WO2006059785A1 - Development of bioactive agent using dendritic cell differentiation-inducing factor - Google Patents

Abstract

The maturation (differentiation) of dendritic cells has a significant meaning in the initiation of an immune response and in a dendritic cell-based vaccine therapy for various cancer types. However, it remains unknown what functions and roles galectins play in dendritic cells. Thus, it is intended to clarify the functions and roles of galectins and develop a technique of regulating the differentiation of dendritic cells. It is found out that Gal-9 has an activity of inducing the differentiation (maturation) of dendritic cells. It is also found out that Gal-8 similarly has an activity of inducing the differentiation (maturation) of dendritic cells though at a lower level than Gal-9. Dendritic cells having been treated with Gal-9 can stimulate and enhance the production of a Th1 type cytokine as well as the production of IL-10 by T cells. Accordingly, it is possible to develop an antiinflammatory agent, an antiallergic agent, an immune suppressor, an antitumor agent and so on by using the activity of inducing dendritic cell differentiation of Gal-8 or Gal-9. Moreover, it is possible to develop an assay reagent, other drugs, an assay, etc. by using the activity of inducing dendritic cell differentiation as described above.

Description

 Development of bioactive agents by dendritic cell differentiation inducers

Technical field

 The present invention relates to a dendritic cell differentiation control technique using a rod-shaped cell differentiation-inducing factor.

In particular, the present invention relates to a technique for controlling differentiation of dendritic cells using galectins such as Gal-9 or a modified form thereof, or galectin 8 or a modified form thereof. The present invention also relates to a technique for utilizing the biological activity associated with the induction of dendritic cell differentiation via the galectins. Background art

 Galectins are animal lectins that exhibit affinity for β-galactosides, which have some conserved sequence portion. To date, 14 types of galectins have been cloned in mammals, and cell adhesion and proliferation (Asakura, H., et al., J. Immunol. 169: 5912 (2002)) Nishi, N., et al., Glycobiology 13: 755 (2003) [Non-Patent Document 2]; Zick, Tsuji, et al., Glycoconj. J. 19: 517 (2004) [Non-Patent Document 3]; Perillo, N., et al., J. Mol. Med. 76: 402 (1998) [Non-patent Document 4]), T cell apoptosis (Perillo, N., et al., Ature 378: 736 (1995) [Non-Patent Document 5]; Kashio, Y., et al., J. Immunol. 170: 3631 (2003) [Non-Patent Document 6]) and immune responses (Liu, FT Clin. Immunol. 97) : 79 (2000) [Non-Patent Document 7]) and other biological processes have been shown to play a role in changing it.

Structurally, galectin-1 (galectin-1; (Gal-1) ² ) is classified as a prototype galectin, whereas Gal-3 is a chimeric type galectin and galectin-8 ( galectin-8; Gal_8) and galectin- Both 9 (galectin-9; Gal-9) are characterized by the tandem repeat subfamily (two different carbohydrate recognition domains (CRD) linked by a linker peptide) ) Belongs to.

 Our group succeeded in cloning human T cell-derived eosinophil migration factor, which resulted in human Gal-9 (Tureci 0. et al., J Biol Chem. 272 (10): 6416-22 (Mar. 7, 1997) [Non-Patent Document 8]) and found to be ekalectin (Matsimioto R. et al., J Biol Chem., 273: 16976-84) (1998) [Non-Patent Document 9]). In addition, the inventors' group has clarified that ekalectin and Gal-9 are the same substance, and human Gal-9 has a short type and medium due to the difference in the length of its link peptide. It was also clarified that there are three types: a mud type and a long type. Gal-9 is a physiologically active substance with activity as a lectin and is expressed in tissue mast cells, eosinophils, macrophages, T cells, B cells, fibroblasts, vascular endothelial cells, various tumor cells, etc. It has been recognized that it is involved in various physiological phenomena, such as a correlation between its expression level and the metastatic potential of tumors. Gal-9 is thought to be involved in various biological activities such as inducing apoptosis of activated T lymphocytes.

 So far, the present inventors have investigated the role of galectins in myeloid lineage cells, and that Gal-3 preferentially acts on macrophages as an activator of migration reaction (Sano, Η, et al. , J. Immunol. 165: 2156 (2000) [Non-patent document 10]), Gal-8 induces adhesion activity and superoxide production in neutrophils (Nishi, N., et al., Glycobiology 13: 755 (2003) [Non-Patent Document 2]), Gal-9 has eosinophil migration activity, induces superoxide production in eosinophils, and prolongs cell survival.

(atsumoto, R., et al., J. Immunol. 168: 1961 (2002) [Non-patent document 11]). Furthermore, Gal-9 is an activated T lymphocyte cell (Kashio, Y., et al., J. I 隱 unol. 170: 3631 (2003) [non-patent document 6]) and tumor cell. It has been shown to induce apoptosis in various cells such as the line (Kageshita, T., et al., Int. J. Cancer 99: 809 (2002) [12]. '

 Galectins are also involved in bone marrow differentiation (Abedin,. J., et al., J. Leukoc. Biol. 73: 650 (2003) [Non-patent Document 13]). The present inventors have shown that Gal-9 expression gradually decreases in the process of eosinophil differentiation, but Gal-10 expression increases.

[Non-patent document 1] Asakura, H., et al., J. Immunol. 169: 5912 (2002) [Non-patent document 2] Nishi, Tsuji, et al., Glycobiology 13: 755 (2003) [Non-patent document] 3) Zick, Y., et al., Glycoconj. J. 19: 517 (2004) [Non-patent document 4] Perillo, Ν · L., Et al., J. Mol. Med. 76: 402 (1998) [Non-patent document 5] Perillo, NL, et al., Nature 378: 736 (1995) [Non-patent document 6] Kashio, Tsuji, et al., J. Immunol. 170: 3631 (2003) [Non-patent document] Reference 7 Liu, FT Clin. Immunol. 97: 79 (2000)

 [Non-Patent Document 8] Tureci 0. et al., J Biol Chem., 272 (10): 6416-22 (Mar. 7, 1997)

 [Non-Patent Document 9] Matsumoto R. et al., J Biol Chem., 273: 16976-84 (1998)

 [Non-patent document 10] Sano, H., et al., J. Immunol. 165: 2156 (2000) [Non-patent document 1 1] Matsumoto, R., et al., J. Immunol. 168: 1961 ( 2002)

 [Non-Patent Document 1 2] Kageshita, Tsuji, et al., Int. J. Cancer 99: 809 (2002)

[Non-Patent Document 1 3] Abedin,. J., et al., J. Leukoc. Biol. 73: 650 (2003) Disclosure of the invention

 Maturation (differentiation) of dendritic cells is important in the initiation of an immune response and in vaccine therapy based on dendritic cells (or dendritic cells; DC) against various cancers. Nevertheless, the functional role of galectins in rod-shaped cells is discussed in terms of Gal-1, Gal-3, and Gal-9 mRNA 1K immature and mature dendritic cells (or not). In the process of dendritic cell maturation, the amount of Gal-9 mRNA increases twice, while Gal-3 mRNA is about 1 There is almost nothing known to the extent that it is reduced to / 3.

 As an immunotherapy for cancer, health foods such as agaricus, propolis, AHCC, and maiko are generally used to activate the immune system, lentinan, krestin, schizophyllan, BCG, 0K432, BRP, etc. Methods using immunostimulants, such as injection of bacterial cell components or transfer of lymphocytes of others, interleukins (I-2, 12, 18, interferon (IF) α, IF y, etc.) Cytokine therapy that administers tocaines, vaccine treatment using cancer cells and their lysates and cancer peptides (eg, MAGE peptide, Muc-1 peptide, etc.) and dendritic cells that are antigen-presenting cells, LAK, CTLs, TILs, dendritic cells (Dendritic Cell (DC))-CTL therapy activates T lymphocytes, which are the center of the immune response that suppresses cancer, while cultivating them outside the body, and the number increases greatly It is a treatment method that focuses on returning to the body of the original patient and the like, and it can activate T lymphocytes very strongly in a test tube fistula and has the side effects of the activator. Because it can be performed without any problem, it is known that activated autolymphocyte therapy has attracted attention, etc. Therefore, it is still necessary to develop a method for controlling the differentiation of rod cells. The

As a result of intensive studies on the role of galectins in rod cells, the present inventors have found that Gal-9 has an activity of inducing differentiation (maturation) of dendritic cells. I found it. Gal-8 is also It was found to have an activity that induces differentiation (maturation) of dendritic cells, although it is less active than Gal-9. Furthermore, the present inventors completed the present invention by finding characteristic phenomena such as that the dendritic cells treated with Gal-9 can stimulate the enhancement of IL-10 production by T cells. The present invention provides the following.

 [1] A tandem repeat type galectin, a mutant protein of the galectin, a nucleic acid encoding them, and a substance selected from the group consisting of degradation products thereof are included as a dendritic cell differentiation inducing factor. A bioactive agent characterized by

 [2] The biological activity is as follows:

 (1) anti-inflammatory activity,

 (2) antiallergic activity,

 (3) immunosuppressive activity,

 (4) antitumor activity,

 (5) IL-10-mediated immunosuppressive activity via Trl cells,

 (6) Dendritic cell differentiationMaturation activity,

 (7) Acquired immunity promoting activity,

 (8) Thl immune response promoting activity,

 (9) Differentiation of dendritic cellsT cell proliferation through maturation and Z or Thl cytokine production promoting activity,

 (10) the activity of stimulating T cells involved in IL-10 synthesis,

 (11) p38 MAPK signaling pathway activation,

 (12) antitumor activity via T lymphocytes (immune system),

 (13) induction of lymphocytes (CTL) having tumor-specific cytotoxic activity,

(14) promoting the anti-tumor activity of CTL, and

 (15) Use for vaccine therapy and / or adoptive immunotherapy to induce CTL outside the body,

The activity according to [1] above, wherein the activity is selected from the group consisting of Product activators.

 [3] The bioactive agent according to [1] above, wherein the tandem repeat type galectin is selected from the group consisting of galectin-8 and galectin-9.

 [4] Tandem lipid type galectins, mutant proteins of the galectins and nucleic acids encoding them, and those selected from the group consisting of degradation products thereof and dendritic cell precursor cells or undifferentiated cells A method for inducing differentiation of dendritic cells, which comprises contacting with dendritic cells.

 [5] A pharmaceutical comprising the bioactive agent according to [1] or [2] above.

 [6] The medicament according to [5] above, which is an antitumor agent, anti-inflammatory agent, antiallergic agent, immunosuppressive agent or autoimmune disease agent.

 [7] Dendritic cell differentiation characterized by containing a tandem repeat type galectin, a mutant protein of the galectin, a nucleic acid encoding them, and a degradation product thereof. Inducer.

 [8] The dendritic cell differentiation inducing agent according to [7] above, wherein the tandem repeat type galectin is selected from the group consisting of galectin-1 8 and galectin-9.

 [9] The dendritic cell differentiation inducing agent according to [7] above, wherein the mutant protein has a sugar chain-binding activity deleted.

 [10] A method for screening a substance that inhibits or promotes dendritic cell differentiation or maturation induced by tandem repeat-type galectins, characterized by using tandem repeat-type galectins and dendritic cells. A way to scream.

 [11] A tandem repeat type galectin, a mutant protein of the galectin, a nucleic acid encoding the same, and a degradation product thereof are selected from the group consisting of the following biological activities:

 (1) anti-inflammatory activity,

(2) antiallergic activity, (3) immunosuppressive activity,

 (4) antitumor activity,

 (5) IL-10-mediated immunosuppressive activity via Trl cells,

 (6) Differentiation of rod-shaped cellsMaturation activity,

 (7) Acquired immunity promoting activity,

 (8) Thl immune response promoting activity,

 (9) Dendritic cell differentiation ・ T cell proliferation and / or Thl cytokine production promoting activity through maturation,

 (10) the activity of stimulating T cells involved in IL-10 synthesis,

 (11) p38 MAPK signaling pathway activation,

 (12) antitumor activity via T lymphocytes (immune system),

 (13) induction of lymphocytes (CTL) having tumor-specific cytotoxic activity,

 (14) promoting the anti-tumor activity of CTL, and

 (15) Vaccine therapy and use for adoptive immunotherapy that induces CTL in vitro or Z,

A bioactive agent characterized by containing as an active ingredient that exhibits an activity selected from the group consisting of:

 [12] A medicament comprising the bioactive agent according to [11] above. The invention's effect

The present invention revealed that tandem repeat galectins, Gal-8 and Gal-9 are involved in the differentiation (maturation) of dendritic cells and promoted the Thl immune reaction. It can be expected that the differentiation (maturation) of dendritic cells can be controlled. Furthermore, since it is possible to control the differentiation (maturation) of dendritic cells, it can be expected that the immune response (inflammatory response) can be regulated. Site-power-in that enhances production from CD4 + T cells by dendritic cells treated with tandem repeat-type galectins, Gal-8, Gal-9 or by co-culture (I-12, IL-10, TNF-α) has anti-tumor activity and differentiated dendritic cells are useful in CTL therapy. Furthermore, differentiated dendritic fine Exosomes released from the vesicle have antitumor activity. Therefore, it is possible to develop anti-inflammatory agents, anti-allergic agents, immunosuppressive agents, anti-tumor agents and the like through dendritic cell differentiation by tan, dem-repeat galectins, Gal-8, and Gal-9. Development of measuring reagents, medicines, and accessories using the induction (maturation) induction activity of dendritic cells by tandem repeat-type galectins, Gal-8 and Gal-9.

 Other objects, features, excellence and viewpoints of the present invention will be apparent to those skilled in the art from the following description. However, the description of the present specification including the following description and the description of specific examples, etc. shows a preferred embodiment of the present invention, and is shown only for explanation. Please understand that there is. Various changes and Z or modifications (or modifications) within the spirit and scope of the present invention disclosed in the present specification can be made by those skilled in the art based on the following description and knowledge from other portions of the present specification. Will be readily apparent. All patent documents and references cited in this specification are cited for illustrative purposes and should be construed as part of this specification. is there.

Brief Description of Drawings

Figure 1 shows 6-day culture in the presence of GM-CSF and IL-4 to induce differentiation into immature dendritic cells, and then GM-CSF and IL to induce maturation. -Expression of various galectins in monocytes, immature dendritic cells (mmmature DCs) and mature dendritic cells (mature DCs) cultured in LPS dg / ml) for an additional 2 days in the presence of 4 The results are shown. Expression of Gal-1, Gal-3, Gal-8 and Gal-9 on the cell surface was examined in monocytes, immature dendritic cells and mature dendritic cells by flow cytometry. Darafu unit fraction attached shadow indicates the stained cells as galectins, part of the colorless graph shows stained with control I _g G cells. Data are for 4 independent experiments. Figure 2 shows the results of the effects of galectins on dendritic cell maturation.

 (A) shows immature dendritic cells cultured for 2 days with the indicated concentrations of Gal-1, Gal-3, Gal-8, Gal-9 or LPS, and then indicated for that cell. It is an analysis of whether or not the molecule being expressed is expressed on the cell surface. Data are shown as mean fluorescence intensity (MFI) for each molecule.

 (B) shows the results of collecting the supernatant of dendritic cells cultured as cultured in (Λ) and assaying for IL-12 by ELISA. Data are expressed as the mean value SD of three sets of one experiment.

 Data in (A) and (B) are representative of five independent experiments.

 Figure 3 shows the results of investigating the effect of Ga-9 on proliferation and cytokine production of T cells drawn by dendritic cells in allogeneic MLR. IFN-g represents IFN-γ, and TNF-a represents TNF-α.

 (Ii) Alodienetic CD4 + T cells are shown for 4 days as indicated ratio with dendritic cells treated with Ga 9 (10 g / ml), LPS (500 ng / ml) or PBS for 2 days Incubated. [] Thymidine was added to the culture for 18 hours at the final time, and [] thymidine incorporation was measured to observe T cell proliferation.

 (B) Allogeneic CD4 + T cells were cultured for 2 days with dendritic cells treated with Gal-9, LPS or PBS as in (A) above, then the culture supernatant was I was asked about the site Caine shown there. The data in (A) and (B) is the average value SD of the three values obtained from one experiment and is representative of four independent experiments. * P 0.05 vs PBS treated dendritic cells.

Figure 4 shows the results of examining the effect of ratatoose on Gal-9-induced -12 production by dendritic cells. Immature dendritic cells have 20 lactose or Incubate with Ga 日間 g dO ^ ug / ml), PS (500 ng / ml) or PBS in the presence of 20 mM sucrose for 2 days, and then culture supernatant is assayed for IL-12. It was. The data is the mean value SD of the three values obtained in one experiment and is representative of three independent experiments.

 Figure 5 shows the results of investigating the effects of Gal-9 CRD mutants on IL-12 production by dendritic cells. Immature dendritic cells can be obtained together with Gal-9 (10 / ig / ml), Gal-9 (R65D) mutant (10 or 30 μg / ml), LPS (500 ng / ml) or PBS. The culture was incubated for days, and then the culture supernatant was assayed for IL-12. The data is the mean value SD of the three values obtained in one experiment, and is representative of three independent experiments. * Gal-9 (R65D) -treated dendritic cells at concentrations of Ρ <0. 05 vs. 10 w g / iTil.

Figure 6 shows the results of examining the effect of Gal-9 on phosphorylation of _P 38 MAPK and EM1 / 2 in dendritic cells. Immature dendritic cells were cultured at the time indicated there together with Gal-9 (10 μg / ml), LPS (500 ng / ml) or PBS, and then the cell lysate was p38 Immunoblotting analysis using antibodies against phosphorylated forms of MAPK or ERK1 / 2 was performed. Blots with antibodies bound to all p38 MAPK or all ERK1 / 2 were probed again. Data are representative of three independent experiments.

 Figure 7 shows the results of examining the effect of signal transduction inhibitors on Gal-9-induced dendritic cell maturation. SB203580 (p38 MAPK inhibitor) and PD98059 (ERK1 / 2 inhibitor) were used as signal transduction inhibitors.

 (A) Immature dendritic cells were incubated with 50 // M PD98059, 50 μM SB203580, or DMS0 (medium) for 1 hour, and then Gal-9 (10 μg / ml), LPS (500 ng / ml) or PBS in the presence of the inhibitor for 2 days, then the cells are expressed on the cell surface by flow cytometry. Was analyzed. The data is representative of four independent experiments.

(B) The amount of IL-12 in the culture supernatant of the cells treated as described in (A) above was measured by ELISA. Data are three values obtained in one experiment The average value of soil SD, which is representative of four independent experiments.

 Figure 8 shows the effect of galectin-9 variant (h-G9NC (null), “ga 卜”) on the cancer peritonitis model inoculated with mouse cancer cells Meth A cells in BALB / c mice (animal survival). Rate).

 Fig. 9 shows the galectin 9 variant (designated h-G9NC (null), "Gal-9 (nul l)") for the cancer peritonitis model inoculated with mouse cancer cells Meth A cells in BALB / c mice. The effect (animal survival curve) is shown.

 Figure 10 shows the effect of the galectin 9 variant (indicated as h-G9NC (null) and “Gal-9 (nul l)) on a cancer peritonitis model inoculated with mouse cancer cells Meth A cells in BALB mice (animals). Survival rate). (A) shows the result of administration of galectin-9 variant (prophylactic effect) simultaneously with cancer cell transplantation. (B) shows the result of administration (treatment effect) of galectin-9 variant after cancer cell transplantation.

 Fig. 11 shows galectin 9 variants (see Fig. 11) for each cancer peritonitis model inoculated with mouse cancer cells Meth A cells in BALB / c mice (A) and nude mice (BALB / c nu-nu mice) (B). h- G9NC (null), “hG9NC (nul l) j” shows the effect (animal survival rate). A is cancer peritonitis BALB mice were treated with galectin modification at the same time as cancer cell transplantation Results (preventive effect) are shown, and B is for cancerous peritonitis nude mice.

 Fig. 12 shows the galectin 9 variant (see Fig. 12) for each cancer peritonitis model in which mouse cancer cells Meth A cells were inoculated into BALB / c mice (A) and nude mice (BALB / c nu-nu mice) (B). h- G9NC (null), shows the effect (animal survival rate) of “hG9NC (null) J”. A shows cancer peritonitis BALB / c mice after cancer cell transplantation, galectin 9 variant The results (therapeutic effect) were administered, and 8 is for malignant peritonitis nude mice.

Fig. 13 shows a galectin-9 variant in a cancer peritonitis model formed by inoculating mouse cancer cells Meth A cells into nude mice (BALB / c nu-nu mice) transfected with spleen cells (T cells). h-G9NC (null)) shows the results (animal survival rate) of the effects of pretreatment. resist: Meth A-resistant mouse spleen cell-transfected nude mouse cancer peritonitis model group, pretreat: Nude Mau transplanted with spleen cells of BALB mice treated with a modified variant of tin 9 Peritonitis model group.

 Figure 14 shows a galectin-9 variant (h-) against a cancerous peritonitis model formed by inoculating mouse cancer cells Meth A cells into nude mice (BALB nu-nu mice) transfected with spleen cells (T cells). G9NC (nul l), “G9j” shows the results of examining the effect of administration (animal survival rate). Resist: Nude mouse cancer peritonitis model group transfected with Meth A-resistant mouse spleen cells, pretreat: Galecti in advance Nude mice transplanted with spleen cells of BALB / c mice treated with 9 variant. Cancer-treated peritonitis model group, nontreat: Nude mouse cancer cells transfected with spleen cells of untreated BALB / c mice as control group Peritonitis model group.

 Fig. 15 shows Fig. 12 (B) and Fig. 13 and 14 in the galectin 9 variant administration group and the non-administration group, and the galectin 9 variant (h-G9NC (nul l), "Gal_9") is administered again. This is a graph of the results (animal survival rate) of investigating the effects of.

 Figure 16 shows the effect of the galectin 9 variant (designated h-G9NC (nul l), “G9”) on a solid tumor model in which MethA cells passaged in vivo were transplanted subcutaneously into BALB / c mice (tumor volume curve) )

 Figure 17 shows the galectin 9 variant (h-G9NC (null), denoted as “G9”) for a solid tumor model subcutaneously transplanted into nude mice (BALB / c nu-ηυ mice) with MethA cells passaged in vivo. The effect (tumor volume curve) is shown.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, it was found that tandem repeat type galectins, in particular, Gal-8 and Gal-9 function as dendritic cell differentiation inducing factors. Therefore, proteins related to the galectins and mutants thereof Alternatively, induction of dendritic cell differentiation using a nucleic acid encoding the substance, such as a modified or derivative Technology to adjust the phenomenon has become possible to develop. The technology may include tandem liby, tomato galectins, particularly those involved in Gal-8 and Gal-9 induced dendritic cell differentiation. For example, we provide screening technology for substances that promote G'a'9-induced dendritic cell differentiation or substances that inhibit Gal-9-induced dendritic cell differentiation, and technologies that use the identified substances for pharmaceutical applications. it can. In the present invention, it is possible to isolate / sequence a predetermined nucleic acid / polynucleotide, etc. using “gene recombination technology”, to produce a recombinant, or to obtain a predetermined protein / peptide. it can. Examples of genes and transformation techniques that can be used in the present specification include those known in the art. For example, J. Sambrook et al., Olecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2nd Edition, 1989 & 3rd Edition, 2001); DM Glover et al. Ed., "DNA Cloning, 2nd ed., Vol. 1 to 3, (The Practical Approach Series), IRL 'Press, Oxford University Press (1995); "Methods in Enzymology series, Academic Press, New Yor For example R. Wu ed.," Methods in Enzymology ", Vol. 68 (Recombinant DNA), Academic Press, New York (1980); Wu et al. Ed., "Methods in Enzymology", Vol. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al. Ed. , Methods in Enzymology ", Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987); R. Wu ed. '"Methods in Enzymology", Vol. 216 (Recombi nant DNA, Part G), Academic Press, New York (1992); R. Wu ed., Methods in Enzymology ", Vol. 217 (Recombinant DNA, Part H) & 218 (Recombinant DNA, Part I), Academic Press, New York (1993); P.. Conn ed., "Methods in Enzymology", Vol. 302 (Green Fluorescent Protein), Academic Press, New York (1999); S. Weissman ed., "Methods in Enzymology", Vol 303 (cDNA Preparation and Characterization), Academic Press, New York (1999), etc., or methods cited in the literature cited there, or methods substantially similar to them or modifications (the descriptions in them) Is included in the disclosure of this specification by reference to it) (hereinafter all referred to as “genetical recombination techniques”). In the present specification, “homology” means each amino acid residue constituting the chain between two chains in a polypeptide sequence (or amino acid sequence) or a polynucleotide sequence (or base sequence). This means the amount (number) of things that can be determined to be the same in the matching relationship between each other or each base, and the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. To do. Homology can be easily calculated. Many methods for measuring the homology of two polynucleotide sequences or polypeptide sequences are known, and the term “homology” (also called “identity”) is well known to those skilled in the art. (Eg Lesk, A.. (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, ( 1993); Grifin, AM & Grifin, HG (Ed.), Computer Analysis of Sequence Data: Part, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York (1987); Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991), etc.). Common methods used to measure the homology of two sequences include Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego, (1994); Carillo, H. & Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), etc., but are not limited thereto. Preferred methods for measuring homology include those designed to obtain a large match between the two sequences being tested. Such a method is What is assembled as a gram. The preferred computer program method for measuring the homology between two sequences is GCG Proghumnokaseshi (Devereux, J. et 'al., Nucleic Acids Research, 12 (1): 387 (1984)), BLASTPヽ BLASTN ゝ FASTA (Atschul, SF et al., J. Mol. Biol., 215: 403 (1990)), etc., are not limited to these, and methods known in the art are used. be able to. As used herein, the term “polypeptide 1” includes

 Five

Any polypeptide may be pointed out. The basic structure of a polypeptide is well known and is described in numerous references and other publications in the art. In view of this, the term “polypeptide” as used herein refers to any peptide containing two or more amino acids that are linked together by peptide bonds or modified peptide bonds. Means peptide or any protein. As used herein, the term “polypeptide” is generally referred to in the art as, for example, peptides, oligopeptides or short-chain, also referred to as peptide oligomers, and proteins. It can usually mean both long chain known ones. Polypeptides often contain amino acids other than the amino acids usually referred to as naturally occurring amino acids (naturally occurring amino acids: or amino acids encoded by genes). Polypeptides, including terminal amino acid residues, are not only made by natural processes where many amino acid residues are translated and processed (or modified) after being translated, but also to those skilled in the art. It will be understood that the above polypeptide can be altered (modified) by well-known chemical modification techniques. Many forms of modifications (modifications) made to the polypeptide are known, and they are described in detail in basic reference books and more detailed papers and numerous research literatures in the field. These are well known to those skilled in the art. Some particularly routine alterations / modifications include, for example, alkylation, acylation, esterification, amidation, glycosylation, lipid binding, Examples include sulfation, phosphorylation, y-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. 1993); BC Johnson (Ed.), Posttranslaional Covalent Modification of Proteins, Academic Press, New York, (1983) (Wold, F., "Posttranslational Protein Modifications: Perspective and Prospects", pp. 1-12); Seifter et al., "Analysis for Protein Modifications and nonprotein cofactors, Methods in Enzymology, 182: 626-646 (1990); Rattan et al.,

Protein Synthesis: Posttranslational Modulation and Aging, Ann. NY Acad. Sci., 663: p. 48-62 (1992). In the present specification, Gal-9 typically includes natural type Gal-9. As natural type Gal-9, long type (L type) Gal-9, medium type (M type) Gal-9 and short type (S type) Gal-9 have been reported. Gal-9 is the N-terminal domain and C-terminal domain linked by the putative link peptide region of SEQ ID NO: 4 disclosed in W0 02/37114 A1, and M-type Gal-9 is An N-terminal domain and a C-terminal domain are linked by a putative link peptide region of SEQ ID NO: 5, and S-type Gal-9 is It is thought that the N-terminal domain and the C-terminal domain are linked. In M-type Ga 卜 9, from the link peptide region of L-type Gal-9, the W0 02/37114 A1 It differs from L-type Gal-9 in that the amino acid residue of the sequence is deleted, and in S-type Gal-9, the corresponding of M-type Gal-9 It differs from M-type Gal-9 in that the amino acid residue of the sequence of SEQ ID NO: 8 of W0 02/37114 A1 is deleted from the link peptide region. It differs from L-type Gal-9 in that the amino acid residue of SEQ ID NO: 9 of W0 02/37114 A1 is deleted from the putative link peptide region. In this specification, Gal-9 is the above-mentioned type Gal-9, M type Gal-9, S type Gal-9, and others. Naturally occurring mutants of the Gal-9 family, as well as artificial mutations in them.

(Ie, one or more amino acid residues deleted, added, modified, inserted, etc.) (mutants or variants, 'or mutins) It may mean one containing peptide fragments. Reference can also be made to Japanese Patent Application No. 2004-287005. The Gal_9 mutant may be a mutin in which the mutation is introduced into the N-terminal CRD and Z or C-terminal CRD, and the sugar chain binding activity is reduced or deleted. In some cases, it may be preferable. Regarding Gal-8, you can refer to JP 2004-215612 publication and Japanese Patent Application No. 2004-175086. Like Gal-9, Gal_8 mutant is mutated to N-terminal CRD and / or C-terminal CRD. May be a mucin in which sugar chain binding activity is reduced or deleted, and in some cases it may be preferred. A representative Gal-9 protein of the present invention includes any one of the amino acid sequences of SEQ ID NOs: 1 to 3 (SEQ ID N0: 1 to 3) in the sequence table of W0 02/37114 A1 or substantially the same. For example, a polypeptide having an equivalent amino acid sequence, eg, at least 5-311, 5-323 or 5-355 contiguous amino acid residues of the amino acid sequence of SEQ ID N0: 1, 2 or 3 Having a group and having substantially equivalent biological activity, such as equivalent antigenicity, etc., or having those characteristics and any of SEQ ID N0: 1, 2 or 3 in the sequence listing At least 50% homology with any one of each domain, or at least 60% homology, or at least 70% homology, or at least 80% homology, or at least 90% higher Homology, or at least And those having a homology of 95% or more, or at least 98% of homology. The Gal-8 protein can be considered almost the same way.

The human Gal-9 polypeptide of the present invention includes a continuous amino acid residue containing all or part of the amino acid sequence of SEQ ID N0: 1 to 3 in the sequence listing of W0 02/37114 A1, Or the amino acid sequence of SEQ ID N0: i, 2 and 3 5 or more, preferably 10 or more, more preferably 20 or more, more preferably 30 or more, more preferably 40 or more, more preferably 50 or more, and more. Preferably 60 or more, more preferably 70 or more, preferably 80 or more, more preferably 90 or more, most preferably 100 or more, and preferably 110 or more. . The human Gal-9-related polypeptide of the present invention may have a part or all of an amino acid sequence selected from the group consisting of SEQ ID ΝΟ: 1, 2 and 3 force ^ (start codon). You may be missing the Met that corresponds to). Anything having such a sequence may be included. The same is true for Gal-8.

Nucleic acids encoding Gal-9 or its constituent domains or fragments thereof may be obtained by coding a peptide having substantially the same biological activity as that of the antigenic equivalent of Gal-9. Any one containing a base sequence with the same effect may be used. Nucleic acids encoding Gal-9 are nucleic acids such as single-stranded DNA, double-stranded DNA, RNA, DNA: RNA hybrid, and synthetic DNA, as well as human genomic DNA, human genomic DNA library, human tissue Either cell-derived cDNA or synthetic DNA may be used. The nucleotide sequence of the nucleic acid encoding Gal-9 can be modified (for example, added, removed, substituted, etc.), and such modified ones can also be included. It may also be a naturally occurring mutant. These nucleic acids may encode a typoid, M-type or S-type Gal-9 peptide or a part thereof, and preferred examples include DNA. The above-mentioned “same base sequence” means, for example, a sequence of 5 sequences out of the base sequence encoding the amino acid sequence of SEQ ID NO: 1 in the sequence listing disclosed in 02/37114 A1 under stringent conditions. An amino acid that hybridizes with at least one nucleotide sequence, preferably at least 10 nucleotide sequences, more preferably at least 15 nucleotide sequences, and even more preferably at least 20 nucleotide sequences, and is substantially equivalent to Gal-9. Examples thereof include those encoding sequences or their complementary chains. The nucleic acid encoding Gal-9 is typically W0 02/37114 SEQ ID N0 in the sequence listing disclosed in Al: a nucleotide sequence encoding a peptide, peptide represented by any one of 1 to 3 and a portion of its continuous amino acid sequence (each characteristic Including a coding sequence only), a codon sequence added with a start codon (codon encoding Met) and a stop codon, and at least 50% of the protein encoded by the base sequence An organism having an amino acid sequence having homology and having at least the characteristic consecutive amino acid residues in the amino acid sequences of SEQ ID N0: 1 to 3, and having substantially the same antigenicity and the like Any peptide may be used as long as it contains a base sequence having the same effect as that of a peptide having a biological activity. The same applies to Gal-8. In the present specification, “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 phos-photoreester method, phospho-diester method, phosphite method, phosphoramidite method, phosphonate method It can be chemically synthesized by such methods. It is known that normal synthesis can be conveniently performed on a modified solid support, for example, commercially available automated synthesizers, such as the Applied Biosystems 3400 DNA synthesizer (Applied Biosystems ), ABI 3900 High-Throughput DNA synthesizer (Applied Biosystems) can be used. The oligonucleotide may contain one or more modified bases, for example, a non-naturally occurring base such as inosine, or a tritylated base, In some cases, it may contain a marked base. In this specification, “polymerase chain reaction J” or “PCR” is generally as described in HA Erlich ed., PCR Technology, Stockton Press, 1989, etc. Point the way, For example, it refers to a method for enzymatically amplifying a desired nucleotide sequence in vitro. In general, the PCR method involves repeating a cycle of primer extension synthesis using two oligonucleotide primers that can preferentially hybridize with a vertical nucleic acid. Typically, primers used in the PCR method can use a primer complementary to the nucleotide sequence to be amplified inside the cage, for example, at the nucleotide sequence to be amplified and at both ends thereof. A force that is complementary, or one that is adjacent to the nucleotide sequence to be amplified can be preferably used. The primer is preferably an oligonucleotide composed of 5 or more bases, more preferably 10 or more bases, more preferably an oligonucleotide composed of 18 to 25 bases.

 The PCR reaction can be carried out by a method known in the art or a method or modification method substantially similar thereto, but in addition to the above documents, for example, R. Saiki, et al., Science, 230: 1350, 1985; R. Saiki, et al., Science, 239: 487, 1988; DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press, Oxford University Press (1995); MA Innis et al. Ed., "PCR Protocols: a guide to methods and applications", Academic Press, New York (1990)); MJ cPherson, P. Quirke and GR Taylor (Ed.), PCR: a practical approach, IRL Press, Oxford (1991); MA Frohman et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (1988), etc. It can be performed according to a modified method. In addition, PCR 3 can be performed using a commercially available kit suitable for it, and can also be performed according to a protocol clarified by a kit manufacturer or a kit distributor.

In a typical case, a PCR reaction consists of, for example, a cage (typically DNA) and a primer designed based on the target nucleic acid. Mix with dNTPs (mixture of doxy nucleoside triphosphate dATP, dGTP, dCTP, dTTP), Taq DNA polymerase and deionized distilled water. Repeat the cycle 25-60 times under typical PCR cycle conditions using an automated thermal cycler such as the GeneAmp ™ PCR system 2700 (Applied Biosystems), but the number of cycles for amplification The number of times can be appropriately set according to the purpose. PCR cycle conditions include, for example, denaturation 90-95 ° C 5-100 seconds, annealing 40-60 ° C 5-150 seconds, extension 65-75 ^C C 30-300 seconds, preferably denaturation A cycle of 94 ° C for 15 seconds, annealing at 58 ° C for 15 seconds, and extension at 72 ° C for 45 seconds can be mentioned, but the reaction temperature and time for annealing can be selected appropriately by experiment, and denaturation reaction and extension reaction The appropriate time can be selected according to the expected chain length of the PCR product. It is usually preferable to change the annealing reaction temperature according to the Tm value of the hybrid of primer and vertical DNA. The length of the extension reaction is usually about I minutes per lOOObp chain length, but it is possible to select a shorter time in some cases. In order to identify a predetermined nucleic acid, a hybridization technique can be used. The hybridization can be performed by the method described in the literature disclosing the “gene recombination technology” or a method or modification method substantially similar thereto, for example, the colony hybridization method, plaque hybrida Use the lysation method, the hybridization method, the translaced assembly method, and the plus and minus methods. For example, in hybridization, 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, washing treatment, etc. Then, 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. To label a probe with a radioactive isotope, use a commercially available standard. Labeling the probe DNA with [a-P] dCTP (Amersham), etc. using a recognition kit such as the random primed DNA labeling kit (Boehringer Mannheim) to obtain a radioactive probe In addition, the labeling can be performed by a method known in the art, for example, digoxigenin, a fluorescent dye, a piotine-avidin system, etc. Hybridization treatment is usually performed. About 35 ° C to about 80 ° C, more preferably about 50 ° C to about 65 ° C for about 15 minutes to about 36 hours, more preferably about 1 hour to about 24 hours, but optimally For example, the hybridization process is performed for about 18 hours at about 55 ° C. As a buffer for hybridization, a buffer commonly used in this field is used. Examples of the modification treatment of the transferred carrier (for example, a membrane) include a method using an alkaline denaturation solution, and it is preferable to treat with a neutralizing solution or a buffer solution after the treatment. In addition, the immobilization treatment of the carrier (for example, a membrane) is usually about 40 ° C to about 100 ° C, more preferably about 70 ° C to about 90 ° C, about 15 minutes to about 24 hours, More preferably, the baking is carried out by baking for about 1 hour to about 4 hours, but can be carried out by appropriately selecting preferable conditions, for example, a carrier such as a filter at about 80 ° C. for about 2 hours. Immobilization is performed by washing the transferred carrier (for example, a membrane) with a washing solution commonly used in the field, such as 1M NaCl, ImM EDTA and 0.1% Sodium Dodecyl. 50 mM Tris- HC1 buffer containing sulfate (SDS), pH 8.0 The carrier including the membrane can be selected from those commonly used in the field, and examples thereof include a nylon filter. The denaturing solution, neutralizing solution, and buffer solution can be selected from those commonly used in the field, and the alkaline denaturing solution contains, for example, 0.5M NaOH and 1.5M NaCl. To give Examples of the neutralizing solution include 1.5M NaCl-containing 0.5M Tris-HCl buffer solution, PH8.0, etc. Examples of the buffer solution include 2 X SSPE.

(0.36 NaCl, 20 mM NaH ₂ P0 ₄ and 2 mM EDTA). Prior to the hybridization treatment, it is preferable to pre-hybridize the transferred carrier (for example, a membrane) as necessary in order to prevent non-specific hybridization reaction. This prehybridization treatment can be performed, for example, by using a prehybridization solution [50% formamide, 5 X Denhardt's solution (0.2% ushi serum albumin, 0.2% po lyv i ny l pyrrol i done), 5 X SSPE, 0.1% SDS, 100 μg / mL heat-denatured salmon sperm DNA], etc., and about 35 ° C to about 50 ° C, preferably about 42 ° C, about Although the reaction can be carried out by reacting for 4 to about 24 hours, preferably about 6 to about 8 hours, those skilled in the art can appropriately experiment by repeating the experiment as appropriate. The denaturation of the labeled probe DNA fragment used in the hybridization is performed, for example, by 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. Etc. 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 with respect to sodium concentration. Preferably about 19 to about 40 mM, more preferably about 19 to about 20 ιιιΜ, and about 35 to about 85 ° C., preferably about 50 to about 70 ° C., more preferably about 60 to about 65 ° C. The conditions of are shown. In the present specification, in the case of “high homology”, depending on the length of the target sequence, for example, 50% or more of homology, further 60% or more, preferably 70% or more, more preferably 80% or more, and In certain cases, it may be 95% or more, particularly preferably 97% or more. The “same-effect base sequence” may, for example, be one that hybridizes to those having the sequence in question under stringent conditions, for example, 5 or more consecutive base groups in the base sequence. A sequence, preferably 10 or more nucleotide sequences, more preferably 15 or more nucleotide sequences, more preferably 20 or more nucleotide sequences, and an amino acid sequence substantially equivalent to the polypeptide. What to code I can get lost. After completion of hybridization, the filter and other carriers can be thoroughly washed, and the labeled probe other than the labeled probe DNA fragment that has undergone a specific hybridization reaction can be removed before detection. . Washing of the 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 (0.1M NaCl, 15mM Quen Acid) It can be carried out by washing with a solution. Hybridized nucleic acids can typically be detected by autoradiography, but various technical methods are known in the field, and should 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 LOMM MgSO ₄ containing 50 mM Tri s-HCl buffer, ρΗ7 · 5) were suspended in such, then the suspension The desired nucleic acid can be isolated, purified and further amplified. The process of screening a target nucleic acid from a nucleic acid sample including a gene library or a cDNA library by a hybridization process can be repeated. Samples carrying a given nucleic acid (eg, phage particles, recombinant plasmids, vector etc.) can be purified and separated by methods commonly used in the art, eg, glycerol gradient over It can be purified by centrifugation (Molecular Clon ing, a laboratory manual, ed. T. amatis, Cola Spring Harbor Laboratory, 2nd ed. 78, 1989). Such as from phage particles, DNA can be purified separated by methods commonly used in the art, for example, resulting phage TM solvent solution (LOMM MgSO ₄ containing 50mM Tri s-HC l buffer, ρΗ7 ・ 8), etc., and after treatment with DNase I and RNase A, add 20 mM EDTA, 50 μg / ml proteinase K and 0.5% SDS, etc. After incubating for 1 hour, this was extracted with phenol-extracted jetyl ether, and then DNA was precipitated by ethanol precipitation. The obtained DNA was then washed with 70% ethanol, dried, and dissolved in TE. Solution (IOmM EDTA-containing lOmM Tris-HCl buffer, pH 8. OH. This can be obtained by dissolving this solution. The target DNA can also be obtained in large quantities by subcloning, for example, subcloning. One Jung can be performed using Escherichia coli as a host and a plasmid vector, etc. DNA obtained by such subcloning can also be obtained by centrifugation, phenol extraction, ethanol precipitation, and the like in the same manner as described above. In this specification, nucleic acids are nucleic acids such as single-stranded DNA, double-stranded DNA, RNA, DNA: RNA hybrid, synthetic DNA, genomic DNA, genomic DNA library, cell origin The nucleic acid can also be obtained by chemical synthesis, in which case the fragments are chemically synthesized and they are fermented. CDNA libraries derived from cloned humans, such as various human tissue or cultured cells (especially chick kidney, brain, pineal gland, lower Pituitary gland, nerve cell, retina, retinal vascular cell, retinal nerve cell, thymus, blood vessel, endothelial cell, vascular smooth muscle cell, blood cell, macrophage, lymphocyte, testis, ovary, uterus, intestine, heart, liver, spleen ) Small intestine, large intestine, gingival-related cells, skin-related cells, glomerular cells, tubule cells, connective tissue cells and other tissues / cells, and various tumor tissues, cancer cells, etc.) cDNA libraries can be used. As a cDNA library used as a cage type, commercially available cDNA libraries from various tissues can be used directly. For example, Stratagene, Invitrogen, Clontech For example, a gene library prepared from human tissue or cells, such as human PI artificial chromosome Genomic Lifecycle (Human Genome Mapping Resource Center). A rabbit tissue cDNA library (available from, for example, Clontech) can be used. Human genomic DNA libraries or human-derived cDNA libraries constructed from various human tissues or cultured cells can be screened using probes. In this specification, the obtained nucleic acid (including DNA) such as a PCR product is usually subjected to 1-2% agarose gel electrophoresis and cut out from the gel as a 'specific band, for example, gene clean kit (Bio 101) Extract using a commercially available extraction kit. The extracted DNA is cleaved with an appropriate restriction enzyme and, if necessary, 'purified or further phosphorylated at the 5' end with T4 polynucleotide kinase, etc., and then a pUC vector such as pUC18. Ligation into an appropriate plasmid vector such as the above, and transform appropriate suitable cells. The base sequence of the cloned PCR product is analyzed. For cloning PCR products, for example, p-Direct (Clontech), pCR-Script ^T '"SK (+) (Stratagene), pGEM-T (Promega), pAmp ™ (Gibco-BRL), etc. Commercially available plasmid vectors can be used to transform host cells, for example using phage vectors, calcium method, noredium / calcium method, calcium / manganese method, TFB high efficiency method, FSB freezing combination It can be carried out by methods known in the art such as the tent cell method, rapid colony method, electoral position method, or substantially similar methods (D. Hanahan, J. Mol. Biol., 166: 557, 1983, etc.) In order to isolate the target DNA, reverse transcription PCR (RT-P and R) and RACE (rapid amplification of cDNA ends) can be applied. For example, MA Innis et al. Ed., "PCR Protocols" (MA Frohman, "a guide to methods and applications"), pp. 28-38, Academic Press, New York (1990), etc. it can.

DNA can be cloned as needed, and for example, plasmid, λ fage, cosmid, P1 phage, F factor, YAC, etc. can be used. Preferred examples include I phage-derived vectors such as Charon 4A, Charon 21A, AgtlO, IgtlADASHII, FIXII, λEMBL3, λZAP11 ™ (Stratagene Etc.) can be used. In addition, the obtained DNA is incorporated into a suitable vector such as plasmid pEX, pMA neo, pG5, as described in detail below, and a suitable host cell as described in detail below. For example, it can be expressed in E. coli, yeast, CH0 cells, COS cells and the like. In addition, the DNA fragment can be used as it is or as a DNA fragment to which an appropriate control sequence is added, or it is incorporated into an appropriate vector and introduced into an animal to create a transgenic animal that expresses the predetermined gene. can do. Examples of animals include mammals, such as mice, rats, rabbits, guinea pigs, and lions. Preferably, the DNA fragment can be introduced into a fertilized egg of an animal such as a mouse to create a transgenic animal. Confirmation of a predetermined gene product can be performed using animal cells, such as 293T cells and COS-1 cells, which have been transfected with the foreign gene. As a method for introducing a foreign gene into an animal cell such as a mammal, a method known in the art or a method substantially similar thereto can be used. For example, a calcium phosphate method (for example, FL Graham et al. , Virology, 52: 456, 1973, etc.), DEAE-dextran method (eg, D. Warden et al., J. Gen. Virol., 3: 371, 1968, etc.), electroporation method (eg, E. Neumann et al., ΕΜΒΟ J, 1: 841, 1982), microinjection, ribosome method, virus infection method, phage particle method, etc. In this way, the gene product produced by an animal cell to which a given gene has been transferred can also be analyzed. As a plasmid into which a predetermined gene or the like (DNA used in the present invention) is incorporated, host cells commonly used in genetic engineering (for example, prokaryotic hosts such as Escherichia coli and Bacillus subtilis, yeast, 293T cells, CH0 cells, Any plasmid can be used as long as it can express the DNA in eukaryotic cell hosts such as COS cells and insect cell hosts such as Sf21. Of course, it is possible to select from the ones attached to commercially available kits and reagents. , Such sequences include, for example, modified codons suitable for proper expression in selected host cells (eg, codons). The target gene, such as a regulatory sequence or facilitating sequence for facilitating the expression of the target gene. Useful linkers, adapters, and other antibiotics, as well as control of nutritional requirements-metabolites, and sequences useful for sorting and detection (hyprid proteins and fusions) Convenient or useful decorations may be applied, including those that encode proteins). Preferably, suitable promoters, such as plasmids hosted by Escherichia coli, are the tritophan fan promoter (trp), the lactose promoter (lac), the tryptophan 'latatoose promoter

(tac) Lipoprotein promoter (lpp), phage P _L promoter, etc., and plasmids using animal cells as the host, SV40 rate promoter, fractional TV LTR promoter, RSV LTR promoter, CMV promoter, SR α promoter, etc. In blastomers using yeast as a host, GAL1, GAL 10 promoters, etc. can be used. In addition, control systems such as CYC1, HIS3, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TP1, A0X1 can be used. An enhancer can be inserted into the vector to promote transcription of the DNA encoding the desired polypeptide, and such an enhancer acts as a promoter and promotes transcription, usually about Examples include elements having a cis action of 10 to 100 bp. Many Hans Hansa are known from mammalian genes such as globin, elastase, anolebumin, human fetoprotein, and insulin. Typically, an enhancer obtained from a eukaryotic cell infectious virus can be suitably used. For example, SV40 enhancer (100-270 bp) in the rate region of the replication origin, the early promoter of cytomegalovirus Examples include the enhancer, the enhancer in the late region of the replication origin of polio, and the enhancer of adenovirus. If necessary, add a signal sequence that matches the host. They can be those well known to those skilled in the art. Examples of plasmids using E. coli as hosts include pBR322, pUC18, pUC19, pUC118, pUC119, pSP64, pSP65, pTZ-18R /-18U, pTZ-19R / -19U, pGEM-3, pGE-4, pGEM-3Z , pGEM-4Z, pGEM-5Zf (-), pBluescript KS ™ (Stratagene). Examples of plasmid vectors suitable for expression in E. coli include pAS, pKK223 (Pharmacia), pC1403, pC931, pKC30, and pRSET-β (Invitrogen). Examples of plasmids that use animal cells as hosts include SV40 vectors, polio-maunolu vector, vaccinia, winores betater, retro uenores vector, etc., specifically pcD, pcD-SR a, CDM8, pCEV4, pME18S, PBC12BI, P SG5 (Stratagene , Inc.), and the like. Examples of plasmids using yeast as a host include Yip type vectors, YEp type vectors, YRp type vectors, YCp type vectors, and the like, for example, pGPD-2. When the host cell is E. coli, examples of host cells include those derived from E. coli K12 strain, such as 例 え ば 533, XLl-Blue, C600, DH1, DH5, DH11S, DH12S, DH5a, DH10B, HB101, C1061 , JM109, STBL2, B834 strains include BL21 (DE3) pLysS. When the host cell is yeast, baker's yeast, fission yeast, and the like may be included. Examples include Saccharomyces cerevisiae, Schizosaccharomyces prombe, Pichia pastoris, Kluyveromyces strains, Candida, Trichoderma reesia, and other yeast strains. When the host cell is an animal cell, for example, COS-7 cells, C0S-1 cells, CV-1 cells, human kidney cell-derived 293 cells, human epidermis cell-derived A431 cells, human cells derived from African green monkey fibroblasts 205 cells derived from colon, COP cells derived from mouse fibroblasts, MOP cells, W0P cells, CH0 cells derived from Chinese hamster cells, CHO DHFR-cells, human HeLa cells, mouse cells derived C127 cells, mouse cells derived NIH 3T3 cells, mouse L cells, 9BHK, HL60, U937, HaK, Jurkat cells, other transformed cell lines, double normal Examples include somatic cells, and Itoda vesicles derived from in vitro primary cultured tissues. Insect cells include Bombyx mori nuclear pol yhedrosis virus, derived from it or other suitable vectors as vectors, Spodoptera frugiperda (caterpi l lar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melangaster (frui tfly), silkworm larvae or silkworm cultured cells such as BM-N cells (for example, Luckow et al., Bio / Technology, 6, 47-55 (1988) Setlow, JK et al. (Eds.), Genetic Engineering, Vol. 8, pp. 277-279, Plenum Publishing, 1986; Maeda et al., Nature, 315, pp. 592-594 (1985)). Plant cells can be used as host cells using Agrobacterium tumefaciens, etc., and they are well known in the art, along with suitable vectors. In the genetic engineering method of the present invention, a restriction enzyme, reverse transcriptase, or DNA fragment that is known or widely used in the art is modified or converted into a structure suitable for cloning. Enzymes such as DNA modifying / degrading enzymes, DNA polymerases, terminal nucleotidyl transferases, and DNA ligases can be used. Examples of restriction enzymes include RJ Roberts, Nucleic Acids Res., 13: rl65, 1985; S. Linn et al. Ed. Nucleases, p. 109, Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982; RJ Roberts, D. Macel is, Nucleic Acids Res., 19: Suppl. 2077, 1991. According to the present invention, a transformant transformed with an expression vector containing a nucleic acid encoding a polypeptide (or protein) can be highly expressed by repeated cloning using an appropriate selection marker as necessary. Can be obtained. For example, in a transformant using an animal cell as a host cell, when the dhfr gene is used as a selection marker, the MTX concentration is gradually increased and cultured, and a resistant strain is selected. Amplify the DNA encoding A cell line capable of obtaining the present can be obtained. The transformant of the present invention can be cultured under conditions capable of expressing the nucleic acid encoding the polypeptide of the present invention, and the target product can be produced and accumulated. The transformant can be cultured in a medium commonly used in the art. For example, a liquid medium can be suitably used for a transformant having a prokaryotic host such as Escherichia coli or Bacillus subtilis or a host such as yeast. The medium contains a carbon source, a nitrogen source, an inorganic substance, and the like necessary for the growth of the transformant. Examples of carbon sources include gnolecose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, cornstrip-liqueur, peptone, casein, meat extract, malt extract, soybean meal Examples of inorganic or organic substances and inorganic substances such as potato extract include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and calcium carbonate. You can also add yeast, vitamins, casamino acids, growth-promoting factors, etc. In addition, a drug such as 3 / 3-indolylacrylic acid can be added to make the promoter work efficiently if necessary. The pH of the medium is preferably about 5 to about 8. For example, E. coli is usually cultured at about 15 to about 45 ° C for about 3 to about 75 hours, and if necessary, aeration or agitation can be added. When cultivating transformants in which the host is an animal cell, examples of the medium include MEM medium containing about 5 to about 20% fetal calf serum, PRMI 1640 medium, DMEM medium, and the like. The pH is preferably from about 6 to about 8. Cultivation is usually carried out at about 30 to about 40 ° C for about 15 to about 72 hours, with aeration and agitation as necessary. A transformant expressing a predetermined gene product can be used as it is, but it can also be used as its cell homogenate, but a predetermined gene product can also be isolated and used. When extracting from the above cultured cells, after culturing, the cells or cells are collected by a known method and suspended in an appropriate buffer, and the cells or cells are obtained by ultrasound, lysozyme, and lysing or freeze-thawing. A method of obtaining a crude extract by centrifugal separation or filtration after breaking can be used as appropriate. Buffer A protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton, X-100 (trade name) or Tween-20 (trade name) may be added to the liquid. When the target product is secreted into the culture solution, the cells or cells are separated from the supernatant by a method known per se after completion of the culture, and the supernatant is collected. The culture supernatant thus obtained or the target product contained in the extract can be purified by appropriately combining known separation / purification methods, such as ammonium sulfate precipitation. Salt filtration, gel filtration using cephadex, etc., for example ion exchange chromatography using a carrier having a jetylaminoethyl group or a carboxymethyl group, for example, hydrophobic groups such as butyl, octyl, and fuunil groups. Purified by hydrophobic chromatography using supported carriers, dye gel chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, high-speed liquid chromatography, etc. Can be obtained. Preferably, it can be purified and separated by treatment with polyacrylamide gel electrophoresis, affinity chromatography with ligands immobilized, etc. Examples include gelatin-agarose affinity chromatography and heparin-agarose chromatography. Further, the obtained polypeptide (or protein) can be modified by amino acid residues contained in a chemical manner, or a peptidase such as pepsin, chymotrypsin, papain, bromelain, endopep It can be modified with enzymes such as thidase and exopeptidase, or it can be partially decomposed into its derivatives. In the polypeptide of the present invention, the C terminus is usually a carboxyl group (—C00H) or carboxylate (—C00—), but the C terminus is an amide (_C0NH ₂ ) or ester (—C00R). Also good. Here, R in the ester is, for example, C 卜₆ alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, C ₈ cycloalkyl group such as cyclopentinole, cyclohexyl, etc. For example, Phenyl, alpha - C _6, such as naphthyl - ₁₂ Ariru group, e.g., benzyl, one, Hue such Enechiru - Le - C Bok ₂ alkyl or alpha - such as naphthylmethyl alpha - naphthyl - - ₂ C such as an alkyl group _{7 In} addition to the ₄ aralkyl group, a bivalyloxymethyl group widely used as an oral ester is used. When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the polypeptide of the present invention includes those in which the carboxyl group is amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.

Further, the polypeptide (or protein) contains an amino group of the N-terminal methionine residue in the above-mentioned polypeptide, such as a protective group (for example, a C- ₅ alkyl monocarboxyl such as formyl group, acetyl). -Protected by a group such as ^-group (^ _ _6- acyl group), Daltamyl group generated by cleavage of the heel end in vivo, and pyroglutamyl substitution, substitution on amino acid side chain of molecular heel groups (e.g., - 0Η, - C00H, amino group, imidazole group, indole group, grayed § - like Gino group) - is protected with a suitable protecting group (such as ₆ Ashiru group such as formyl group, such as Asechiru group) Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound etc. Furthermore, it is commonly used in genetic engineering based on the base sequence of the gene according to the present invention. By using this method, the corresponding polypeptide introduced with mutations such as substitution, deletion, insertion, transfer or addition of one or more amino acids as appropriate in the amino acid sequence of a given polypeptide is produced. Examples of such mutations ■ conversion / modification methods include, for example, the Japan Biochemical Society, “Second Life Chemistry Experiment Course 1, Genetic Research Method II”, P105 (Susumu Hirose), Tokyo Chemical Doujin (1986); Edited by the Biochemical Society of Japan, “Seminar in Experimental Chemistry 2, Nucleic Acid III (Recombinant DNA Technology)”, p233 (Susumu Hirose), Tokyo Chemical Doujin (1992); R. Wu, Shi Grossman, ed., Methods in Enzymology, Vol 154, p. 350 & p. 367, Academic Press, New York (1987); R. Wu, L. Grossman, ed., "Methods in Enzymology, Vol. 100, p. 457 & p. 468, Academic Press. , New York (1983); JA Wells et al., Gene, 34: 315, 1985; T. Grundstroem et, al., Nucleic Acids Res., 13: 3305, 1985; J. Taylor et al., Nucleic Acids Res., 13 : 8765, 1985; R. Wu ed., "Methods in Enzymology", Vol. 155, p. 568, Academic Press, New York (1987); AR Oliphant et al., Gene, 44: 177, 1986, etc. The method is mentioned. For example, site-directed mutagenesis using synthetic oligonucleotides (site-directed mutagenesis) (Zoller et al., Ucl. Acids Res., 10: 6487, 1987; Carter et al., Nucl. Acids Res , 13: 4331, 1986), cassette mutagenesis: Wells et al., Gene, 34: 315, 1985, restriction selection mutagenesis: Wells et al., Philos Trans. R. So London Ser A, 317: 415, 1986), alanine-scanning method (Cunningham & Wells, Science, 244: 1081-1085, 1989), PCR mutagenesis, Kunkel method, dNTP [c Examples include the S] method (Eckstein) and the region-directed mutagenesis method using sulfite and nitrous acid. In addition, it is expressed as a fusion polypeptide (fusion protein) when it is produced by a genetic recombination method, and has biological activity substantially equivalent to that of the desired polypeptide in vivo or in vitro. Convert to ■ May be processed. Although fusion production methods commonly used in genetic engineering can be used, such fusion polypeptides can be purified by affinity chromatography using the fusion part. These fusion polypeptides include those fused to a histidine tag, or -galactosidase (/ 3-gal), maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX) Alternatively, those fused to the amino acid sequence of Cre Recombinase can be mentioned. 'Similarly, the polypeptide can be tagged with a heterogeneous epitope and can be purified by immunoaffinity chromatography using an antibody that specifically binds to the epitope. . In a more suitable embodiment, poly-histidine (poly-His) or poly-histidine-glycine (poly-His- Gly) tag, and such epitope tag, for example, AU5, c-Myc, CruzTag, 09, CruzTag 22, CruzTag 41, Glu-Glu, HA, Ha. 11, KT3, FLAG (registered trademark, Sigma-Aldrich), Omni -Probe, S-probe, T7, Lex A, V5, VP 16, GAL4, VSV-G (Field et al., Molecular and Cellular Biology, 8: pp. 2159-2165 (1988); Evan et al., Molecular and Cellular Biology, 5: pp. 3610-3616 (1985); Paborsky et al., Protein Engineering, 3 (6): pp. 547-5¾3 (1990); Hopp et al., BioTechnology, 6: pp. 1204-1210 (1988); Martin et al., Science, 255: pp. 192-194 (1992); Skinner et al., J. Biol. Chem., 266: pp. 15163-15166 (1991) Lutz-Freyermuth et al., Pro Natl. Acad. Sci. USA, 87: pp. 6393-6397 (1990)). A two-hybrid method using yeast can also be used. Furthermore, the fusion polypeptide may be one with a marker that becomes a detectable protein. In a more preferred embodiment, the detectable marker may be a biotin Avi Tag based on the biotin / streptavidin system, a fluorescent substance, or the like. The fluorescent substances include luminescent jellyfishes such as Aequorea victorea, green fluorescent protein (GFP), and a modified variant (GFP variant) such as EGFP ( Enhanced-humanized GFP), rsGFP (red-shift GFP), yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (cyan) Examples include fluorescent protein (CFP), blue fluorescent protein (BFP), and GFP derived from Renilla reniformis. Laboratory Lecture 3—GFP and Bioimaging, Yodosha (2000)). Detection can also be performed using an antibody that specifically recognizes the fusion tag (including a monoclonal antibody and its fragment). The expression and purification of such fusion polypeptides is a suitable commercial kit. It can also be performed according to protocols that have been identified by kit manufacturers or kit distributors.

 The resulting protein (which may contain peptides or polypeptides) can be immobilized by binding it to an appropriate carrier or solid phase using known techniques such as enzyme immunoassay. it can. Solid-phased proteins and solid-phased peptides can be conveniently used for screening of binding materials. The modification and modification of the structure of the polypeptide protein is described in, for example, the Japanese Biochemical Society, “New Chemistry Experiment Course 1, Protein VI I, Protein Engineering”, Tokyo Kagaku Dojin (1993). Alternatively, it can be carried out by the methods described in the literature cited therein, or in a substantially similar manner. As described below, the biological activity may include immunological activity, eg, antigenicity. Among these modifications, deamination, hydroxylation, carboxylation, phosphorylation, sulfation, alkylation such as methylation, acylation such as acetylation, esterification, amidation, ring opening , Ring closure, glycosylation, changing the type of sugar chain to be different, increasing or decreasing the number of sugar chains, lipid binding, substitution to D-form amino acid residues, etc. These methods are known in the art (eg, TE and reighton, Proteins' ■ Structure and Molecular Properties, pp. I 9-86 WH Freeman & Co., San Franc i sco, USA (1983 ) etc) .

The peptide or polypeptide (or protein) derived from the human has one or more amino acid residues that differ from the natural ones in terms of identity, and the position of one or more amino acid residues differs from the natural one. It may be different. The peptide derived from the human of the present invention has one or more amino acid residues peculiar to human Gal-9 (including L, M, and S) proteins (for example, 1 to 80, preferably 1 to 60, more preferably 1-40, more preferably 1-20, especially 1-10, etc.) Deletion analogues missing, one or more of the unique amino acid residues (eg 1 to 80, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially 1 to 10) are substituted with other residues. 1 or more (for example, 1 to 80, preferably 1, to 60, more preferably 1 to 40, more preferably 1 to 20, particularly 1 to 10) It also includes additional analogs to which an amino acid residue is added. As long as the domain structure that is characteristic of natural human Gal-9 protein is maintained, the sugar chain binding activity may be lost, and all the mutants as described above are included in the active components of the present invention. The In addition, the peptide or polypeptide may include a primary structure conformation substantially equivalent to that of natural human Gal-9 protein or a part thereof having a primary structure conformation. It may be included that has substantially the same biological activity as the above. It can also be one of naturally occurring variants. The protein (or peptide or polypeptide) derived from the human is, for example, 60% of the amino acid sequence selected from the group consisting of SEQ ID N0: 1-3 in the sequence listing of W0 02/37114 A1. Some of them have homology higher than 70%, more preferably those having homologous amino acid sequences of 80% or 90% or more. The part of the protein derived from the human is a peptide of a part of the protein derived from the human (that is, a partial peptide of the protein), and the Gal-9 of the present invention. Any substance may be used as long as it has an activity substantially equivalent to that of the protein (the sugar chain binding activity may be reduced or deleted). For example, the partial peptide of the protein is at least 5 or more, preferably 20 or more, more preferably 50 or more, more preferably 70 or more of the constituent amino acid sequences of the human Gal-9. More preferred are peptides having a sequence of 100 or more amino acids, and in some cases 200 or more amino acids, and preferably they correspond to consecutive amino acid residues or, for example, Among the amino acid sequences shown in SEQ ID N0: 1 to 3 in the sequence listing of W0 02/37114 A1, those having the same homology as mentioned above can be mentioned with respect to the homology to the corresponding region. Human Gal-8 protein However, it can be understood similarly. The protein (or peptide or poly, peptide) may be a mutin having a mutation as described above in the CRD region or may be preferable. · In this specification, “substantially equivalent” means protein activity, for example, differentiation activity of a given dendritic cell (maturation) induction activity, biological activity associated with the dendritic cell differentiation (maturation) induction activity (For example, cytotoxic activity or its promoting activity, apoptosis-inducing activity or its promoting activity, anti-inflammatory activity, antiallergic activity, immunosuppressive activity, antitumor activity), dendritic cell differentiation (maturation) inducing activity It means that the accompanying physiological activity is substantially the same. Furthermore, the meaning of the term may include a case where the activity has substantially the same quality, and the activity of substantially the same quality includes the activity of inducing differentiation (maturation) of dendritic cells, Biological activity associated with the dendritic cell differentiation (maturation) inducing activity (eg, cytotoxic activity or its promoting activity, apoptosis inducing activity or its promoting activity, anti-inflammatory activity, antiallergic activity, immunosuppressive activity, antitumor activity ), And physiological activity associated with the dendritic cell differentiation (maturation) inducing activity. The substantially homogeneous activity means that these activities are qualitatively homogeneous, for example, physiologically, pharmacologically, or biologically homogeneous. For example, dendritic cell differentiation (maturation) inducing activity, dendritic cell differentiation (maturation) inducing activity, cytotoxic activity, antitumor activity, etc. are equivalent (for example, about 0.001 to about 1000 times) Preferably about 0.01 to about 100 times, more preferably about 0.1 to about 20 times, more preferably about 0.5 to about 2 times), but the degree of these activities Quantitative factors such as protein molecular weight may be different. Secondly, amino acid substitutions, deletions, or insertions often do not cause significant changes in the physiological and chemical properties of the polypeptide, and in some cases give favorable changes. In such cases, the polypeptide to which the substitution, deletion, or insertion has been made is not subject to such substitution, deletion, or insertion. Would be substantially the same as the one not. Substantially identical substitutions of amino acids in the amino acid sequence can be selected from other amino acids of the class to which the amino acids belong. For example, non-polar (hydrophobic) amino acids include alanine, phenylalanine, oral isine, isoleucine, valine, proline, tryptophan, methionine, etc., and polar (neutral) include glycine, serine, threonine, Cysteine, tyrosine, asparagine, glutamine, and the like. Examples of positively charged amino acids (basic amino acids) include arginine, lysine, and histidine, and negatively charged amino acids (acidic amino acids) include asparagine. Acid, glutamic acid and the like.

For the synthesis of the protein of the present invention and a part of the peptide, methods known in the peptide synthesis field, for example, chemical synthesis methods such as liquid phase synthesis method and solid phase synthesis method can be used. In such a method, for example, a protein or a peptide synthesis resin is used, and an appropriately protected amino acid is sequentially bonded to the desired amino acid sequence on the resin by various condensation methods known per se. For the condensation reaction, various activating reagents known per se are preferably used, and as such a reagent, for example, carbodiimides such as dicyclohexylcarbodiimide can be preferably used. When the product has a protecting group, the desired product can be obtained by removing the protecting group as appropriate. When the peptide (or polypeptide) is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and also obtained as a salt. In this case, it can be converted into a free form or other salt by a method known per se or a method analogous thereto. The peptide (or polypeptide) salt is preferably physiologically acceptable or pharmaceutically acceptable, but is not limited thereto. Examples of such salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, such as acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, citrate, tartaric acid, malic acid. , Benzoic acid, methanesulfur Examples thereof include salts with organic acids such as phonic acid, p-toluenesulfonic acid, and benzenesulfonic acid. Further, examples of the salts include ammonium salts such as salts with organic bases such as ethylamine, dimethylamine, trimethylamine, and hydroxetylamine. Galectins, especially Gal-8 and Gal-9 expressed genes (including DNA such as cDNA and RNA such as niRNA) can be used to express specific genes in the field according to the above-mentioned “genetical recombination technology”. Known methods for detection and measurement, such as in situ hybridization, Northern blotting, dot blot, RNase protection assay, RT-PCR, Real-Time PCR (Journal of Molecular Endocrinology, 25, 169-193 (2000) and references cited therein), DNA array analysis (Mark Shena, Microarray Biochip Technology ", Eaton Publishing (March 2000)), etc. Galectins disclosed in this specification, in particular, phenomena related to the biological activity of Gal-8 and Gal-9 can be detected, etc. Galectins using these technologies, especially Gal-8 and Gal -9 expression inheritance Measurement systems, reagents, methods, processes, and the like used for them are all included in the technology of the present invention and the systems used for the in situ hybridization, for example, non-RI in situ hybridization, for example. This may include, for example, direct and indirect methods, which include, for example, those in which a detectable molecule (reporter) is directly bound to a nucleic acid probe. In the indirect method, the signal is amplified using, for example, an antibody against a reporter molecule, etc. The oligonucleotide in the nucleic acid probe has a functional group (for example, a primary aliphatic amino group). , SH groups, etc.) may be introduced, and haptens, fluorescent dyes, enzymes, etc. may be bound to these functional groups. Typical examples include digoxigenin (DIG), piotin, fluorescein, etc., but can be appropriately selected from the labels described above for the antibody as described above, and multiple labeling can also be used. And labeled antibodies are also available The The nucleic acid probe labeling method can be selected and used as appropriate from methods known in the art. For example, the random prime method, nick translation method, DNA amplification by PCR, labeling tiling method, in Examples include in vitro transcription. For the observation of the treated sample, it can be used by appropriately selecting from methods known in the field. For example, a field microscope, a phase contrast microscope, a reflection contrast microscope, a fluorescence microscope, a digital imaging microscope, an electron A microscope can be used, and flow cytometry can be used. In the present specification, malignant cells may include tumor cells that metastasize. Tumors that metastasize are malignant tumors. Generally, the malignant tumors are classified into epithelial and non-epithelial types. In some cases, they may be considered as cancer, sarcoma, leukemia, etc. When it is simply called “cancer”, it generally refers to a malignant tumor in the general public. As used herein, “cancer” may be interpreted in a broad sense and should not be interpreted simply as an epithelial malignancy. As used herein, “cancer” may include epithelial malignant tumors and non-epithelial malignant tumors (including those that are tumorigenic and non-plastic), and skin cancer (including melanoma). Breast cancer, ovarian cancer, uterine cancer, testicular malignant tumor, prostate cancer, bladder cancer, kidney cancer, thyroid cancer, pharyngeal / laryngeal cancer, tongue cancer, maxillary cancer, esophageal cancer, stomach cancer, colon / rectal cancer, Lung 'bronchial cancer, liver cancer (including hepatocellular carcinoma, intrahepatic bile duct cancer), extrahepatic bile duct / gallbladder cancer, pancreas cancer, leukemia, malignant lymphoma, plasmacytoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, Rhabdomyosarcoma, liposarcoma, fibrosarcoma, malignant hemangioma, malignant hemangioendothelioma, brain tumor (including Meningioma, Dario Ima, Was Higuchi Cytoma, etc.) Not be done In addition, it should be understood that all the objects of the present invention using the activities of Gal-8 and Gal-9 are included and not particularly limited. Using the functions of Gal-8 and Ga 卜 9 clarified in the present invention, Functions such as predetermined biological activity of galectins such as Gal-9 (eg, dendritic cell differentiation-inducing activity, cytotoxic activity associated with dendritic cell differentiation induction, anti-inflammatory activity, anti-allergic activity, immunosuppression Compounds that promote activity, antitumor activity, etc., compounds that inhibit (antagonist), or salts thereof can be screened. This also means providing the screening reagent. Thus, a variety of dendritic cell differentiation-inducing activities exhibited by the polypeptide such as Gal-8 or Gal-9 protein, a part of the peptide or their salts elucidated in the present invention are used. In relation to substances, compounds that promote a predetermined function such as the biological activity of the Gal-9 protein of the present invention, some of its peptides or their salts, etc. Also provided is a screening method for compounds (antagonists) or salts thereof.

In the screening, for example, (i) Gal-8 or Ga-9 protein, a part of the peptide or a salt thereof (which may contain a transformant expressing the protein, the same shall apply hereinafter), etc. Comparison is made between the case where the test sample is brought into contact with the case where (ii) the test sample in question does not exist in the protein of the present invention, a part of the peptide or a salt thereof. Usually performed in the presence of dendritic cell precursor cells, monocytes, or undifferentiated dendritic cells (immature dendritic cells), but the presence of differentiated dendritic cells (mature dendritic cells) May be done below. Specifically, in the above screening, the biological activity (for example, activity related to the interaction between each Gal-8 or Gal-9 protein and a biological component) is measured and compared. To do. An appropriate detection substrate may be present in the screening system for convenience of measurement. As the substrate, any substrate can be used as long as it can be effectively used for measurement. For example, a known substrate can be selected and used, but a synthesized compound can be preferably used. The substrate can be used as it is, but preferably a substrate labeled with a fluorescence such as fluorescein, an enzyme or a radioactive substance can be used. Examples of the test sample include proteins, peptides, non-peptide compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts such as animals, and cell extracts. Testing used for test samples Examples of the compound may preferably include an anti-galectin antibody, an enzyme inhibitor, a cytokine, a compound having various inhibitory activities, particularly a synthetic compound. These compounds may be novel compounds or known compounds. The screening can be performed according to a usual method for measuring binding activity or enzyme activity. For example, the screening can be performed with reference to methods known in the art. In addition, various labels, buffer solutions and other suitable reagents can be used, and the procedure described there can be performed. The peptide used can be treated with an activator, or its precursor or latent type can be converted into the active type in advance. The measurement is usually performed in a buffer solution that does not adversely affect the reaction, such as Tris-HCl buffer solution, phosphate buffer solution, etc., for example, pH of about 4 to about 10 (preferably pH of about 6 to about 8) Can be done. In these individual screenings, the normal conditions and procedures in each method are added to the ordinary technical considerations of those skilled in the art, and the protein of the present invention or a polynucleotide having substantially the same activity as that of the present invention. What is necessary is just to construct a measurement system related to peptide or peptide. For the details of these general technical means, refer to review articles, written books, etc. L, see eg Methods in Enzymology, Academic Press f ± (USA)). According to the present invention, the elucidated structure of Gal-9 gene and the knowledge about the DNA sequence are used to screen the target genomic DNA, mRNA, and Gal-9 expression activity, Gal-9 activity. Furthermore, it is possible to design probes and blimmers for detecting dendritic cell differentiation-inducing activity by Gal-9. Any probe or primer for specific detection may be used as long as it substantially allows specific detection of dendritic cell differentiation-inducing activity by Gal-9. A typical example is one that makes it possible to detect a characteristic sequence portion of the gene disclosed in WO 02/37114 (0 02/37114 A1). As long as it is useful for specific detection, it is acceptable to detect a part of the Gal-9 gene. For example, to obtain human Gal-9 by PCR,

 Gal-9 sense sequence: CAGGCACCCATGGCTCAAACTAC [SEQ ID NO: 1] 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 given gene. In cases where detection is effective, those that are effectively bound in the form of hybridization are preferred. For such purposes, for example, oligonucleotides containing 5 or 10 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. An oligo (or poly) nucleotide having a base sequence capable of effectively hybridizing to a target sequence may have another nucleotide or nucleotide chain added to one or both ends of the selected base sequence, A label (including a marker or a reporter) described in the present specification may be bound. The label may be incorporated, for example, during PCR. As the label, those widely used in the field can be used, and for example, radioactive substances, fluorescent substances, luminescent substances, enzymes, etc., and also a piotin-avidin system may be used. Preferably, the probe may be labeled to facilitate detection. For gene isolation, PCR and PCR using reverse transcriptase (RT) (RT-PCR) can be used. Competitive PCR can also be performed for quantitative measurements. For example, if a predetermined cDNA is used as a probe, a specific gene in a cell can be detected and measured by Northern blotting, Southern blotting, in situ hybridization, etc., for example. The same applies to galectin-8. In order to specifically amplify a given gene for detection, primers and In this case, one consisting of a pair of oligonucleotides that define both ends of the sequence to be amplified is used. However, the oligonucleotide disclosed in this specification or one of the specific oligonucleotides defined in the present invention is universal. Those composed of a pair of oligonucleotides composed of one of the primers can also be used.

This primer is used to initiate the extension of the sequence to be amplified, and can be used not only in the PCR method but also in amplification methods such as the LCR method and the TAS method. The use of the primer is not limited to a specific nucleic acid amplification method, and can be used for various purposes-application-purposes. In this detection method, a nucleic acid sample is obtained by the method described in “Gene Recombination Technique” above, and if necessary, an amplification reaction is performed using a primer capable of specifically amplifying the target gene. Test whether it 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 or RT-PCR method. The product from the amplification operation is detected by, for example, electrophoresis, for example, agarose gel electrophoresis, and the presence or absence of the amplified DNA by a conventional method, for example, staining with ethidium bumb amide stain and then UV irradiation. be able to. Alternatively, it can be detected by a predetermined probe. For example, if Gal-9 gene expression is not present in the test sample, amplification does not occur or is at a low level.For example, there is no separation of amplification products such as blotting and reverse blotting. A detection method can also be used. The relevant proteins or polypeptides targeted in this specification, fragments thereof, and nucleic acids including DNA (including mRNA oligonucleotides) can be used alone or organically, and further It can be applied to genomics and proteomics technologies in combination with sense technology, antibodies including monoclonal antibodies, and recombinant cells (transformants). Gene expression analysis using nucleic acid array, protein array, gene function solution Analysis, protein-protein interaction analysis, and related gene analysis. For example, in the nucleic acid array technology, a cDNA library is used, or DNA obtained by PCR technology is placed on a substrate with a spotting device at a high density, and analysis of the sample is performed using high hybridization. The arraying is performed by attaching DNA to each unique position of a substrate such as a glass slide, a silicon plate, or a plastic plate using a needle or a pin, or using an ink jet printing technique. Can be implemented. Data is acquired by observing signals obtained as a result of hybridization on the nucleic acid array. The signal may be obtained from a label such as a fluorescent dye (for example, Cy3, Cy5, BODIPY, FITC, Alexa Fluor dyes (trade name), Texas red (trade name), etc.). A laser scanner or the like can be used for detection, and the obtained data can be processed by a computer system equipped with a program according to an appropriate algorithm. In protein array technology, tagged recombinant protein products may be used, including two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) including enzymatic digestion fragments (including Technologies such as electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI) are included. MALDI-T0F analyzer, ESI-3 quadrupole analyzer, ESI-ion trap analyzer, etc.), staining technology, isotope labeling and analysis, and image processing technology can be used. wear. Accordingly, the present invention may also include the enzyme gene system obtained or used above, and software, databases, etc. relating to antibodies against the enzyme gene system. Detection / 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, radioimmunoassay (RIA), FIA , LIA, EIA, ELISA, etc. can be used, and BF separation can be performed or the measurement can be performed with or without. RIA, EIA, FIA and LIA are preferable, and sandwich type assembly is also preferable. For example, in a sandwich type assay, one is an antibody against the Gal-8 or Gal-9 polypeptide of the present invention or an antibody against a Gal-8 or Gal-9 related peptide fragment, and the other is Gal-8 or Gal-9. The antibody against the C-terminal residue of -9 is labeled, and one of them is detectably labeled (of course, other combinations are possible and can be designed appropriately according to the purpose). Other antibodies capable of recognizing the same antigen are immobilized on the solid phase. Incubate the sample, labeled antibody, and solid-phased antibody to react sequentially as necessary. After separating the unbound antibody, the labeled product is measured. The amount of label measured is proportional to the amount of antigen, ie Gal-8 or Gal-9 polypeptide antigen. In this assembly, it is called a simultaneous sandwich type assembly, a forward sandwich type assembly or a reverse sandwich type assembly depending on 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 particular reagent, buffer, etc., temperature or incubation time can be varied according to factors such as the concentration of antigen in the sample and the nature of the sample. A person skilled in the art can perform the measurement by appropriately selecting optimum conditions effective for each measurement using a normal experimental method.

As a measurement system for Gal-8 or Gal-9, for example, for tissues, immunostaining (METHODS, 24, 289-296 (2001); J Immunol Methods, 47 (2), 129-144 (1981); ibid , 150 (1-2), 5-21, 23-32 & 151-158 (1992); Cancer J, 7 (1), 24-31 (2001), etc.), immunoelectron microscope (Mo 1 Biotechnol, 7 (2), 145-151 (1997); J Electron Microsc Tech., 19 (1), 57-63 & 64-79 (1991); ibid., 19 (3), 305-315 (1991), etc.) Expression gene measurement systems such as protein measurement system and in situ hybridization are available for tissue extracts such as EIA, RIA, FIA, LIA, Western blotting (J Electron Microsc (Tokyo), 45 (2), 119-127 (1996); Methods Biochem Anal., 33, 1-58 (1988) Methods Enzymol., 271, 177-203 (1996); ibid., 305, 333-345 (2000); J Immunol Methods, 152 (2), 227-236 (1992); ibid., 170 (2), 177-184 (1994); ibid., 195 (1-2), 149-152 (1996); Yoshiyuki Kuchino Other editions, such as “gene-protein, experimental manipulation blotting method”, Soft Science Co., Ltd., published on November 10, 1987), Northern blotting, dot blot, RNase protection RT-PCR, reverse transcription polymerase chain reaction, Real-iime PCR (Cl inical Chemistry, 46: 11, 1738 -38 743 (2000)) Can advantageously utilize protein measurement systems such as EIA, RIA, FIA, LIA, and Western blotting. In addition, a measurement system for directly inducing differentiation of Gal-9-induced dendritic cells can be constructed and used advantageously.

In the EIA measurement system, for example, in the competition method, an anti-Gal-8 or Gal-9 antibody is used as a solid-phased antibody, and labeled and unlabeled antigens (antigens include Gal-8 or Gal-9 or a fragment thereof In addition, non-competitive methods such as sandwich methods can use immobilized anti-Gal-9 antibody and labeled anti-Gal-8 or Gal-9 antibody, and anti-Gal- Alternatively, the 8 or Gal-9 antibody can be directly labeled, or the antibody against the anti-Gal-8 or Gal-9 antibody can be labeled or immobilized without immobilization. Sensitivity amplification methods include, for example, a combination of a non-enzyme labeled primary antibody that uses a high molecular weight polymer, an enzyme, and a primary antibody (envision reagent applied; Enhanced polymer one-step staining (EPOS)). In combination with non-enzyme labeled secondary antibody, for example, a combination of enzyme and anti-enzyme antibody complex such as PAP (peroxidase-antiperoxidase) method, biotin labeled secondary antibody such as SABC (avidin-biotinylated peroxidase complex) method A combination of a biotin-labeled enzyme and a streptavidin complex with a biotin-labeled enzyme, such as ABC (streptavidin-biotin complex) method, LSAB (labeled streptavidin-biotin) method, CSA (catalyzed signal ampl ification) SABC and Piotin standards! (3) A combination of tyramide and an enzyme and labeled streptavidin, a polymer polymer labeled with a secondary antibody and an enzyme, and the like. · In the measurement method of the present invention, an immunological measurement method is preferably used. In this case, the solid phase carrier is made of polystyrene, polycarbonate, polypropylene or polybule that adsorbs proteins such as antibodies well. Various materials and forms such as balls, microplates, sticks, microparticles or test tubes can be arbitrarily selected and used.

 The measurement can be performed in a suitable buffer solution system so as to maintain an optimum pH, for example, a pH of about 4 to about 9. Particularly suitable buffers include, for example, phosphate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer, Tris-hydrochloric acid buffer, veronal buffer, etc. The buffering agents can be used by mixing with each other at an arbitrary ratio. The antigen-antibody reaction is preferably performed at a temperature between about 0 ° C and about 60 ° C.

In applying various analysis / quantification methods including these individual immunological measurement methods to the measurement method of the present invention, it is not necessary to set special conditions and operations. By adding the usual technical considerations of those skilled in the art to the usual conditions and operation methods in each method, a measurement system related to the target substance of the present invention or a substance having substantially the same activity can be constructed. Good. For details of these general technical means, refer to review articles, books, etc. [For example, Hiroshi Irie, “Radio Imnoassy”, Kodansha, published in 1974; Hiroshi Irie, "Continuing Radio Imno Assy", Kodansha, published in 1979; Eiji Ishikawa et al., Edited by "Enzyme Immunoassay", Medical School, published in 1978; Eiji Ishikawa et al., "Enzyme Immunoassay" (No. 2nd edition), Medical School, published in 1982; Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition), Medical School, published in 1987; HV Vunakis et al. (Ed.), Methods in Enzymology, Vol. / 0 (Immunochemical Techniques, Part A), Academic Press, New York

(1980); J. J. Langone et al. (Ed.), Methods in Enzymology ^, Vol.

73 (Immunochemical Techniques, Part B), Academic Press, New York

(1981); JJ Langone et al. (Ed.), "Methods in Enzymology ^{, /} , Vol.

74 (Immunochemical Techniques, Part C), Academic Press, New York (1981); JJ Langone et al. (Ed.), "Methods in Enzymology Vol. 84 (Immunochemical Techniques, Part D: Selected Immunoassays) Academic Press, New York (1982); JJ Langone et al. (Ed.),

Methods in Enzymology Vol. 92 (Immunochemical Techniques, Part E: Monoclonal Antibodies and General Immunoassay Methods) Academic Press, New York (1983); J. J. Langone et al. (Ed.),

Methods in Enzymology ^, Vol. 121 (Immunochemical Techniques, Part I: Hybridoma Technology and Monoclonal Antibodies) _t Academic Press New York (丄 986); JJ Langone et al, (ed.), "Methods in Enzymology ^, Vol. 178 (Antibodies, Antigens, and Molecular Mimicry), Academic Press, New York (1989); M. Wilchek et al. (Ed.) ^ Methods in Enzymology ^, Vol. 184 (Avidin-Biotin Technology) Academic Press, New York (1990); JJ Langone et al. (Ed.),

Methods in Enzymology, Vol. 203 (Molecular Design and Modeling: Concepts and Applications, Part B: Anibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs) Academic Press, New York (1991) or references cited there ( The description contained therein is included in the disclosure of this specification by reference to it)].

Methods known to detect and measure the expression of specific genes in the field according to the above-mentioned “gene recombination technology” for Gal-9 expressing genes (including DNA such as cDNA and RNA such as mRNA) in situ hybridization, Northern blotting, dot blotting, RNase protection assay, RT-PCR Real-Time PCR (Journal of Molecular Endocrinology, 25, 169-193 (2000) and references cited therein), DNA array angles? Detecting and measuring Gal-9-induced dendritic cell differentiation-inducing activity by analysis method (Mark Shena edition, “Microarray Biochip Technology, Eaton Publishing” (March 2000)). The Gal-9 expression gene measurement system, the reagents, methods, and processes used for it are all Gal-9-induced dendritic cell differentiation-inducing activity detection agent, Gal-9-induced dendritic cell differentiation-inducing activity detection method and In situ hybridization may include, for example, non-RI in situ hybridization, including direct and indirect methods, for example. The direct method uses, for example, a molecule in which a detectable molecule (reporter) is directly bound to a nucleic acid probe, and the indirect method uses, for example, an antibody against the reporter molecule. Signal Functional groups (for example, primary aliphatic amino groups, SH groups, etc.) are introduced into the oligonucleotides in the nucleic acid probe, and haptens, 螢Photo dyes, enzymes, etc., may be bound to.Neutral forces for labeling nucleic acid probes include digoxigenin (DIG), piotin, fluorescein, etc. Labels described above for antibodies The labeling method of the nucleic acid probe can be appropriately selected from methods known in the art and used. For example, random prime method, nick 'translation method, DNA amplification by PCR, labeling Z tee ring method, in vitro transcr For example, it is possible to use a method selected from methods known in the art, such as an observation field microscope, a phase contrast microscope, a reflection contrast microscope, a fluorescence microscope, and the like. A digital imaging microscope, an electron microscope, etc. can also be used, and it can also be based on a flow cytometry etc. In this invention, Gal-9 and a Gal-9 expression gene can be used as a marker of a dendritic cell differentiation inducing factor. Various forms of Gal-9-induced dendritic cell differentiation-inducing activity detection agent or Gal-9-induced dendritic cell differentiation induction detection and / or measurement agent, Gal-9-induced dendritic cell differentiation inducing activity detection method or Gal-9-induced dendritic cell differentiation induction detection and / or measurement method, Gal-9-induced dendritic cell differentiation inducing activity detection or dendritic cell differentiation inducing factor Detection and Z or measurement reagent sets or systems can be created and not only useful in purification, identification, isolation-utilization of Gal-9 induced dendritic cell differentiation induction, they are excellent. In the present invention, a method for obtaining a cancer cell metastasis action or a cancer metastasis inhibitory action related to induction of dendritic cell differentiation by Gal-8 or Gal-9, a reagent, kit, system (detection- Including measurement system). Anti-tumor agent, anti-allergic agent, immunosuppressive agent, agent for autoimmune disease, anti-inflammatory agent and corticosteroid hormone active ingredient agent by controlling Gal-8 or Gal-9 induced dendritic cell differentiation Can provide. In addition, using Gal-9-induced induction of dendritic cell differentiation, it can be applied to fields using the pharmacological action / biological activity of darcocorticoid. Allergies and autoimmune diseases are caused by hyperimmune reactions of CD4 + T lymphocytes, and steroids and immunosuppressants are used to treat refractory allergies and autoimmune diseases. Since Gal-8 or Gal-9 induced dendritic cell differentiation is thought to be involved in these reactions, Gal-9 induced dendritic cell differentiation is induced by immunosuppression, anti-inflammatory action, It can be expected to show allergic activity, and can be used for the development of antitumor agents, antiallergic agents, immunosuppressive agents, autoimmune disease agents, anti-inflammatory agents, and corticosteroid hormone substitutes.

The technique of the present invention can be effectively used in dendritic cell therapy. Dendritic cell therapies include: (1) Autologous dendritic cell tumor local injection therapy (DCI), (2) Self-cancer extracted antigen-presenting dendritic cell vaccine therapy (TP-DC), (3) Artificial antigen-presenting dendritic Cellular vaccine therapy (PP-DC). In dendritic cell therapy, (a) cancer cells or cancer tissues are processed to obtain extracts, etc., or artificial antigens that are produced by artificially producing some of the proteins produced by cancer are prepared, while (b) blood collection. Prepared dendritic cells obtained by culturing leukocytes, etc. obtained together with specific cytokines (not recognizing cancer), and then (c) the dendritic cells in the body Above to recognize the cancer This is a technique performed using the item (a). Autologous dendritic cell tumor local injection therapy was developed as a dendritic cell therapy that can be performed when autologous cancer is not preserved or when artificial antigens cannot be used. When injecting dendritic cells directly into the autologous cancer, the dendritic cells eat and digest part of the autologous cancer, present its features on the surface, move to the lymph nodes, and normal lymphocytes in the lymph nodes It helps to change the cancer to attack. Generally, the tumor is caught by ultrasonography, endoscopy, or CT examination, and dendritic cells are injected using a needle in it. It is a technique to do. Self-cancer extracted antigen-presented dendritic cell vaccine therapy (TP-DC) is a therapy that uses autologous cancer as a textbook used by dendritic cells. A cancer that has been removed by surgery, mixed with dendritic cells outside the body, and injected subcutaneously (vaccine). The same principle as in vaccination in childhood. The purpose is to derive the power to remove cancer or to suppress the growth of cancer. When autologous cancer and dendritic cells are mixed, the dendritic cells eat and digest the autologous cancer, present the characteristics of the cancer on the surface, and when this dendritic cell is injected subcutaneously as an acupuncture, it educates lymphocytes. However, lymphocytes that have been taught the characteristics of cancer travel around the body and attack cancer. Research is being conducted on skin cancer, prevention of recurrence of kidney cancer, liver cancer, spleen cancer, and colon cancer. Artificial antigen-presenting dendritic cell vaccine therapy (PP-DC) is a therapy that uses artificial antigens to show cancer characteristics in rod cells when autologous cancer is not preserved. Artificial antigens that target abnormal substances that cause cancer, such as melanoma, have been found, and dendritic cell vaccine therapy using these human antigens is currently being developed. Vaccine therapy using artificial antigen is a therapy that uses the apparent characteristics of cancer. When artificial antigen and dendritic cells are mixed outside the body and injected subcutaneously, the dendritic cells are transformed into lymphocytes and the apparent characteristics of cancer. The lymphocytes that have been taught are a method of finding and attacking cancer based on its apparent characteristics, and even if the self-cancer is not preserved, it is only necessary to use an artificial antigen that is already available. However, it cannot be used unless the apparent characteristics of autologous cancer and artificial antigen are known. Also people There are types of dendritic cells that can utilize antigens. When the active ingredient of the present invention [for example, a substance involved in the induction of differentiation of Gal-9-induced dendritic cells, a liquid containing the same, etc.] is used as a medicament, it is usually used alone or pharmaceutically acceptable in various formulations. It can be mixed with an agent and administered as a pharmaceutical composition or pharmaceutical preparation. Preferably, it is administered in the form of a pharmaceutical preparation suitable for use such as oral administration, topical administration or parenteral administration, and any dosage form (inhalation method or rectal administration is included depending on the purpose) ). The active ingredients of the present invention may be used as a mixture, for example, a mixture of galectin 9 and a galectin 9 variant, a mixture of galectin 8 and a galectin 9 variant, and the like.

In addition, the active ingredient of the present invention includes various medicaments such as antitumor agents (anticancer agents), cancer cytotoxic agents, tumor metastasis inhibitors, thrombus formation inhibitors, joint destruction therapeutic agents, analgesics, anti-inflammatory agents and It can also be used in combination with κ or immunosuppressants, and they can be used without limitation as long as they have an advantageous function, for example, can be selected from those known in the field . And 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 suitable. Preferably, orally or parenterally to 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 forms of preparations 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, microcapsules, embeddings, powders, powders, granules, fine granules, injections, solutions, elixirs, emulsions, irrigants, syrups Agent, solution, emulsion, suspension, liniment, lotion, aerosol, spray, inhalant, Sprays, ointments, plasters, patches, pasta, poultice creams, oils, suppositories (eg rectal suppositories), tinctures, skin drops, eye drops, nasal drops, ear drops, Examples include powders for coating agents, infusions, liquids for injection, freeze-dried preparations, gel preparations, and the like.

 The pharmaceutical composition can be formulated according to a usual method. 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, suspending agent , Dispersant, Thickener, Gelling agent, Hardener, Absorber, Adhesive, Elasticizer, Plasticizer, Disintegrant, Propellant, Preservative, Antioxidant, Sunscreen, Moisturizer, Relaxant, Charge By using an inhibitor, a soothing agent or the like alone or in combination with the protein of the present invention and the like, it can be produced into a unit dosage form required for the practice of generally accepted formulations.

Formulations suitable for parenteral use include sterile solutions or suspensions of the active ingredient and water or other pharmaceutically acceptable media such as injections. In general, water, saline, aqueous dexrose solution, other related sugar solutions, and glycols such as ethanol, propylene glycol, and polyethylene glycol are listed as preferred liquid carriers for injections. When preparing an injection, a solution known in the art using a carrier such as distilled water, Ringer's solution, physiological saline, an appropriate dispersing agent or wetting agent, and a suspending agent can be used. Prepare in an injectable form such as a suspension or emulsion. Examples of 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 interfaces It may be used in combination with an activator (for example, polysorbate 80 ™, HC0-50, etc.). Examples of 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. In addition, a buffer (for example, phosphate buffer, sodium acetate buffer, etc.) or a reagent for adjusting osmotic pressure, a soothing agent (for example, benzalkonium chloride, hydrochloric acid hydrochloride, etc.), a stabilizer (for example, Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, fuyunol, etc.), antioxidants such as ascorbic acid, absorption enhancers and the like. The prepared injection is usually filled in a suitable ampoule. For parenteral administration, 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. Oily vehicles or solvents used in the formulation include natural, synthetic or semi-synthetic mono-, di- or triglycerides, natural, semi-synthetic or synthetic fats or fatty acids, such as peanuts Vegetable oils such as oil, corn oil, soybean oil and sesame oil. For example, 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. mouthwashes, dentifrices, oral sprays, inhalants, ointments, dental fillers, dental coatings, dental pastes, suppositories 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 method for tti 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, for example, buffering agents such as sodium bisulfite or disodium edetoxalate; antiseptics including antibacterial and antifungal agents such as acetic acid or phenylmercuric nitrate, benzalkonium chloride, or oral hexidine And thickeners 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, etc., but it 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 can be suspended or dissolved in the excipient. Adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. Examples of suitable preparations for oral use include solid compositions such as tablets, pills, capsules, powders, granules, and troches, and liquid compositions such as liquids, syrups, and suspensions. Is mentioned. When preparing the formulation, use formulation adjuvants known in the art. Tablets and pills can also be manufactured with an enteric coating. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the material of the above type. In addition, when the active ingredient is a protein or polypeptide, polyethylene glycol (PEG) is particularly useful in conjugation because it is extremely toxic in mammals. In addition, when PEG is conjugated, 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 include α-amino groups of amino-terminal amino acids, f-amino groups of lysine side chains, carboxyl groups of aspartic acid or glutamic acid side chains, It can be conveniently attached to the activated derivative of the daricosyl chain attached to the α-carboxyl group of the carboxy-terminal amino acid, or to certain asparagine, serine or threonine residues.

 Many activated forms of PEG that are suitable for direct reaction with proteins are known. PEG reagents useful for reacting with amino groups of proteins include active esters of carboxylic acids and carbonate derivatives, especially those with a leaving group of N-hydroxysuccinimide, p-nitrophenol, imidazole, Or 1-hydroxy-2-nitrobenzene-4-sulfonate. Similarly, PEG reagents containing an amino hydrazine or hydrazide group are useful for reaction with aldehydes formed by periodate oxidation in proteins.

The active ingredient of the present invention can be administered over a wide range of dosages, and the dosage and frequency of administration are determined by the sex, age, weight, general health condition, diet, administration time, administration method of the patient being treated. Depending on the rate of excretion, the combination of drugs, and the condition of the patient being treated at that time, these or other factors may be 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 Shoten Co., Ltd. ; Ichibanse, et al. Development of pharmaceuticals, Volume 12 (Formulation base [1]), issued on October 15, 1990, Yodogawa Shoten Co., Ltd .; Development of pharmaceuticals, Volume 12 (Formulation material [II]) It is possible to select and apply as needed from among them, referring to the description of Yodogawa Shoten Co., Ltd. issued on October 28th.

The active ingredient of the present invention is as described herein: (a) the biological activity borne by Gal-8 or Gal-9 through Gal-8 or Gal-9 induced dendritic cell differentiation -Promising as an anti-tumor agent, anti-allergic agent, immunosuppressive agent, autoimmune disease agent, anti-inflammatory agent, and a drug that uses the same activity as corticosteroid hormones is there. In the description and drawings, the terms are either according to IUPAC-IUB Cosition on, Biochemical Nomenclature, or based on the meaning of terms commonly used in the field.

By using Gal-8 or Gal-9 induced dendritic cell differentiation inducing action, the function of Ga 該 8 or Gal-9 is related to Gal-8 or Gal-9 binding. It is possible to search for and identify molecules, especially proteins involved in dendritic cell differentiation inducing action. 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 Gal-8 or Gal-9 dendritic cells from candidate proteins, for example, the following methods can be selected and applied. By knowing the interacting proteins, it is possible to know novel functions of target proteins as well as regulatory mechanisms such as those via Gal-8 or Gal-9. Available methods are (1) immunoprecipitation, (2) Wes 卜 Western method (West Western method or Far Western method: Far Western method, including ligand and blotting method), (3) Intermolecular cross-linking (4) Expression cloning method, (5) Two-hybrid system, (6) Phage display method, (7) Surface plasmon resonance method, (8) Fluorescence polarization method However, the present invention is not limited to these methods, and methods known in the art and modifications thereof can be applied. The immunoprecipitation method adds an antibody specific to a target protein to various sample protein solutions and immunoprecipitates the protein interacting with the target protein as an immune complex. It is effective to investigate the presence of proteins that interact with the target protein by any method once isolated as a product and identified by SDS-PAGE or Western blotting. Here, a specific antibody against the target protein or a specific antibody against a known protein, a resin that traps an antibody such as protein A or protein G cephalose, or the like can be used. It is preferable that a protein solution labeled with throat can be prepared. Commercially available kits can also be used, such as Affi-Prep 10, Affi-Gel Hz (BIO-RAD), NHS Sepharose HP (Pharmacia).

As the protein to be measured, a fusion protein prepared and purified can be used. In this case, antibodies against Tag can be used effectively, and the recombinant protein can be a host such as E. coli, yeast, or mammalian cells. In addition to using expression systems, it is also possible to use cell-free translation systems for reticulocytes. It is also possible to use co-purification when purifying the fusion protein simply by adding an excess amount of the target fusion protein to the protein solution. These methods are described in, for example, Rudner, DZ et al., Ol, Cell Biol., 116, 1765-1773, 1998 (using His-tag), Cleve land, dw et al., J. Mol. Biol. , 116, 207-225, 1977 (using tau (microtubule-associated protein)). 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 the cloning of expression libraries and used for cDNA cloning (Akira Nomura, et al., “Immunity '92” (cDNA cloning using protein probes), Nakayama Shoten, pl69. -175 (1992)). Any protein that can be phosphorylated and labeled with porotin kinase can be used effectively as a probe. The ligand-blotting method is a method for analyzing a protein that binds to a ligand, which is a type of far-western plot that uses a RI-labeled ligand to detect the biotin-ligand. Use strep 卜 avidin. Conjugate antibody, detection using unlabeled ligand, specific antibody and labeled secondary antibody. Signs and It is also possible to use non-RI labels. This method can be referred to, for example, Soutar, AK & Wade, DP, Protein function: a practical approach (Creighton, TE eds.), IRL press, pp55-76, 1989. 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 together with Western blotting and immunoprecipitation methods. This is an effective technique for analyzing structures, examining interacting proteins or nearby proteins (or domains), and analyzing subunit structures such as receptors. For information on chemical crosslinking agents, etc., refer to the PIERCE website (http: AAvww. Piercenet. Coni /). This method can be referred to hermanson, GT, Bioconjugate Techniques, Academic Press, 1996, for example.

The expression cloning method involves the expression of any of the cDM pools in a cell, and the target protein or tagged protein as a probe. The specific cDNA is cloned from the cDNA group used and analyzed through cloning of receptors, ligands, and the like. Expression clones are known in the art, such as expression systems using prokaryotic cells such as colon bacteria, expression systems using cultured cells such as mammalian cells, and expression systems using Xenopus laevis cells. An expression system is available. This method can be referred to, for example, Hiromi Sasaki, “Special Edition of Invincible Biotechnical Series' Proceeding with Bio-Experiment”, Chapter 6, Yodosha, 1997. In expression cloning in E. coli, etc., cDNA libraries such as Agtll, A ZAPII can be screened by transforming into E. coli, etc. Made. In addition, in expression cloning in cultured cells, an expression vector with a specific cDNA is transferred into the cultured cells and bound to the target protein from the cells in which expression is observed. Screening can be done by selecting and crawling things. The sorting is not limited to this, but an ELISA method or FACS (Fluorescence Activated Cell Sorting) method can be suitably used. In addition, iriRNA is synthesized in vitro from specific cDNA and microinjected into Xenopus laevis eggs, and the protein expressed by the interaction with the target protein is used. It is also possible to select and clone. The two-hybrid system utilizes the phenomenon of functional recovery of a transcriptional activator constructed so that a reporter gene is expressed when the domain structure of the protein of interest or its protein interacts with an independent protein domain. This is a crawling system that searches for proteins that interact with each other. Commercially available pre-made libraries can be used in this method, and examples include, but are not limited to, MATCHMAKER GAL4 cDNA LIBRARY and MATCHMAKER LexA cDNA LIBRARY (Clontech). In addition, for creating a self-made library, for example, HybriZAP Two-hybrid vector system (Stratagene) can be mentioned. This method can be found in IJ J. Masaru Yamamoto, Biomanual Series 1. Basic Technology of Genetic Engineering, Yodosha, 1993, Downward, J., FEBL Lett., 338, 113-117, 1994, etc. it can.

The phage display method is a procedure in which a library having a random amino acid sequence of 5 to 7 residues on the surface of a phage is used to collect phages that bind to the target protein and propagate the phages. This is a method for searching for highly specific amino acid sequences. It is also possible to search a protein database from the obtained results and select a corresponding protein from known proteins. For example, Smith, GP & Scott, JK, Methods in Enzymology, Vol. 217, pp228-257, 1993 can be referred to for this method. The surface plasmon resonance method is typically a method developed for the purpose of monitoring the interaction between biomolecules on a sensor chip in real time using the BIAC0RE ™ system. In the BIAC0RE ™, when the light is applied to the sensor chip (gold thin film) on the side where biomolecules are not immobilized so as to be totally reflected, a portion where the reflected light intensity is reduced is observed as a part of the reflected light. (SPR signal is generated). This change in the refractive index of the angle at which the light part appears) is dependent on the mass on the sensor chip. Sensor chip CM5: Carboxymethyldextran surface sensor chip SA: Streptavidin immobilized in advance Sensor chip NTA: NTA immobilized, chelate with nickel And those capable of immobilizing poly-His fusion proteins. This method is, for example, Setsuko Hashimoto, Bunseki 5, Analysis of biomolecular interactions using surface plasmon resonance phenomenon, PP362-368, 1997, Toru Natsume, Biomanual UP series, Protein molecular interaction test method Pp211-230, Yodosha, 1996.

Fluorescence polarization uses the phenomenon that when a molecule with a fluorescent label excited by plane-polarized light undergoes a movement such as rotation during the excited state, the emitted fluorescence becomes a plane different from that of the excitation light. However, since the purity of a molecule is affected by its size, it will remain polarized when it becomes a polymer due to complex formation, etc., and it will be depolarized if it is small and has high mobility. Therefore, the degree of polarization can be measured to know the interaction. Various fluorescent labels can be used. For example, FS, FITC, FXS, etc. can be used. The measurement can be performed using, for example, FBEAC0N ™. The affinity column can be used to search for and identify a binding active substance for a specific target substance. Synthetic peptides, fusion proteins, antibodies, etc. can be used as ligands for affinity columns.

Using the induction of differentiation and / or differentiation promotion phenomenon observed between human Gal-9 or Ga 又 は 8 and dendritic cells, the above (1) immunoprecipitation method, (2) West Estan 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 method Applying the system (Two-Hybrid system), (6) Phage display method, (7) Surface plasmon resonance method, (8) Fluorescence polarization method, etc. It can be identified and the properties of the identified thing can be examined in detail.

Example

 The present invention will be specifically described below with reference to examples. However, these examples are provided merely for the purpose of explaining the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application. In the present invention, it should be understood that various embodiments based on the thought S of this specification are possible.

All examples were performed or can be performed using standard techniques, except as otherwise described in detail, and are well known and routine to those skilled in the art. is there. In the following examples, unless otherwise indicated, specific procedures and treatment conditions are described in DNA cloning by J. Sambrook, t. F. Fritsch & T. Manitos, Molecular Cloning; 2nd. Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); J. Sambrook et al., "Molecular Cloning: A Laboratory Manual ', 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001) J. Sambrook & DW Russel, Molecular Cloning ", 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (2001) and DM Glover et al. Ed.," DNA Cloning, 2nd ed., Vol. 1 to 4, （The Practical Approach Series), IRL Press, Oxford University Press (1995); HA Erlich ed., PCR Technology, Stockton Press, 1989; DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press, Oxford University Press (1995) and MA Innis et al. Ed., "PCR Protocols", Academic Press, New York (1990), or when using commercially available reagents or kits. They use attached protocols (protocols) and attached chemicals.

 G9NC (null) is disclosed in W02005 / 093064 and manufactured and acquired in Example 1 of W02005 / 093064. Example 1

 [Expression and purification of recombinant protein]

 Human recombinants Gal-1, Gal-3, Gal ~ 8, and Gal-9 were added to Nishi, N., et al., Glycobiology 13: 755 (2003); Matsushita, N., et al., J. Biol: It was expressed in Escherichia coli BL-21 cells as described in Chem. 275: 8355 (2000). A cDNA for Gal-8 containing the mutations R69H and R233H was prepared by introducing the mutation R233H into Gal-8 (R69H) (N. Nishi, et al., Glycobiology 13: 755 (2003)). It is difficult to obtain a stable expression of a Gal-9 mutant in which both CRDs have lost glycan binding activity.In consideration of the case, the N-terminal CRD has lost glycan binding activity. A mutant Gal-9 (R65D) was also produced. This mutant, Gal_9 (R65D), was able to detect eosinophil migration ¾ "sex (eosinophi l chemoattractant activity: ECA) ¾: none (Matsushita, N., et al., J. Biol. Chem. 275: 8355 (2000)) All the mutants were confirmed by DNA sequencing (recombinant protein was ratatose-agarose column (Biochemistry, Tokyo, Japan)) Purified by affinity chromatography on a thione-sepharose column (Amersham Biosciences, Uppsala, Sweden).

Protein concentration is determined using BCA Atsey reagent (Pierce, Rockford, IL). And BSA was determined as the standard. All galectin preparations were checked for endotoxin contamination by Limulus Atsey (Bio Whittaker, Walkersville, MD). If present, all contaminating LPS is made of polymer particles as described in Hirayama, C., et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 781: 419 (2002). And removed.

[Purification of monocyte-derived dendritic cells]

 Simple; | ¾-derived dendritic noncell derived dendritic cel ls) is purified as described in ■ Sal lusto, F., et al., J. Exp. Med. 179: 1109 (1994). It was. That is, human PBMCs from healthy human donors were isolated using the interface of the density gradient Amersham Biosciences. Monocytes were purified from the PBMCs by positive selection using a MACS column (Miltenyi Biotec, Bergisch Gladbach, Gerrhany) with antibody (Ab) binding microbeads against CD14. The purity of the isolated CD14 + monocytes was> 90% as measured by a single cytometry.

In order to differentiate into dendritic cells (DC), the purified CD14 + monocytes were treated with 10% FBS, ImM glutamine, penicillin (100 U / ml), streptomycin (100 〃 g / ml), human rGM. -5% C0 at 37 ° C in RPMI 1640 with CSF (50 ng / ml, PeproTech, Rocky Hill, NJ) and human rlL-4 (100 ng / ml, R & D Systems, Minneapol is, MN) Incubated in humid air containing ₂ . Every two days, half of the medium was replaced with fresh medium. After 6 days in culture, the cells show typical immature dendritic cell morphology and flow cytometry, which shows a cell surface phenotype of CD14—, CD80 ^low , CD83 ”, and CD86 ^seml. To induce the cells to become more mature dendritic cells, E. coli LPS (500 ng / ml, Sigma-Aldrich, St. Louis, MO), Gal-1, Gal- 3, Gal-8 or Gal_9 is added to the medium and the cells are cultured for another 2 days did. Apoptosis occurred in various types of cells when cultured with 1 μΜ (~ 33 g / ml) of Gal-9 (Kashio, Y., et al., J. Immunol. 170: 3631). (2003), Kageshita, T., et al., Int. J. Cancer 99: 809 (2002)). After immature dendritic cells were cultured with Gal_9 at a concentration of 33 / ig / ml for 2 days, the number of cells was only 10% of those cultured without Gal-9. In contrast, the number of cells was not affected when cultured with Gal_9 at a concentration of lO g / ml. Thus, experiments were conducted using Gal-9 at a maximum concentration of 10 μg / ml. Where indicated, 20 mM lactose was added to the medium along with sucrose serving as the same concentration of control. In experiments using SB203580 (Calbiochem, San Diego, Calif.), PD98059 (Calbiochem), wortmannin (Sigma-Aldrich), the cells were incubated for 1 hour at 37 ° C before exposure to LPS or Gal-9. Treated with each inhibitor. Considering that the inhibitor was dissolved in DMS0, DMS0 at a concentration of 0.1% (ν / ν) was used as a control.

[Flow cytometry analysis]

 The amount of galectins on the cell surface was determined as described in Abedin, M. J. et al, J. Leukoc. Biol. 73: 650 (2003). Specifically, after washing the cells with PBS containing 0.01% sodium azide and 2% FBS, a rabbit antibody against human Gal-1, human Gal-3, human Gal-8 or human Gal-9 (25 μm g / ml) and incubated for 1 hour on ice. After washing with PBS, the cells were incubated with water for 45 minutes in water with FITC-conjugated (conjugated) goat antibody (25 μg / ml, Santa Cruz Biotechnology, Santa Cruz, Calif.) To rabbit IgG.

The expression of costimulatory molecules and HLA-DR on dendritic cells was examined by four-color flow cytometry analysis. FITC-binding mAb for CD80 (mouse IgM, clone BB1), PE-binding mAb for CD86 (mouse IgG2b, clone IT2.2), Alofikocani for CD83 Binding mAb (mouse IgGl, clone HB15e), PE binding mAb to CD14,

(Mouse IgG2a, clone M5E2), and arophycosinin-binding mAb (mouse IgG2a, clone G46-6) to HLA-DR were obtained from BD Biosciences PharMingen (San Diego, Calif.). Here, mAb refers to a monoclonal antibody (hereinafter the same). FITC-conjugated mAbs to CD54 (mouse IgGl, clone 84H10), PE-conjugated mAbs to CD40 (mouse IgGl, clone AB89) and 7-amino-actinomycin D were obtained from Beckmati-Coulter Immunotech (Marseille, France). Before staining with antibody (Ab), dendritic cells block on non-specific binding of the antibody to FcRs, so on ice with human IgG (lOO yU g / ml, Chemicon, Temecula, CA) And incubated for 10 minutes. Cells were analyzed with a FACS Calibur instrument (BD Biosciences, San Jose, CA). 7-amino-actinomycin!) Dead cells were excluded from the analysis based on staining.

[ELISA]

 The amount of IL-12 (p70) and IL-10 released into the culture of dendritic cells was measured using an ELISA kit (Pierce Endogen, Rockford, IL). The detection limit for both IL-12 and IL-10 was 15.4 pg / ml.

[Alogenetic MLR]

T cells were isolated using MACS columns (Miltenyi Biotec) with microbeads conjugated with antibodies to CD4 from healthy donor PBMCs for use as responder cells. Mitomycin C {Q ^ g / m Kyowa醱酵, Tokyo, dendritic cells with Japan) was treated with 37 ^Q C 30 min, then washed three times for use as Sutimyure Ichita single cell. Purified alogenetic T cells (> 90% CD4 ⁺ cells) were dispensed at a concentration of IX 10 ⁵ cells / well into U-bottom 96-well microphone mouthplates. Next, mitomycin C-treated dendritic cells were added in various numbers to the wainole and the cell mixture was cultured at 37 ° C under 5% CO. 3 3 culture And then into each well 0. [H] thymidine (Amersham Pharmacia,

Biotech, Piscataway, NJ) was added and the cells were incubated for an additional 18 hours. The cells were then harvested with a glass fiber filter and examined for radioactivity on the cells by scintillation spectroscopy equipped with TopCount (Packard, eriden, CT). The results were expressed as counts per minute, and the average of three culture experiments was taken.

(Production of Thl and Th2 cytokines)

 Dendritic cells and T cells were cultured at a ratio of 1:10 for 48 hours, and the culture supernatant was collected and stored frozen at -30 ° C until analysis. The amount of Thl (IFN-γ, IL-2, TNF-α) and the amount of Th2 (IL-4, IL-5, IL-10) cytokines in the culture supernatant is determined by the cytometric 'bead'. Determined using an assay kit (BD Biosciences). That is, the supernatant was added with PE detection reagent, and mixed with human cytokine capture beads before incubating at room temperature for 3 hours. After washing, the beads were analyzed using FACS Calibur instrument and CBA software (BD Biosciences).

(Imknob mouth analysis)

 Immature dendritic cells are exposed to LPS (500 ng / ml) or Gal-9 (10 μg / ml), washed twice with ice-cold PBS, and lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, ImM PMSF, leupeptin (10 mg / ml), pepstatin A (l mg / ml), aprotinin (1 mg / ml), ImM sodium orthovanadate, 50 mM NaF, 2 mM sodium pyrophosphate, 1% Nonidet P40) was subjected to sonication. The resulting cell lysate was centrifuged at 10,000 xg for 15 minutes, and the resulting supernatant was subjected to SDS-PAGE under reducing conditions. The separated protein was immobilized on Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, Was transferred to MA).

The resulting membrane (membrene) was blocked in a solution containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.05% Tween-20 to block nonspecific binding. Incubate with 5% skimmed milk. The membrane is then incubated at 4 ° C with mitogen-activated protein kinases (MAPKs) p38 or antibodies specific for ERK1 or ERK2 phosphorylated species. It was processed. The antibodies were then examined for total p38 or total ERK1 / 2 (all antibodies were obtained from Cel Signaling Technology, Beverly MA). To examine again, the membrane was agitated for 30 minutes at 50 ° C in stripping buffer (62.5 mM Tris-HCl (H 6.7), lOOmM 2-mercaptoethanol, 2% SDS). However, it was exposed to the antibody and incubated. The blot was developed using the Phototope-HRP Western Blot Detection System (Cell Signaling Technology).

(Statistical analysis)

 All data are shown as means soil SD. Differences between groups were analyzed by Mann- Whitney U test using Stat View software (Abacus Concepts Inc., California, USA). p straight <0. 05 was considered statistically significant.

[Result]

 [Human monocyte-derived dendritic cell differentiation · Galectin expression during maturation and the effect of galectin on dendritic cell maturation (differentiation)]

 First, changes in the expression of galactins (Gal-1, Gal-3, Gal-8, and Ga.1-9) on the cell surface during LPS-induced maturation of human monocyte-derived dendritic cells I investigated. Monocytes expressed Gal-1 on the cell surface, but the level of expression did not change significantly during dendritic cell maturation (Figure 1).

The expression of Gal-3 was clearly low in monocytes, but compared to this, expression of Gal-3 was increased in immature dendritic cells. Moreover, mature dendritic cells were up-regulated. In monocytes and immature dendritic cells, the expression of Gal-9 was not clear, but dendritic cells After maturation, it was up-regulated. On the other hand, monocytes showed> only a little expression of Gal-8 or no expression of Gal-8. And for immature or mature dendritic cells, there was clearly a slight change.

 Next, we examined the effects of externally added galectins on the maturation of dendritic cells. When immature dendritic cells are cultured with Gal-9 for 2 days in the presence of GM-CSF or IL-4, CD40, CD54 CD80, CD83, CD86, and HLA-DR are expressed on the cell surface. Remarkably up-regulated (Figure 2A). Gal-8 also increased the expression of these proteins on the cell surface, but less. On the other hand, Gal-1 and Gal-3 did not show any action effect.

In addition, Gal-9 greatly increased the production of IL-12 (p70) by dendritic cells in a concentration-dependent manner (Fig. 2B). It was lower. Moreover, Gal-8 induced Mel so Shi not increase IL- 12 production a little but, Gal- 1 and Ga Bok 3 had no operational effect any (Fig. 2 B) ₀

[Gal-9 induced enhancement of antigen presentation (or presentation) activity and Thl cytokine secretion in allogeneic MLR]

 The activity of inducing proliferation of allogeneic T cells is a functional characteristic of dendritic cells in vitro.

 Therefore, we compared the allostimulatory activity of rod cells before and after induction of maturation by LPS or treatment with Gal-9. As a result, T-cell proliferation was enhanced, although less than that observed with LPS-treated dendritic cells (Fig. 3A), indicating that Gal-9 was expressed in allogeneic MLR. This indicates that the antigen (Ag) presenting activity (antigen presenting activity) of dendritic cells is increased.

We also examined the release of cytokines into the culture supernatant during MLR. Allogeneic to the same extent as observed in dendritic cells treated with Ga 9 The secretion of Thl cytokines IFN-γ, TNF-α, and I2 from CD4 ⁺ T cells was elevated (Fig. 3B).

 Dendritic cells treated with Gal-9 had no effect on IL-4 or IL-5 production, but induced a marked increase in IL-10 production. Gal-8, like Gal-1 or Gal-3, is similar to that of Gal-9 in terms of the activity of dendritic cells in MLR, but it is less prominent than that. Indicated.

[: Effects of lactose on the maturation of Gal-9-induced dendritic cells]

 Considering that most of the biological effects of galectins are via glycan binding activity, it is necessary to determine whether Gal-9 is necessary for the activity of inducing dendritic cell growth. Examined. Lactose had only a small inhibitory effect on Gal-9-induced upregulation of co-stimulatory molecules on the cell surface of dendritic cells as well as HLA-DR expression (Table 1).

Table 1 shows the results of the effect of ratatoose on Gal-9-induced upregulation of costimulatory molecules and HLA-DR on the cell surface of rod-shaped cells (DC).

table 1

 MFI

 Cell surface marker treatment Sucrose lactose

 CD80 PBS 30.5 29.1

 Gal-9 61.5 54.7

 LPS 62.6 84.3

 CD83 PBS 10.3 8.8

 Gal-9 48.7 46.4

 LPS 45.7 59.4

 C086 PBS 18.9 18.9

 Gal-9 196.3 167.1

 LPS 191.9 207.9

 CD40 PBS 58.8 59.4

 Gal-9 187.7 137.7

 LPS 224.6 239.9

 HLA-DR PBS 46.6 47.5

 Gal-9 162.5 147. 7

 LPS 253.7 254.9

In Table 1, immature dendritic cells were cultured for 2 days with Gal-9 (10 ^ ug / ml), LPS (500 ng / ml) or PBS in the presence of 20 mM lactose or 20 mM sucrose. The flow cytometry was then used to analyze the expression of the indicated molecule on the cell surface. Here the data is representative of three independent experiments.

 Furthermore, the Gal-9-induced increase in IL-12 production by dendritic cells was slightly reduced with lactose (Figure 4). This suggests that the activity of Gal-9 in the galectin that induces maturation of dendritic cells is not essential.

[Induction of dendritic cell maturation by Gal-9 mutant lacking sugar chain binding activity] In order to confirm that the activity of Gal-9 that induces maturation of dendritic cells is not related to its lectin properties, the Gal- that lacked the 3-galactosidic binding activity The effect of 9 mutants on this process was investigated. When dendritic cells were treated with Gal-9 (R65D) mutant (10 μg / ml), expression of costimulatory molecules and HLA-DR on the cell surface was increased in dendritic cells. Wild-type Gal-9 had no noticeable effect (Table 2). Table 2 shows the results of investigating the effect of Gal-9 CRD mutants on the cell surface phenotype of dendritic cells.

Table 2

 MFI

 Gal-9 Gal-9 (R65D) Gal-9 (R65D)

 Cell surface marker PBS (10 (10 με / ml) (30 Mg / ml) LPS

 g / ral)

 CD80 5.8 38.5 24.3 39.1 39.6

 CD83 3.5 24.7 15.6 20.4 28.8

 CD86 4.6 69.2 39.9 72.4 79.8

 CD40 40.3 124.1 84.7 116.5 155.4

 HLA-DR 25.9 85.4 58.7 84.6 109.5

In Table 2, immature dendritic cells are combined with Gal-9 (10 μg / ml), Gal_9 (R65D) mutant (10 or 30 μg / ml), LPS (500 ng / ml) or PBS. And then analyzed by flow cytometry for the expression of the proteins shown there on the cell surface. Data are representative of 4 independent experiments. Higher concentrations of Gal-9 (R65D) (30 μg / tnl) have the effect that wild-type Gal-9 expresses these same molecules on the cell surface as observed at a concentration of 10 μg / ml. Induced. In addition, Gal-9 (R65D) at a concentration of 30 g / ml increases I 12 production by dendritic cells to the same extent when wild-type galectin is shown at lO g / ml. (Fig. 5). This clearly indicates that the activity of this galectin to induce maturation of Gal-9 dendritic cells does not require the / 3-galactosid binding activity. ·

[P38 MAPK and ERK1 / 2 phosphorylation during Gal-9-induced dendritic cell maturation]

Those that induce maturation of dendritic cells, such as LPS and TNF-α, cause phosphorylation of MAPKs p38 and ERK1 / 2 (Arrighi, JF, et al., J. Immunol. 166: 3837 ( 2001)) ₀ Thus, immunoblot analysis was conducted to examine the effect of Gal-9 on the phosphorylation of these signaling molecules in terminal-mature dendritic cells. Treatment of immature dendritic cells with Gal-9 induced phosphorylation of both p38MAPK and ERK1 / 2 within 10 minutes (Figure 6).

(Effects of signal transduction inhibitors on Gal-9-induced dendritic cell maturation) To see if MAPK signaling pathway is involved in Gal-9-induced dendritic cell maturation, immature dendritic cells were Inhibitors, ie PD98059 (ERK1 / 2 inhibitor) (Alessi, DR, et al., J. Biol. Chem. 270: 27489 (1995)), SB203580 (p38 MAPK inhibitor) (Lee, JC, et al., Nature 372: 739 (1994)) or wortmannin (phosphatidinoreinositol 3_kinase inhibitor) was examined to determine the effect of this process on CD40 induced by Gal-9. Upregulation of CD80, CD83, CD86 and HLA-DR was disturbed by pretreatment of immature dendritic cells with SB203580 (Fig. 7A), treated with PD98059 (Fig. 7A), It was not treated with wortmannin.

Furthermore, Gal-12-induced IL-12 production in dendritic cells was almost completely inhibited by SB203580 (Fig. 7B), unaffected by PD98059 (Fig. 7B), and also by vrortmannin. Was not. [Examination evaluation]

 Gal-9 matures in vitro (differentiates) as demonstrated by up-regulation of costimulatory and HLA molecules on the cell surface and IL-12 production. It was shown here that it has an activity to induce It was also possible to show that this activity of Gal-9 is independent of its lectin properties. Gal-8 has also been found to induce maturation of dendritic cells, although its effect is inferior to that of Gal-9.

 Expression of galectins in monocyte lineage cells such as macrophages and dendritic cells has been shown so far. For example, fresh and isolated monocytes express Gal-3, and that the level of Gal-3 expression is greatly increased after it has matured into macrophages. It has been shown (Liu, FT, et al., Am. J. Pathol. 147: 1016 (1995)). Macrophage cell line THP-1 expresses Gal-9 on the cell surface and in the cytoplasm (Hirashima,., Et al., Glycoconj. J. 19: 593 (2004)).

Here, Gal-3, Gal-8 and Gal-9 are only expressed at low levels, even on the surface of human monocytes, whereas Gal-1 is easily detected. It has been shown that it can. So far, hybridization by microarray has shown that both immature dendritic cells and mature dendritic cells contain Gal-1 mRNA, Gal-3 mRNA, and Gal-9 mRNA. . And the amount of Gal-9 mRNA is higher in mature dendritic cells than in immature dendritic cells, and the amount of Gal-3 mRNA is more mature in dendritic cells than in immature dendritic cells. Less in cells. From the data obtained in the above example, the expression of Gal-3 in immature dendritic cells is increased compared to that in monocytes, whereas the expression of Gal-1, Gal-8 and Gal_9 Shows that there was virtually no difference between both types of cells. However, the expression of both Gal-3 and Gal-9 on the cell surface is upregulated during dendritic cell maturation. Chillon had been.

Each of Gal-9 and Gal-8 depends on the concentration, as reflected in the upregulation of costimulatory and HLA molecule expression on the cell surface and the upregulation of IL-12 production. It was shown here that it induces the maturation of dendritic cells in the same manner. And it is shown here that the effect of Gal-9 is more remarkable than that of Gal-8. Furthermore, dendritic cells treated with Gal-9 have been shown to be able to promote both T cell proliferation and production of Thl cytokines by these cells in allogeneic MLR.

In contrast to LPS-treated dendritic cells, Gal-9-treated dendritic cells enhanced the production of IL-10 (Th2 site force-in) by T cells. This is a dendritic crescent package treated with darcocorticoids or prostaglandin E ₂ (Vieira, PL, et al., J. Immunol. 161: 5245 (1998); Kal inski, P., "et al , J. Immunol. 159: 28 (1997)), mature dendritic cells in response to Gal-9 can stimulate T cells involved in IL-10 synthesis. It is a suggestion.

Gal-9 is a specific antigen (Matsumoto, R., et al., J. Biol. Chem. 273: 16976 (1998)), ConA (Nagai, H., et al., Int. Arch. Alergy Immunol 104 (suppl. L): 12 (1994)), some relases to release from T cells in response to stimulation with PMA (Chabot, S., et al., Glycobiology 12: 111 (2002)). It is done. The release is associated with the activity of a matrix-metaprotein proteinase that has not yet been identified (Chabot, S., et al., Glycobiology 12: 111 (2002)) _D

In addition, various types of cells, such as endothelial cells, fibroblasts, tissue macrophages, mast cells, and eosinophils, express Gal-9 both in the cytoplasm and on the cell surface. Yes. A number of stimuli have been found to regulate the expression of Gal-9 (Hirashima, M. et al., Immunol. Lett. 36: 27332) (1993)) ₀

 Taken together, these observations suggest that Gal-9 is a natural inducer candidate for dendritic cell maturation in vitro. Galectins play an important role in the natural immunity by altering the function of Fecta cells. For example, Gal-3 shows macrophage chemoattractant activity (Sano, H., et al., J. Immunol. 165: 2156 (2000)), Gal -9 originally was eosinic acid It was identified as an eosinophil chemoattractant (Matsumoto,., Et al., J. Biol. Chem. 273: 16976 (1998)).

 In addition, the functions of various eosinophils are activated by Gal-9, and Gal-9 expressed on the surface of fibroblasts and endothelial cells mediates adhesion to eosinophils (Asakura, H , et al., J. Immunol. 169: 5912 (2002)).

In addition, Ga 卜 8 mediates neutrophil adhesion and induces superoxide production by these cells through interaction with integrins and up-regulation of pro-matrix metalloproteins "^^-lase-9" (Nishi, N., et al., Glycobiology 13: 755 (2003)) The effect of Gal-9 on eosinophils depends on the properties of lectin (Matsushita, N., et al , J. Biol. Chem. 275: 8355 (2000)) In contrast, Gal-9's activity to induce dendritic cell maturation is independent of its lectin properties. Lactose had minimal inhibitory effects on Gal-9-induced dendritic cell maturation, and a CRD variant of Gal-9, namely Gal-9 (R65D) [It lacks galactoside binding activity] is slightly higher in concentration However, the effect was similar to that of the wild-type Gal-9 protein, and lactose did not interfere with the induction of maturation of dendritic cells by Gal-8. However, the CRD mutant of Gal-8, namely Gal-8 (R69H, R233H), also induced maturation of dendritic cells. Thus, the 3-galactosidic binding activity of galectins is not necessary for the activity of inducing dendritic cell maturation. Only the reduced form of Gal_l has lectin activity, while the oxidized form of Gal-1 shows different biological activities and promotes axonal regeneration in peripheral nerves (Horie , H., et al., J. Neurosci. 19: 9964 (1999); Inagaki, Y. 'et al., Eur. J. Biochem. 267: 2955 (2000)). Think.

Maturation of dendritic cells induced by Gal-9 depends on the signal ing action of p38 MAPK, but maturation induced by LPS or CD40 ligand (Arrighi, JF, et al. al., J. Immunol. 166: 3837 (2001)), which is not dependent on that of ERK1 / 2.

 Gal-4 stimulates CD4 + T cells in the small intestine in mice and produces IL-6 through binding at synaptic sites involved in immunity (Hokama, A., et al., Immunity 20: 681 (2004)).

 Gal-4, like Gal-9, is a tandem repeat galectin containing two CRDs (Hadari, YR, et al., J. Biol. Chem. 270: 3447 (1995); Averbeck, M. , et al., Eur. J. Immunol. 34: 2708 (2004)). Thus, Gal-9 binds to dendritic cells at the synapse involved in immunity, and Gal-9 induces maturation of dendritic cells (the binding of Gal-9 causes p38 to bind). This effect may be initiated through activation of the MAP signaling pathway.

Gal-4 induced IL-6 production by CD4 + T cells appears to be highly dependent on signal transduction by protein kinases. However, this suggests that the signaling molecule activated by Gal-9 is different from the signaling molecule activated by Gal-4. The synapse formation and cell fistula signaling by the Ding cells has been studied relatively well. Those by dendritic cells are not yet well understood (Averbeck, M., et al., Eur. J. Immunol. 34: 2708 (2004)) _0. The maturation process of dendritic cells is not only induced by LPS and Gal-9 ', but also various cytokines. Caused by prostaglandins such as IFN-γ IL-1 and TNF-α, poly (1: 0, bacteria, etc. (Sallusto, F., et al., J. Exp. Med. 179: 1109 (1994); Kalinski, P., et al., J. Immunol. 159: 28 (1997); Banchereau, J., et al., Nature 392: 245 (1998); Verdi jk, RM, et al., J Immunol 163: 57 (1999); Bender, A., et al., J Immunol Methods 196: 121 (1996); Luft, T., et al., J Immunol 161: 1947 (1998)).

 Many of these factors have previously been shown to up-regulate Ga 9 expression in various cell types (Asakura, H., et al., J. Immunol. 169: 5912). (2002); Ishika a, A., et al., Immunol. Cell Biol. 82: 410 (2004); Yoshida, H., et al., NeuroReport 12: 3755 (2001)).

 Therefore, it is possible that Ga 卜 9 is up-regulated on the cell surface of dendritic cells and other cells in response to such factors, which may be induced by the maturation of dendritic cells. These results of the present invention are not only based on the role of Gal-9 in the natural immune response due to the function of granulocytes, but also by inducing the maturation of rod cells. This suggests that it plays an important role in the role of the acquired immune response.

 Thus, this Gal-9 may contribute to the mutual arrest between innate and acquired immunity.

 Dendritic cells are specialized antigen presenting cells (ABC) required for the initiation of the immune response (Steinman, RM, et al., Adv Exp. Med. Biol. 329: 1 (1993)).

 Thus, they are essential for inducing the initial CTL (cytolytic T lymphocyte) response to tumor cells (Thurner, B., et al.,

ί J. Exp. Med. 190: 1669 (1999)). Dendritic cell immunotherapy has been applied to various cancers such as malignant meonoma, non-Hodgkins lymphoma, multiple melanoma, prostate cancer, rectal cancer, colon cancer, and non-small cell lung cancer (Engleman, EG, Semin. Oncol. 30: 23 (2003)).

 In addition, dendritic cells have the property of secreting exosomes, but the exosomes themselves are considered to be mediators useful for tumor treatment (Zitvogel, et al., Nature Med. 4: 594 (1998); Chaput, N., et al., Bull. Cancer 90: 695 (2003); Andre, F., et al., Adv. Exp. Med. Biol. 495: 349 (2001)). Proteomic analysis shows that exosomal proteins include Gal-3, thioredoxin peroxidase II, Alix, etc. (Thery, C., et al., J. Immunol. 166). : 7309 (2001)).

Gal-9 has been shown not only to induce apoptosis in activated CM + T cells, but also to induce apoptosis in various types of malignant tumor cells, including malignant melanoma and lymphoma cells. (Kashio, Y., et al., J. Immunol. 170: 3631 (2003); Kageshita, T., et al., Int. J. Cancer 99: 809 (2002)).

 The apoptosis-inducing activity of Gal-9 (proapoptotic activity) is greater than that of Gal-1 (Tsuchiyama, Y., et al., Kidney Int. 58: 1941 (2000)). Gal-9 does not induce apoptosis in resting T cells or normal cells at concentrations that act on activated T cells or cancer cells (Kashio, Y., et al., J Immunol. 170: 3631 (2003)).

Thus, galectins, especially Gal-9, are substances that have potential as active agents in applications in anti-tumor immunity therapy, such as when used with dendritic cells. Example 2

 [Bioactivity of Galectin 9 variant]

Experiment ①.

 1) Cell culture method

Meth A cells were cultured in RPMI (SIGMA) medium supplemented with 10% FBS (JRH). 2 × 10 ⁵ cells were seeded in a 75 cm ² flask (SUMIL0N), collected 2 days later, and subcultured. Grows about 10 times in 2 days.

 2) Judgment experiment of galectin 9 variant in cancer peritonitis model

 Collect Meth A cells cultured by the method in 1), wash twice with PBS (-) (1500 rpm, 5 min centrifugation), and adjust the cell concentration to 5 X 10 "/ 200 L PBS (-). It was prepared with.

The prepared Meth A cells (5 10 ⁵ cells / 200 cells / mouse) were inoculated into the peritoneal cavity of BALB mice (SLC; 6-week-old female) using a 26 gauge (26G) injection ^ ". Immediately use the galectin 9 variant prepared to the working concentration (333 g / mL) at 300 i L / 10 / ig / animal, 300 μL / 30 / ig / animal or 300 μL / 100 ig / animal, or the same amount PBS (-) was administered intraperitoneally with a 26-gauge injection needle every day until 18 days after cell inoculation.

 3) Statistical processing

 The survival rate obtained in 2) was analyzed by Kaplan-Meier method. The results are shown in Figure 8. Experiment ②

Meth A cells: Cells (5 × 10 ⁵ cells / 100 / x L) prepared with PBS (−) were inoculated into the peritoneal cavity of BALB mouse (SLC, 6-week-old female, n = 10).

Galectin 9 variant (h-G9NC (null), 100 g / 300 _ML ) was administered daily up to 18 days after intraperitoneal cell inoculation. (1) Immediately after MethA cell inoculation (Day 0), (2) Three days later (Day 3), (3) Seven days later (Day 7), (4) Ten days later (Day 10) Divided into 4 groups (n = 10) and compared survival rates. The results are shown in Fig. 9. FIG. 9 is a survival curve showing that galectin-9 variant has an antitumor effect in a cancer peritonitis model with Meth A cells. Experiment ③

 A. Simultaneous administration (prevention experiment) ''

Meth A cells (5 Χ 10 ⁵ β / 200 _μ were inoculated into the abdominal cavity of BALB / c mice (SLC; 6 weeks old female) using a 26 gauge (26G) needle. Immediately after inoculation ΙΟΟ μ g Similarly, the galectin-9 variant prepared in PBS (-) was administered intraperitoneally with a 26G injection needle to a volume of 300 L. After administration, 1 day after cell inoculation, 2 days later, 12 days every other day The survival rate was compared with the control group.

 B. Post-transplant administration (treatment experiment)

Meth A cells (5 10 ⁵ cells / 200 cells) were inoculated into the abdominal cavity of BALB / c mice (SLC, 6 weeks old female) using a 26 gauge (26G) needle. Seven days after cell inoculation, the modified galectin 9 prepared in PBS (-) so as to be ΙΟΟ μg / 300 μL was similarly administered intraperitoneally with a 26G injection needle. Thereafter, administration was carried out every other day until day IV. Survival was compared with the control group. Figure 10 shows the results of Λ and B. Experiment ④

The cultured Meth A cells were collected, washed twice with PBS (−) (centrifuged at 1500 rpm and 5 min), and prepared with PBS (−) so that the cell concentration was ⁵ × 10 ⁵ cells / 200 / L. Prepared Meth A cells (5 X 10 ⁵ cells / 200 L / mouse) in BALB / mouse and BALB / c nu- rm mice

(Nude mice) (Claire Japan, 6 week old female) was inoculated into the abdominal cavity using a 26 gauge needle. Immediately after inoculation, 100/300 L of Galectin 9 variant or the same amount of PBS (-) was intraperitoneally administered with a 26 gauge (26G) injection needle. Continuous throwing was performed, and survival rates were compared. The results are shown in FIG. Experiment ⑤

Prepared Meth A cells (5 × 10 ⁵ cells / 200 μs / mouse) in 26 abdominal cavity of BALB / mouse and BALB / c nu -nu mouse (nude mouse) (Claire Japan, 6 week old female) Inoculation was performed using a (26G) needle. After cell inoculation 7 golectin 9 variant was injected intraperitoneally with lOO g / 300 / i L / mouse or the same volume of PBS (-) with a 26G needle. Gave. After that, repeated throwing was performed and the survival rate was compared. The results are shown in FIG. Experiment ⑥ '

 1) T cell purification

 eth A-resistant mouse spleen cells: Meth A transplanted into the peritoneal cavity and simultaneously administered galectin 9 variant (lOO ^ u g), surviving to over 50 S BALB spleen cells

 Galectin 9 variant pre-treatment: BALB / c mouse spleen cells pre-treated with galectin 9 variant intraperitoneally for more than 14 days

 No treat: spleen cells of BALB / c mice of the same age without treatment as a control group

 Donor: 2 BALB-resistant Meth A resistant, 2 no treated 2 Stabilized galactin 9 pretreated Use 2 each at a time, adoptive immunization with spleen cells a total of 4 times It was.

 After BALB mice in each treatment group were dislocated, the spleen was removed aseptically in a clean bench. The cell suspension obtained by grinding the spleen with a glass slide was removed from the petri dish with a micropipette, the tissue piece was removed through a nylon mesh, collected in a 15 mL tube and hemolyzed. After washing with PBS (−), the spleen cells of each group were prepared with PBS (−) so that the count was 1 × 10 ′ / body.

2) T cell transplantation

Meth A resistant spleen cells, untreated (no treat) spleen cells, galectin-9 variants pretreated (pretreated) splenocytes, respectively 1 X 10 ^7/500 1 / animal Day- 1, 3, 7, and 11 BALB / c nu-nu was administered intraperitoneally.

Prepared Meth A resistant spleen cells, untreated (m> treat) spleen cells, galectin 9 pre-treated spleen cells on Day-1, 3, 7, and 11 BALB / c nu-nu (CLEA Japan) 6 weeks old Ϋ Each group η = 6) was intraperitoneally administered with IX 10 '/ animal 26 gauge (26G) injection needle. Meth A cells (5 × 10 ⁵ cells / 200 1 / mouse) prepared on DayO were intraperitoneally transplanted into mice with a 26G injection needle. Immediately after Meth A transplant On the same day, administration of Galectin 9 variant (10 (^ g / 450 / il) or the same amount of PBS (-) was started with a 26G needle. Galectin 9 variant and PBS (-) were administered on Day The results were shown in Fig. 13 and 14. Fig. 12 (B) and Fig. 13 and 14 were renewed in the galectin 9 variant administration group and the non-administration group. This is shown in Fig. 15. As a result, it was revealed that, if spleen cells were present, the life-prolonging effect was recognized by the modified galectin 9. That is, CD4 and CD8 that do not exist in nude mice It was suggested that NKT cells are involved in the life-prolonging effect of the modified galectin 9. Statistical analysis was performed by the log rank test.

Meth A cells are 10 ° /. The cells were cultured in RPMI (SIGMA) medium supplemented with FBS (JRH). Inoculate 2 X 10 ° cells into a 75 cm ² flask (SUMIL0N), collect after 2 days, and subculture. It grows about 10 times in 2 days.

The vitro passaged Meth A cells were harvested, PBS (-) was washed with (1500 rpm, 5min ^{centrifugation), 1 Χ 10 6/200} μ prepared intraperitoneally administered to do BALB 1 to so as PBS, 8 days Meth A was collected from the mouse abdominal cavity.

MethA cells passaged in vivo were collected, washed twice with PBS (-) (centrifuged at 1500 rpni for 5 min), and prepared with PBS (-) so that the cell concentration was 10 ⁷ cells / 100 / l. Needle inoculation 27 gauge dorsal skin of the prepared Meth A cells (1 X 10 ⁷ cells / 100 / i 1 / mouse) and BALB / c and BALB / c nu- nu mice (SLC, 6 weeks old female) did. On the fifth day after inoculation, the tumor size was determined by the following calculation method, and the mice were divided into groups so that the tumor sizes were averaged.

 Galectin 9 variant 30 mg / 100 M 1 (n = 5), control group of BALB / c and BALB nu-nu mice of the same age as PBS (- ) Was administered from the tail vein with a 27G needle. From day 5 after Meth A transplantation, the galectin 9 variant was administered every day (15 administrations). Tumor size (total 7 times) was measured 3 times a week. Tumor volume (V) is:

V = 1/2 X long axis X short axis square Calculated by The results are shown in FIGS. .

[Examination evaluation] ''

 1) When Meth A cells were transplanted into the peritoneal cavity of BALB / c mice and galectin-9 variant was administered immediately after transplantation, the survival time was significantly prolonged depending on the dose dependence of galectin-9 variant (Fig. 8). This life-prolonging effect was also observed in the system in which Meth A cells were transplanted into the peritoneal cavity of BALB mice and then galectin-9 variant was administered immediately after transplantation, 3, 7, and 10 days later (Fig. 9). . That is, the prevention and treatment effect of galectin-9 variant was recognized in a cancer peritonitis model using MethA cells. In addition, galectin-9 variants have also been observed every other day in prophylactic and therapeutic experiments (Figure 10). However, when Meth A cells were transplanted into the peritoneal cavity of BALB / c nu-nu (nude mice) lacking T lymphocytes, the prevention experiment system (Fig. 11) and treatment experiment system (Figure 12) were used. No extension of survival was observed. This suggests that the antitumor activity of galectin 9 variants (and galectin 9) is mediated by T lymphocytes.

 2) Transplantation of Meth A cells into the peritoneal cavity of BALB mice and simultaneous administration of galectin 9 variant to the spleen cells of surviving mice, and peritoneal cavity of BALB / c nu-nu (nude mice) transplanted with Meth A cells Co-administration with the drug prolonged survival (Figure 13). However, the spleen cells of mice not treated with Meth A cells and treated with galectin-9 variant and untreated spleen cells were ineffective. This suggests that the modified galectin 9 may be involved in the induction of lymphocytes (CT1) with tumor-specific cytotoxic activity.

 3) When the modified galectin 9 was administered to the system of 2), the survival time was further extended. This suggests that the galectin 9 variant promotes the antitumor effect of CTL (FIGS. 14 and 15). '

 4) From the above, it was suggested that the galectin 9 variant is useful for vaccine therapy using an antigen peptide and adoptive immunotherapy that induces CTL outside the body.

5) In addition, MethA cells were transplanted subcutaneously into BALB / c mice. When the galectin-9 variant was administered intravenously, inhibition of tumor growth was observed (Fig. 16). However, suppression of tumor growth was not observed in the galectin-9 variant in a system in which Meth A cells were similarly tumor-bearing under the back of BALB nu-nu (nude mice) (Fig. 17). This suggests that T cells are involved in the antitumor activity of galectin-9 variants not only in the cancer peritonitis model of 1) but also in the subcutaneous transplant model, ie, the solid tumor model. Example 3

 The modified galectin 9 used in the above examples is also known as stabilized galectin 9, and is G9NC (nul l) produced and obtained in Example 1 of W02005 / 093064 (SEQ ID NO: 1 of W02005 / 093064). The polypeptide having the amino acid sequence represented by SEQ ID NO: 2 was used. The stable galectin-9, that is, G9NC (nul). Can also be prepared as follows. The expression of stable galectin-9 (G9NC (null)) was induced by E. coli transformed with pET-G9NC (null) by electroporation (

BL2KDE3)) 2 ° /. (w / v) Incubate in 2 x YT medium containing glucose and 100 μg / ml ampicillin. When the absorbance at 600 nm reaches 0.7, the final concentration of isopropyl-β-D-thiogalatatopyranoside is 0. I went with 15 rnM. After culturing at 20 ° C for 22.5 hours, the cells were collected by centrifugation and stored at -80 ° C until purification. Cryopreserved cells were 10 mM Tris-HC1 (pH 7.5), 0.5 M NaCl, 1 mM DTT, 1 mM PMSF, 10 m MgCl _2> 25 μg / ml DNase, 0.2 mg / ml egg white After well suspending in lysozyme, Triton X-100 was added to a final concentration of 1% and sonicated for 18 minutes. After centrifuging at 18,800 xg for 75 minutes, the recombinant protein in the obtained supernatant was purified by affinity chromatography using ratato-agarose, and then the buffer was replaced by PBS by dialysis. After removing the precipitate through 0.22 w ni sterilization filter, endotoxin was removed by Cell Mouth Fine ET-Clean L treatment, and bacteria and sediment were further removed through 0.22 / m sterilization filter. The final standard of G9NC (null). G9NC prepared in this way (null) Has no contaminating band on protein polyacrylamide electrophoresis, and the endotoxin content is less than 0.5 EU / ml. Stable to freezing and thawing, and retains biological activity stably for more than half a year even at 4 ° C storage.

 As a galectin-9, a recombinant body obtained by expressing a native non-type galectin-9, M-type galectin-9 and S-type galectin-9 in a host cell using “genetical recombination technology” is used. May be.

Industrial applicability

 In the present invention, galectins, particularly Gal-9, induces differentiation of dendritic cells, and Gal-9-induced differentiated dendritic cells exhibit various characteristic properties. It can be used to develop pharmaceuticals, especially anti-tumor agents, anti-inflammatory agents, anti-allergic agents, immunosuppressive agents, or agents for autoimmune diseases. In addition, it is possible to develop various activators and reagents, Atsey methods, Atsey reagents, measuring reagents such as screening methods and reagents, pharmaceuticals, and accessories.

 It will be apparent that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and thus are within the scope of the claims appended hereto.

[Sequence Listing Free Text]

SEQ 丄 D NO: 1, Ol igonucleotide to act as a primer for PCR

SEQ ID NO: 2, Oligonucleotide to act as a primer for PCR

Claims

1. A tandem repeat-type galectin, a mutant protein of the galectin, a nucleic acid encoding them, and a substance selected from the group consisting of degradation products thereof are characterized as containing a dendritic cell differentiation inducing factor. To say Bioactive agent. 2. The biological activity is as follows:  (1) Anti-inflammatory activity, ί car  (2) Anti-allergic activity,  (3) immunosuppressive activity,  (4) antitumor activity,  (5) IL-10-mediated immunosuppressive activity via Trl cells,  (6) Dendritic cell differentiationMaturation activity,  (7) Acquired immunity promoting activity,  (8) TM immune response promoting activity,  (9) Dendritic cell differentiationT cell proliferation through maturation and activity to promote Z- or Thl-site force-in production,  (10) the activity of stimulating T cells involved in I-10 synthesis,  (11) p38 MAPK signaling pathway activation,  (12) antitumor activity via T lymphocytes (immune system),  (13) induction of lymphocytes (CTL) having tumor-specific cytotoxic activity, (14) promoting the anti-tumor activity of CTL, and  (15) Use for acupuncture and / or adoptive immunotherapy to induce CTL outside the body, The bioactive agent according to claim 1, wherein the bioactive agent is selected from the group consisting of: 3. Tandem repeat type galectin is galectin-8 and galectin The bioactive agent according to claim 1, wherein the bioactive agent is selected from the group consisting of N-9. 4. a tandem repeat type galectin, a mutant protein of the galectin, a nucleic acid encoding the same, and a degradation product thereof, a dendritic cell precursor cell or an undifferentiated rod-shaped cell; A method for inducing differentiation of dendritic cells, which comprises contacting the cells. 5. A pharmaceutical comprising the bioactive agent according to claim 1 or 2 6. The medicament according to claim 5, which is a pile tumor agent, an anti-inflammatory agent, an antiallergic agent, an immunosuppressive agent, or an autoimmune disease agent. 7. Dendritic cell differentiation characterized in that it contains a tandem repeat type galectin, a mutant protein of the galectin, a nucleic acid encoding them, and a degradation product thereof. Inducer. 8. The dendritic cell differentiation inducing agent according to claim 7, wherein the tandem repeat type galectin is selected from the group consisting of galectin 1 and galectin-9. 9. The dendritic cell differentiation inducing agent according to claim 7, wherein the mutant protein is one lacking sugar chain binding activity. 10. A method of screening for a substance that inhibits or promotes tandem repeat galectin-induced dendritic cell differentiation or maturation, characterized by using a tandem repeat galectin and dendritic cells. Ning method. 11. A tandem repeat type galectin, a mutant protein of the galectin, a nucleic acid encoding them, and a degradation product thereof are selected from the group consisting of the following biological activities:  (1) anti-inflammatory activity,  (2) antiallergic activity,  (3) immunosuppressive activity,  (4) antitumor activity,  (5) Immunosuppressive activity via Trl cells by I 10  (6) Differentiation of rod-shaped cellsMaturation activity,  (7) Acquired immunity promoting activity,  (8) Thl immune response promoting activity,  (9) Dendritic cell differentiation · T cell proliferation and maturation through maturation, or Thl site force-in production promoting activity,  (10) activity to stimulate T cells involved in IL-10 synthesis,  (11) Activation of ρ38 ΜΛΡΚ signaling pathway,  (12) antitumor activity via T lymphocytes (immune system),  (13) induction of lymphocytes (CTL) having tumor-specific cytotoxic activity,  (14) promoting the anti-tumor activity of CTL, and  (15) Use for acupuncture and / or adoptive immunotherapy to induce CTL outside the body, A bioactive agent characterized by containing as an active ingredient that exhibits an activity selected from the group consisting of: 12. A pharmaceutical comprising the bioactive agent according to claim 11.