of the peptide (or polypeptide) of the present invention is preferably a physiologically acceptable salt or a pharmaceutically acceptable salt, but is not limited thereto.
  • salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, such as acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, citrate, tartaric acid, malic acid, benzoic acid. Acid, methanesulfonic acid
  • salts with organic acids such as p-toluenesulfonic acid and benzenesulfonic acid.
  • examples of the salts include ammonium salts such as salts with organic bases such as ethylamine, dimethyl / leamine, trimethylamine, and hydroxychetilamine.
  • ammonium salts such as salts with organic bases such as ethylamine, dimethyl / leamine, trimethylamine, and hydroxychetilamine.
  • in situ hybridization may include, for example, non-RI in situ hybridization, including, for example, direct and indirect methods.
  • the direct method uses, for example, a molecule (reporter) directly bound to a nucleic acid probe
  • the indirect method uses, for example, an antibody against a reporter molecule.
  • the signal is amplified.
  • Functional groups for example, primary aliphatic amino groups, SH groups, etc.
  • haptens, fluorescent dyes, enzymes, etc. are bound to these functional groups. Cough It may be done.
  • the labeling of the nucleic acid probe typically includes digoxigenin (DIG), biotin, fluorescein, etc., but can be used by appropriately selecting from the label described in the antibody as described above. Multiple labeling can be used, and labeled antibodies can also be used.
  • Nucleic acid probe labeling methods can be selected from methods known in the art as appropriate. For example, random prime method, nick 'translation method, DNA amplification by PCR, labeling Z-tiling method, in vitro Examples include transcription.
  • random prime method for the observation of the processed sample, it can be used by appropriately selecting from methods known in the field.
  • malignant cells may include tumor cells that metastasize. Tumors that metastasize are malignant tumors. Generally, the malignant tumors are classified into epithelial and non-epithelial types.
  • cancer In some cases, they may be considered as cancer, sarcoma, leukemia, etc.
  • cancer When it is simply called “cancer”, it generally refers to a malignant tumor in the general public.
  • cancer may be interpreted in a broad sense, and should not be simply interpreted as an epithelial malignant tumor.
  • cancer may include epithelial malignant tumors and non-epithelial malignant tumors (including both tumorigenic and non-forming ones), and skin cancer (including melanoma).
  • breast cancer ovarian cancer
  • uterine cancer testicular malignant tumor
  • prostate cancer bladder cancer
  • kidney cancer thyroid cancer
  • pharyngeal / laryngeal cancer tongue cancer
  • maxillary cancer esophageal cancer
  • stomach cancer colon / rectal cancer
  • Lung 'bronchial cancer liver cancer (including hepatocellular carcinoma, intrahepatic cholangiocarcinoma), extrahepatic bile duct' gallbladder cancer, knee cancer, leukemia, malignant lymphoma, plasmacytoma, osteosarcoma, chondrosarcoma, smooth muscle , Rhabdomyosarcoma, liposarcoma, fibrosarcoma, malignant hemangioma, malignant hemangioendothelioma, brain tumor (including meningioma, glioma, astrocytoma), etc.
  • galectin 9 and galectin 9 binding protein are used interchangeably.
  • functions such as a predetermined biological activity of galectin 9 (for example, cytotoxic activity, Apoptosis-inducing activity, activity related to darcocorticoid, activity to suppress metastasis of malignant cells, etc.) Screening for compounds (agonist), inhibitors (antagonist) or their salts, galectin Screening compounds that promote interaction between 9 and galectin 9 binding protein and biological activity induced by Z or binding (agonist), inhibitor (antagonist) or their salts You can do it.
  • a predetermined biological activity of galectin 9 for example, cytotoxic activity, Apoptosis-inducing activity, activity related to darcocorticoid, activity to suppress metastasis of malignant cells, etc.
  • the present invention relates to various substances using the activity shown in the interaction and / or binding between the galectin 9 and the galectin 9-binding protein elucidated in the present invention, and the galectin 9 and galectin 9 of the present invention.
  • screening compounds agonists
  • inhibitors antagonists
  • salts thereof that promote a given function, such as the interaction between binding proteins and / or biological activity exerted by the binding.
  • Galectin 9-binding protein, Galectin 9 protein, a part of the peptide or a salt thereof which may contain a transformant expressing the protein, the same shall apply hereinafter
  • a suitable test sample is brought into contact
  • the biological activity for example, activity related to the interaction between each galectin-9 protein and a biological component
  • An appropriate detection substrate may be present in the screening system for convenience of measurement. As the substrate, any substrate can be used as long as it can be effectively used for measurement.
  • a known substrate can be selected and used, but a synthesized compound or the like can be preferably used.
  • the substrate can be used as it is, but preferably a substrate labeled with fluorescence such as fluorescein, enzyme or radioactive substance can be used.
  • the test sample include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts such as animals, and cell extracts.
  • test compounds used in test samples include anti-galectin antibodies, anti-galectin 9-binding protein antibodies, anti-galactin 9-binding protein sugar chain antibodies, enzyme inhibitors, cytokines, and various inhibitor activities. It may contain compounds, especially synthetic compounds. These compounds may be novel compounds or known compounds.
  • the screening can be performed according to the usual method for measuring the binding activity or enzyme activity.
  • ⁇ can be performed with reference to methods known in the art, various labels, buffer systems, and other appropriate methods. Can be carried out in accordance with the procedures described there.
  • the peptide used can be treated with an activator, or its precursor or latent type can be converted into an active type in advance. Measurement is usually in a buffer that does not adversely affect the reaction such as Tris-HC1 buffer, phosphate buffer, etc., for example, pH about 4 to about 10 (preferably pH about 6 to about 8) Can be done.
  • the compound obtained by using the screening method or screening kit of the present invention or a salt thereof is the above-mentioned test compound, for example, peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extraction It is a compound selected from a liquid, a plant extract, an animal tissue extract, etc., and a compound that promotes or inhibits the protein interaction and functions of Z or binding of the present invention.
  • the salt of the compound include pharmaceutically acceptable salts.
  • Examples include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like.
  • Preferable examples of the salt with an inorganic base include sodium metal salts such as sodium salts and strength salts, alkaline earth metal salts such as calcium salts and magnesium salts, and aluminum salts and ammonium salts. .
  • the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2, 6_ / retidine, ethanolanolamine, diethanol / reamine, triethanolamine, cyclohexylamine, And salts with dicyclohexylamine, ⁇ , ⁇ '-dibenzylethylenediamine, and the like.
  • the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like.
  • Preferable examples of the salt with an organic acid include formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, succinic acid, malic acid, malic acid, methanesulphonic acid, and benzenesulphonic acid.
  • salts with benzoic acid and the like Preferable examples of the salt with basic amino acid include, for example, salts with arginine, lysine, ornithine and the like, and preferable examples of the salt with acidic amino acid include, for example, aspartic acid, Examples include salts with glutamic acid.
  • anti-galectin 9 antibody, anti-galectin 9-binding protein antibody An antibody such as an anti-galectin 9-binding protein saccharide antibody can be obtained as a polyclonal or monoclonal nanore antibody using known means.
  • antibody may be used in a broad sense, and it may be a single monoclonal antibody against a desired protein or its constituent polypeptides and related peptide fragments. It may be an antibody composition having specificity for an epitope, and also comprises a monovalent or multivalent antibody as well as a polyclonal antibody and a monoclonal antibody, and also represents an intact molecule and fragments and derivatives thereof.
  • a 'chimeric or hybrid antibody including fragments such as F (ab') 2 , Fab 'and Tab, and further having at least two antigens or epitope binding sites, or, for example, Bispecific recombinant antibodies such as quadrome and triome, interspecific hybrid antibodies, anti-diotites Antibodies, or those that are chemically modified or processed and considered to be derivatives thereof, obtained by applying known cell fusion or hybridoma technology or antibody engineering, or using synthetic or semi-synthetic technology
  • Antibodies antibodies prepared using conventional techniques known from the viewpoint of antibody production, or antibodies prepared using DNA recombination techniques, are target antigenic substances described and defined herein, or are neutralized with respect to target epitopes It may include antibodies that have properties or antibodies that have binding properties.
  • a protein or fragment thereof, a part of the peptide, or a sugar chain is immunized to mammals, birds, etc., and antiserum is collected from the mammals, birds, etc.
  • a polyclonal antibody contained in this antiserum can be used.
  • Mammals to be immunized with a sensitizing antigen are not particularly limited, but are generally rodent animals such as mice, rats, hamsters, rabbits, hidges, goats, Primates such as Ushi, Uma, Pig, Inu, Cat, Sal, etc.-Birds such as ⁇ birds are used.
  • rodent animals such as mice, rats, hamsters, rabbits, hidges, goats, Primates such as Ushi, Uma, Pig, Inu, Cat, Sal, etc.-Birds such as ⁇ birds are used.
  • the sensitizing antigen is injected intraperitoneally or subcutaneously in a mammal or the like.
  • An appropriate carrier can also be used during immunization with the sensitizing antigen.
  • the antiserum containing the polyclonal antibody can be prepared from blood collected from the animal after breeding the immunized animal for a predetermined period.
  • the obtained antiserum is used as a predetermined active ingredient of the present invention after confirming that it specifically recognizes the target antigen or a specific site thereof.
  • the antibody obtained as a monoclonal antibody derived from a mammal can also be used.
  • Monoclonal antibodies generated against antigenic material are produced using any method that can provide for the production of antibody molecules by a series of cell lines in culture.
  • the modifier “monoclonal” refers to the nature of the antibody as being derived from a substantially homogeneous population of antibodies, and it is assumed that the antibody needs to be produced by some specific method. Must not.
  • Each monoclonal antibody contains a population of antibodies that are identical, except that only a small amount of naturally occurring variants may be present.
  • Monoclonal antibodies have high specificity and are directed against sites with a single antigenicity. In contrast to normal (polyclonal) antibody preparations that typically contain different antibodies directed against different antigenic determinants (epitotops), each monoclonal antibody is a single antibody on that antigen.
  • monoclonal antibodies are also superior in that they are synthesized by hybridoma culture and are free from or less contaminated by other immunoglobulins.
  • Monoclonal antibodies include hybrid antibodies and recombinant antibodies. As long as they show the desired biological activity, regardless of their origin, type of immunoglobulin class or subclass, the variable region domain can be replaced with a constant region domain (for example, a humanized antibody), or light-weighted can be replaced with heavyweight. Replace one chain or another It can be obtained by replacing it with a chain of species or by fusing it with a heterogeneous protein (eg, US Pat. No.
  • the monoclonal antibodies according to the present invention have corresponding sequences of antibodies in which a part of the heavy chain and / or light chain is derived from a specific species or belongs to a specific antibody class or subclass. While the rest of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, Specifically encompassing “chimeric” antibodies (immunoglobulins) (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81, pp. 6851-6855 (1984)).
  • Examples of the monoclonal antibody according to the present invention include those produced by mammal-derived hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques.
  • . -Monoclonal antibody-producing hybridomas that produce specific antibodies can be produced using cell fusion technology using myeloma cells as follows. That is, the target protein or fragment thereof is used as a sensitizing antigen, and this is immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. This method can be prepared by screening monoclonal antibody-producing cells by the screening method described above.
  • the method for preparing galectin 9 or a fragment thereof and the method for immunizing mammals can be carried out in accordance with the above-described method for preparing antiserum containing polyclonal antibodies.
  • immune cells are collected from the mammal which has been confirmed to increase the desired antibody level in the serum and subjected to cell fusion.
  • spleen cells As the other parent cell to be fused with the immune cell, mammalian myeloma Use cells.
  • Various known cell lines can be used as the myeloma cell.
  • Cell fusion between immune cells and myeloma cells is basically performed by known methods such as the Keller and Milstein method (Kohler. G. and Milstein, C-, Methods Enzymol. (1981) 73, 3- 46) etc.
  • the antibody of the present invention may be a monoclonal antibody obtained by utilizing cell fusion technology using myeloma cells, and can be produced, for example, by the following steps.
  • An immunogenic antigen can be prepared as follows. As described above, as described above, the target protein or its constituent polypeptide or sugar chain or a fragment derived therefrom (some domain polypeptides, link polypeptides, fragments, some Ability to use isolated peptides (synthetic polypeptides, peptides, and synthetic sugar chains) Based on the amino acid sequence information of the determined protein, chemistry of the appropriate oligopeptide or oligosaccharide It can be synthesized and used as an antigen. The antigen can be used as it is to immunize animals by mixing it with an appropriate adjuvant, but it may be an immunogenic conjugate.
  • the antigen used as an immunogen is a fragment of a protein, or a synthetic polypeptide fragment obtained by chemical synthesis by selecting a characteristic sequence region based on the amino acid sequence and designing the polypeptide. It may be.
  • the fragments are combined with various carrier proteins via an appropriate condensing agent to form an immunogenic conjugate such as a hapten monoprotein, which is used for specific arrangement.
  • a cysteine residue or the like can be added in advance to the designed polypeptide to facilitate the preparation of the immunogenic conjugate.
  • the carrier proteins In binding to carrier proteins, the carrier proteins can first be activated. For such activation, introduction of an activated linking group can be mentioned.
  • active bond groups include (1) activated ester or activated carboxyl group, such as nitrophenyl ester group, pentafluorophenyl ester group, 1-benzotriazole ester group, N-succinimide ester. (2) An activated dithio group such as a 2-pyridyldithio group.
  • Carrier proteins include keyhole-limpet hemocyanin
  • KLH bovine serum albumin
  • BSA bovine serum albumin
  • glopurin egg white anolebumin
  • polylysine polypeptides
  • bacterial cell components such as BCG
  • Immunization of animals with immunogenic antigens can be performed as follows. Immunization can be carried out by methods known to those skilled in the art, such as Shigeru Muramatsu, et al., Laboratory Biology Course 14, Immunobiology, Maruzen Co., Ltd. Experimental Lecture 5, Immunobiochemical Research Method, Tokyo Kagaku Doujin, 1986, Japan Biochemical Society, New Chemistry Experimental Laboratory 12, Molecular Immunology III, Antigen, Antibody-Complement, Tokyo Kagaku Doujin, 1992 It can be done according to the method. Immunization is achieved by injecting the immunizing agent (with adipant as needed) into the mammal one or more times.
  • the immunizing agent and Z or adjuvant are injected into a mammal multiple times by subcutaneous injection or intraperitoneal injection.
  • the immunizing agent include those containing the above antigen peptide or a related peptide fragment thereof.
  • the immunizing agent may be used in the form of a conjugate with a protein known to be immunogenic in the mammal to be immunized (for example, the above carrier proteins). For example, Jupiter's complete Freund's adjuvant, Ribia 13792
  • mice such as BALB / c, hamsters, and other suitable animals.
  • the dose of the antigen is, for example, about 1 to 400 g Z animal for mice, generally injected into the peritoneal cavity or subcutaneously of the host animal, and thereafter every 1 to 4 weeks, preferably every 1 to 2 weeks. Repeat booster immunization 2-10 times intraperitoneally, subcutaneously, intravenously or intramuscularly.
  • immunization mice BALB / c mice, F1 mice of BALB / c mice and other mice, and the like can be used.
  • an antibody titer measurement system can be prepared and the antibody titer measured to confirm the degree of animal immunity.
  • the antibody of the present invention may be obtained from an immunized animal thus obtained and includes, for example, antiserum and polyclonal antibody.
  • Myeloma cells can be prepared as follows.
  • the infinitely proliferative strain (tumor cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulin, such as P3-NS-1-Ag4-1 (NS-1, Eur. J. Immunol., 6: 511-519, 1976), SP ⁇ 2 / 0-Agl4 (SP-2,
  • 8-azaguanine-resistant mouse myeloma cell lines include cell culture medium such as Dulbecco's MEM medium (DMEM medium) and RPMI-1640 medium, antibiotics such as benicillin and amikacin, and fetal calf serum (FCS).
  • the cells are subcultured in a medium supplemented with 8--zaguanine (for example, 5-45 / zg / ml), but the cells are subcultured in a normal medium 2-5 days before cell fusion.
  • a medium supplemented with 8--zaguanine for example, 5-45 / zg / ml
  • the cells are subcultured in a normal medium 2-5 days before cell fusion.
  • the cell line used was prepared by thawing the cryopreserved strain completely at approximately 37 ° C, washing it with normal medium such as RPMI-1640 medium three times or more, and then culturing in normal medium to prepare the required number of cell lines. It may be. 2005/013792
  • Cell fusion between antibody-producing cells and myeloma cells can be performed as follows. Animals immunized according to the above step (2), for example mice, are excised 2 to 5 days after the final immunization, and then a spleen cell suspension is obtained. In addition to spleen cells, lymph node cells from various parts of the body can be obtained and used for cell fusion. More specifically, cell fusion is performed, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. As the culture solution used for the cell fusion, for example, RPMI1640 ⁇ sound culture solution suitable for growth of the myeloma cell line, MEM culture solution, and other normal culture solutions used for this kind of cell culture can be used.
  • fetal calf serum FCS
  • FCS fetal calf serum
  • a cell medium such as minimum essential medium (MEM medium), DMEM medium, RPMI-1640 medium, etc.
  • MEM medium minimum essential medium
  • DMEM medium DMEM medium
  • RPMI-1640 medium RPMI-1640 medium
  • a cell fusion promoter such as polyethylene glycol.
  • Various other cell fusion promoters known in the art can be used. : Hemagglutinating Virus of Japan).
  • polyethylene glycol having a molecular weight of 1,000 to 8,000 can be used, and a polyethylene render having a molecular weight of 1,000 to 4,000 can be more preferably used.
  • concentration of polyethylene glycol in the fusion medium is preferably 30 to 60%, for example.
  • the proportion of immunocytes and myeloma cells used that is, the spleen cells (lymphocytes) used for fusion: the proportion of myeloma cell lines can be arbitrarily set, for example 1: 1 to 20: 1 More preferably, it can be 4: 1 to 10: 1.
  • a predetermined amount of immune cells and myeloma cells are mixed well in the culture medium, and a PEG solution (for example, an average molecular weight of about 1000-6000) that has been pre-warmed to about 37 ° C is usually 30-
  • the target fused cell is formed by adding and mixing at a concentration of 60% (w / v). Then add the appropriate culture solution sequentially. Add and centrifuge to remove the supernatant to remove cell fusion agents that are undesirable for the growth of high-pridoma.
  • fusion reaction for 1 to 10 minutes, then add cell culture medium such as RPMI-1640 medium.
  • the fusion reaction treatment can be performed multiple times. After the fusion reaction treatment, the cells are separated by centrifugation and transferred to the selection medium.
  • Hypridoma (fusion cells) selection and monocloning can be performed as follows.
  • the selection medium include normal selection culture media such as FCS-containing MEM medium and RPMI-1640 medium containing hypoxanthine, aminopterin and thymidine, so-called HAT medium.
  • Cultivation with the above HAT culture solution is continued for a sufficient period of time (usually several days to several weeks) to kill cells (non-fusion cells) other than the desired hybridoma.
  • the method of exchanging the selective medium is generally the same as the volume dispensed on the culture plate the next day, and after that, every 1 to 3 days, half the volume is replaced with HAT medium. You can also make changes to.
  • the medium can be changed every 1 to 4 days in a so-called HT medium excluding aminobuterin.
  • mouse thymocytes can be used as a feeder, which may be preferable.
  • Culture supernatants of hypridoma-growing culture wells such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (FIA), luminescent immunoassay (LIA), Western blotting, etc.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescent immunoassay
  • LIA luminescent immunoassay
  • FACS fluorescence-induced cell separator
  • the production of the monoclonal antibody can be carried out as follows.
  • the hyperidoma is cultured according to a normal method and obtained as a culture supernatant, or the hyperidoma is administered to a mammal that is compatible therewith to proliferate.
  • the method of obtaining the ascites is adopted.
  • the former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.
  • the obtained hybridoma strain can be cultured in an appropriate growth medium such as an FCS-containing MEM medium or RPMI-1640 medium, and a desired monoclonal antibody can be obtained from the medium supernatant.
  • an appropriate growth medium such as an FCS-containing MEM medium or RPMI-1640 medium
  • ascites can be used to make hyperpridoma.
  • each hybridoma is transplanted and proliferated in the abdominal cavity of a histocompatibility animal syngeneic with the animal derived from myeoma cells, and each hybridoma is transplanted and proliferated, for example, in a nude mouse.
  • the monoclonal antibody produced in the ascites of the animal can be recovered and obtained.
  • animals Prior to transplantation of hyperidoma, animals can be administered intraperitoneally with mineral oils such as pristane (2, 6, 10, 14-tetramethylpentadecane). Can also be collected. Ascites fluid is used as it is, or conventionally known methods such as salt precipitation such as ammonium sulfate precipitation, gel filtration using cephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity ⁇ It can be purified and used as a monoclonal antibody by a chromatography method, high-speed: night body matrix chromatography method, or the like.
  • mineral oils such as pristane (2, 6, 10, 14-tetramethylpentadecane).
  • Ascites fluid is used as it is, or conventionally known methods such as salt precipitation such as ammonium sulfate precipitation, gel filtration using cephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity ⁇ It can be pur
  • the ascites containing the monoclonal antibody can be purified and separated by treatment with anion exchange gel such as DEAE-Sepharose and an affinity column such as Putin A column after fractionation with ammonium sulfate.
  • anion exchange gel such as DEAE-Sepharose
  • an affinity column such as Putin A column after fractionation with ammonium sulfate.
  • the affinity chromatography on which an antigen or antigen fragment (for example, a synthetic peptide, recombinant antigen protein or peptide, or a site specifically recognized by an antibody) is immobilized, or a protein A is immobilized. Tee 'chromatograph T / JP2005 / 013792
  • Transgenic mice or other organisms such as other mammals can also be used to express antibodies such as humanized antibodies against the immunogenic polypeptide product of the present invention.
  • nucleic acid sequence encoding the monoclonal antibody can be isolated and sequenced by conventional techniques such as using an oligonucleotide probe that can specifically bind to a gene encoding the heavy chain or light chain of a mouse antibody. .
  • the DNA can be placed in an expression vector and placed in a host cell such as CHO or COS.
  • the DNA can be modified, for example, by replacing it with a sequence encoding a human heavy chain or light chain constant region domain in place of the homogeneous mouse sequence (Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6581,
  • the antibody can be modified by applying a chemical protein synthesis technique including the use of a condensing agent as described below to prepare a chimeric antibody or a hybrid antibody.
  • Humanized antibodies can be performed by techniques known in the art (eg, Jones et al., Nature, 321: pp. 522-525 (1986); Riechmann et al., Nature, 332: pp 323-327 (1988); Verhoeyen et al., Science,
  • Human monoclonal antibodies can also be performed by techniques known in the art, and human myeloma cells and human 'mouse heteromyeloma cells for producing human monoclonal antibodies are known in the field ( Kozbot, J. Immunol., 133, pp. 3001 (1984);
  • bispecific antibodies are also known in the art (Millstein et al., Nature, 305: pp. 537-539 (1983); W093 / 08829; Traunecker et al., EMBO J., 10: pp. 3655-3659 (1991); Suresh et al., "Methods in Enzymology ", Vol. 121, pp. 210 (1986)).
  • these antibodies may be used as antibody fragments such as Fab, Fab ′, F (ab ′) 2 obtained by treating with an enzyme such as trypsin, papain, pepsin and, if necessary, reduction.
  • the antibodies can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 ( CRC Press, Inc., 1987).
  • any method known in the art can be used to conjugated the antibody to each detectable group, for example, David et al., Biochemistry, 13 ⁇ , pages 1014-1021 (1974); Pain et al, J. Immunol. Meth., 40: pp. 219-231 (1981); and “Methods in Enzymology”, Vol. 184, pp. 138-163 (1990);
  • an antibody to which a labeling substance is given an IgG fraction, or a specific binding part Fab ′ obtained by reduction after pepsin digestion can be used.
  • enzymes peroxidase, alkaline phosphatase or 3-D-galactosidase
  • chemical substances fluorescent substances
  • radioisotopes etc.
  • the detection in the present invention can be measured by immunostaining, eg tissue or cell staining. , Immunity electron microscope, Imunoatsuse E.g. competitive type immunoassay or non-competition Radioimmunoassay (RIA), FIA, LIA,
  • EIA, ELISA, etc. can be used, and the measurement can be performed with or without BF separation.
  • RIA, EIA, FIA, and LIA are preferable, and a sandwich type assembly is also preferable.
  • the amount of label measured is proportional to the amount of antigen, ie galectin-9 polypeptide antigen.
  • this assembly it is called a simultaneous sandwich type assay, a forward sandwich assay or a reverse sandwich assay depending on the order of addition of the insolubilized antibody or the labeled antibody.
  • washing, agitation, shaking, filtration, or pre-extraction of antigen are appropriately employed in the measurement process under specific circumstances.
  • Other measurement conditions such as the concentration of a specific reagent, buffer, etc., temperature or incubation time can be varied according to factors such as the concentration of antigen in the sample and the nature of the sample.
  • a person skilled in the art can perform the measurement by appropriately selecting the optimum conditions effective for each measurement using a normal experimental method.
  • Various carriers capable of immobilizing an antigen or antibody are known, and can be appropriately selected and used in the present invention.
  • Various carriers used for antigen-antibody reactions and the like are known.
  • these carriers can be selected and used.
  • Particularly preferably used are, for example, glass, activated glass such as amino alkyl silyl glass, porous glass, silica gel, silica-alumina, anolemina, magnetized iron, magnetized alloy and other inorganic materials, polyethylene , Polypropylene, Polyvinyl chloride, Polyvinylidene fluoride, Polyvinyl, Poly (vinyl acetate), Polycarbonate, Polymetatalylate, Polystyrene, Styrene-butadiene copolymer, Polyacrylamide, Cross-linked polyacrylamide, Styrene-one methacrylate copolymer, Polyglycidyl metatalylate, acrolein ethylene glycol dimethacrylate copolymer, etc., cross-linked
  • filter paper beads, tubes, cuvettes, inner walls of test vessels, for example, test tubes, titer plates, titerwells, microplates, glass cells, cells made of synthetic materials such as synthetic resin cells, glass rods, synthetic materials And rods with thickened or thinned ends, rods with rounded or flattened ends, and surfaces of solid substances (objects) such as thin-plate-shaped rods.
  • Antibodies can be bound to these carriers, and preferably antibodies (including antisera and purified antibodies) that react specifically with the antigen obtained in the present invention can be bound.
  • the binding between the carrier and those involved in the antigen-antibody reaction may be performed by a physical method such as adsorption, a chemical method using a condensing agent or the like, an activated one, etc. It can be performed by a method using a mutual chemical bonding reaction.
  • Labels include enzymes, enzyme substrates, enzyme inhibitors, prosthetic molecules, proenzymes, enzyme precursors, apoenzymes, fluorescent materials, dye materials, chemiluminescent compounds, luminescent materials, color materials, magnetic materials, metal particles Examples thereof include gold colloids, non-metallic element particles such as selenium colloids, and radioactive materials.
  • enzymes include oxidoreductases such as dehydrogenases, reductases, and oxidases, such as transferases that catalyze the transfer of amino groups, carboxyl groups, methyl groups, acyl groups, phosphate groups, and the like. Examples include hydrolyzing enzymes that hydrolyze bonds, glycoside bonds, ether bonds, peptide bonds, lyases, isomerases, ligases, and the like. Enzymes can also be used for detection by using multiple enzymes in combination. For example, enzymatic cycling can be used. The labeling the Kuraitai elements typical radioactive materials, [32 ⁇ ], ⁇ [ 125 1], [131 1], [3 ⁇ 4], [ "C], which we like [3 3 ⁇ 4].
  • Typical enzyme labels include peroxidases such as horseradish rust peroxidase, galactosidases such as Escherichia coli j3-D-galactosidase, maleate dehydrogenase, glucose-6-phosphodedehyde JP2005 / 013792
  • Alogenous phosphatase such as drogenase, gnorecoxoxidase, g / recoamylase, acetylenorecholinesterase, catalase, urinary intestinal alkaline phosphatase, and Escherichia coli alkaline phosphatase.
  • alkaline phosphatase is used, umbelliferone derivatives such as 4-methyl umbelliferyl phosphate, phosphorylated phenol derivatives such as two-mouth phenol phosphate, enzymatic cyclization system using NADP, luciferin derivatives, It can be measured by using the substrate such as dioxetane derivatives and the resulting fluorescence and luminescence.
  • Luciferin and luciferase systems can also be used. When catalase is used, it reacts with hydrogen peroxide to produce oxygen, which can be detected with an electrode.
  • the electrode may be a glass electrode, an ion electrode using a hardly soluble salt film, a liquid film type electrode, a polymer film electrode or the like. Enzyme labeling can be replaced with piotin labeling and enzyme-labeled avidin (streptavidin).
  • a sensitivity enhancement method known in the art can be appropriately employed, such as using a biotin-avidin system, or using a secondary antibody such as an antibody against an anti-galectin antibody.
  • the sign may use several different kinds of signs.
  • BCIP BCIP
  • a PAK TM DAK0
  • Ampl i Q TM DAK0
  • Al force phosphatase 4-methyl umbelliferyl- / 3-D-galactoside, etc. 05 013792
  • a combination of enzyme reagents such as rugalatatoside, 0-ditropuunol-3-D-galactoside, etc., fenilgaratatoside, ⁇ -D-galactosidase, glucose-6-phosphate'dehydrogenase, ABTS, etc. and glucose oxidase
  • enzyme reagents such as rugalatatoside, 0-ditropuunol-3-D-galactoside, etc., fenilgaratatoside, ⁇ -D-galactosidase, glucose-6-phosphate'dehydrogenase, ABTS, etc. and glucose oxidase
  • quinol compounds such as hydroquinone, hydroxybenzoquinone, and hydroxyanthraquinone
  • thiol compounds such as lipoic acid and glutathione, phenolate derivatives, and phenocene derivatives can be used by the action of enzymes and the like.
  • Fluorescein isothiocyanate such as rhodamine B isothiocyanate, tetramethylrhodamine isothiocyanate (RITC), tetramethylrhodamine isothiocyanate isomer
  • R Rhodamine derivatives such as (TRITC), 7-amino-4_coumarin-3-acetic acid, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, ataridinium salt, lumiferin, norecipherase, ecequorin, etc. Imidazonole, oxalic acid ester, rare earth chelate compound, coumarin derivative and the like.
  • Detection of signals generated including color development and fluorescent light can be performed visually, but a known device can also be used. For example, a fluorescent photometer or a plate reader can be used. In addition, a known apparatus can be used to detect a signal emitted from a radioisotope (isotope). For example, a gamma counter or a scintillation can be used.
  • the reaction can be carried out using a reaction between a pyridine group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, etc. In addition, it can be applied by appropriately selecting from the modified methods.
  • condensing agent that can be used for preparing the immunogenic complex
  • a condensing agent that can be used for binding to a carrier and the like
  • condensing agents include formaldehyde, dartaldehyde, hexamethyle. PT / JP2005 / 013792
  • N-sulfosuccinimidyl 4- N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl) Yes minobenzoate, N-succinimidyl 4- (1 -Maleimidophenyl) butyrate, N- (E-maleimidocaproyloxy) succinate, acid imide (EMCS), iminothiolane, S-acetylmertotosuccinic anhydride, methyl-3- (4'-dithiopyridyl) Examples include propionimidate, methyl-4-mercaptoptyryl imidate, methyl-3-mercaptopropionimidate, N-succinimidyl-S-acetyl mercaptoate.
  • a labeled antibody reagent such as an antiserum, a purified antibody, or a monoclonal antibody in which a substance to be measured is labeled with an enzyme, and an antibody bound to a carrier can be reacted sequentially. It is also possible to react at the same time.
  • the order in which reagents are added depends on the type of carrier system selected.
  • the labeled antiserum, purified antibody, or labeled antibody reagent such as monoclonal antibody should be placed together with the sample sample containing the substance to be measured together in the appropriate test tube.
  • an immunological measurement method is used, and the solid phase carrier used in the measurement can be an antibody.
  • the solid phase carrier used in the measurement can be an antibody.
  • Various materials and forms such as polystyrene, polycarbonate, polypropylene or polyvinyl balls, microplates, sticks, fine particles or test tubes that adsorb proteins well can be selected and used. JP2005 / 013792
  • the measurement can be carried out in an appropriate buffer system so as to maintain an optimum pH, for example, a pH of about 4 to about 9.
  • buffers include, for example, phosphate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer. , Tris-HCl buffer, Veronal buffer and the like.
  • the buffering agents can be mixed and used in any ratio.
  • the antigen-antibody reaction is preferably performed at a temperature between about .0 ° C and about 60 ° C.
  • Anti-serum labeled with enzymes, purified antibodies, antibody reagents such as monoclonal antibodies, antibody reagents bound to carriers, and incubation of substances to be measured should be performed until equilibrium is reached.
  • the reaction can be stopped after a limited incubation process by separating the solid phase and the liquid phase at a time point earlier than the equilibrium of the antigen-antibody reaction is achieved. It is possible to measure the degree of the presence of a label such as an enzyme.
  • the measurement operation can be performed using an automated measurement device, and the measurement is performed by detecting the display signal generated by converting the substrate by the action of the enzyme using a luminescence detector, a photo detector, or the like. It can also be determined.
  • EDTA ethylenediamine tetraacetate
  • a blocking treatment to prevent non-specific binding reactions that are commonly employed in the field or known to those skilled in the art may be applied, such as normal serum and serum proteins such as mammals, albumin, hemoglobin, ovo It can be treated with albumin (OVA), skim milk, fermented milk material, collagen, gelatin and the like. As long as the purpose is to prevent non-specific binding reactions 5 013792
  • Tween 20 trade name
  • Tween 80 trade name
  • NP-40 trade name
  • Triton X100 trade name
  • nonionic surfactants such as Briji
  • amphoteric surfactants such as CHAPS
  • cationic surfactants Those selected from the group consisting of anionic surfactants can be added and used.
  • any form of solution solution, non-fluid sample and the like can be used, preferably a biological sample such as thymus, testis, intestine, kidney, All organs and tissues such as brain, breast tissue, ovary, uterus, prostate, intestine-rectum, stomach, lung, bronchi, spleen cancer, liver, malignant tumors of those organs / tissues, leukemia cells, blood Serum, plasma, joint fluid, cerebrospinal fluid, sputum fluid, bile fluid, saliva, amniotic fluid, urine, other body fluids, cell culture fluid, tissue culture fluid, tissue homogenate, biopsy sample, tissue, cells, etc. .
  • a biological sample such as thymus, testis, intestine, kidney, All organs and tissues such as brain, breast tissue, ovary, uterus, prostate, intestine-rectum, stomach, lung, bronchi, spleen cancer, liver, malignant tumors of those organs / tissues, leukemia cells, blood
  • Protein measurement systems such as EIA, RIA, FIA, LIA, and Western plotting can be used advantageously.
  • a predetermined antibody is used as a solid-phase antibody, and a labeled antigen and an unlabeled antigen (an antigen includes a target protein or a fragment peptide thereof) are used.
  • a labeled antigen and an unlabeled antigen an antigen includes a target protein or a fragment peptide thereof
  • the sadditch method in addition to using immobilized antibodies and labeled antibodies, antibodies can be directly labeled, or antibodies against antibodies can be labeled without being immobilized, It can also be carried out in a solid phase.
  • a sensitivity amplification method for example, in combination with a non-enzyme-labeled primary antibody, a polymer polymer, an enzyme, and a primary antibody are used (envision reagent applied; Enhanced polymer one-step staining (EPOS)).
  • provision reagent applied Enhanced polymer one-step staining (EPOS)
  • EPOS Enhanced polymer one-step staining
  • non-enzyme labeled secondary antibody for example, PAP (peroxidase-antiperoxidase) method and other enzyme and anti-enzyme antibody complex combinations, SABC (avidin-biotinylated peroxidase complex) method, etc.
  • Biological activities involving gal9 include cytotoxic activity against malignant tumor cells, apoptosis-inducing activity against malignant tumor cells, anti-tumor activity against malignant tumor cells (anti-cancer activity), and apoptosis of activated T cells Inducing activity, in particular, apoptosis inducing activity of CD4 positive T cells, immunosuppressive activity, anti-inflammatory effect, anti-allergy effect, etc.
  • protein groups such as Hsp70, Hsp90, and other molecular chaperones, include protein folding or import assisting activity, apoptotic activity, immunoregulatory activity, and chaperone complex forming activity. It is done.
  • Gal9 or gal9 binding protein mentioned above is capable of detecting or measuring the interaction of either be cowpea to detecting or measuring the g Al9 and g Al9 binding protein bioactivity involved.
  • this technology it is possible to screen for inhibitors or promoters of interactions and / or binding between gal9 and gal9-binding proteins, and drug development is possible using the identified inhibitors or promoters. It is.
  • this technology it becomes possible to regulate the binding between gal9 and gal9-binding protein and to control the interaction between them.
  • Physiological phenomena ⁇ Bioactive phenomena can be studied, and pharmaceuticals related to them can be developed.
  • the present invention provides a method for screening a compound that inhibits or promotes the interaction and / or binding between gal9 and a gal9-binding protein. 2005/013792
  • the screening method comprises: (a) (1) HSP70, (2) ATP1B3, and (3) a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence of a protein selected from the group consisting of HSP90 Or a polypeptide, a partial peptide thereof or a salt thereof and (b) a polypeptide containing the same or substantially the same amino acid sequence as that of the gal9 or gal9 variant, or a partial peptide thereof
  • the screening test sample is allowed to coexist in the presence of one polypeptide selected from the group consisting of salts.
  • the substance in group (a) or the polypeptide in group (b) those obtained as recombinant proteins by genetic recombination technology are used, but these polypeptides are co-expressed. Cells or extracts thereof can also be used.
  • the substance of group (a) refers to a substance that can bind to gal9.
  • a test sample can be screened by various methods. For example, (l) (a) HSP70,
  • a method of bringing three members into contact at the same time a method of bringing three members into contact at the same time; Alternatively, a method of contacting with the polypeptide (b) may be considered. Even when cells co-expressing these are used, these methods can be selected by adjusting the expression time of each cell and the contact time between the test sample and the cell.
  • the substance of group (a) is not particularly limited as long as it has an activity of binding to the wild-type gal9 protein.
  • As the g Al9 variants as long has the activity to bind to the wild-type g Al9 binding protein, in the amino acid sequence of gal9 protein wild type, substitution of amino acid residues as described above, deletions and Or a polypeptide having an amino acid sequence modified by addition.
  • test samples include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts such as animals, and cell extracts.
  • test compounds used in test samples are preferably anti-gal9 antibodies, anti-gal9-binding protein antibodies, anti-gal9-binding protein glycan antibodies, enzyme inhibitors, cytokines, compounds with various inhibitor activities. In particular, it may contain synthetic compounds and the like. These compounds may be novel compounds or known compounds.
  • the screening can be carried out according to a usual method for measuring binding activity or biological activity.
  • One aspect of the screening method of the present invention comprises contacting any substance or active polypeptide of group (a), any of the polypeptides of group (b) and a test sample, Next, the interaction between both groups of substances or polypeptides is detected by detecting or measuring the biological activity involving gal9 or gal9-binding protein, and a compound that promotes or inhibits the interaction is selected.
  • the selection step may be omitted.
  • Another aspect of the screening method of the present invention is the substance or active polypeptide of any one of group (a), the polypeptide of any of group (b) and 2005/013792
  • Substances used in the present invention can be used by binding to a support.
  • T in the combination of the purified substance or the roughly purified substance of the group (a) and the polypeptide of the group (b) is bound to a support.
  • the substance may be immobilized on the support by a standard method.
  • the support to which a substance such as a polypeptide is bound include insoluble polysaccharides such as agarose, dextran, cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon. More specifically, commercially available beads and plates produced using these as raw materials are used.
  • a column packed with these may be used.
  • examples include multiwell plates (96-well multiwell plates, etc.) and biosensor chips.
  • a commonly used method using chemical bonding, physical adsorption, or the like may be used.
  • an antibody that specifically recognizes a polypeptide can be bound to a support in advance, and the antibody can be bound to a polypeptide. Furthermore, it can be bound via avidin-piotin.
  • the binding between the substance in group (a) and the polypeptide in group (b) is usually carried out in a buffer solution.
  • a buffer solution for example, phosphate buffer solution, Tris buffer solution and the like are used.
  • Incubation conditions are those that are already used, for example, from 4 ° C to room temperature for 1 second to 3 hours, preferably 3 seconds to 2 hours.
  • incubation is performed for 10 seconds to 30 minutes. Washing after incubation is not particularly limited as long as it does not interfere with protein binding.
  • a buffer containing a surfactant is used.
  • 0.05% Tween20 is used as the surfactant.
  • the substance in group (a) and the polypeptide in group (b) and the test sample are incubated under appropriate conditions and then washed. Binding and non-specific binding can be separated. Then, the binding state between the substance in group (a) and the polypeptide in group (b) may be evaluated.
  • the substance such as a polypeptide to be bound to the support may be either the substance of group (a) or the polypeptide of group (b). That is, when the substance of group (a) is bound to a support, the substance of group (a) is solid-phased, and then the polypeptide of group (b) and a test sample are mixed beforehand. Alternatively, after adding the test sample, the polypeptide of group (b) may be added. In addition, when the polypeptide of group (b) is immobilized on a support, similarly, the substance of group (a) and a test sample are mixed in advance, or after addition of the test sample ( a) A group of substances may be added.
  • a control group may be installed together with a group in which the test sample is brought into contact with a substance such as a polypeptide.
  • a control group there can be a negative control group not containing a test sample, a positive control misalignment group, or both groups.
  • the bound substance may be simply detected, or the bound substance may be measured quantitatively. In these cases, the results obtained in the negative control group without the test sample, the results obtained in the group with the test sample and / or the positive control. JP2005 / 013792
  • the target compound can be detected by comparing the results obtained in the 86 control group.
  • the activity of the target compound can also be quantitatively measured.
  • the target compound can be detected by comparing the value obtained in the negative control group not containing the test sample with the value obtained in the group to which the test sample was applied. If the value obtained is increased or decreased compared to the negative control, it can be determined that the test sample contains the compound of interest.
  • a biosensor using the surface plasmon resonance phenomenon can be used as a means for detecting or measuring the bound substance. Biosensors using the surface plasmon resonance phenomenon can observe the interaction between polypeptides in real time as a surface plasmon resonance signal without labeling with a small amount of polypeptide (for example,
  • biosensor such as BIAcore. That is, the sensor chip on which one of the combination of the substance of the group (a) and the polypeptide of the group (b) is immobilized is contacted with a substance such as the other polypeptide of the combination, and the immobilized one It is intended to detect substances such as polypeptides that bind to the substance as changes in the resonance signal. 2005/013792
  • the sensor chip was activated with an EDC / HS aqueous solution (200 mM EDC (N-ethyl-N '-(3-imethylammopropyl) carbonate hydrochloride), 50 m NHS (N-hydroxysuccinimide)), and then the HBS buffer was activated. Wash the sensor chip with (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% Tween20). Next, an appropriate amount of polypeptide dissolved in HBS buffer is brought into contact with the sensor chip and immobilized.
  • EDC EDC
  • NHS N-hydroxysuccinimide
  • a test sample is injected following the other polypeptide having an interaction with one polypeptide.
  • a control group may be set up together with the group injecting the test sample.
  • a control group there can be a negative control group and / or a positive control group that does not include a test sample.
  • the bound polypeptide can be quantitatively measured as the amount of change in resonance signal. In this case, by comparing the results obtained in the negative control group without the test sample, the results obtained in the group containing the test sample, and the results obtained in the sputum or positive control group, the target Compounds can be detected and determined.
  • any polypeptide can be labeled and the label of the bound polypeptide can be used. For example, in the screening method described above, the other sample is brought into contact with one polypeptide together with the test sample. JP2005 / 013792
  • the test sample and the other labeled polypeptide are preferably brought into contact with one polypeptide bound to the support. After incubation, washing may be performed to detect or measure the bound polypeptide label.
  • the polypeptide used in the present invention can be labeled by a generally known method. Examples of labeling substances include radioisotopes, enzymes, fluorescent substances, biotin z avidin, and the like. Commercially available labeled substances can be used for these labeled substances. Examples of radioisotopes include 32 P, 33 P, m I, 125 I,% “c, 3 3 ⁇ 4.
  • enzymes include alkaline phosphatase, horseradish peroxidase (HRP), 3— Examples include galatatosidase, / 3-Darcosidase, etc.
  • fluorescent substances include fluorescein sothiocyanate (FITC) and rhodamine.
  • the substrate is added, and enzymatic changes of the substrate, such as color development, are detected or measured with an absorptiometer.
  • an absorptiometer In the case of fluorescent substances, detect or measure with a fluorimeter.
  • the target compound can be determined by comparing these results with the values obtained in the control mouth group.
  • Primary antibodies that specifically recognize peptides or sugar can be used.
  • one polypeptide is brought into contact with the test sample and the other polypeptide, incubated with the test sample, and then washed to specifically recognize the bound polypeptide.
  • Detect or measure with primary antibody That is, the test sample and the other polypeptide are preferably brought into contact with one of the polypeptides bonded to the support. After incubation, washing is performed, and the bound polypeptide may be detected or measured with a primary antibody that specifically recognizes the polypeptide.
  • the primary antibody is preferably labeled with a labeling substance.
  • this can be done as follows. That is, a solution containing any of the polypeptides is added to the plate and left overnight. After washing the plate, block it with eg BSA to prevent non-specific binding of the polypeptide. Wash again and add the test sample and the other polypeptide to the plate. At the same time, place a negative control group without test sample and a Z or positive control group, and incubate them. After incubate
  • a primary antibody that specifically recognizes and recognizes the polypeptide for detection or measurement, in the case of a radioisotope, it is detected or measured by liquid scintillation.
  • the substrate is added, and the enzymatic change of the substrate, for example, color development is detected or measured with an absorptiometer.
  • an absorptiometer In the case of fluorescent substances, detect or measure with a fluorometer.
  • the target compound can be determined by comparing these results with the values obtained in the control mouth group.
  • a primary antibody that specifically recognizes another peptide fused with the polypeptide used in the present invention can be used as a means for detecting or measuring the bound polypeptide. For example, the screening method described above 2005/013792
  • one polypeptide is contacted with the other polypeptide together with the test sample, incubated with the test sample, washed, and the other polypeptide fused with the polypeptide is washed. It is detected or measured with a primary antibody that specifically recognizes and recognizes the peptide. That is, the test sample and the other polypeptide are preferably brought into contact with one polypeptide bound to the support. After incubation, washing is performed, and the bound polypeptide may be detected or measured with a primary antibody that specifically recognizes another peptide fused with the polypeptide.
  • the primary antibody is preferably labeled with a labeling substance. Specifically, this can be done as follows.
  • a solution containing any of the polypeptides is added to the plate and left overnight. After washing the plate, block it with eg BSA to prevent non-specific binding of the polypeptide. Wash again and add to the plate the other polypeptide fused to the test sample and the other peptide. At the same time, place negative control group and / or positive control without test sample and incubate them. After incubation, wash and add antibodies against other peptides fused to the added polypeptide along with the test sample. After appropriate incubation, the plate is washed and the polypeptide is detected or measured with a primary antibody that specifically recognizes other peptides fused to the polypeptide.
  • a radioisotope it is detected or measured by liquid scintillation.
  • the substrate is added, and enzymatic changes of the substrate, for example, color development, are detected or measured with an absorptiometer.
  • Detect or measure with a fluorimeter The target compound can be determined by comparing these results with the values obtained in the control group.
  • a primary antibody specifically recognizing the polypeptide used in the present invention and a secondary antibody specifically recognizing the primary antibody are used as means for detecting or measuring the bound polypeptide. Can do.
  • one of the polypeptides is brought into contact with the other sample along with the test sample, incubated with the test sample, then washed and bound to the polypeptide.
  • the peptide is detected or measured with a primary antibody that specifically recognizes the polypeptide and a secondary antibody that specifically recognizes the primary antibody. That is, the test sample and the other polypeptide are preferably contacted with one of the polypeptides bound to the support.
  • the pre-purified polypeptide may be detected or measured with a primary antibody that specifically recognizes the polypeptide and a secondary antibody that specifically recognizes the primary antibody.
  • the secondary antibody is preferably labeled with a labeling substance. Specifically, this can be done as follows.
  • a solution containing either polypeptide is added to the plate and left overnight. After washing the plate, it is probed with eg BSA to prevent non-specific binding of the polypeptide. Wash again and add the test sample and the other polypeptide to the plate. At the same time, place a negative control group and Z or positive control group that do not contain the test sample, and incubate them. After incubation, wash and add primary antibody against other peptides fused with the added polypeptide along with the test sample. After moderate incubation, the plate is washed and then a secondary antibody that specifically recognizes the primary antibody is added.
  • the polypeptide is detected or measured by a secondary antibody that specifically recognizes the primary antibody that specifically recognizes the polypeptide.
  • a secondary antibody that specifically recognizes the primary antibody that specifically recognizes the polypeptide.
  • the substrate is added, and enzymatic changes of the substrate, for example, color development, are detected or measured with an absorptiometer.
  • an absorptiometer In the case of fluorescent substances, detect or measure with a fluorimeter.
  • the target compound can be selected by comparing these results with the values obtained in the control mouth group.
  • a primary antibody specifically recognizing another peptide fused with the polypeptide and a secondary antibody specifically recognizing the primary antibody it is possible to use.
  • one of the polypeptides is brought into contact with the other polypeptide together with the test sample, and incubated with the test sample. After washing, the detected or bound polypeptide is detected by a primary antibody that specifically recognizes another peptide fused with the polypeptide and a secondary antibody that specifically recognizes the primary antibody. taking measurement. That is, the test sample and the other polypeptide are preferably brought into contact with one polypeptide bonded to the support.
  • the secondary antibody is preferably labeled with a labeling substance.
  • this can be done as follows. That is, a solution containing either polypeptide is added to the plate and left overnight. After washing the plate, block it with eg BSA to prevent non-specific binding of the polypeptide. Wash again and add the test polypeptide and the other polypeptide fused to the other peptide to the plate. At the same time, place negative control group and Z or positive control group without test sample, and incubate them. After incubation, wash and add primary antibody against other peptides fused to the polypeptide added with the test sample. After moderate incubation, the plate is washed and then a secondary antibody that specifically recognizes the primary antibody is added.
  • the polypeptide is detected or measured with a secondary antibody that specifically recognizes a primary antibody that specifically recognizes another peptide fused with the polypeptide.
  • a secondary antibody that specifically recognizes a primary antibody that specifically recognizes another peptide fused with the polypeptide.
  • a radioisotope it is detected or measured by liquid scintillation.
  • the substrate is added, and enzymatic changes of the substrate, such as color development, are detected or measured with an absorptiometer.
  • an absorptiometer In the case of a fluorescent substance, it is detected or measured by a fluorometer.
  • the target compound can be determined by comparing these results with the values obtained for the control group. More particularly, the present invention is particularly preferably an ELISA (Enzyme-linked
  • Immunosorbent Assay can be performed as follows. In other words, the protein of group (a) fused with another peptide, such as 6 XHis, is fixed. Dilute with phase buffer (0.1 M NaHC0 3 , 0.02% NaN 3 , pH 9.6). Add an appropriate amount of this diluted solution to each well of a 96-well immunoplate (Nunc) and incubate at 4 ° C.
  • phase buffer 0.1 M NaHC0 3 , 0.02% NaN 3 , pH 9.6
  • wash each well 3 times with a washing puffer prepared to 0.05% Tween 20 in PBS
  • dilution buffer 1% BSA, 0.5% Twreen20, PBS
  • incubate at 4 ° C. to room temperature for 1 second to 3 hours, preferably 3 seconds to 2 hours, more preferably 10 seconds to 30 minutes.
  • wash each well 3 times with washing buffer add mouse anti-gal9 antibody (manufactured by Galfama Co., Ltd.) diluted to 3 ⁇ g / ml with buffer, and incubate for 1 hour at room temperature To do.
  • Wash each well 3 times with wash buffer add 100 ⁇ l Alfifphosphatase-labeled goat anti-mouse IgG antibody (ZYMED) diluted 1000-fold with dilution buffer, and incubate for 1 hour at room temperature Cuprate.
  • the process up to blocking is performed manually, and the subsequent reaction is performed by a robot, which can be automated to achieve high throughput screening. That is, the polypeptide of group (a) fused with another peptide, for example, 6 X His Dilute with (0.1 NaHC0 3 , 0.02% NaN 3 , pH 9.6). Add an appropriate amount of this diluted aqueous solution to each hole of 96-well Imunoblend (Nunc) and incubate at 4 ° C. After washing 3 times each well with wash buffer (PBS to 0 ⁇ 05% Tween20 become as those prepared), 5% BSA dissolved in PBS (manufactured by SIGMA) solution 200 mu 1 was added, 4. Block with C.
  • Biomek 2000 dispenser (Beckman) or Multipipette 96-hole simultaneous dispenser (Sagian) can be used to dispense or remove the solution into each hole of the immunoplate. It can.
  • An EL404 microplate washer (manufactured by Bio Tek) can be used to wash each hole in the immunoplate.
  • a SPECTRA max 250 plate reader (manufactured by Molecular Devices) can be used for measuring the absorbance.
  • the program is set to perform the following operations.
  • each well was washed 3 times with a washing buffer, and fused with the test sample and another peptide diluted with a dilution buffer (1% BSA, 0.5% Tween20, PBS), for example, MBP (maltose binding polypeptide).
  • a dilution buffer 1% BSA, 0.5% Tween20, PBS
  • MBP maltose binding polypeptide
  • wash each well 3 times with wash buffer add 100 ⁇ l of rabbit anti-MBP antiserum (New England Biolabs) diluted 5000-fold with dilution buffer to each well, and incubate at room temperature for 1 hour.
  • Wash each well 3 times with wash buffer add Alfifphosphatase-labeled goat anti-usagi IgG antibody (TAG0) diluted 5000 times with dilution buffer to each well, and incubate for 1 hour at room temperature .
  • TAG0 Alfifphosphatase-labeled goat anti-usagi IgG antibody
  • an antibody used in the present invention a commercially available antibody or an antibody contained in a commercially available kit can be used, or a monoclonal antibody or a polyclonal antibody obtained by a known means can be used.
  • Monoclonal antibodies are immunized using the desired sensitizing antigen according to the usual immunization method, and the resulting immune cells are fused with known parental cells by the usual cell fusion method. It can be prepared by screening a monoclonal nanore antibody-producing cell by a screening method.
  • Antibodies against the galectin 9 binding site of a protein selected from the group consisting of HS? 70, ATPIB3 and HSP90 can also be used.
  • the galectin 9 binding site is a signal sequence that crosses the N-terminal ATPase domain or the endoplasmic reticulum membrane, the substrate recognition domain on the C-terminal side, or the protein in the endoplasmic reticulum.
  • the signal “KDEL” may be selected from the sequence region, or the other region may be a characteristic sugar chain added.
  • the combination of the polypeptide in group (a) and the polypeptide in group (b) by fusion Poribe peptide with specific fluorescent proteins can mosquito 3 ⁇ 4 possible use Rereru fluorescence resonance energy transfer between fluorescent proteins (FRET ⁇ fluorescent resonance energy transfer) (A.
  • the compound that regulates the binding of the polypeptides in groups (a) and (b) by comparing the intensity of the radiation in the presence and absence of the test sample, etc. Will be screened.
  • the fluorescence resonance energy transfer is carried out in the same manner as in (a) a polypeptide of group (A) and a yellow fluorescent protein (YFP), and (b) group of polypeptides. This occurs even in molecules where blue fluorescent protein (CFP) exists across the spacer, and the detection sensitivity in this case can be adjusted by adjusting the length of the spacer.
  • the antibodies against the respective polypeptides are labeled with YFP and CFP, respectively.
  • these forms can also be used in the screening method of the present study.
  • the fusion polypeptide with the fluorescent protein is expressed in cells (co-expression in the case of a bimolecular system). Cells can be used for the screening of the present invention. In this case, it is possible to simultaneously detect or measure the binding of the polypeptides in groups (a) and (b) and to detect or measure the biological activity involving p30.
  • the antibody can be applied to genomics and proteomics technologies by combining with antibodies, transgenic animals, etc.
  • RNAi RNA interference
  • dsR A double-stranded RNA
  • gene polymorphism analysis centered on single nucleotide polymorphisms (SNP), nucleic acid array, gene expression analysis using protein array, gene function analysis, protein-protein interaction analysis, related genes Analysis and pesticide analysis are possible.
  • SNP single nucleotide polymorphisms
  • a cDNA library is used, or DNA obtained by PCR technology is placed on a substrate with a spotting device at a high density, and the sample is analyzed using hybridization.
  • the arraying is performed by attaching DNA to each unique position of a substrate such as a slide glass, a silicon plate, or a plastic plate using a needle or a pin, or using ink jet printing technology. Can be implemented. Data is acquired by observing signals obtained as a result of hybridization on the nucleic acid array.
  • the signal may be obtained from a label such as a fluorescent dye (for example, Cy3, Cy5, BODIPY, FITC, Alexa Fluor dyes (trade name) Texas red (trade name), etc.).
  • a laser scanner or the like can be used for detection, and the obtained data can be processed by a computer system equipped with a program according to an appropriate algorithm.
  • Protein array technology may also use tagged protein products that are expressed in two-dimensional electrophoresis.
  • MS mass spectrometry including enzymatic digestion fragments (MS) (this includes electrospray ionization (ESI), matrix-assisted laser desorption ionization (matrix- assisted laser
  • the software of the present invention is related to the gal9, g al9 binding proteins and their related analogs, derivatives, etc., and antibodies thereto Databases etc. may also be included.
  • DNA eg, DNA encoding gal9 or gal9-binding protein
  • it is used as a DNA fragment or linked downstream of a promoter capable of expressing the DNA in animal cells. Is generally advantageous.
  • a gene construct that is linked downstream of various promoters capable of expressing the DNA derived from an animal with high homology to the animal is used as a fertilized egg of the target animal, such as a mouse.
  • a fertilized egg of the target animal such as a mouse.
  • the mouse is not particularly limited to a pure mouse, and examples thereof include C57BL / 6, Balb / C, C3H, (C57BL / 6 X DBA / 2) F and (OF,).
  • this promoter for example, a virus-derived promoter, a ubiquitous expression promoter such as meta-chinin, etc. can be preferably used.
  • the DNA when the DNA is introduced, it can be recombined into a recombinant retrovirus and used.
  • a mouse fertilized egg into which the target DNA has been introduced can be grown using a foster parent mouse such as ICR.
  • DNA eg, DNA encoding gal9 or gal9-binding protein
  • the presence of the DNA encoding the protein in the germ cell of the animal after DNA transfer means that all the offspring of the animal have the DNA encoding the protein in all the germ cells and somatic cells. .
  • the offspring of this type of animal that inherited the gene has the potential to express the protein in all of its germ cells and somatic cells. '
  • the DNA-introduced animal can be reared in a normal breeding environment as the DNA-bearing animal after confirming that the gene is stably retained by mating. Furthermore, by introducing male and female animals carrying the target DNA, JP2005 / 013792
  • an animal into which the DNA has been introduced is useful as an animal for screening an inhibitor of the protein because the protein is highly expressed. It is also useful as an antisense oligonucleotide capable of inhibiting the expression of the gene, for example, an animal for screening for antisense DNA and the like.
  • This transgenic animal can also be used as a cell source for tissue culture. For example, by analyzing DNA or RA directly in the tissues of transgenic mice, or by analyzing proteins expressed by the gene, such as proteins related to gal9 or gal9-binding proteins. Can do.
  • Cells of the tissue producing the protein are cultured by standard tissue culture techniques, and these are used, for example, blood cells such as brain, thymus, and vascular endothelial cells, blood cells, testis, brain, intestine, kidney and others It is possible to study the function of cells derived from these tissues. Also, by using the cells, it is possible to contribute to drug development that enhances the functions of various tissues. If there is a high-expressing cell line, it is possible to isolate and purify the protein. For example, Brinster, R. L., et al.,; Pro
  • Active ingredient of the present invention e.g., (a) the galectin 9 polypeptide, galectin 9 variant, a partial peptide thereof or a salt thereof, a peptide related thereto, etc. (b) the galectin 9 or galectin 9 variant Or nucleic acid such as DNA encoding the related polypeptide, etc. (c) Galectin 9 binding JP2005 / 013792
  • a predetermined antibody of the present invention a partial fragment thereof (including a monoclonal antibody) or a derivative thereof, (induced by interaction and / or binding between the galectin 9 and galectin 9-binding protein)
  • a compound that controls the activity in question cytotoxic activity, apoptosis-inducing activity, a phenomenon that promotes or inhibits the activity of a specific effect without adversely affecting normal cells, or a tissue or protein
  • a pharmaceutical composition or pharmaceutical preparation, etc. usually alone or mixed with various pharmacologically acceptable formulation aids
  • it is administered in the form of a pharmaceutical preparation suitable for use such as oral administration, topical administration, or parenteral administration, and any dosage form (inhalation method or rectal administration) is used depending on the purpose. Is also included).
  • the active ingredient of the present invention includes various drugs such as an antitumor agent (anticancer agent), a tumor transfer inhibitor, a thrombus formation inhibitor, a joint antipyretic agent, an analgesic, an anti-inflammatory agent and / or an immunosuppressive agent.
  • drugs such as an antitumor agent (anticancer agent), a tumor transfer inhibitor, a thrombus formation inhibitor, a joint antipyretic agent, an analgesic, an anti-inflammatory agent and / or an immunosuppressive agent.
  • drugs can be used without limitation as long as they have advantageous functions, and can be selected from, for example, those known in the art.
  • Parenteral dosage forms may include topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal administration, but can also be administered directly to the affected area. Is also preferred.
  • mammals including humans (eg, intracellular, tissue, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathoracic, intrathecal, infusion, injection Administration to the intestine, rectum, ear drops, eye drops, nose, teeth, skin and mucous membranes, etc.).
  • humans eg, intracellular, tissue, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathoracic, intrathecal, infusion, injection Administration to the intestine, rectum, ear drops, eye drops, nose, teeth, skin and mucous membranes, etc.
  • Specific formulation preparation forms include solution preparations, dispersion preparations, semi-solid preparations, granular preparations, molded preparations, leachable preparations, etc., for example, tablets, coated tablets, sugar-coated preparations, Pills, lozenges, hard capsules, soft capsules, mic mouth capsules, implants, powders, powders, granules, fine granules, injections, liquids 5 013792
  • 101 agents elixirs, emulsions, irrigants, syrups, solutions, emulsions, suspensions, liniments, lotions, aerosols, sprays, inhalants, sprays, ointments, plasters, patches , Pasta, cataplasm, cream, oil, suppository (eg rectal suppository), tincture, skin solution, eye drops, nasal drop, ear drops, coating agent, infusion solution, injection solution Powder preparations for freezing, freeze-dried preparations, gel preparations and the like.
  • suppository eg rectal suppository
  • the pharmaceutical thread and composition can be formulated according to a conventional method.
  • physiologically acceptable carriers for example, as needed, physiologically acceptable carriers, pharmaceutically acceptable carriers, adjuvants, excipients, excipients, diluents, flavoring agents, flavoring agents, sweetening agents
  • Vehicle preservative, stabilizer, binder, pH adjuster, buffer, surfactant, base, solvent, filler, extender, solubilizer, solubilizer, tonicity agent, Emulsifying agent, suspending agent, dispersing agent, thickening agent, gelling agent, curing agent, absorbent, adhesive, elastic agent, plasticizer, disintegrating agent, propellant, preservative, antioxidant, shading Unit dosage form generally required for the practice of a pharmaceutical preparation by using an agent, moisturizer, emollient, antistatic agent, soothing agent, etc. alone or in combination with the protein of the present invention. Can be manufactured.
  • Formulations suitable for parenteral use include sterile solutions or suspensions of the active ingredient and water or other pharmaceutically acceptable media such as injections.
  • water, saline, aqueous dextrose solution, other related sugar solutions, and daricols such as ethanol, propylene glycol, and polyethylene glycol are preferable liquid carriers for injections.
  • a carrier such as distilled water, Ringer's solution, physiological saline, an appropriate dispersing agent or wetting agent and suspending agent, etc., using a method known in the art, Prepare in the form of an injection such as a solution, suspension, or emulsion.
  • aqueous liquids for injection examples include physiological saline, isotonic solutions containing buducose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and are pharmacologically acceptable.
  • a suitable solubilizer 2005/013792
  • alcohol eg, ethanol
  • polyalcohol eg, propylene glycol, polyethylene glycol
  • nonionic surfactant eg, polysonolate 80 TM, HC0-50, etc.
  • oily liquid examples include sesame oil and soybean oil, and it may be used in combination with benzyl benzoate, benzyl alcohol or the like as a solubilizing agent.
  • a buffer for example, phosphate buffer, sodium acetate buffer, etc.
  • a reagent for adjusting osmotic pressure for example, a soothing agent (for example, salt benzalkonium, hydrochloric acid hydrochloride, etc.)
  • a soothing agent for example, salt benzalkonium, hydrochloric acid hydrochloride, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • acid inhibitors such as ascorbic acid, absorption enhancers, etc. Les.
  • the prepared injection solution is usually filled in a suitable sample.
  • solutions in sterile pharmaceutically acceptable liquids such as water, ethanol or oil, with or without the addition of surfactants and other pharmaceutically acceptable auxiliaries
  • oily vehicles or solvents used in the formulation include natural, synthetic or semi-synthetic mono-, di- or triglycerides, natural, semi-synthetic or synthetic fats or fatty acids, such as peanuts And vegetable oils such as oil, corn oil, soybean oil and sesame 3 ⁇ 4.
  • this injection can be prepared so that it usually contains about 0.1 to 10% by weight of the compound of the present invention.
  • Formulations suitable for topical, e.g. oral, or rectal use include, for example, mouth washes, dentifrices, oral sprays, inhalants, ointments, dental fillers, dental coatings, dental pastes, seats Agents and the like. Mouthwashes and other dental preparations are prepared by the 'I Award' method using a pharmacologically acceptable carrier.
  • a pharmacologically acceptable carrier As an oral spray and an inhalant, the compound of the present invention itself or a pharmacologically acceptable inert carrier can be dissolved in an aerosol or nebulizer solution, or administered as an inhalable powder to teeth.
  • Ointments are commonly used bases such as ointment bases (white petrolatum, paraffin, JP2005 / 013792
  • Additives include, for example, buffering agents such as sodium bisulfite or disodium edetate; antiseptics including antibacterial and antifungal agents such as acetic acid or phenylmercuric nitrate, benzalkonium chloride or oral hexidine A thickening agent such as hypromelrose.
  • Suppositories are carriers well known in the art, preferably suitable non-irritating excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc., preferably solid at room temperature but intestinal At temperature, it is prepared by a conventional method using a liquid that melts in the rectum to release the drug, etc., but usually prepared to contain about 0.1 to 95% by weight of the compound of the present invention. Is done. Depending on the excipient and concentration used, the drug can be suspended or dissolved in the excipient. Adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • suitable non-irritating excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc.
  • suitable non-irritating excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc.,
  • Preparations suitable for oral use include solid compositions such as tablets, pills, capsules, powders, granules, troches, and liquid compositions such as liquids, syrups, and suspensions. Etc.
  • formulation adjuvants known in the art are used. Tablets and pills can also be manufactured with an enteric coating.
  • the dispensing unit form is a capsule, the material of the above type can further contain a liquid carrier such as fat or oil.
  • the nucleic acid such as the DNA of the present invention is used as a therapeutic and / or prophylactic agent as described above, the nucleic acid can be used alone or in a genetic recombination technique as described above. It can be used by binding to a suitable vector such as a virus-derived vector such as a retrovirus-derived vector. Nucleic acids such as the DNA of the present invention are generally known. JP2005 / 013792
  • the drug can be administered as it is, or it can be used as it is or, for example, in combination with an appropriate adjuvant or physiologically acceptable carrier so as to promote uptake into cells. It can be administered as a pharmaceutical composition or a pharmaceutical preparation as described above. A method known as gene therapy can also be applied.
  • the active ingredient of the present invention can be administered in a wide range of dosages, and the dosage and frequency of administration are determined by sex, age, weight, general health condition, diet, administration time, Depending on the method of administration, excretion rate, combination of drugs and the condition of the patient being treated at that time, it will be determined in consideration of these and other factors.
  • the additives and preparation methods are described in, for example, the Japanese Pharmacopoeia Editorial Committee, 14th revised Japanese Pharmacopoeia, June 27, 2001.
  • the active ingredient of the present invention has, as described herein, (a) galectin 9 and a variant thereof, (b) galectin 9, a variant thereof and a biological activity substantially equivalent thereto.
  • a polynucleotide encoding a polypeptide (c) a substance that promotes interaction and / or binding between galectin 9 and galectin 9 binding protein 'compound, (anti-galectin 9 binding protein antibody, (e) galectin 9 binding Antibodies against protein sugar chains, etc., which show that human galectin 9 has no cytotoxic activity on normal cells but has cytotoxic activity on tumor cells, induces apoptosis on tumor cells However, it does not induce apoptosis in normal cells, suppresses metastasis of malignant cells, activated immune cells, especially activated CD4 Katsusei to induce apoptosis sex T cells (contrary resting T cells In particular, it is useful for utilizing apoptosis of CD4 positive T cells (helper
  • PBS (-) is a phosphate buffered saline solution without Ca 2+ and Mg 2+ , pH 7.4 (phosphate-buffered saline (pH 7.4), Ca 2+ , Mg 2+ free) Yes
  • BSA is bovine serum albumin (bovine serum albumin).
  • CD4 + T cells and CD8 + T cells were separately obtained by MACS (magnetic activated cell sorting) (MACS is Miltenyi Biotec, Auburn,
  • Multisort Kit CD4 CD4 MultiSort Kit: Daiichi Kagaku (Miltenyi Biotec) contains MultiSort CD4 MicroBeads, MultiSort Release Reagent and tiltiSort Stop Reagent
  • Multisort Kit CD8 CD8 MultiSort Kit: Daiichi Kagaku (Miltenyi Biotec) contains MultiSort CD8 MicroBeads, MultiSort Release Reagent and MultiSort Stop Reagent.
  • MNL is separated from human peripheral blood and transferred to a microtube.
  • MNL 10 7/80 1 become as 0. 5% BSA / PBS - add ().
  • MNL is 10 7/20/1 and made by sea urchin MultiSort CD4 MicroBeads ⁇ monoclonal mouse anti- human CD4
  • M0LT-4 T cell-derived cell line
  • ATCC American 'Type' Culture-Collection
  • Cell lines were maintained at 37 ° C. in 5% CO 2 in RPMI-1640 medium (Sigma, St Louis, MO, USA) supplemented with 10% FCS.
  • M0LT-4 was cultured in a flask and used for the experiment. T cells were isolated from CD4 / 8, stimulated with anti-CD3 antibody, and collected with a scraper.
  • Galectin 9 is (His) 6 -Galectin 9 (short type) C (His) 6 -gal-9 (S): PCT / JP03 / 10587 (filing date: August 21, 2003) Disclosure] and modified galectin lacking a linker peptide 9 Cgal9NC (null); plasmid obtained by expression in Escherichia coli (BL21 (DE3)) with pET-G9NC (null) Application No. 2004-94401 (filed on March 29, 2004) was used.
  • the cells should be 10 5 cells / 500 / zl / well (48 well plate, SUMIL0N, Sumitomo Beichi Kite Co., Ltd.), and the final concentration ⁇ ⁇ ⁇ 0 ⁇ 3) 6 319) Ii gal9NC (null) was added and stimulated for 24 hours.
  • the cells were collected, fixed with ethanol, and analyzed for apoptosis by staining with propidium iodide (PI).
  • PI propidium iodide
  • Anti-CD3 monoclonal antibody (a CD3) was obtained from IMMI 0TECH, Marseille, FR.
  • the CD3 solid phase plate was prepared as follows. 3 Prepared at 3 ⁇ g / mL a Add CD3-containing TBS to each well, lniL / wel (24 well plate, CORNING or FALCON), cover with plate seal, and adsorb to plate at 4 ° C. After the reaction, a CD3 is removed, and 1. Wash each well with 2 mL of cold PBS (-). Do this twice.
  • T cell activation was performed as follows. 10% FBS (fetal bovine
  • Serum T cells (CD4, CD8) prepared at 1 X 10 6 cells / ml in RPMI-1640 medium are seeded on a CD3 solid-phase plate, and 20 ⁇ in a 5% C0 2 incubator at 37 ° C. Incubate for 24 hours.
  • TBS refers to 50 nM Tris-HCl (pH 8.0). Distilled water is added appropriately to 5 ml of X 10 TBS (10 mM TBS), adjusted to pH 8.0 with NaOH, and adjusted to 50 ml with distilled water.
  • X 10 TBS 10 mM TBS
  • ImM TBS, pH 8.0 obtained from ImM TBS, pH 8.0 2ral and PBS 38ral
  • Total-A isolation from cells is performed using reagents: TRI z . TM (GIBC0) was used.
  • RNA samples from CD4 + T cells isolated from peripheral blood in [Step 2] and [Step 5] below, and the RNA samples from MOLT-4 cells in [Step 5] are prepared by this method.
  • RNA interference RNA interference
  • the siR A transfection was performed as follows.
  • Opti-MEM I Reduced-Serum Medium (GIBCO) to 5 X 10 4 cells / 500 ⁇ l per well (approx. 10 5 cells / ml) (10 5 / well, 50-80% confluent).
  • the galectin 9-binding molecule was purified from the apoptotic-sensitive cell membrane fraction, and 53 proteins were detected. . That is, obtain the active membrane fraction from the T cell line cultured cell MOLT-4, then obtain the fraction that binds to the gal9CT column, determine the sequence, and select 53 candidate proteins as galectin 9 binding substances I was able to. Sequencing of the proteins in the gal9CT column-bound fraction was carried out by entrusting it to the AproScience.
  • CD4 + T cells were used as cells.
  • CM + T cells were isolated from peripheral blood. The resulting CD4 + T cells were activated with an anti-CD3 antibody (-CD3). This cell was used as a CD4 T cell (+), and a non-activated CD4 T cell ( ⁇ ) was compared between +/ ⁇ .
  • Total RNA was extracted from CD4 T cells (+) and CD4 T cells ( ⁇ ), respectively. Extracted total RNA
  • DNA array analysis was performed. DNA array analysis was performed by Takara Bio Inc. According to Takara Bio Inc., the total RNA of the sample is Agilent
  • RNA of the GeneChip TM expression analysis sample synthesizes cDNA using a primer with a T7 promoter, and then synthesizes CRA by in vitro transcription (IVT) using T7RA polymerase. Labeled by incorporation of nucleotides. Generated cRNA is fragmented and hybridized to GeneChip probe array (using Hybridization Oven), dedicated device
  • Galectin 9 receptor (1) heat shock 70kDa protein 5: HSP70, (2) Sodium / potassium-transporting ATPase beta-3 chain: ATP1B3, (3) 4F2 heavy chaim antigen: 4F2hc (T cell antigen), (4) heat shock 90kDa protein: Four proteins of HSP90 were selected. Since HSP was also present in the cell membrane, intracellular HSP90 was also left as a trap.
  • HSP70 Hs00607129_gH
  • Real-time PCR extracts (1) total RNA from CD4 + T cells isolated from peripheral blood from CD4 T cells activated with anti-CD3 antibody (+) and activated CD4 T cells (-). (2) Experiments comparing total RNA extracted from M0LT-4 cells (induced apoptosis by gal9) and HMC-1 cells (not induced by gal9) And went. Table 2 Cell T cell activation (-/ +) MOLT-4> HMC-1
  • RNAi In order to examine the effect of RNAi on the above candidate genes by knocking down a given gene, prepare siR As for each candidate gene and induce apoptosis using M0LT-4 cells (induced by gal9). The effect was examined. MOLT-4 cells were treated with siRNA of each sequestered gene, and gal9 was added to cells knocked down with a predetermined gene (Knock Down: KD), and apoptotic cells were measured after 24 hours of culture. If there is a knockdown (KD) MOLT-4 cell in which apoptosis induced by gal9 is suppressed, the protein produced by the KD gene is related to apoptosis by galectin-9.
  • KD knockdown
  • M0LT-4 cells KD of 4F2hc gene did not show a significant difference in normal apoptosis from normal M0LT-4 cells.
  • M0LT-4 cells KD of HSP70, HSP90, and ATP1B3 each increased the number of cells that were apoptotic without any stimulation (PBS) compared to M0LT-4 cells that were not genetically manipulated. Admitted. This is expected to indicate that these three proteins function as apoptosis-suppressing substances in cells. Therefore, remove 4F2hc from the galectin 9 receptor
  • HSP70, ATP1B3, and HSP90 were candidates. Table 3
  • Apoptosis induced by gal9 was slightly enhanced in 4F2hc gene KD cells compared to non-genetically engineered cells.
  • MOLT-4 cells KD of HSP70, HSP90, and ATP1B3 all the apoptosis induced by gal9 was suppressed to some extent.
  • HSP70, HSP90, and ATP1B3 are gal9-binding proteins related to apoptosis by galectin-9. That is, it is clear that galectin 9 is a factor that regulates apoptosis of malignant tumor cells, activated T cells and the like via those selected from HSP70, HSP90 and ATP1B3. .
  • HSP70 Tanhaku poor name: heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa), source: human, gene name: HSPA5 (or GRP78 / B1P ), Protein Accession No. NP—005338, mRNA Accession No. NM 005347,
  • ATP1B3 protein name: Sodium / potassium-transporting ATPase Deta-3 chain
  • HSP90 heat shock 90kDa protein 1, beta
  • source human
  • 4F2hc protein Name: 4F2 heavy chain antigen, source: human, gene name: 4F ⁇ hc, or CD98), Protein Accession No. NP 1002385, mRNA Accession No. Ran — 002394.
  • PCT / JP03 / 10587 claiming priority of Japanese Patent Application 2003-16076 filed on January 24, 2003 (Filing date: January 2003)
  • the contents of the disclosure of PCT / JP03 / 10587 and the contents shown in the drawings are all referred to in this specification. Included as part of Similarly, the contents disclosed in this specification are closely related to and based on the contents disclosed in Japanese Patent Application No. 2004-94401 filed on March 29, 2004.
  • Cancer 2 00 4 - all 944 contents shown in bright Saisho and drawing 01 is included as part of the present specification by reference.
  • the candidate galectin 9-binding molecules identified above can be analyzed by analyzing the interaction with galectin 9 as follows, and the most suitable galectin 9-binding molecule can be identified. Can be analyzed and clarified.
  • the cells After adding galectin 9 and reacting with the cells, the cells are lysed and a cell extract is prepared. If a substance that binds to galectin 9 is present in the cell, it will be present in the cell extract in a state of binding to galectin 9.
  • the obtained cell extract is reacted with an anti-galectin 9 antibody attached with a gel or the like to bind the anti-galectin 9 antibody.
  • Electrophorese cell protein extract and transfer to membrane eg P V DF.
  • the distribution of the protein to which galectin 9 binds can be determined (by using cells derived from any tissue, it can be determined in which tissue the binding protein is present). (Use fractions of cytoplasm, cell membrane, etc.) and know molecular weight.
  • galectin 9 For the labeling of galectin 9, a known method can be used without limitation, and for example, there are the following methods.
  • Galectin 9 is labeled with phosphate and detected with anti-phosphorylated antibody (can be phosphorylated using protein kinase).
  • E. Tag Flag, His, GST, etc. is fused to galectin 9 and detected with an antibody against Tag.
  • the target protein is detected by immunoprecipitation or the like using Western glotting with anti-galectin 9 antibody.
  • the labeling method for detection can be the same as the above-mentioned West Western method.
  • the cDNA of the protein that binds to galectin 9 is known from the recombinant phage.
  • galectin 9 Add galectin 9 to the cultured cells, and after culturing, wash the cells to remove nonspecifically bound galectin 9.
  • This system detects the interaction between two proteins in yeast using the transcriptional activity of the reporter gene as an index.
  • the feature of this method is that it can detect interactions between proteins in vivo in yeast, and can perform a sensitive assay that can detect weak temporary interactions.
  • the operation procedure is,
  • MA-AD Transcriptional activating gene
  • reporter genes that detect transcriptional activity are integrated into the yeast chromosome, and various types have been developed and used, but they can be used without limitation.
  • the reporter gene is usually a trophic function gene or lacZ gene.
  • the interaction is detected by restoring auxotrophy on the medium plate and measuring the [3] galactosidase activity.
  • the yeast cells proliferate when they interact with each other, and the conditions are such that the yeast does not increase otherwise.
  • Phage display is used to identify proteins that interact with other molecules.
  • a phage that interacts with a 96-well plate coated with galectin 9 is used to select an phage that expresses a protein that binds more strongly based on the intensity of color development. it can.
  • This Biacore detection system uses surface plasmon resonance (Surface Plasraon resonance).
  • Candidate protein (a) is added to galectin 9 (ligand) on the sensor chip surface 2005/013792
  • a solution containing a candidate protein (referred to as “analyte”) that is likely to act is fed onto the sensor chip at a constant flow rate.
  • Fluorescence polarization is a method that does not fix the interaction between sugar chain-protein, antigen-antibody, or protein in solution or separate tracer molecules.
  • the interaction can be analyzed directly.
  • the principle of this fluorescence polarization method is that when nine fluorescently labeled galectin molecules in a liquid are excited by plane polarized light, they emit polarized fluorescence in the same plane. However, when it is rotated by Brownian motion during the excited state, fluorescence is emitted to a plane different from the excitation plane, and the fluorescence polarization is canceled. In other words, the degree of fluorescence polarization represents the degree to which the fluorescent molecule rotates from when excited until it emits fluorescence. In general, small molecules labeled with fluorescent probes are stimulated by Brownian motion in solution. 3792
  • the identified galectin-9-binding molecule can be used to search for substances that control it according to the technology disclosed in this specification.
  • the present invention provides a technique that utilizes the knowledge that the expression of biological activities such as apoptosis is controlled by the interaction between human galectin 9 and galectin 9 binding protein.
  • human galectin 9 It is possible to screen for interaction regulators between gal9 and gal9 binding proteins, such as compounds that inhibit the binding between proteins, and to develop reagents used for them, and between gal9 and gal9 binding proteins such as identified inhibitors
  • Binding regulators can be expected as pharmaceuticals, and can be used as anti-inflammatory drugs, anti-allergy drugs, immunosuppressants, transplantation immunosuppressants, anticancer drugs, and the like.
  • modified gal9-related peptides and nucleic acids as well as anti-gal9-binding protein antibodies and anti-gal9-binding protein glycosylated antibodies are expected to be useful as active substances for controlling the interaction between gal9-gal9-binding proteins.
  • This technology-Knowledge can be used to develop reagents and bioassays for biological function analysis.

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Abstract

La galectine-9 possède différentes fonctions dépendant de sa localisation. D'autre part, on considère que la galectine-9 participe à diverses fonctions biologiques. Ainsi, il était nécessaire de préciser dans les détails les activités biologiques de la galectine-9 et de développer des techniques comprenant le développement de médicaments. Afin de préciser ces activités, il est nécessaire d'isoler et d'identifier une protéine de liaison à la galectine-9. Comme candidat pour une protéine biologique capable de se lier à la galectine-9 humaine, on peut utiliser une protéine spécifique, c'est à dire, une protéine choisie parmi le groupe constitué de (1) HSP70, (2) ATP1B3 et (3) HSP90. Grâce à l'analyse de l'interaction et/ou de la liaison de la protéine candidate avec la galectine-9, il est possible de développer un agent antitumoral, un agent antiallergique, un immunosuppresseur, un médicament pour des maladies auto-immunes, un agent anti-inflammatoire et un agent de principe actif utilisable comme un substitut pour l'hormone adrénocorticostéroïde.
PCT/JP2005/013792 2004-07-23 2005-07-21 Facteur de proteine de liaison a la galectine-9 WO2006009309A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2434103B (en) * 2006-01-12 2009-11-25 Owen Mumford Ltd Lancet firing device

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002089831A1 (fr) * 2001-04-26 2002-11-14 Protegene Inc. Medicaments preventifs et curatifs contre la nephrite
JP2003189874A (ja) * 2001-12-28 2003-07-08 Galpharma Co Ltd ガレクチン−９活性制御剤
WO2004064857A1 (fr) * 2003-01-24 2004-08-05 Galpharma Co., Ltd. Medicaments contenant galectine 9

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002089831A1 (fr) * 2001-04-26 2002-11-14 Protegene Inc. Medicaments preventifs et curatifs contre la nephrite
JP2003189874A (ja) * 2001-12-28 2003-07-08 Galpharma Co Ltd ガレクチン−９活性制御剤
WO2004064857A1 (fr) * 2003-01-24 2004-08-05 Galpharma Co., Ltd. Medicaments contenant galectine 9

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2434103B (en) * 2006-01-12 2009-11-25 Owen Mumford Ltd Lancet firing device

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