WO2007060747A1 - Facteur d'induction de galectine-9 - Google Patents
Facteur d'induction de galectine-9 Download PDFInfo
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- WO2007060747A1 WO2007060747A1 PCT/JP2005/021942 JP2005021942W WO2007060747A1 WO 2007060747 A1 WO2007060747 A1 WO 2007060747A1 JP 2005021942 W JP2005021942 W JP 2005021942W WO 2007060747 A1 WO2007060747 A1 WO 2007060747A1
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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- 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
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- 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 a factor having a galectin 9-inducing activity, that is, a galectin-9-inducing factor, and particularly to a mammalian galectin-9-inducing factor containing a human galectin-9-inducing factor.
- the present invention also relates to a technique for using the galectin-9 inducer.
- Non-patent Document 1 ekalectin reported by Tureci et al.
- Non-Patent Document 2 ekalectin reported by Tureci et al.
- the inventors' group clarified that ecalectin and galectin 9 are the same substance, and potato galectin 9 has three types: short type, medium type, and long type, depending on the length of the linked peptide. It was also revealed that there are types.
- Non-Patent Document 1 Tureci 0. et al., J Biol Chem., Mar. 7, 1997, 272 (10): 6416-22
- Non-Patent Document 2 Matsumoto R. et al., J Biol Chem., 1998,
- Galectin-9 is a physiologically active substance having activity as a lectin, tissue mast cells, eosinophils, macrophages, T cells, B cells, fibroblasts Its expression has been observed in vascular endothelial cells, various tumor cells, etc., and it is predicted to be involved in various physiological phenomena, such as a correlation between the expression level and the metastatic potential of tumors. It's getting on. Substances that can control the production and release of the galectin-9 can be expected to exhibit activities such as antitumor effects and induction of anti-inflammatory effects. Galectin 9 is thought to be involved in physiological activities important for various living organisms, such as inducing apoptosis of activated T lymphocytes.
- Galectin-9 various physiological phenomena and biological activity phenomena may be controlled.
- Factors that can control the amount of galectin 9 and the expression and release of galectin 9 in the body are expected to be promising as pharmaceuticals.
- the present inventors induced galectin 9 (hereinafter referred to as “Gal-9j ′”) production-release in a certain cell membrane solubilized fraction (hereinafter referred to as “”).
- Ga-9j ′ induced galectin 9
- a factor that induces Ga ⁇ 9 production / release exists in the solubilized fraction of the tumor cell membrane, and that in the mf, Gal-9 producing cells are present at the administration site.
- glycosin 9 inducer there is a factor that induces invasion of cells and the production and release of Gal-9 from those cells, which will be referred to herein as “galectin 9 inducer”.
- the factor by using the factor, it becomes possible to induce an anti-tumor effect or an anti-inflammatory effect.
- the present invention provides the following.
- B cell lymphoma-derived cell line BAL is a galectin-9 inducer that can be identified as having its biological activity in a cell membrane solubilized fraction obtained from one cell, and the bioactivity of the galectin-9 inducer is At least the following:
- Histopathological examination consists of injected eosinophils and mononuclear cells, granulation tissue with a small number of neutrophils is observed,
- a human-derived galectin-9 inducer characterized in that it can be identified by a substance selected from the group consisting of
- [4] Cell membrane solubilized fraction obtained from a cell line derived from B cell lymphoma, consisting of column chromatography such as concanaparin A column chromatography, anion column chromatography, and hydroxylate column chromatography Purification and treatment selected from the group Or galectin 9-inducing factor according to any one of [1] to [3] above, which can be concentrated.
- column chromatography such as concanaparin A column chromatography, anion column chromatography, and hydroxylate column chromatography
- a reagent that induces galectin 9 in a cell comprising the galectin 9-inducing factor according to any one of [1] to [4].
- a method for inducing galectin 9 into a cell comprising bringing the galectin 9-inducing factor according to any one of [1] to [4] above into contact with the cell.
- a pharmaceutical comprising the galectin 9-inducing factor according to any one of [1] to [4] above.
- the galectin-9 inducing factor according to any one of [1] to [4] above, which is derived from human.
- the present invention provides the following.
- Histopathological examination consists of eosinophils and mononuclear cells, and granulation tissue with a small number of neutrophils is observed.
- bioactive agent according to [A] above which is a galectin-9 inducer having an activity selected from the group consisting of
- a reagent that induces galectin 9 in cells which contains the galectin 9 inducing factor described in [A] or [B].
- [D] A method for inducing galectin 9 in a cell, which comprises contacting the cell with a galectin 9-inducing factor according to [A] or [B] above.
- a pharmaceutical comprising the galectin-9 inducer described in [A] or [B] above.
- the galectin 9 inducer was identified and purified according to the present invention, drug development using the purified galectin 9 inducer, physiological phenomena involved in galectin 9, and research and development on biological activity Will progress.
- the galectin 9 inducer can be obtained by concentrating the cell membrane solubilized fraction and the fraction that retains the activity by using a concanavalin A adsorption fraction, Resource Q TM ion exchange column, hydroxypatite column, etc. As obtained. By administering this factor, it is possible to obtain biological activities such as NK-like activity enhancing activity and anti-tumor activity, so that it is possible to develop measuring reagents, pharmaceuticals, and Atsusei using the galectin 9-inducing activity become.
- Figure 1 shows the test results for the antitumor activity of BALL-mf.
- (b) shows the test results on tumor rejection in Meth-A tumor-bearing mice. Note: Number of animals treated with BALL-mf with tumor rejection. Garden: Number of animals treated with Daudi-mf with tumor rejection.
- Fig. 2 is a photograph showing the morphology of a biological tissue stained with Giemsa after excision of the skin at 27 days after treatment of a histopathological examination: Meth-A-bearing mouse.
- BALL-mf processing (a).
- Daudi-mf processing (b).
- Eosinophils are indicated by arrows with an E.
- Fig. 3 shows histopathological examination: The tumor site was excised 27 days after BALL-mf treatment of Meth-A tumor-bearing mouse, and after fixation, Giemsa staining (a) or toluidine blue staining (b) It is the photograph which shows the form of the biological tissue which did. Similarly, a photograph showing the morphology of a biological tissue that was excised from a tumor site of a Daudi-mf-treated mouse, fixed, and stained with Giemsa (c) is also shown.
- Eosinophils are indicated by an arrow with an E
- fertile cells are indicated by an arrow with an M
- neutrophils are indicated by an arrow with an N
- Meth-A cells indicating nuclear enrichment are indicated by an arrow alone. It is shown.
- FIG. 4 is a photograph showing the form of a biological tissue showing the result of histological examination after collecting tissue 24 hours after injection of BALL-mf into the back skin of a mouse and Giemsa staining. Significant eosinophil infiltration is seen with lymphocytes and ⁇ & woven mast cells. When infiltrating cells were examined, many mast cells and eosinophils (arrows) and a small number of lymphocytes were infiltrated.
- Figure 5 shows that when Daudi-mf was injected into the back of the mouse as a control, the infiltration of cells of the lymphocyte system was remarkable, but the infiltration of eosinophils was not observed. No infiltration of mast cells or eosinophils was observed, and infiltration of lymphocytes was prominent
- Figure 6 shows in situ to examine whether the infiltrating cells after BALL-mf injection have galectin-9 mRNA Hybridization was performed. The results were: (a) Full cells had large amounts of galectin 9 mRNA. Eosinophils, macrophages and fibroblasts also had mild. (B) Mast cell infiltration just above the panniculus carnosus muscle and a large amount of galectin 9 mRNA.
- FIG. 7 shows the in vivo effect of the galectin 9 inducer.
- in situ hybridization (A) and immunostaining (B) were examined.
- galectin-9-producing cells were mainly mast cells, and fibroblasts, lymphocytes, eosinophils, etc. had the galectin-9 gene.
- immunostaining (B) was also found by immunostaining (B) that the above cells retain galectin 9 in the cytoplasm.
- Fig. 8 shows the results of examination on the release effect of galectin 9 produced by BALL-mf.
- mRNA was extracted and the amount of galectin-9 niRNA was examined by RT-PCR. It was found that galectin-9 mRNA expression was slightly enhanced by BALL-mf. I understood.
- Western blotting and FACS analysis were performed. FACS analysis revealed that cytoplasmic galectin-9 protein was decreased in BALL-mf stimulated cells. This result suggests that BALL-mf enhances galectin 9 production, but rather induces galectin 9 release.
- Fig. 9 shows the results of examination on the release effect of galectin 9 produced by BALL-mf.
- eosinophil migration activity was measured. As a result, it was found that eosinophil migration activity was enhanced, and that the activity was absorbed by the anti-galectin 9 antibody column, so that release of galectin 9 was enhanced.
- the ability of eosinophil migration by BALL-mf to be absorbed by anti-galectin 9 antibody is not absorbed by anti-galectin 8 antibody. Therefore, it is considered that the action is due to the galactin 9 inducer.
- Galectin 9 production was induced not only by mast cell line cells but also by galectin 9 inducers in eosinophil, macrophage and T cell lines.
- FIG. 10 shows the results of investigating the antitumor activity of BAL mf in vivo.
- BALL-mf was found to inhibit Meth-A tumor engraftment and growth.
- % Square In the test 29 out of 35 animals in the control group (PBS-treated group) and 22 out of 25 animals in the Daudi-mf-treated group were engrafted and proliferated, but 24 out of 30 animals were excluded in the BALL-mf-treated group. Power to be engrafted.
- Fig. 11 is a photograph showing the morphology of the tissue showing the results of immunohistochemical analysis of the tumor tissue.
- immunohistochemical staining was performed using anti-galectin 9 antibody
- BALL-mf-treated group infiltration of mast cells with marked galectin 9 was observed in the surrounding area (A), and mast cells also infiltrated into the tumor.
- Galectin 9 was also expressed in tumor cells (A).
- Daudi-mf treatment group invasion of galectin-9-expressing cells was not observed, and expression in tumor cells was hardly observed.
- Fig. 12 shows the results of examining the apoptosis-inducing activity of Meth-A tumor cells by galectin-9. Galectin 9 induces apoptosis of Meth-A cells.
- Fig. 13 shows the results of examining BALL-mf for purification of galectin 9-inducing factor and its antitumor effect by lentil-lectin affinity.
- galectin 9-inducing activity was mainly observed in the adsorbed fraction.
- the adsorbed fraction showed an antitumor activity comparable to the original.
- the infiltration of eosinophils and mast cells was the same as the original.
- Figure 14 shows the results of examining the isoelectric focusing and antitumor activity of the inducer.
- FIG. 15 shows the results of examining the antitumor activity of the fraction obtained by isoelectric focusing shown in FIG. Strong antitumor activity is induced in F-2 and F-3. F-1 and F-4 are similar to PBS or, conversely, increase tumor cell growth. Anti-tumor activity is seen in the inducers contained in F-2 and F-3. Tissue staining Infiltration of eosinophils and mast cells was observed.
- Figure 16 shows the result of purifying BALL-mf by Con A affinity column chromatography.
- BALL-mf was non-adsorbed with a Con A column, fractionated into adsorbed fractions, and subjected to SDS-PAGE. As a result, different protein bands were observed.
- Fig. 17 shows the results of examining the antitumor effect of the fraction fractionated on the Con A column. Strong antitumor activity was observed by the adsorption fraction. From this, it was found that the inducer exhibiting an antitumor effect is a glycoprotein having mannose or glucose.
- Fig. 18 is a photograph of the tissue showing the results of examining the cytotoxic activity of the BALL-ttif Con A column adsorption fraction.
- FIG. 19 shows the results of examining the antitumor effect of each fraction (A to G) for the fraction obtained by purifying the Con A column adsorption fraction with an anion column (RESOURCE Q).
- FIG. 20 shows the results of examining the antitumor activity by changing the concentration of fraction D obtained as a result of fractionation of the Con A column adsorption fraction using an anion column. Antitumor activity is observed in a concentration-dependent manner.
- Figure 21 shows the fraction obtained after purification by anion column (RESOURCE Q)! ) Is a ligation (elution pattern) fractionated with a hydroxyapatite column (CHT2-1), and the results of electrophoresis of each fraction obtained.
- FIG. 22 shows the results of examining the antitumor activity of each fraction obtained by purification using a hydroxylate column (CHT2-1). At the same time, a photograph showing the result of electrophoresis of fraction D is also shown.
- FIG. 23 is a photograph showing the results (elution pattern) of fractionation on an anion column (RESOURCE Q) and the electrophoresis results of each fraction obtained.
- Figure 24 shows the results (elution pattern) of fractionation of Con A elution fraction using an anion column (RESOURCE Q).
- FIG. 25 is a photograph (Silver stain) showing the result of electrophoresis of a fraction obtained by applying the Con A elution fraction to an anion column (RESOURCE Q).
- FIG. 26 shows the Con A elution fraction on an anion column (RESOURCE Q). It is the photograph (SYPRO ORANGE) which shows the result of the electrophoresis of the obtained fraction.
- FIG. 27 is a photograph showing the results of Western plotting of the fraction obtained by applying the Con A elution fraction to an anion column (RESOURCE Q).
- FIG. 28 is a photograph (Silver stain) showing the result of electrophoresis of a fraction obtained by applying the RESOURCE Q elution fraction to a hydroxypatite column (CHT2-I).
- FIG. 29 is a photograph showing the results of Western blotting of a fraction obtained by applying the RESOURCE Q elution fraction to a hydroxypatite column (CHT2-I).
- FIG. 30 is a photograph showing the results of Western plotting of the fraction obtained by applying the RESOURCE Q elution fraction to the hydroxypatite column (CHT2-I).
- Figure 31 shows the antitumor activity and electrophoresis pattern (photo) results of the RESOURCE Q fraction.
- Figure 32 shows the antitumor activity results of RESOURCE Q fraction D.
- FIG. 33 shows the correspondence between the bands obtained as a result of electrolysis of the fraction eluted with hydroxypatite column (CHT2-1) and each galectin-9 inducer candidate protein. .
- FIG. 34 shows the correspondence between the protein obtained by LC-MS analysis of a fraction sample eluted with a hydroxypatite column (CHT2-I) and the electrophoresis band of the sample.
- Gal-9 producing / free cells include mast cells, eosinophils, macrophages, T cells, B cells, fibroblasts, vascular endothelial cells, various tumor cells, and the like.
- the galectin 9-inducing factor includes mf derived from B cell line (for example, human cell lineine: BALL-l established from human acute lymphoblastoid leukemia (ALL)), its concanalin A absorption Examples include mf eluted in the fraction, mf eluted from the Resource Q ' M ion exchange column, and fraction eluted from the hydroxypatite column.
- the galectin 9-inducing factor can be confirmed by detecting and measuring its biological activity, for example, galectin 9-inducing activity in vitro or in vivo.
- galectin-9-inducing activity in the in vitro mouth can be obtained by stimulating Gal-9 production / free cells as described above with mf, followed by RT-PCR, Western blotting, flow cytometry, immunohistochemical staining, Measured by quantitative or qualitative analysis of Gal-9 mRNA or Gal-9 protein by ELISA, ELISPOT, or RIA.
- Using the cell culture solution of the cells quantitatively determine the Gal-9 protein by RT-PCR, Western blotting, flow cytometry, immunohistochemical staining, ELISA, ELISPOT, RIA, etc.
- galectin-9-inducing activity is also measured by infusion of galectin-9-producing cells and enhancement of Gal-9 release after administration of mf to animals such as mice, rats, guinea pigs, rabbits, and monkeys. It can be measured. It can also be measured using the direct or indirect enhancement of Gal-9 in tumor cells as an indicator. Examples of such animals include experimental animals, and examples of administration methods include intradermal, subdermal, intramuscular, intravenous or arterial, intraperitoneal injection, and eating and drinking.
- the galectin-9 inducing factor is characterized by having an activity of inducing galectin-9 expression.
- the factor is characterized by significantly inducing the expression of galectin 9 by its presence or its expression.
- the factor expresses various physiological activities and / or biological activities through inducing galectin-9.
- the galectin 9-inducing factor of the present invention can be obtained from, for example, a B cell lymphoma-derived cell line BALL-1 cells that have been irradiated with radiation.
- BALL-1 cells that can be used to obtain the galectin-9 inducer of the present invention are independent of the American Type Culture Collection (ATCC), Manassas, Virginia, USA.
- the cell line is cultured in a common medium used for culturing human-derived cells such as RPMI 1640 medium containing 10% fetal calf serum (FCS).
- FCS fetal calf serum
- the culture medium is not particularly limited as long as the cell line can grow.
- a liquid nutrient medium containing saccharides, amino acids, vitamins, other organic nutrients, trace inorganic salts, and the like can be used. .
- the cells are irradiated with radiation as necessary.
- the cells are collected (for example, collected by centrifugation), disrupted in a buffer solution (for example, The cell-free extract is obtained by physical disruption using glass beads, sonication, or biochemical methods using enzymes.
- a buffer solution for example, The cell-free extract is obtained by physical disruption using glass beads, sonication, or biochemical methods using enzymes.
- the galectin-9 inducer of the present invention can be concentrated or separated and purified or purified from a cell membrane solubilized fraction obtained from BALL-1 cells.
- the cell membrane solubilized fraction is obtained by, for example, solubilizing BALL-1 cells by homogenizing with a surfactant in the presence of a protease inhibitor (eg, phenylmethylsulfonyl fluoride), and then centrifuging.
- a protease inhibitor eg, phenylmethylsulfonyl fluoride
- the galectin 9-inducing factor of the present invention can be obtained from the solubilized fraction of the cell membrane, fractionated by the solubility of the protein (precipitation with organic solvent, salting out by ammonium sulfate, etc.), dialysis, cation exchange chromatography, anion exchange. It can be purified by chromatography, gel filtration, hydrophobic chromatography, and affinity chromatography using chelates, dyes, antibodies, etc. alone or in appropriate combination.
- anion exchange chromatography using DEAE-Sepharose affinity chromatography using pull ⁇ -Sepharose, Mono Q HR 5/5 (FPLC system, Amersham Pharmacia Biotech) It can be electrophoretically purified to almost a single band through a high performance liquid chromatography system such as In a typical case, it can be obtained as a nearly single band by polyacrylamide gel electrophoresis.
- the galectin-9 inducer of the present invention is obtained from the cell membrane solubilized fraction of BALL-1 cells by using concanaparin A column chromatography, anion column column chromatography, and hydroxypatite column chromatography.
- Purification and Z or concentration can be achieved by treatment selected from the group consisting of Protein quantification is performed using a commercially available protein assay, and can be performed, for example, by a dye binding method, or can be performed using a protein autoanalyzer.
- the DNA encoding the galectin-9 inducer of the present invention can be subjected to an isolation treatment by the following method, for example.
- the N-terminal amino acid sequence is analyzed.
- the purified product was cleaved with an enzyme such as lysyl endopeptidase or V8 protease, if necessary, and then reverse phase liquid chromatography or the like.
- the amino acid sequence is analyzed using a protein sequencer. In sequence analysis, amino acid sequences can be determined using multiple peptide fragments. Based on the determined amino acid sequence, a primer for PCR is designed, and the chromosomal DNA or cDNA library of the inducer-producing cell
- a part of the DNA of the present invention can be obtained by performing PCR using a PCR primer designed from the amino acid sequence.
- the human genome database GenBank TM, DNA Data Bank of Japan (DDBJ), etc.
- an appropriate program eg, BLAST program, etc.
- a restriction enzyme digest of chromosomal DNA of the inducer-producing cells was introduced into phages, plasmids, etc., and a library or cDNA library obtained by transforming E. coli was used. Utilizing it, desired DNA can be obtained by colony hybridization, plaque hybridization, and the like.
- the base sequence of the DNA fragment obtained by PCR is analyzed, and from the obtained sequence, a PCR primer is designed to extend outside the known DNA, and the chromosomal DNA of the inducer-producing cell is appropriately used.
- a PCR primer is designed to extend outside the known DNA, and the chromosomal DNA of the inducer-producing cell is appropriately used.
- the desired DNA can be obtained by synthesis in addition to genomic DNA or cDNA cloned by the above method.
- the galectin-9 inducing factor of the present invention has at least the following biological activity: (1) galectin 9-inducing activity,
- Histopathological examination consists of eosinophils and mononuclear cells, and granulation tissue with a small number of neutrophils is observed,
- galectin 9-inducing activity can be used as an index. Galectin 9-inducing activity is determined by the change in the amount of galectin 9 present, the change in galectin 9 activity, and the change in galectin 9 expression activity when the galectin 9-inducing factor of the present invention is added and when it is not added. Changes in the amount of galectin-9 mRNA can be identified by caution. Galectin 9 and galectin 9 expression activity can be performed with reference to the assay method disclosed in, for example, WO 02/37114 Pamphlet (W0 02/37114 A1).
- a predetermined nucleic acid such as a polynucleotide using “gene recombination technology”, produce a recombinant, or obtain a predetermined protein / peptide. it can.
- gene recombination techniques that can be used in the present specification include those known in the art.
- oligonucleotide is a relatively short single-stranded or double-stranded polynucleotide, preferably a polydioxynucleotide, Angew. Chem. Int. Ed. Engl. Vol. 28, p. 716-734 (1989), known methods such as phosphotriester method, phosphodiester method, phosphite method, phosphoramidite method, phosphonate method It can be chemically synthesized by such methods.
- oligonucleotide may contain one or more modified bases, for example, It may contain a non-naturally occurring base such as inosine or a tritylated base, and in some cases, it may contain a marker-attached base.
- reaction polymerase chain reaction
- PCR generally refers to the method described in HA Erlich ed., PCR Technology, Stockton Press, 1989, etc.
- a PCR method consists of two oligonucleotide primers that can preferentially hybridize with a truncated nucleic acid. This includes the repeated cycle of primer extension synthesis, typically using primers for the nucleotide sequence to be amplified within the saddle.
- Complementary primers can be used, for example, preferably the nucleotide sequence to be amplified is complementary to both ends thereof or adjacent to the nucleotide sequence to be amplified.
- the primer is preferably an oligo consisting of 5 or more bases, more preferably 10 or more bases.
- Nucleotide, more preferably al include oligonucleotides consisting of between 18 and 25 bases It is.
- the PCR reaction can be carried out by a method known in the art or substantially the same method or modified method.
- a method known in the art for example, Saiki, et al., Science, 230: 1350, 1985; Saiki, et al., Science, 239: 487, 1988; DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IR, Press, Oxford University Press ( 1995);. A. Innis et al. Ed., "PCR Protocols: a guide to methods and applications, Academic Press, New York (1990)); MJ McPherson, P.
- PCR 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.
- the PCR method can be performed using a commercially available kit suitable for the kit manufacturer or kit distributor. It may be thus carried out in the protocol that is more apparent.
- PCR reactions typically involve, for example, a cage (typically DNA) and a primer designed based on the nucleic acid of interest, 10X reaction buffer (attached to Taq DNA polymerase) , DNTPs (mixture of doxynucleoside triphosphates dATP, dGTP, dCTP, dTTP), Taq DNA polymerase and deionized distilled water.
- DNTPs mixture of doxynucleoside triphosphates dATP, dGTP, dCTP, dTTP
- Taq DNA polymerase deionized distilled water.
- PCR cycle conditions include, for example, denaturation 90-95 ° C 5-100 seconds, annealing 40-60 ° C 5-150 seconds, extension 65-75 ° C 30-300 seconds, preferably denaturation 94 ° C
- a cycle of 15 seconds, annealing 58 ° C 15 seconds, extension 72 ° C 45 seconds can be mentioned, but the reaction temperature and time of annealing can be selected appropriately by experiment, and the time of sex reaction and extension reaction Expected PCR An appropriate value can be selected according to the chain length of the product. It is preferable to change the annealing reaction temperature according to the Tm value of the hybrid of the normal primer and the vertical DNA.
- the extension time is usually about 1 minute per lOOOb'p chain length, but a shorter time can be selected in some cases.
- a hybridization technique can be used.
- the hybridization can be carried out by the method described in the literature disclosing the “gene recombination technology” or a method or modification method substantially similar thereto, such as the colony hybridization method.
- the plaque hybridization method, the hybridization.translation assembly method, and the plus / minus method can be used.
- samples containing nucleic acids such as DNA are transferred to a carrier (including a membrane such as a nylon filter), and if necessary, transformation treatment, immobilization treatment, and washing treatment are performed.
- the product transcribed on the carrier (for example, a membrane) is reacted with a labeled probe DNA fragment modified as necessary in a hybridization buffer.
- a labeling kit such as a random primed DNA labeling kit
- the hybridization treatment is usually about 35 ° C to about 80 ° C, more preferably about 50 ° C to about 65 ° C, about 15 minutes to about 36 hours, more preferably about 1 hour to about 24 hours. However, the optimum conditions can be selected as appropriate. For example, the hybridization treatment is performed at about 55 ° C for about 18 hours.
- Hybridize As the buffer for the cyon, it can be selected from those commonly used in the field.
- Examples of the modification treatment of the transferred carrier include a method using an alkali-denatured 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 a temperature of about 40 ° C to about 100T, more preferably about 70 ° C to about 90 ° C, and about 15 minutes to about 24 hours. Is performed by baking for about 1 hour to about 4 hours, but can be carried out by appropriately selecting preferable conditions. For example, the immobilization is performed by baking a carrier such as a filter at about 80 ° C. for about 2 hours.
- a washing solution commonly used in the field such as 50 mM Tris-HCl buffer containing 1M NaCl, ImM EDTA and 0.1% Sodium Dodecyl sulfate (SDS), It can be performed by washing with pH 8.0 or the like.
- the carrier including the membrane can be selected from those commonly used in the field, for example, a nylon filter.
- the alkali-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 L 5M NaCl. Examples of the neutralizing solution include 1.5M NaCl-containing 0.5M Tris-HCl buffer solution, PH8.0, and the like.
- the buffer solution examples include 2 X SSPE (0.36 M NaCl, 20 mM NaH 2 P0 4 and 2 mM EDTA).
- the transferred carrier for example, a membrane
- This prehybridization treatment is performed, for example, by using a prehybridization solution [50% formamide, 5 X Denhardt's solution (0.2% ushi serum albumin, 0.2% polyvinyl pyrrolidone), 5 X SSPE, 0.1.
- the denaturation of the labeled probe DNA fragment used for hybridization is, for example, heating at about 70 ° C to about 100 ° C, preferably about 100 ° C, for about 1 minute to about 60 minutes, preferably about 5 minutes. And so on.
- the hybridization can be carried out by a method known per se or a method analogous thereto, but the stringent conditions in this specification are, for example, about 15 to about 50 mM, preferably about 19 to about 40 mM, more preferably about 19 to about 20 mM, temperature about 35 to about 85 ° C., preferably about 50 to about 70 ° C., more preferably about 60 to about 65 ° C. Indicates.
- 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 the detection process.
- Washing treatment of a carrier such as a filter can be performed by selecting from those commonly used in the field, for example, containing 0.1% SDS 0.5 X SSC (0.15M NaCl, 15m It can be carried out by washing with a solution such as kenic acid.
- Hybridized nucleic acids can typically be detected by autoradiography. However, various technical methods are known in the field, and can be appropriately selected from these methods and used for detection. You can also.
- the nucleic acid bands corresponding to the detected sheet Gunaru a suitable buffer, for example, SM solution (lOOmM NaCl and 10 mM MgSO 4 containing 50 mM Tris-HCl buffer, pH 7.
- nucleic acid can be isolated and purified and subjected to further amplification.
- Samples carrying a given nucleic acid eg, phage particles, recombinant plasmids or vectors
- a given nucleic acid eg, phage particles, recombinant plasmids or vectors
- glycerol gradient ultracentrifugation Molecular cloning, a laboratory manual, ed. T. aniatis, old spring Harbor Laboratory, 2nd ed. 78, 1989.
- DNA can be purified and isolated by methods commonly used in the field.
- ⁇ -out for example, Taumyu and resulting phage solution (10 mM MgSO 4 containing 50 mM Tri s-HCl buffer, pH 7. 8) were suspended in such, after treatment with a DNase I and RNase A, 20mM EDTA, 50 ⁇ Add g / ml Proteinase IV and 0.5% SDS mixture and incubate at 65 ° C for approximately 1 hour. After extraction with phenol extraction jetyl ether, precipitate the DNA by ethanol precipitation. The DNA obtained is washed with 70% ethanol, dried, and dissolved in TE solution (lOmM EDTA-containing lOmM Tris-HCl buffer, pH 8.0). The target DNA can also be obtained in large quantities by subcloning.
- a nucleic acid is a nucleic acid such as single-stranded DNA, double-stranded DNA, RNA, DNA: RNA hybrid, or synthetic DNA, and also genomic DNA, genomic DNA library, cell-derived cDNA, or synthetic DNA. Either may be used.
- the genomic DNA, mRNA screening and galectin 9 expression activity, galectin 9 activity, and even galectin of the target are utilized by utilizing the elucidated structure of the galectin 9 gene and the knowledge of the DNA sequence.
- 9 Probes and primers can be designed to detect inductive activity. As a specific detection probe and primer, it should be possible to specifically detect galectin-9-inducing activity specifically.
- a characteristic sequence portion can be mentioned.
- a portion of the galectin 9 gene can be detected if it is useful for specific detection. What you do is also acceptable. For example, to obtain human lectin 9 by PCR:
- Gal-9 sense sequence CAGGCACCCATGGCTCAAACTAC [SEQ ID NO: 1J
- Antisense sequence TATCAGACTCGGTAACGGGGGT [SEQ ID NO: 2]
- the primer set described in the examples can be used.
- Probes and primers used for detection are preferably nucleic acid fragments or oligonucleotides, which are required to specifically hybridize to a predetermined gene. In cases where detection is effective, those that are effectively bound by hybridization are preferred, and for such purposes, for example, oligonucleotides containing 5 or 10 or more consecutive bases, preferably 15 Or an oligonucleotide containing 25 or more consecutive bases, more preferably an oligo (or poly) nucleotide containing 30 or 50 or more consecutive bases.
- the oligo (or poly) nucleotide having a base sequence capable of effectively hybridizing to the target sequence may have another nucleotide or nucleotide chain added to one or both ends of the selected base sequence.
- Labels may be attached.
- the label may be incorporated, for example, during the PCR process.
- those widely used in the relevant field can be used, and for example, a radioactive substance, a fluorescent substance, a luminescent substance, an enzyme, and the like, and a piotin-avidin system may be used.
- the probe may be labeled for ease of detection.
- PCR and PCR using reverse transcriptase (RT) RT-PCR
- Competitive PCR can also be performed for quantitative measurements.
- a primer consists of a pair of oligonucleotides that define the ends of the sequence to be amplified. From a pair of oligonucleotides composed of one of the oligonucleotides disclosed herein or one of the specific oligonucleotides defined in the present invention and one of the universal primers. It can also be used.
- the primer is used for initiating chain extension of a sequence to be amplified, and can be used not only in PCR methods but also in amplification methods such as LCR method and TAS method.
- the use of the primer is not limited to a specific nucleic acid amplification method, and can be used for various purposes and applications.
- a nucleic acid sample is obtained by the method described in “Gene Recombination Technology”, and if necessary, an amplification reaction is performed using a primer capable of specifically amplifying the target gene. Test whether or not this occurred. Therefore, in the method of the present invention, known nucleic acid extraction methods such as DNA and mRNA or other suitable nucleic acid extraction methods can be used.
- Amplification of the extracted nucleic acid such as DNA or mRNA can be performed by any amplification method such as PCR method, RT-PCR method and the like.
- the product from the amplification operation is detected by, for example, electrophoresis, for example, agarose gel electrophoresis, and the presence or absence of amplified DNA by a conventional method, for example, staining with ethidium bumb amide stain and then UV irradiation. be able to.
- it can be detected by a predetermined probe. For example, when the expression of the galectin 9 gene is not present in the test sample, amplification does not occur or is at a low level.
- a detection method that does not involve separation of amplification products such as blotting and reverse blotting Can also be used.
- Related proteins or polypeptides targeted in this specification, fragments thereof, and nucleic acids (including mRNAs and oligonucleotides) including DNA can be used alone or organically, and even antisense technology can be used. It can be applied to genomics and proteomics technologies in combination with antibodies, including monoclonal antibodies, and recombinant cells (transformants). It is possible to perform gene expression analysis, gene function analysis, protein-protein interaction analysis, and related gene analysis using nucleic acid arrays and protein arrays.
- nucleic acid array technology a single cDNA library is used, or DNA obtained by PCR technology is placed on a substrate with high density using a spotting device, and sample analysis is performed using hybridization. Is done.
- the array is formed by attaching DNA to each unique position on a substrate such as a slide glass, a silicon plate, or a plastic plate using a needle or pin, or using ink jet printing technology. Can be implemented. Data is acquired by observing a signal obtained as a result of high pre-crystallization 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 can also use tagged protein products, such as two-dimensional electrophoresis (2-DE), mass spectrometry including enzymatic digestion fragments (MS) Electrospray ionization (ESI), matrix-assisted laser (matrix-assisted laser)
- the present invention may also include an enzyme gene system obtained or usable as described above, and software and databases related to antibodies against it. Detection and measurement in the present invention can be performed by immunostaining such as tissue or cell staining, immunoelectron microscopy, immunoassay such as competitive immunoassay or non-competitive immunoassay, and radioimmunoassay (RIA), FIA , LIA, EIA, ELISA etc.
- immunostaining such as tissue or cell staining, immunoelectron microscopy, immunoassay such as competitive immunoassay or non-competitive immunoassay, and radioimmunoassay (RIA), FIA , LIA, EIA, ELISA etc.
- sandwich type assembly one is an antibody against the galectin-9 polypeptide of the present invention or an antibody against the related peptide fragment of galectin 9, the other is an antibody against the C-terminal residue of galectin 9, and one is Label detectably (of course, other combinations are possible and can be designed as appropriate depending on the purpose).
- Other antibodies that can recognize the same antigen are immobilized on the solid phase. Incubate the sample to react with the labeled antibody and the solid-phased antibody sequentially as necessary. After separating the unbound antibody, measure the labeled product.
- 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 assembly, a forward sandwich-type assembly or a reverse sandwich-type assembly according to the order of addition of the insolubilized antibody or the labeled antibody. For example, 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 measurement by appropriately selecting optimum conditions effective for each measurement using a normal experimental method.
- a measurement system for galectin-9 for example, tissue immunostaining
- the competition method uses an anti-galectin 9 antibody as a solid-phase antibody, and uses a labeled antigen and an unlabeled antigen (such as galactin 9 or its fragment peptide).
- an unlabeled antigen such as galactin 9 or its fragment peptide.
- the non-competitive method for example, the sandwich method, the immobilized anti-galectin 9 antibody and the labeled anti-galectin 9 antibody can be used, and the anti-galectin 9 antibody can be directly labeled or not immobilized.
- An antibody against the anti-galectin 9 antibody can be labeled or immobilized.
- a polymer polymer, an enzyme, and a primary antibody are used.
- a non-enzyme labeled secondary antibody for example, a combination of enzyme and anti-enzyme antibody complex such as PAP (peroxidase-antiperoxidase) method, SABC (avidin-biotinylated peroxidase complex) method, etc.
- PAP peroxidase-antiperoxidase
- SABC avidin-biotinylated peroxidase complex
- Biotin-labeled secondary antibody and piotin-labeled enzyme-avidin complex Biotin-labeled secondary antibody and piotin-labeled enzyme-avidin complex, ABC i ⁇ streptavidin-biotin complex) method, LSAB labeled streptavidin-biotin) method, etc.
- Combination of avidin complex CSA (catalyzed signal amplification) method, etc.
- an immunological measurement method is preferably used.
- the solid phase carrier is made of polystyrene that adsorbs proteins such as antibodies well, made of polystrength, and made of polypropylene.
- various materials and forms such as polyvinyl balls, microplates, sticks, fine particles or test tubes can be arbitrarily selected and used.
- 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-monohydrochloride buffer, Naruguchi 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.
- galectin 9 inducer activity can be detected.
- the galectin 9 expression gene measurement system using these technologies, the reagents, methods, and processes used for it are all included in the galectin 9 inducer activity detection agent, galectin 9 inducer activity detection method of the present invention, and the system used therefor It is.
- Such in situ hybridization may include, for example, non-RI in situ hybridization, which may include, for example, direct and indirect methods.
- 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. Is.
- Functional groups for example, primary aliphatic amino groups, SH groups, etc.
- oligonucleotide for example, oligonucleotide in the nucleic acid probe, and haptens, fluorescent dyes, enzymes, etc. are bound to these functional groups. It may be done.
- Typical examples of the label of the nucleic acid probe include digoxigenin (DIG), piotin, fluorescein, etc., but can be appropriately selected from the labels described in the antibody as described above. Labeling can be used, and labeled antibodies can also be used.
- the nucleic acid probe labeling method can be appropriately selected from methods known in the art. For example, random prime method, nick-translation method, DNA amplification by PCR, labeling Z-tiling method, Examples include in vitro transcription.
- the processed sample it can be used by appropriately selecting from methods known in the art.
- a fluorescent microscope, a phase contrast microscope, a reflection contrast microscope, a fluorescent microscope, a digital imaging microscope, an electron microscope, etc. Can also be used, and flow cytometry can also be used.
- galectin 9 and a galectin 9-expressing gene can be used as a marker for galectin 9 inducer, thereby detecting various forms of galectin 9 inducer activity or galectin 9 inducer detection and Z or measuring agent, galectin 9 inducer Galectin 9 inducer detection and / or measurement method, galectin 9 inducer activity detection or galectin 9 inducer detection and Z or measurement reagent set or system can be created, and purification of galectin 9 inducer ⁇ Identification ⁇ Isolation ⁇ Not only useful in use, they are excellent.
- a method for obtaining a cancer transfer inhibitory effect by inducing the production and release of galectin 9, a reagent, kit, system (including a detection and measurement system), etc. used therefor. Provides antitumor agents, antiallergic agents, immunosuppressive agents, agents for autoimmune diseases, anti-inflammatory agents, and active ingredient agents to replace corticosteroid hormones by controlling the concentration or expression of galectin 9 in vivo it can.
- galectin-9 inducer it can be applied to fields using pharmacological action / biological activity of darcocorticoid.
- Allergies and autoimmune diseases are caused by hyperimmune reactions of CD4 positive T lymphocytes, and steroids and immunosuppressants are used to treat refractory allergies and autoimmune diseases. Since galectin 9 is clearly involved in these reactions, galectin 9 inducer can be expected to show immunosuppressive action, anti-inflammatory action, antiallergic activity, antitumor agent, antiallergic agent, immunity It can be used as an inhibitor, an autoimmune disease agent, an anti-inflammatory agent, and a corticosteroid hormone substitute.
- the active ingredient of the present invention for example, galectin 9-inducing factor, liquid containing the same, etc.
- it is usually used alone or mixed with various pharmacologically acceptable pharmaceutical adjuvants to prepare a pharmaceutical composition or medicine It can be administered as a preparation.
- a pharmaceutical preparation suitable for use such as oral administration, topical administration, or parenteral administration, and depending on the purpose, any dosage form (including inhalation or rectal administration) is used. Also good.
- the active ingredient of the present invention includes various drugs such as antitumor agents (pile cancer agents), tumor transfer inhibitors, thrombus formation inhibitors, joint destruction treatment agents, analgesics, anti-inflammatory agents and Can be used without limitation as long as they have an advantageous function, 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 containing humans eg, intracellular, tissue, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathoracic, intrathecal, infusion, It can be administered to the enema, transrectum, ear drops, eye drops, nose, teeth, skin, 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, elixirs, emulsions, irrigants Syrup, water, emulsion, suspension, liniment, ij, lotion, aerosol, spray, inhalant, spray, ointment, plaster, patch, pasta, cataplasm, cream , Oils, suppositories (eg rectal suppositories), tinctures, skin solutions, eye drops, nasal drops, ear drops, coatings, infusions, liquids for injections, freeze drying Preparations, gel preparations, etc. Can be mentioned.
- the pharmaceutical composition can be formulated according to a usual method.
- physiologically acceptable carriers for example, as needed, physiologically acceptable carriers, pharmaceutically acceptable carriers, adjuvants, excipients, excipients, diluents, flavoring agents, fragrances, sweeteners, vehicles, preservatives, Stabilizer, binder, pH regulator, buffer, surfactant, base, solvent, filler, extender, solubilizer, solubilizer, tonicity agent, emulsifier, suspension Agents, dispersants, thickeners, gelling agents, curing agents, absorbents, adhesives, elastic agents, plasticizers, disintegrants, propellants, preservatives, antioxidants, sunscreen agents, moisturizers, relaxation agents, It is generally accepted by using an antistatic agent, a soothing agent, etc.
- Preparations 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, dextrose aqueous solution, other related sugar solutions, and darikols 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 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 solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.), and are pharmacologically acceptable.
- Suitable solubilizers such as alcohols (eg ethanol), polyalcohols (eg propylene glycol, polyethylene glycol etc.), nonionic surfactants (eg polysorbate 80 TM, HCO-50 etc.) May be used together.
- the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol or the like as a solubilizer.
- a buffer for example, phosphate buffer, sodium acetate buffer, etc.
- a reagent for adjusting osmotic pressure for example, a soothing agent (for example, benzalkonium chloride, hydrochloric acid hydrochloride, etc.), a stabilizer (for example, Human albumin, polyethylene dallicol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants such as ascorbic acid, absorption enhancers, etc.
- the prepared injection is usually filled in a suitable ampoule.
- solutions in sterile pharmaceutically acceptable liquids such as water, ethanol or oil, with or without the addition of surfactants and other pharmaceutically acceptable auxiliaries Alternatively, it is formulated in the form of a suspension.
- the oily vehicle or solvent used may be natural, synthetic or semi-synthetic mono-, di- or triglycerides, natural, semi-synthetic or synthetic fats or fatty acids, such as peanut oil. , Vegetable oils such as corn oil, soybean oil and sesame oil.
- this injection can be prepared so that it usually contains about 0.1 to 10% by weight of the compound of the present invention.
- Preparations suitable for topical, e.g. oral, or rectal use include e.g. mouthwash, dentifrice, oral spray, inhalant, ointment, dental filler, dental coating, dental paste, suppository Etc. Mouthwashes and other dental preparations are prepared by conventional methods using pharmacologically acceptable carriers.
- the oral spray and inhalant can be dissolved in a solution for aerosol or nebulizer together with the compound of the present invention itself or a pharmacologically acceptable inert carrier, or can be administered to teeth as a fine powder for inhalation.
- the ointment is prepared by a conventional method by adding a commonly used base such as an ointment base (white petrolatum, paraffin, olive oil, Macrogol 400, Macrogol ointment, etc.).
- Medications for topical application to teeth and skin can be formulated into solutions or suspensions in appropriately sterilized water or non-aqueous excipients.
- Additives include buffering agents such as sodium bisulfite or disodium edetate; A thickening agent such as hypromelrose.
- Suppositories are carriers well known in the art, preferably non-irritating suitable excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc., preferably solid at normal temperature but intestinal At temperature, it is prepared by a conventional method using a liquid that melts in the rectum to release the drug, but is 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 is suspended in the excipient. ⁇ Can be dissolved or dissolved. Adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
- suitable excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc.
- suitable excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc.
- suitable excipients such
- preparations suitable for oral use include solid compositions such as tablets, pills, capsules, powders, granules, and troches, and liquid compositions such as liquids, syrups, and suspensions. Can be mentioned.
- 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 active ingredient is a protein or polypeptide
- PEG polyethylene glycol
- the immunogenicity and antigenicity of the heterologous compound may be effectively reduced.
- the compound may be provided in a microcapsule device.
- Polymers such as PEG can be ⁇ -amino group of amino terminal amino acid, ⁇ -amino group of lysine side chain, carboxyl group of aspartic acid or glutamic acid side chain, ⁇ -carboxyl group of amino acid of carboxy terminal, or some kind of It can be conveniently attached to activated derivatives of daricosyl chains attached to asparagine, serine or threonine residues.
- PEG reagents useful for reacting with protein amino groups include carboxylic acids, activated esters of carbonate derivatives, in particular the leaving group is N-hydroxysuccinimide, p-nitrotropenol, imidazole, Alternatively, 1-hydroxy-2-butoxybenzene-4-sulfonate may be mentioned.
- PEG reagents containing aminohydrazine or hydrazide groups are useful for reaction with aldehydes generated by periodate oxidation in proteins.
- the active ingredient of the present invention can be administered over a wide range of doses.
- the dose and the number of doses are determined according to the sex, age, body weight, general Depending on the patient's physical condition, diet, time of administration, method of administration, rate of excretion, combination of drugs, .. condition of the patient being treated at the time, these and other factors are taken into account. '' In pharmaceutical manufacturing, the additives and preparation methods are described in, for example, the Japanese Pharmacopoeia Editorial Committee, 14th revised Japanese Pharmacopoeia, June 27, 2001, Yodogawa Co., Ltd.
- the active ingredient of the present invention is as described herein: (a) By controlling the biological activity of galectin 9 through induction of galectin 9 production and release, for example, human galectin 9 is present in normal cells. It shows no cytotoxic activity, shows cytotoxic activity against tumor cells, induces apoptosis against tumor cells, does not induce apoptosis in normal cells, and suppresses metastatic properties of malignant cells Properties, activity to induce apoptosis of activated immune cells, especially activated CD4 positive T cells (in contrast to induction of apoptosis of resting T cells, especially CD4 positive resting T cells (helper T cells)) It does not induce properties) and is useful on using antitumor agents, antiallergic agents, immunosuppressive agents, autoimmune disease agents Anti-inflammatory agents, is promising as a drug utilizing activity similar adrenocortical steroids hormones.
- the cytotoxicity of natural killer cells can be measured.
- NK natural killer
- a typical method for measuring cytotoxicity is from cells The cell damage is measured by measuring the amount of lactate dehydrogenase (LDH) released.
- LDH lactate dehydrogenase
- the released LDH is then assayed based on the activity of dehydrogenating the lactic acid to produce pyruvic acid and NADH.
- the produced NADH reduces the tetrazolium salt with a diaphorase catalyst to form a red formazan dye having an absorption of 490 nm, so that an increase in the amount of absorbance at 490 nm can measure the activity of LDH.
- the number of cells that are damaged by dead cells or cell membranes appears as an increase in LDH enzyme activity in the culture supernatant, and thus the cytotoxic activity is measured.
- mononuclear leukocytes acquire (mononuclear leukocyte MNL), stimulate or control the cell (3 X 10 6 cells / mL) with the active substance (e.g., BALL-mf, such as IL-2) Containing 10% FCS supplemented with an appropriate medium (for example, antibacterial antimycotic solution (Sigma chemicals, St. Louis, MO, USA)). Incubate in RPMI 1640 medium). After culturing, the cells are used as effector cells for the target cells.
- the active substance e.g., BALL-mf, such as IL-2
- FCS fetal bovine serum
- an appropriate medium for example, antibacterial antimycotic solution (Sigma chemicals, St. Louis, MO, USA)
- target cells K562 is Na 2 51 Cr0 4 (Dai ichi Radioisotope Laboratories, East; specific ⁇ , lmCi / mL) label was treated with a (50 Ci / lO 6 cells). Wash the cells twice and incubate at 37 ° C for 30 minutes. Cells are washed and resuspended to 1 X 10 5 cells / mL. Place the labeled cells in each well of a 96-well round bottom microtiter plate (1 x 10 4 cells / well, 3 sets), effector cell: target cell ratio (E: T ratio) 10 ⁇ Incubate with effector cells at 40.
- E T ratio
- galectin-9 By using a galectin-9 inducer, search for and identify galectin-9-binding molecules involved in the function of galectin-9, especially apoptosis-related galectin-9-binding proteins be able to.
- the search target is not particularly limited, and various biological materials can be used.
- As a method for searching for interacting molecules various methods known in the art can be used alone or in any combination. For example, to identify a protein that interacts with galectin 9 from candidate proteins, for example, it can be selected from the following methods and applied. By knowing the interacting protein, it is possible to know the novel function of the target protein as well as the regulatory mechanism, eg, via galectin-9.
- Available methods include (1) immunoprecipitation method, (2) West Western method (West Western method or Far Western method: Far Western method, including ligand 'blotting method), (3) intermolecular Crosslinking method, (4) Expression cloning method, (5) Two-hybrid system, (6) Phage display method, (7) Surface plasmon resonance method, (8) Fluorescence polarization method, etc.
- the present invention is not limited to these methods, and methods known in the art and modifications thereof can be applied.
- Immunoprecipitation is a method in which an antibody specific to a target protein is added to various protein solutions as samples to interact with the target protein.
- the protein is isolated as an immune complex as immunoprecipitation 3 ⁇ 4 / and is identified by SDS-PAGE or Western blotting to determine the presence of a protein that interacts with the target protein. It is effective.
- a specific antibody against the target protein or a specific antibody against a known protein, a resin that wraps an antibody such as protein A or protein G sepharose can be used, and preferably a protein labeled with RI or the like. It is preferable that a solution can be prepared.
- kits can also be used, such as Affi-Prep 10, Affi-Gel Hz (BIO-RAD), NHS Sepharose HP (Pharmacia).
- a fusion protein produced and purified can be used as a protein to be measured.
- antibodies against Tag can be used effectively, and as a recombinant protein, hosts such as E. coli, yeast, and mammalian cells can be used.
- hosts such as E. coli, yeast, and mammalian cells can be used.
- a cell-free translation system for reticulocytes it is also possible to use co-purification when the fusion protein is simply purified by adding an excess amount of the target fusion protein to the protein solution.
- the West Western or Far Western method is a modification of the Western method that uses a probe protein such as a labeled target protein or a known protein instead of an antibody to bind to the protein transferred to the membrane. By doing this, detection and measurement are performed. 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 (Teraki Nomura, et al., "Immunity '92" (cDNA cloning using protein probes), Nakayama. Bookstore, ppl69-175 (1992)). Any protein that can be phosphorylated and labeled with porotin kinase can be used effectively as a probe.
- -Blotting is a method for analyzing proteins that bind to a ligand. It is a method that refers to a type of first western blot, and is detected by RI using a ligand labeled with RI. Detection using streptavidin. Conjugate antibody, detection using a specific antibody and a labeled secondary antibody using an unlabeled ligand. For signs, non-RI signs can be used. For example, Soutar, A. K. Wade, D. P., Protein funct ion: a pract i cal 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 blotting and immunoprecipitation methods. This is an effective technique for analyzing proteins, studying interacting proteins or neighboring proteins (or domains), and analyzing subunit structures such as receptors.
- chemical cross-linking agents see the website of PI ERCE
- any of in the cDNA pools was expressed in cells, the purpose of the protein Takara ⁇ Rui tag (Tag): with a: tan ⁇ click proteins used by professional blanking, interaction
- a specific cDNA is cloned from the used cDNA group, 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. are known in the art. Expression systems are available.
- This method can be referred to, for example, Hiromi Sasaki, “Invincible Biotechnical Series Special Edition / Proceeding with Bio-Experiment”, Chapter 6, Yodosha, 1997.
- a cDNA library such as Agtll, ⁇ ZAP II is transformed into E. coli, etc. It can be screened with a method, and basically it is done by the Far Eastern method.
- an expression vector with a specific cDNA is transferred to the cultured cells, and those that bind to the target protein are selected from the cells in which expression is observed, and then cloned. Can be screened.
- an ELISA method or a FACS (Fluorescence Activated Cell Sorting) method can be suitably used.
- FACS Fluorescence Activated Cell Sorting
- the two-hybrid system utilizes the phenomenon of functional recovery of a 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
- HybriZAP Two-hybrid vector system (Stratagene) can be cited as an example. This method can be referred to, for example, Masaru Yamamoto, Bio Manual Series 1. Basic Technology of Genetic Engineering, Yodosha, 1993, Downward, J., FEBL Lett., 338, 113-117, 1994, etc. .
- the phage display method is to repeat the operation of collecting phages that bind to the target protein using a library with 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 highly specific amino acid sequences. Also, search the protein database from the obtained results, and respond to known proteins It is also possible to choose what to do. This method can be referred to, for example, Smith, GP 6, Scott, J.., Methods in Enzymology, Vol. 217, pp228-257, 1993.
- the surface plasmon resonance method is typically a method developed for the purpose of monitoring the interaction between biomolecules on the sensor chip in real time using the BIAC0RE TM system.
- Sensor chip CM5 Carboxymethyldextran surface sensor chip SA: Streptavidin immobilized in advance
- Sensor chip NTA NTA immobilized, chelated with nickel, poly -What can immobilize His fusion proteins.
- This method is, for example, Setsuko Hashimoto, Bunseki 5, Analysis of biomolecular interaction using surface plasmon resonance phenomenon, pp362-368, 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 in the excited state, the emitted fluorescence becomes a plane different from that of the excitation light.
- the purity of a molecule is affected by its size, it becomes polarized when it becomes a polymer due to the formation of a complex, etc., and the mobility is low when it is a low molecule. Therefore, it is possible to know the interaction by measuring this degree of polarization.
- 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. Search for binding active substances for specific target substances.
- ⁇ Identification can be performed using the affinity column.
- Synthetic peptides, fusion proteins, antibodies, etc. can be used as ligands for affinity columns.
- Protein 801 (-H, GRP94, GRP78, GRP58, and S100 calcium-binding protein, and those selected from the group consisting of their degradation products were used as a galectin-9 inducer derived from humans, 1) Immunoprecipitation, (2) West Western method (West Western method, or Far Western method: Far Western method, including ligand ⁇ blotting method), ( 3 ) Intermolecular crosslinking method, (4) Expression The functions of Galectin 9 by applying cloning methods, (5) Two-hybrid system, (6) Phage display method, (7) Surface plasmon resonance method, (8) Fluorescence polarization method, etc. And properties of proteins that interact with galectin 9 can be examined in detail.
- BALL-1 cells were prepared using B cell lymphoma-derived cell lines, BALL-1 cells and Daudi cells as tumor cells. Solubilization treatment is described in Hirashima, M. et al., Immunol. Letters, 36: 273-281 (1993) and Seki, M. et al., Int. Arch. Allergy Immunol., 114: 2-5 (1997). The method described in) was modified. BALL-1 cells cultured in RPMI 1640 medium containing 10% FCS were used as starting materials.
- Cycle% 50 on ice for 4 minutes (approximately 2 minutes).
- the sonicator treatment was performed for 2 minutes, then rested, and again for 2 minutes.
- the resulting crushed material was centrifuged. Centrifugation is 100,000 G, 1 hour, 4. It carried out on condition of C.
- the pellets obtained by centrifugation were treated with 50 mM Tris-HCl (pH 8.2), 1 mM EDTA, and 1% in the same amount as when the above BALL-1 cells were resuspended in 1 mM PMSF-PBS. It was resuspended in a solution consisting of CHAPS and homogenized. The homogenization treatment was performed for several minutes on ice using a 10 mL or 20 mL Teflon (registered trademark) ⁇ glass homogenizer until the pellets disappeared completely on ice. The resulting product was centrifuged. Centrifugation was performed at 20,000 G (15,000 rpm) for 30 minutes at 4 ° C. The supernatant (Sup (MF)) was collected.
- Optical Density OD
- a solution composed of 50 mM Tris-, HC1 ( ⁇ 8.2), 1 mM EDTA and 1% CHAPS was used.
- the resulting supernatant is thoroughly dialyzed against PBS and then passed through a 0.2 mL pore size filter to obtain a tumor cell membrane solubilized fraction (mf) (BALL-mf fraction). ). It was stored at -80 ° C until use.
- Daudi cells (2 X 10 8 cells / mL) cultured in RPMI 1640 medium containing 20% FCS were treated in the same manner to obtain mf (Daudi- m f fraction) until it was used. Stored at 80 ° C.
- Meth-A sarcoma was used as the target tumor cell.
- Meth-A sarcoma cells are maintained in RPMI 1640 medium containing 10% ushi fetal serum (FBS), lOOU / mL penicillin, 100 ⁇ g / mL streptomycin and 0.25 ⁇ g / mL amphotericin B. It was.
- the cultured cells (1 ⁇ 10 6 cells / 100 in PBS) were inoculated subcutaneously on the back of Balb / c mice. After 3 weeks, the grown tumor was excised, cut into 2 cm pieces, and 10% FBS supplemented with 1 mg / mL collagenase (Type I, Sigme C-0130; Sigma, St. Louis, MO, USA) Contains RPMI 1640 in medium.
- the mixture is homogenized with magnetic stirrer at 37 ° C for 1.5 hours, passed through cotton gauze, then washed twice with PBS, then
- 100 ng / 200 / L BALL-tnf or Daudi-mf was injected subcutaneously around the tumor cell inoculation site of the animal (100 / L / site) PBS was used as a control The injection was repeated every 3 weeks.
- the body weight of the animal and the size of the tumor (short axis, a and long axis, b) were measured 3 times, and the tumor volume (V) was measured by Attia et al., Cancer Res., 26: 1787-
- Sense sequence, hG9S CGTCAATGGCTCTGTGCAGCTGTC [SEQ ID NO: 3]
- a sense sequence mG9SQl: GGTCAGAGTTCAAGGTGATGGTGA [SEQ ID NO: 5]
- Purified polyclonal antibodies (antibodies against human Gal-9) were obtained from rabbits immunized with the C-terminal domain of human Gal-9. The antibody was purified using Gal-9 C-terminal domain-bound Sepharose, and the antibody was confirmed to recognize mouse Gal-9.
- Antiserum ammonium fraction preparation of crude IgG fraction
- the obtained liquid was transferred to a centrifuge tube and centrifuged at 13,000 rpm (RPR-16 rotor, high-speed centrifuge, Hitachi Machine Co., Ltd.) for 30 minutes. The supernatant was discarded and the precipitate was dissolved in PBS (50 mL). The solution was put into a dialysis tube (Dialysis System 27, Wako Pure Chemical Industries, Ltd.) and dialyzed against PBS. After dialysis, transfer the liquid in the dialysis tube to a centrifuge tube. Centrifuge for 30 minutes at 13,000 rpm (RPR-16 rotor). 0.05 mL of 10% (w / v) sodium azide was added per 10 mL of supernatant and stored in a plastic bottle at 4 ° C (crude IgG fraction).
- the diluted crude IgG fraction was poured onto the equilibrated column (flow rate: 1 ml / min), the first 5 mL flowing out of the column was discarded, and the subsequent effluent was collected in a plastic bottle.
- the crude IgG fraction was run, another 5 mL of PBS was run and the effluent was collected in the same plastic bottle.
- the liquid in the plastic bottle was again flowed through the column under the same conditions, and the effluent at that time was also collected in the plastic bottle.
- the column is then washed with 50 mL of PBS (containing 20 olol / L lactose) (flow rate: 2 mL / min).
- GST-recombinant galectin-7 (5-10 mg) immobilized high trap NHS-activation column (5 m, Amersham Biosciences) was connected to a peristaltic pump and washed with 20 mL PBS (flow rate: every minute 2 ml).
- Cell pellets were treated with lysis buffer (10 mM Tris-HCl, 0.15 NaCl, 2 mM EDTA, 2 mM EGTA, and freshly added 0.5 mM PMSF, 10 ⁇ g / mL leupeptin, antipain, pepstatin A and Cell lysate was prepared by sonicating with 1 mM DTT).
- lysis buffer 10 mM Tris-HCl, 0.15 NaCl, 2 mM EDTA, 2 mM EGTA, and freshly added 0.5 mM PMSF, 10 ⁇ g / mL leupeptin, antipain, pepstatin A and Cell lysate was prepared by sonicating with 1 mM DTT).
- the PVDF membrane was washed and incubated for 45 minutes with PBS-T containing peroxidase-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotech).
- the PVDF membrane was immersed in an ECL-HRP substrate solution in an ELC kit (Amersham Pharmacia Biotech), exposed to XJB-1 X-ray film (Kodak), and the band was visualized.
- the cells are collected by centrifugation and washed with PBS (0.05+) containing 0.05% NaN 3 and 2% fetal calf serum (FCS). Incubation was performed for 30 minutes on ice in the presence of / ig / mL of Usagi anti-human Gal_9 antibody. Cells were washed several times with PBS + and incubated with ice for 30 minutes with FITC-conjugated rabbit anti-rabbit IgG antibody (Santa Cruz Biotechnology).
- PBS 0.05+
- FCS fetal calf serum
- Tumor cells were inoculated on day 27 and tumors were excised and weighed. After immobilizing the histopathological sample with 10% neutral buffered formaldehyde solution, the paraffin-embedded tissue was cut into 4 ⁇ thick sections, deparaffinized, rehydrated, and Stained with hematoxylin and eosin or Giemsa reagent (Giemsa s reagen).
- In situ hybridization was performed to examine whether cells accumulated at the site where BALL-mf was injected contained galectin-9 mRNA.
- RNA probes labeled with digoxigenin were synthesized by in vitro transcription using the DIG RNA labeling kit (SP6 / T7; Roche Molecular Biochetnicals, Mannheim, Germany).
- DIG RNA labeling kit SP6 / T7; Roche Molecular Biochetnicals, Mannheim, Germany.
- a PCR-amplified galectin 9 cDNA fragment (bases 500 to 1208 of the nucleotide sequence; Matsumoto, R. et al., J. Biol. Chem., 273: 16976-16984 (1998)) -T Easy Vector (Promege, Madison, WI, USA), cloned and linearized plasmid DNA was used as vertical DNA for in vitro transcription.
- a sense probe and an antisense probe were synthesized, and a sense probe was used as a negative control.
- the hybridization protocol was applied to 4 ⁇ paraffin sections and performed according to the reagent manufacturer's protocol. After digestion with proteinase K for 2 hours at 37 ° C, the hybridization was carried out at 43 ° C with a 1 / ig / mL probe in 20 L of hybridization solution under a coverslip. After washing under stringent conditions, digoxigenin rabenole can be obtained using the digoxigenin detection kit (Roche Molecular Biochemicals). Visualized. As a control, the case where the sense probe was used and the case where the probe was excluded were used.
- the tumor size began to decrease after 2 weeks, and after 18 days, the tumor size was significantly greater than in the Daudi-mf-treated and PBS-treated mice. It was getting smaller (Fig. L (a)).
- Fig. L (a) the tumor size was observed to be rejected on day 20 after treatment with BALL-mf, and on day 22, another 3 mice, day 25 Eyes were also observed in each of four additional mice. Tumors were completely rejected on day 27 in 8 out of 10 BALL-mf treated mice, but only 1 out of 10 in PBS and Daudi-mf treated mice Only one animal was rejected.
- mice injected with BALL-mf consisted mainly of eosinophils (arrows with an E) and mononuclear cells at the site of injection, with a small number of neutrophils. Granulation tissue was observed. Granulation tissue was also observed in mice treated with Daudi-mf, but infiltrating cells were mainly mononuclear cells and not eosinophils (Fig. 2b).
- mice treated with BALL-mf showed inflammatory cells (mainly eosinophils [arrows marked with E]) and some mast cells [marked with M] around the tumor or in the tumor tissue. ), But not neutrophils) (Fig. 3a). Tumor cells showing nuclear enrichment (marked with arrows only) were also found (Fig. 3a). As shown in FIG. 3b, accumulation of mast cells showing metachromatism around the tumor or in the tumor tissue was confirmed.
- the tissue surrounding the tumors of mice treated with Daudi-mf showed significant intracellular infiltration (Figure 3c), but surprisingly, countless neutrophils (N Arrows) and mononuclear cells were found around the tumor tissue. A small number of eosinophils and mast cells were detected at the site, and tumor cells showing nuclear enrichment could not be found (FIG. 3c).
- In situ hybridization was performed to determine the type of cells expressing galectin 9 at the injection site.
- the subcutaneous muscle plate panniculus carnosus muscle
- eosinophils mainly mast cells, other fibroblasts, lymphocytes, and eosinophils produced galectin 9 (Fig. 6a).
- Fig. 6a Normally, no hypertrophic cells were found in the vicinity of the muscle plate, but BALL-mf injection showed infiltration of mast cells with galectin-9 in the muscle plate (Fig. 6b).
- Fig. 6a cells expressing Gal-9 mRNA were found at the site where BALL-mf was injected.
- Meth-A sarcoma tumor-bearing mouse treated with BALL-mf in vivo tumor elimination is seen, the tumor elimination is probably due to natural 'killer (NK) cell activation and galectin 9 production ⁇ release enhancement It is.
- eosinophilia has occurred in the tissue surrounding the tumor. It is well known that there is a correlation between the prognosis of a malignant tumor and the type of cells that infiltrate the tumor supporting tissue. For example, patients with lymphocyte infiltration around the tumor have a good prognosis. This may be the result of lymphoin production and activation of Z or NK cells.
- eosinophils are more cytotoxic than neutrophils, possibly due to the generation of hydroxyl radicals that depend on eosinophil peroxidase. It has also been shown that the attachment of tumoricidal eosinophils to tumor cells is associated with protein kinase activation. In mice treated with BALL-mf, infiltration of eosinophils but not neutrophils was found in the tissue surrounding the tumor. On the other hand, in mice treated with Daudi-mf, neutrophil infiltration was mainly induced (Fig. 2a and Fig. 2b).
- galectin 9 belongs to the galectin family and is a novel and powerful eosinophil chemotractant.
- mast cell infiltration is induced in the tumor and surrounding tissues around the injection site of BALL-mf ( Figures 2 and 6).
- mast cells may be associated with good prognosis as well as eosinophils.
- eosinophils may be involved in antitumor activity mediated by IL-4.
- the present inventors have shown that galectin-9 production by the induction of PPD has been increased by short-term stimulation with IL-4, but IL-5 production from peripheral blood mononuclear cells is suppressed. Show. Since mast cells may be a major source of IL-4 at the site of inflammation, the mast cells at that site may be involved in eosinophil accumulation. Also, based on in situ hybridization results, mast cells appear to be an important source of galectin-9 at the BALL-mf treatment site (Fig. 6).
- NK activity tumor cell line K562 (NK-sensitive cell) and other tumor cell lines (for example, both LAK-sensitive cells Daudi, KMG-2 (Darioblastoma cells), KAT0II I (gastric cancer), etc.) are enhanced.
- NK cell line K562 NK-sensitive cell
- other tumor cell lines For example, both LAK-sensitive cells Daudi, KMG-2 (Darioblastoma cells), KAT0II I (gastric cancer), etc.
- galectin 9 enhances cytotoxicity and NK-like activity (although its activity is low) against Meth-A.
- mice treated with BALL-mf have found nuclear enrichment in some of the tumor cells in close proximity to the fibrous tissue surrounding the tumor, whereas mice treated with Daudi-mf do not.
- the cells shown were not seen ( Figures 3a and 3c).
- galectins play an important role in apoptosis.
- galectin 1 has been shown to induce apoptosis of T cells
- galectin 3 has also been shown to prevent cell death.
- overexpression of galectin 7 may be involved in the apoptotic process of sunburned keratinocytes induced by UVB.
- galectin 9 it has been reported that mouse galectin 9 induces apoptosis of thymocytes and activated T lymphocytes.
- BALL-mf Stimulation of tumor cells with BALL-mf showed no expression of galectin-9 or induction of apoptosis. Galectin 9 itself induced tumor cell apoptosis. Therefore, BALL-mf does not exhibit an antitumor effect by acting directly on tumor cells, but exhibits an antitumor effect by inducing the expression and release of galectin 9 in T cells and mast cells. Was suggested.
- BALL-1 cell-derived membrane solubilized fraction (BALL mf) obtained in Example 1 was used as a starting material for purification.
- the lentil lectin column was divided into a non-adsorbed fraction and an adsorbed fraction, galectin 9-inducing activity was mainly observed in the adsorbed fraction.
- an antitumor activity comparable to the original was observed in the adsorption fraction. Eosinophils and mast cell infiltration were similar to the original results.
- BALL-1 cells cultured in RPMI 1640 medium containing 10% FCS were used as starting materials.
- the sonicator treatment was performed for 2 minutes, then rested, and again for 2 minutes.
- the resulting crushed material was centrifuged. Centrifugation was performed at 100,000 G for 1 hour at 4 ° C.
- the pellets obtained by centrifugation were treated with the same amount of 50 mM Tris-HCl (H8.2), 1 mM EDTA and 1 BALL-1 cells resuspended in 1 mM PMSF-PBS. Resuspended in a solution consisting of% CHAPS and homogenized. The homogenization treatment was performed for several minutes on ice using a 10 mL or 20 mL Teflon (registered trademark) ⁇ glass homogenizer until the pellets disappeared completely on ice. The resulting product was centrifuged. Centrifugation was performed at 20,000 G for 30 minutes at 4 ° C.
- the supernatant (MF) was collected. Absorbance (Optical Density: 0D) was measured. As the blank, a solution consisting of 50 mM Tris-HCl (pH 8.2), 1 mM EDTA and 1% CHAPS was used.
- the eluted Ft was collected. WASH flowed the equilibration buffer in the same amount as the apply volume. Apply Elution buffer, cap and keep at room temperature for 20 minutes. Collect 1 mL each in a tube every 5 minutes. Check 0D (280 nm).
- Buffer A, 10 mM Tris-HCl (pH 7.5), 0.03% CHAPS
- Buffer A, 10 mM Na-Pi (pH 6.8), 0.03% CHAPS, 0.05% NaN 3
- Buffer A, 10 mM Na-Pi (pH 6.8), 0.03% CHAPS
- Buffer B, 500 mM Na-Pi (pH 6.8), 0.03% CHAPS
- Samples were concentrated (X 40) using Strata Clean Resin (Stratagene, CA, USA). Concentrated sample is SDS-PAGE: 12% gel, SYPRO Orange (Mol ecular Probes, Inc., USA)
- BALL-mf was fractionated into a non-adsorbed fraction and an adsorbed fraction using a Con A column, and electrophoresed by SDS-PAGE, and different protein bands were observed (Fig. 16).
- A adsorbed fraction
- B non-adsorbed fraction
- tumor growth was suppressed as compared to PBS, but the tumor was not excluded (Table 1).
- GRP78 Bip / BiP (HSP70 family);
- GRP58 ERp57, PDI (protein disul fide isomerase);
- 80K-H a substrate for protein kinase C, glucosidase II b—subunit, AGE—receptor, participation in FGF signal ing
- GRP94 For GRP94, GRP78, GRP58, 80K-H, which are candidate proteins for galectin 9 inducer, commercially available antibodies that recognize each of them, whether the candidate substance exists in the purified fraction of CHT2-I column Confirmed it. Recognized antibodies include the following.
- Anti-GRP94 MoAb Stressgen, SPA-850
- Anti-GRP94 PoAb Santa Cruz, sc-11402
- Anti-GRP78 PoAb Santa Cruz, sc-13968
- Fig. 33 shows the correspondence between SDS-PAGE bands of fractions obtained by CH 2-1 column fractionation and each galectin-9 inducer candidate protein.
- the pellets obtained by centrifugation were treated with 50 mM Tris-HC1 (pH 8.2), 1 mM EDTA, and 1 in the same amount as when the BALL-1 cells were resuspended in 1 mM PSF-PBS. Resuspended in a solution consisting of% CHAPS and homogenized. The homogenization treatment was carried out for several minutes on ice using a 10 mL or 20 mL Teflon (registered trademark) glass homogenizer until the pellets disappeared completely on ice. The resulting product was centrifuged. Centrifugation was performed at 20,000 G (15,000 rpm) for 30 minutes at 4 ° C (using a SIGMA tabletop centrifuge and spinning into 2 mL tubes).
- BALL-1 mf obtained above was subjected to force ram chromatography using a carrier with Con A as a ligand. Mix Con A Sepharose beads and BALL-l mf and rotate at 4 ° C, 0 / N. BALL-1 mf was diluted 2 times with PBS (-) and mixed. This was then applied to the column. The same amount of equilibration buffer as Wash was applied as Wash. First, the elution buffer was applied to a 2 mL column, held at 4 ° C for 1 hour, and then eluted. Column conditions:
- the BALL-1 mf Con A-fraction sample obtained above was dialyzed with a solution consisting of 10 mM Tris-HCl (pH 7.5) and 0.03% CHAPS.
- Buffer A 10 mM Tris-HCl (pH 7.5), 0.03% CHAPS
- Fig. 26 shows the results of SDS-PAGE SYPR0 ORANGE (M: protein marker, 2-212 kDa; S.
- Buffer A, 10 mM Na-Pi (pH 6.8), 0.03% CHAPS
- M pre-stained protein marker, 6 to 175 kDa
- ST Banol-1 mf Con A-fraction post-dialysis sampnore
- a 280 0.20, 2 ⁇ g / 10 ⁇ L / lane.
- fraction numbers 25-30 a full-length band was observed near 80 kDa.
- bands appearing to be degradation products derived from 80K-H are also seen at around 43 kDa and 36 kDa.
- Figure 34 shows the sample SDS-PAGE bands and the protein names obtained by LC-MS analysis.
- Fraction D which showed the highest antitumor activity among the five fractions (AE) obtained by the hydroxylate column chromatography, had 80K-H as the main component and GRP94 (TRA as the minor component).
- -l / gp96 / E p99 / endoplasmin) GRP78 (BiP), GP58 (ERp57 / PDI), S-100 protein, etc. were included.
- 80K-H was first identified as a protein kinase C substrate in vitro (and later proved not to be a physiological substrate). 80K-H is also glucosidase I I / 3-subunit, AGE-receptor
- 80K-H may be the main body of the inducer (antitumor activity)
- GRP94, GRP78, and GRP58 are endoplasmic reticulum proteins, and there are reports that GRP94 is also present on the cell surface, Similar to 80K-H, it has a glutamate-rich sequence, so these components may also contribute to the activity of fraction D, or may act cooperatively with 80K-H.
- S-100 is a calcium-binding protein and is functionally similar to the EF-hand structure (calcium-binding motif) present in 80K-H. Also, Since Latation D includes those that are considered to be degradation products of these components, it cannot be denied that a specific fragment has a strong activity.
- proteins 80K-H, GRP94, GRP78, GRP58, and S100 calcium-binding protein are provided as factors having galectin 9-inducing activity (galectin 9-inducing factor) or candidate substances thereof.
- galectin 9 inducer the gene sequence (base sequence) and the amino acid sequence encoded thereby can be referred to as follows. A simple test 'analysis can be performed.
- S100 calcium binding protein A8 (calgranulin A) (S100A8)
- S100 calcium binding protein A9 (calgranulin B) (S100A9)
- GRP94 glucose-regulated protein 94
- gp96 tumor rejection antigen 1 (TRAl)
- tumor rejection antigen gp96 tumor rejection antigen 1 [Mus musculus]
- Homo sapiens heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) (HSPA5), mRNA
- heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa); Heat-shock 70kD protein-5 (glucose-regulated protein, 78kD); heat shock 70kD protein 5 (glucose-regulated protein, 78kD) [Homo sapiens]
- Homo sapiens protein kinase C substrate 80K-H PRKCSH
- transcript variant 2 mRNA
- Homo sapiens protein kinase C substrate 80K-H PRKCSH
- transcript variant 1 mRNA
- protein Kinase C substrate 801 (-H isoform 1; glucosidase I I, beta subunit; AGE-binding receptor 2; hepatocystin; 80K-H protein;
- galectin-9 inducer was identified and purified, drug development using the purified galectin-9 inducer, Physiological phenomena involving Kuching 9, which can be used for the development of physical activity,
- galectin 9-inducing factor can be obtained by using cell membrane solubilized fraction 3 ⁇ 45 ⁇ and concentra- tion of concentrated activity by using concanavalin A adsorbed fraction, Resource Q TM ion exchange column, ⁇ hydroxypatite column, etc. Obtained as a picture. By administering this factor, it is possible to obtain biological activities such as activity that enhances NK-like activity and pile tumor activity, so that it is possible to develop measuring reagents, medicines, and accessories using the galectin 9-inducing activity It is.
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Abstract
La galectine-9 est une substance physiologiquement active ayant une activité en tant que lectine, dont l'expression est observée dans différentes cellules. Etant donné qu'il existe une corrélation entre la quantité d'expression de galectine-9 et la capacité de métastase d'une tumeur, on suppose que la galectine-9 est impliquée dans différents phénomènes physiologiques. On s’attend à ce qu'une substance capable de réguler la production/libération de galectine-9 ait des activités telles qu'une activité d'induction d'effet antitumoral et d'effet anti-inflammatoire et il existe une demande pour identifier une telle substance. On a découvert qu'un facteur capable d'induire la production/libération de galectine-9 ('facteur d'induction de galectine-9') est présent dans une certaine fraction solubilisée de membranes de cellules tumorales. Le facteur peut être administré sous la forme d'une fraction adsorbée à la concanavaline A ou une fraction conservant l'activité concentrée en utilisant une colonne d'échange d'ions Resource Q™, une colonne d'hydroxyapatite ou similaire. Il devient alors possible de développer un réactif de mesure, un produit pharmaceutique, un dosage ou similaire en utilisant l'activité d'induction de galectine-9 du facteur.
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Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999028461A2 (fr) * | 1997-12-03 | 1999-06-10 | Leadd B.V. | Dispositif et procede d'induction de l'apoptose en faisant interferer des proteines de type bip |
WO2001075177A2 (fr) * | 2000-04-03 | 2001-10-11 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Marqueurs tumoraux pour le cancer des ovaires |
WO2002037114A1 (fr) * | 2000-11-01 | 2002-05-10 | Galpharma Co., Ltd. | Agent permettant de detecter l'aptitude d'une tumeur cancereuse a se metastaser |
JP2002542168A (ja) * | 1999-04-07 | 2002-12-10 | フーゴ・アー・カトゥス | 心不全の治療 |
JP2003189874A (ja) * | 2001-12-28 | 2003-07-08 | Galpharma Co Ltd | ガレクチン−9活性制御剤 |
JP2004244411A (ja) * | 2003-01-24 | 2004-09-02 | Galpharma Co Ltd | ガレクチン9含有医薬 |
JP2004346068A (ja) * | 2003-04-28 | 2004-12-09 | Galpharma Co Ltd | ガレクチン9誘導因子 |
JP2005529848A (ja) * | 2002-02-13 | 2005-10-06 | デューク・ユニバーシティ | 非ペプチド結合ストレス応答性ポリペプチドによる免疫応答の調節 |
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2005
- 2005-11-22 WO PCT/JP2005/021942 patent/WO2007060747A1/fr active Application Filing
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WO1999028461A2 (fr) * | 1997-12-03 | 1999-06-10 | Leadd B.V. | Dispositif et procede d'induction de l'apoptose en faisant interferer des proteines de type bip |
JP2002542168A (ja) * | 1999-04-07 | 2002-12-10 | フーゴ・アー・カトゥス | 心不全の治療 |
WO2001075177A2 (fr) * | 2000-04-03 | 2001-10-11 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Marqueurs tumoraux pour le cancer des ovaires |
WO2002037114A1 (fr) * | 2000-11-01 | 2002-05-10 | Galpharma Co., Ltd. | Agent permettant de detecter l'aptitude d'une tumeur cancereuse a se metastaser |
JP2003189874A (ja) * | 2001-12-28 | 2003-07-08 | Galpharma Co Ltd | ガレクチン−9活性制御剤 |
JP2005529848A (ja) * | 2002-02-13 | 2005-10-06 | デューク・ユニバーシティ | 非ペプチド結合ストレス応答性ポリペプチドによる免疫応答の調節 |
JP2004244411A (ja) * | 2003-01-24 | 2004-09-02 | Galpharma Co Ltd | ガレクチン9含有医薬 |
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