JP2003189874A - Agent for regulating galectin-9 activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material capable of controlling a galectin-9 activities by elucidating the functions performed by a carbohydrate recognition domain (CRD) and a link peptide domain of the galectin-9. <P>SOLUTION: A mutant protein associated with the CRD of the galectin-9 is constructed, and elucidated to play an important role in the exhibition of the activities. A sugar chain structure having activities for specifically bonding to the CRD is revealed. An antagonist and an agonist of the galectin-9 are developed and a method for developing a medicine is provided by utilizing the information about the sugar chain structure. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an agent for controlling galectin-9 activity, a method therefor, a screening method therefor, a screening reagent, a system and the like. The present invention relates to a novel polynucleotide (or nucleic acid) and polypeptide (or protein) (or part thereof) derived from galectin-9 (or a part thereof) or a salt thereof;
Galectin-9 variants and derivatives; Method for producing the variants or derivatives polynucleotides (or nucleic acids) and polypeptides (or proteins); Sugar chains recognized by the galectin-9 or analogs thereof (or protein derivatives) Antibodies against (or part thereof), immunological assay methods using the antibodies and reagents used therefor; Galectin-9 agonists and antagonists, screening methods and screening kits related thereto;
It also relates to the use of the polynucleotide (or nucleic acid), polypeptide (or protein), mutant, derivative, agonist and antagonist.

[0002]

BACKGROUND OF THE INVENTION Galectins refer to a family of animal-derived lectins that have a specific β-galactoside binding activity. So far, nine human galectin families have been identified, namely galectin-1 to 4 (galectin-1 to -4),
Galectin-7 to 10 (galectin-7 to -10), and galectin-12 (galectin-12). The members of each family can be further divided into three subtypes according to their structure (Hirabayashi, J. et al., Gl.
ycobiology, 3: 297-304 (1993)). Galectin-1, -2,
-7 and -10 have one CRD consisting of approximately 130 to 140 amino acid residues and belong to the prototype. Galectin-3 has a carboxy-terminal CRD and a different type of amino-terminal region and is the only chimeric type. Galectin-4, -8, -9
And -12 are two of the two linked by a linker peptide.
It has a CRD and is classified as a tandem repeat type.

Prototype and chimeric type galectins have one CRD, of which galectin-1, -2 and -3, and possibly galectin-7.
It is known that -10 and -10 also form dimers / multimers and have a polyvalent sugar-binding ability (Cho, M. et al., Biochemistry, 35: 13081-130
88 (1996); Gitt, M. et al., J. Biol. Chem., 267: 1
0601-10606 (1992); Hsu, DK et al., J. Biol. Che
m., 267: 14167-14174 (1992); Gitt, M. et al., J. Bi.
ol. Chem., 270: 5032-5038 (1995); Pfeifer, K. et a
L., Glycobiology, 3: 179-184 (1993)). Therefore, it is considered that most of the galectins have a polyvalent binding function and have an ability to crosslink a glycoconjugate.

Human galectin-9 was first identified as a potential autoimmune antigen in patients with Hodgkin's disease, and was suggested to play a role in controlling cell-cell interactions in the immune system. (Tureci, O.
et al., J. Biol. Chem., 272: 6416-6422 (1997)). Mouse galectin-9 was cloned by Wada et al. (J. Biol. Chem., 272: 6078-6086 (1997)), and its properties were investigated, and as a result, the ability to induce thymocyte apoptosis in vitro was confirmed. Have been shown (Wada, J. e
T al., J. Clin. Invest., 99: 2452-2461 (1997)).

[0005]

[Problems to be Solved by the Invention] These findings and the discovery that human ecalectin / galectin-9 has strong eosinophil chemoattractant (ECA) activity (Matsumoto, R. et. al., J. Biol. Che
m., 273: 16976-16984 (1998)), galectin-9 seems to have various roles in the immune system. In order to understand the mechanism by which galectins regulate the immune system, it is necessary to clarify the function of each CRD and elucidate their sugar-binding specificity.

[0006]

[Means for Solving the Problems] The present inventors' group has so far shown that eosinophil migration activity seems to be a galectin-9-specific activity (Matsushita et al., J. B.
iol. Chem., 275: 8355-8360 (2000)). That is, almost no eosinophil migration activity was observed in galectin-1 and -3,
Galectin-8 showed low eosinophil migration activity (ie, galectin-9 no more than 10%). The group of the present inventors has also revealed that the sugar-binding activity of galectin-9 is essential for eosinophil migration activity. In addition, N-terminal CRD and C-terminal C of galectin-9
From experiments using recombinant (recombinant) proteins consisting only of RD, these CRDs, at least a part of which may exist as dimers and / or multimers, showed similar eosinophil migration. Although it shows activity, it has also been revealed that its eosinophil migration activity is 10% or less of that of full-length galectin-9.

[0007] In the process of elucidating the characteristics of T cell-derived factors having eosinophil-specific migration activity at the molecular level, the present inventors have found that ecalectin is a variant of human galectin-9. , A new and potent eosinophil chemotactic factor (Matsumoto, R. et al., J. B.
iol. Chem., 273: 16976-16984 (1998)). Subsequently, the present inventors determined that ecarectin and human galectin-9 are the same substance, determined the DNA sequence using a galectin-9 cDNA clone derived from Jurkat cell, and prepared a database of EST sequences. Confirmed by computer analysis (Matsushita, N. et al., J. Biol. Che
m., 275: 8355-8360 (2000)). Furthermore, we have also clarified the basic characteristics of galectin-9 in eosinophil migration activity. That is, in other members of the human galectin family, almost no eosinophil chemotactic activity is observed (galectin-1 and -3) or very low eosinophil chemotactic activity (galectin-8). , N-terminal CRD and C of galectin-9 compared to full-length galectin-9
-Although the terminal CRD is low, it shows significant eosinophil migration activity, and the galectin-9 eosinophil migration activity is inhibited by lactose. Revealed to lose. These results indicate that the interaction between the two CRDs of galectin-9 and cell surface glycoconjugates is essential for eosinophil migration.

From the above, the group of the present inventors does not show that galectin-9 exerts its eosinophil migration activity by cross-linking the galactoside-containing glycoconjugate on the surface of eosinophils. I came to have a hypothesis of a heel. There are three possible interactions between two CRDs and cell-surface glycoconjugates: 1) the two regions (domains) bind to two different glycoconjugates; 2) the two One domain binds to two identical glycoconjugates; in this case galectin-9 exerts its function via homodimer / multimer formation of the target glycoconjugate (galectin-9 receptor) And 3) the two regions (domains) bind to two different oligosaccharide chain moieties on the same glycoconjugate molecule.

Human galectin-9 is composed of two sugar chain recognition regions.
It is a β-galactoside binding protein consisting of CRD and a linker peptide (also called a connecting region: link peptide). To date, the present inventors have confirmed that galectin-9 has been used as an eosinophil chemotactic factor (e) produced by activated T cells.
osinophil chemoattractants: ECAs). The sugar-binding activity of galectin-9 is essential for eosinophil migration, and although both N-terminal CRD and C-terminal CRD show similar eosinophil migration activity, It has been shown that the chemotactic activity is substantially lower than that of full-length galectin-9 (which has both CRDs).

In the present invention, these possibilities are examined by using a mutant galectin-9 protein having two N-terminal CRDs and a mutant galectin-9 protein having two C-terminal CRDs. At the same time, the sugar binding specificity of the two CRDs was investigated. Furthermore, in the present invention, the significance of the linker peptide structure for eosinophil chemotactic activity was investigated using three galectin-9 isoforms each having a different linker peptide. The three isoforms of galectin-9, galectin-9S, galectin-9M and galectin-9L, are expressed in Jurkat cells and they differ from each other only in the linker peptide.

The inventors of the present invention have confirmed that the human galectin-9
Role of CRD in eosinophil migration activity and its associated C
Further research was conducted on the properties of RD for sugar binding.
In addition to this, the present inventors have compared the eosinophil migration activity of three galectin-9 isoforms in order to clarify the significance of the presence of the linker peptide. Protein composed of two N-terminal CRDs (galectin-9NN: galectin-9NN), two C-terminal CRDs
A protein composed of (galectin-9CC: galectin
-9CC), and three isoforms of galectin-9 (sh
ort type: galectin-9S, intermediate type: galectin-9M and lo
ng type: galectin-9L) was prepared and prepared as a recombinant protein. The recombinant proteins are all major wild-type galectin-9 galectins.
It had a hemagglutination activity comparable to that of -9M. Galectin
-9NN and galectin-9CC showed eosinophil migration which was indistinguishable from galectin-9M. Galectin-9L, which is the galectin-9 isoform with the longest linker peptide, is less soluble than the other isoforms, but all three isoforms are comparable in the concentration range tested. It showed eosinophil migration activity.

Further, frontal affinity chromatography technology (Hirabayashi et al., J. Chromatogr.
A, 890, 261-271 (2000); Arata et al., J. Biol. Che
m., 276: 3068-3077 (2001)), galectin-9 N-
Terminal CRD and C-terminal CRD and various glycoproteins (glycoprotei
The interaction between sugar chains derived from ns) and sugar chains derived from glycolipids was investigated. As a result, any CRD
Was also found to have an affinity for branched sugar chains.
In particular, it was revealed that the two CRDs have high affinity for 3-branched sugar chains and 4-branched sugar chains having an N-acetyllactosamine structure. The 3-branched and 4-branched sugar chains inhibited galectin-9 eosinophil migration activity at low concentrations. From the above results, N-terminal CRD and C of galectin-9
-Terminal CRD is considered to exert eosinophil chemotactic activity by binding to the 3-branched and 4-branched sugar chains on the glycoconjugate present on eosinophils or sugar chains very similar to these. To be Thus, the present invention provides a galectin-9 activity control agent, a control method, a cleaning method therefor, and means, reagents, and techniques for the development of various drugs. In a preferred embodiment, the present invention provides [1] a carrier characterized by immobilizing N-terminal CRD or C-terminal CRD of galectin-9; [2] using the carrier according to [1] above. , Galectin-9
A screening method characterized by screening a sugar chain or an analog thereof recognized by the bacterium; [3] comprising a substance which is obtained by the screening method described in [2] above and has a galectin-9 binding activity Galectin-9 activity inhibitor or regulator; [4] Galectin-9 using the carrier according to [1] above.
And a screening kit for a compound that promotes or inhibits the biological activity of the above; [5] A substance obtained by the screening method according to the above [2] and containing a substance having a binding activity to galectin-9. Medicine; [6] (a) Mutant protein having N-terminal CRD of galectin-9 and not having C-terminal CRD, and (b) C-terminal CRD of galectin-9 and having N-terminal A polypeptide or a salt thereof, which is selected from the group consisting of mutant proteins not having CRD; [7] having a nucleotide sequence encoding the polypeptide according to [6] above [8] A vector comprising the nucleic acid according to [7] above;

[9] A host cell characterized by being transformed with the nucleic acid according to [7] above or the vector according to [8] above; [10] above

The above-mentioned [6], wherein the host cell according to [9] is cultured in a nutrient medium, the polypeptide according to the above-mentioned [6] is expressed in the host cell, and the expressed polypeptide is recovered. [11] An antibody against a sugar chain or an analogue thereof obtained by the screening method described in [2] and recognized by galectin-9; [12] A sugar chain or an analogue thereof recognized by galectin-9. An agent which promotes or inhibits the biological activity of galectin-9 characterized by containing; and [13] an antibody against a sugar chain or an analog thereof recognized by galectin-9 or a mimetic thereof, and galectin
-9, an agent having a biological activity equivalent to that of -9, having a part of the activity, lacking a part of the activity, or inhibiting a part or all of the activity. ing.

Other objects, features, excellences, and aspects possessed by the present invention will be apparent to those skilled in the art from the following description. However, it is understood that the description of the present specification, including the description below and the description of specific examples and the like, shows the preferred embodiments of the present invention and is provided only for the purpose of explanation. I want to. Various changes and / or alterations (or modifications) within the spirit and scope of the invention disclosed herein can be made by those of ordinary skill in the art based on knowledge from the following description and other portions of the specification. Would be readily apparent. All patent and literature references cited in this specification are cited for the purpose of illustration and are to be construed as part of this specification, the contents of which are included herein. is there.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, a mutant capable of selectively utilizing a region predicted to be a CRD of human galectin-9S, -9M and -9L belonging to the galectin-9 subclass among human-derived galectins. Will be provided. Galectin-9
Has an N-terminal CRD and a C-terminal CRD, but in the present invention,
Linearly linked N-terminal CRDs, linearly linked C-terminal CRDs, N-terminal CRD (or C-terminal
There is provided a form in which CRD) can be bound to a carrier or the like and immobilized. The human-derived galectin-9 CRD polypeptide or a peptide having a homology of at least 60% with the amino acid sequence of the galectin-9 CRD polypeptide and having sugar chain binding activity or equivalent antigenicity, or a salt thereof, Characteristic partial peptides of peptides or salts thereof, genes encoding them, such as DNA and RNA, vectors or plasmids containing the genes so that they can be manipulated by gene recombination technology,
A method for producing the polypeptide or a salt thereof by culturing a host cell transformed with such a vector, and further the transformed cell, and the characteristic features of the polypeptide or a salt thereof or the polypeptide thus obtained Sugar chain and its analogs obtained by screening with various partial peptides or salts thereof, and further antibodies against the sugar chain or its analogs, particularly monoclonal antibodies, hybridoma cells producing the antibodies, and the mutants. Or the identified sugar chain and its analog,
Furthermore, a measuring / diagnosing means and a reagent using the antibody are provided. Furthermore, the use of the active ingredient disclosed and described in the present specification is provided, for example, a drug or a reagent containing the active ingredient is provided, and a disease, a disease or an abnormal condition using the active ingredient is provided. Treatment of and /
Alternatively, a preventive method and a screening method are provided. In particular, a galectin-9 activity control agent and control method, as well as a cleaning method and means, reagents, and techniques for the development of various drugs are provided.

As used herein, the term "polypeptide" may refer to any polypeptide as described below. The basic structure of polypeptides is well known and is described in numerous reference books and other publications in the art. In view of these, the term “polypeptide” as used herein means any peptide or any protein containing two or more amino acids which are linked to each other by peptide bonds or modified peptide bonds. To do. As used herein, the term “polypeptide” is commonly referred to in the art as a short chain, also commonly referred to as a peptide, oligopeptide or peptide oligomer, and as a protein, which is known in many forms. It may mean both of the long chains mentioned. Polypeptides may often contain amino acids other than those commonly referred to as the 20 naturally occurring amino acids (naturally occurring amino acids: or gene-encoded amino acids). Polypeptides may also be subjected to natural processes such as processing and other alterations (or modifications) after translation of many amino acid residues, including terminal amino acid residues, as well as chemistry well known to those skilled in the art. It will be understood that the above-mentioned polypeptide can be modified (modified) also by a technical modification technique. There are many known forms of alterations (modifications) to be made to the polypeptide, which are described in detail in basic reference books and more detailed papers in this field as well as in many research literatures. However, these are well known to those skilled in the art.

Some particularly conventional alterations / modifications include, for example, glycosylation, lipid binding, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for example TE Creighton. , Protein
s-Structure and Molecular Properties, Second Editio
n, WH Freeman and Company, New York, (1993); B.
C. Johnson (Ed.), Posttranslational Covalent Modif
ication of Proteins, Academic Press, New York, (19
83) (Wold, F., "Posttranslational ProteinModificat
ions: Perspective and Prospects ", pp.1-12); Seifte
r et al., "Analysis for Protein Modifications and
nonprotein cofactors ", Meth. Enzymol., 182: 626-64
6 (1990); Rattan et al., "Protein Synthesis: Postt
ranslational Modification and Aging ", Ann. NY A
cad. Sci., 663: p.48-62 (1992) etc. can be referred to. The “polypeptide” of the present invention includes, in particular, CRD-related variants of human galectin-9S, -9M and -9L and related polypeptides thereof. Human galectin
-9, a homology of at least 60% with one of the amino acid sequences of human galectin-9 CRD, preferably 70% or more homology, with a focus on the biological activity of CRD.
More preferably 80% or more homology, and preferably 85
% Or more homology, more preferably 90% or more homology,
More preferably 95% or more homology, particularly preferably 97%
All of those having the above-mentioned homology and having an amino acid sequence having substantially the same biological activity such as sugar chain binding activity or equivalent antigenicity are included.

In the present specification, the term "homology" refers to a residue of each amino acid constituting two chains in a polypeptide sequence (or amino acid sequence) or a polynucleotide sequence (or base sequence). Meaning the amount (number) of groups or groups that can be determined to be identical in the mutual correspondence relationship of the bases, and the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences To do. Homology can be easily calculated. There are many known methods for measuring homology between two polynucleotide or polypeptide sequences, the term "homology".
The term (also referred to as “identity”) is well known to those of skill in the art (eg, Lesk, AM (Ed.), Computational
Molecular Biology, Oxford University Press, New Y
ork, (1988); Smith, DW (Ed.), Biocomputing: Info
rmatics and Genome Projects, Academic Press, New Y
ork, (1993); Grifin, AM & Grifin, HG (Ed.),
Computer Analysis of Sequence Data: Part I, 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 Yo
rk, (1991) etc.). A general method used to measure the homology of two sequences is Martin, J. Bishop (Ed.), G.
uide to Huge Computers, Academic Press, San Diego,
(1994); Carillo, H. & Lipman, D., SIAM J. Applied
Math., 48: 1073 (1988) and the like, but are not limited thereto. Preferred methods to measure homology include those designed to give the largest match between the two sequences tested. Such methods include those assembled as a computer program. A preferred computer program method for determining homology between two sequences includes the GCG program package (Devereux, J. et al., Nucleic Acids Resea
rch, 12 (1): 387 (1984)), BLASTP, BLASTN, FASTA (A
tschul, SF et al., J. Molec. Biol., 215: 403 (1
990)) and the like, but the method is not limited thereto, and a method known in the art can be used.

The genes encoding human galectin-9S, -9M and -9L are known and can be suitably used as a template for obtaining the mutant of the present invention, but the gene is expressed separately such as Jurkat cells. You can use the cloned one. Typically, by using the nucleotide sequences represented by SEQ ID NOs: 1 to 8 in the sequence listing as a primer, a predetermined coding gene portion can be obtained and used. The variant polynucleotide (or nucleic acid) of the present invention has a start codon in its base sequence, for example, a codon encoding Met (and a stop codon, for example,
TGA, TAA or TAG) may be present or absent and added later, and it has an amino acid sequence having at least 80% homology to the protein encoded by the nucleotide sequence and has a sugar It may be any as long as it has a nucleotide sequence having the same effect as that of having a chain binding activity or encoding a peptide having substantially the same biological activity as the antigenicity or the like. . The polynucleotide (or nucleic acid)
Is a nucleic acid such as single-stranded DNA, double-stranded DNA, RNA, DNA: RNA hybrid, synthetic DNA, and human genomic DNA.
, Human genomic DNA library, cDNA derived from human tissues / cells, or synthetic DNA. The base sequence of the polynucleotide (or nucleic acid) can be modified (eg, added, removed, substituted, etc.),
Such modifications may also be included. Moreover,
As described below, the nucleic acid of the present invention may encode the mutant peptide of the present invention or a part thereof, and preferred examples thereof include DNA. Further, the above-mentioned "equal nucleotide sequence" means, for example, 5 or more consecutive nucleotide sequences among CRD nucleotide sequences under stringent conditions, preferably 10 or more nucleotide sequences, more preferably 15 nucleotide sequences.
One or more base sequences, more preferably 20 or more base sequences, which hybridize with each other and encode a substantially equivalent amino acid sequence in terms of sugar chain binding, and the like can be mentioned.

The gene recombination technique of the present invention is described in, for example, J.
Sambrook, EF Fritsch & T. Maniatis, "Molecular
Cloning: A Laboratory Manual (2nd edition) ", Cold
Spring Harbor Laboratory Press, Cold Spring Harbo
r, New York (1989); DM Glover et al. ed., "DNA
Cloning ", 2nd ed., Vol. 1 to 4, (The Practical App
roach Series), IRL Press, Oxford University Press
(1995); The Biochemical Society of Japan, "Sequel to Biochemistry Experiment Course 1, Gene Research Method II", Tokyo Kagaku Dojin (1986);
"Shinsei Chemistry Laboratory 2, Nucleic Acid III (Recombinant DNA Technology)", Tokyo Kagaku Dojin (1992); R. Wu ed., "Methods i
n Enzymology ", Vol. 68 (Recombinant DNA), Academic
Press, New York (1980); R. Wu et al. Ed., "Method
s in Enzymology ", Vol. 100 (Recombinant DNA, Part
B) & 101 (Recombinant DNA, Part C), Academic Pres
s, New York (1983); R. Wu et al. ed., "Methods in
Enzymology ", Vol. 153 (Recombinant DNA, Part D), 1
54 (Recombinant DNA, Part E) & 155 (Recombinant DN
A, Part F), Academic Press, New York (1987); JH
Miller ed., "Methods in Enzymology", Vol. 204, Ac
ademic Press, New York (1991); R. Wu et al. ed., "
Methods in Enzymology ", Vol. 218, Academic Press, N
The method described in ew York (1993) or the method described in the literature cited therein, or a method substantially similar thereto or a modified method can be used (for the description in them, refer to it). Included in the disclosure herein).

In the present specification, "polymerase chain.
Reaction (Polymerase Chain Reaction) "or" PC
“R 2” generally refers to methods such as those described in US Pat. No. 4,683,195 and the like, eg, for in vitro enzymatic amplification of a desired nucleotide sequence. In general, the PCR method involves repeating a cycle for primer extension synthesis using two oligonucleotide primers capable of preferentially hybridizing with a template nucleic acid.
Typically, the primer used in the PCR method can be a primer complementary to the nucleotide sequence to be amplified inside the template, and for example, the nucleotide sequence to be amplified and the complementary nucleotides at both ends thereof can be used. Those that are target or are adjacent to the nucleotide sequence to be amplified can be preferably used. The PCR reaction is
It can be carried out by a method known in the art or a method substantially similar thereto or a modified method, and for example, R. Saik
i, et al., Science, 230: 1350, 1985; R. Saiki, et a
l., Science, 239: 487, 1988; HA Erliched., PCR
Technology, Stockton Press, 1989; DM Glover e
t al. ed., "DNA Cloning", 2nd ed., Vol. 1, (The Pra
ctical Approach Series), IRLPress, Oxford Universi
ty Press (1995); MA Innis et al. ed., "PCRProt
ocols: a guide to methods and applications ", Acade
mic Press, New York (1990)); MJ McPherson, P. Q
uirke and GR Taylor (Ed.), PCR: a practical app
roach, IRL Press, Oxford (1991); MA Frohman et
al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (19
88) etc. or modified it,
It can be performed according to a modified method. In addition, the PCR method can be performed using a commercially available kit suitable for the PCR method, or can be performed according to the protocol disclosed by the kit manufacturer or the kit vendor.

In a typical case, for example, a template DNA and a primer are mixed with 10 × reaction buffer (attached to a DNA polymerase such as Taq DNA polymerase), dNTPs (deoxynucleoside triphosphate dATP, dGTP, dCTP, dTTP). Mixture), Taq DNA polymerase and deionized distilled water. The 5'-end side primer contains at least a start codon or is selected so as to be amplified including the start codon, and the 3'-end side primer contains at least a stop codon, or It is preferable to select such that the stop codon can be included in the amplification. The mixture is for example GeneAmp 2400 PCR s
Using an automatic thermal cycler such as ystem, Perkin-Elmer / Cetus, etc., the cycle is repeated 25 to 60 times under general PCR cycle conditions, but the number of cycles for amplification is set appropriately according to the purpose. be able to. PCR cycle conditions include, for example, denaturation 90-95 ° C 5-100
Second, annealing 40-60 ℃ 5 ~ 150 seconds, extension 65 ~ 75 ℃ 3
0 to 300 seconds cycle, preferably denaturation 94 ° C for 15 seconds,
A cycle of annealing at 58 ° C for 15 seconds and extension at 72 ° C for 45 seconds can be mentioned. The annealing reaction temperature and time can be selected appropriately by experiments, and the denaturation reaction and extension reaction times can also be estimated as expected PCR products. An appropriate value can be selected according to the chain length of the. The reaction temperature for annealing is
Usually, it is preferable to change according to the Tm value of the hybrid between the primer and the template DNA. The extension reaction time is usually 10
About 1 minute per chain length of 00 bp is a rough guide,
It is possible in some cases to choose a shorter time.

The obtained PCR product is usually subjected to 1 to 2% agarose gel electrophoresis and cut out from the gel as a specific band, and the DNA is extracted using a commercially available extraction kit such as gene clean kit (Bio 101). To do. Extracted
Cleave the DNA with an appropriate restriction enzyme, purify if necessary, and further phosphorylate the 5'end with T4 polynucleotide kinase if necessary, then pUC18 or other pUC
A suitable plasmid vector such as a system vector is ligated, and an appropriate competent cell is transformed. The base sequence of the cloned PCR product can be analyzed appropriately. For cloning PCR products, for example, p-Direct (Clontech), pCR-Script ^TM SK
(+) (Stratagene), pGEM-T (Promega), pAmp ^TM (Gibco
-BRL) and other commercially available plasmid vectors can be used. The nucleotide sequence can be determined by the dideoxy method such as M
13 Although it can be performed using the Maxam-Gilbert method such as the dideoxy method, a commercially available sequencing kit,
For example, using Taq Die Primer Cycle Sequencing Kit (Applied Biosystems), Sequenase v 2.0 kit, or an automatic nucleotide sequencer such as fluorescent DNA
Sequencer instrument (e.g. Model 373A, Applied Bios
ystems) and so on. Examples of the polymerase used in the dideoxy method include Klenow fragment of DNA polymerase I, AMV reverse transcriptase, Taq DNA polymerase, T7 DNA polymerase, and modification.
Examples include T7 DNA polymerase.

In the present specification, the "oligonucleotide" is a relatively short single-stranded or double-stranded polynucleotide, preferably polydeoxynucleotide,
Angew. Chem. Int. Ed. Engl., Vol.28, p.716-734 (19
89), and known methods such as triester method, phosphite method, phosphoamidite method and phosphonate method can be used for chemical synthesis. It is generally known that the synthesis can be conveniently carried out on a modified solid support, for example using an automated synthesizer, which is commercially available. The oligonucleotide may contain one or more modified bases, for example:
It may contain an unnatural base such as inosine or a tritylated base.

Hybridization is carried out using a predetermined insertion DN.
The plaque formed by a microorganism that retains A (nucleic acid itself may be transferred) is transferred to a membrane such as a nylon filter, and if necessary, denaturation treatment, immobilization treatment, washing treatment, etc., and then the membrane It is carried out by reacting the one transcribed into the labeled probe DNA fragment, which has been denatured if necessary, in a hybridization buffer. The hybridization treatment is usually about 35 ° C to about 80 ° C.
C., more preferably about 50.degree. C. to about 65.degree. C., for about 15 minutes to about 36 hours, more preferably for about 1 hour to about 24 hours, but can be performed by selecting optimal conditions as appropriate. . For example, the hybridization treatment is performed at about 55 ° C. for about 18 hours. The hybridization buffer can be selected from those commonly used in the art and used, for example, Rapid hybridization buffer (Amersh
am) and the like can be used. Examples of the modification treatment of the transferred film include a method using an alkali denaturing solution,
After the treatment, it is preferable to treat with a neutralizing solution or a buffer solution. The immobilization treatment of the membrane is usually about 40 ° C to about 100 ° C, more preferably about 70 ° C to about 90 ° C for about 15 minutes to about 24 hours, more preferably about 1 hour to about 4 hours. It is carried out by baking, but it can be carried out by appropriately selecting preferable conditions. For example, the immobilization is performed by baking the filter at about 80 ° C for about 2 hours. As the washing treatment of the transferred film, a washing solution commonly used in this field, for example, 1M NaCl, 1 mM EDTA and 0.1% sodium dodecyl is used.
It can be performed by washing with 50 mM Tris-HC1 buffer containing sulfate (SDS), pH 8.0 and the like. Membranes such as nylon filters can be selected and used from those commonly used in the art, and examples thereof include nylon filters [Hybond-N, Amersham].

The alkali denaturing solution, the neutralizing solution, and the buffer solution may be selected from those commonly used in the art, and examples of the alkali denaturing solution include:
A solution containing 0.5M NaOH and 1.5M NaCl can be mentioned. Examples of the neutralizing solution include 1.5M NaCl.
0.5M Tris-HCl buffer solution, pH 8.0 and the like can be mentioned. Examples of the buffer solution include 2 × SSPE (0.36M NaCl,
20 mM NaH ₂ PO ₄ and 2 mM EDTA) and the like. In addition, prior to the hybridization treatment, it is preferable to pre-hybridize the transferred film in order to prevent non-specific hybridization reaction. This prehybridization treatment can be performed, for example, by prehybridization solution [50%
formamide, 5 x Denhardt's solution (0.2% bovine serum albumin, 0.2% polyvinyl pyrrolidone), 5 x SSPE, 0.
1% SDS, 100 μg / ml heat-denatured salmon sperm DNA]
The reaction can be carried out at about 35 ° C. to about 50 ° C., preferably about 42 ° C. for about 4 to about 24 hours, preferably about 6 to about 8 hours. By repeating the above, more preferable conditions can be determined. Labeled probe DNA used for hybridization
The fragment can be denatured, 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. Hybridization can be performed by a method known per se or a method analogous thereto, but the stringent conditions in the present specification include, for example, sodium concentration of about 15
To about 50 mM, preferably about 19 to about 40 mM, more preferably about
The temperature is about 19 to about 20 mM, and the temperature is about 35 to about 85 ° C, preferably about 50 to about 70 ° C, more preferably about 60 to about 65 ° C.

After the hybridization is completed, the filter is thoroughly washed to remove the labeled probe other than the labeled probe DNA fragment which has undergone the specific hybridization reaction. The filter can be washed by using one selected from those commonly used in the art. For example, 0.5 × SSC containing 0.1% SDS (O.15M NaCl,
It can be carried out by washing with a 15 mM citric acid) solution or the like. The hybridized plaque (nucleic acid) can be typically detected by autoradiography, but can also be appropriately selected from the methods used in the art and used for plaque (nucleic acid) detection. The plaque (nucleic acid) corresponding to the detected signal is transferred to an appropriate buffer,
For example, SM solution (50 mM containing 100 mM NaCl and 10 mM MgSO ₄
Suspend in Tris-HCl buffer, pH 7.5, etc., and then appropriately dilute this phage (which may include the nucleic acid itself) suspension to infect E. coli and culture the resulting E. coli,
A target recombinant phage (nucleic acid) is obtained from the cultured Escherichia coli. The above-mentioned probe DNA may be used, if necessary, to perform a process of screening a target recombinant phage (nucleic acid) from a gene library or a cDNA library by a hybridization process, which can be repeated. The target recombinant phage (nucleic acid) is
It can be obtained by subjecting cultured Escherichia coli to extraction treatment, centrifugation treatment, or the like.

The obtained phage particles can be purified and separated by a method commonly used in the art.
Glycerol gradient ultracentrifugation method (Molecular cl
oning, a laboratory manual, ed. T. Maniatis, Cold
Spring Harbor Laboratory, 2nd ed. 78, 1989) and the like. From the phage particles, DNA can be purified and separated by a method commonly used in the art. For example, the obtained phage can be isolated in TM solution (10 mM MgS
Suspend in O ₄ containing 50 mM Tris-HCl buffer, pH 7.8, etc.,
After treatment with DNase I and RNase A, etc., 20 mM EDTA, 50
Add μg / ml Proteinase K and 0.5% SDS mixture, etc., and incubate at about 65 ° C for about 1 hour, then extract with phenol and extract with diethyl ether, then precipitate with DNA to precipitate DNA.
And then the DNA obtained is washed with 70% ethanol, dried, and dissolved in a TE solution (10 mM Tris-HC1 buffer containing 10 mM EDTA, pH 8.0). Further, the target DNA can be obtained in a large amount by subcloning or the like. For example, the subcloning can be performed using E. coli as a host and using a plasmid vector or the like. The DNA obtained by such subcloning can also be purified and separated by a method such as centrifugation, phenol extraction and ethanol precipitation in the same manner as above. Thus, according to the present invention, a clone (for example, as a recombinant phage etc.) containing the target DNA can be obtained.

The nucleic acid having all or part of the nucleotide sequence of the desired mutant of the present invention can be obtained by chemical synthesis. In that case, the fragments may be chemically synthesized and then they are enzymatically linked. It is also possible to obtain a target sequence by using the chemically synthesized fragment as a primer or a probe as described above. The primer used in the PCR method is not particularly limited as long as it can amplify the DNA fragment containing the above site. Typically, the primer is (a) an oligonucleotide having a nucleotide sequence corresponding to an arbitrary region of the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing, and (b) SEQ ID NO: 1 of the sequence listing. It is possible to use an oligonucleotide having a complementary nucleotide sequence to any region of the indicated nucleotide sequence, more preferably (1) N-terminal or C-terminal CR
An oligonucleotide having a nucleotide sequence corresponding to an arbitrary region on the 5'end side of the D nucleotide sequence and (2) N-terminal or C-
An oligonucleotide having a complementary base sequence to any region on the 3′-end side of the terminal CRD base sequence can be used, and for example, 3 to 100, preferably 10 to 50,
More preferably, those containing 15 to 35 nucleotides can be mentioned.

The DNA fragment obtained according to the present invention can be transformed into a suitable vector, such as a plasmid, as described in detail below.
It can be incorporated into a vector such as pEX, pMAMneo or pKG5 and expressed in a suitable host cell as described in detail below, for example, Escherichia coli, yeast, CHO cell, COS cell and the like. The DNA fragment may be used as it is, or as a DNA fragment to which an appropriate regulatory sequence is added, or it may be incorporated into an appropriate vector and then introduced into an animal to prepare galectin-9NN.
Alternatively, transgenic animals expressing mutants such as galectin-9CC can be created. Examples of animals include mammals, such as mice, rats, rabbits, guinea pigs, and cows. Preferably, the DNA fragment can be introduced into a fertilized egg of an animal such as a mouse to prepare a transgenic animal.
The exogenous nucleic acid can be introduced into animal cells such as mammals by a method known in the art or a method substantially similar thereto, for example, calcium phosphate method (for example, FL Graham et al., Virology, 52:
456, 1973), DEAE-dextran method (eg D.
Warden et al., J. Gen. Virol., 3: 371, 1968, etc.), electroporation method (eg E. Neumann)
et al., EMBO J, 1: 841, 1982), microinjection method, ribosome method, virus infection method, phage particle method and the like. The gene product produced by the animal cell thus transfected with the mutant nucleic acid can also be analyzed.

As a plasmid incorporating a nucleic acid such as DNA obtained in the present invention, a host cell commonly used in genetic engineering (for example, prokaryotic host such as Escherichia coli or Bacillus subtilis, yeast, CH
Any plasmid may be used as long as it can express the DNA in a eukaryotic host such as O cell and COS cell, and an insect cell host such as Sf21). Within such a sequence, for example, codons suitably modified for expression in the selected host cell may be contained, restriction enzyme sites may be provided, and the desired gene Regulatory sequences and facilitating sequences for facilitating expression, such as linkers and adapters that help bind the target gene, antibiotic resistance, metabolism control, selection, etc. It may contain useful sequences (including those encoding hybrid proteins and fusion proteins) and the like. Preferably, a suitable promoter, such as a tryptophan promoter (trp) or a lactose promoter in a plasmid having E. coli as a host
(lac), tryptophan lactose promoter (ta
c), lipoprotein promoter (lpp), lambda phage
For plasmids that use the P _L promoter, etc. as an animal cell host, SV40 rate promoter, MMTV LTR promoter, RSV LTR promoter, CMV promoter, SRα promoter, etc.
L1, GAL10 promoter and the like can be used.

As a plasmid using E. coli as a host,
For example pBR322, pUC18, pUC19, pUC118, pUC119, pSP6
4, pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGEM-3, pG
EM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf (-), pBluescrip
t KS ^™ , (Stratagene) and the like. Suitable plasmid vectors for expression in E. coli include pAS and pKK2
23 (Pharmacia), pMC1403, pMC931, pKC30, pRSET-B
(Invitrogen) is also included. Examples of plasmids using animal cells as hosts include SV40 vector, polyoma virus vector, vaccinia virus vector, retrovirus vector, and the like, for example, pcD, pcD-SR.
α, CDM8, pCEV4, pME18S, pBC12BI, pSG5 (Stratage
ne) and so on. Examples of the plasmid using yeast as a host include YIp type vector, YEp type vector, YRp type vector, YCp type vector, and the like, for example, pGPD-2. When the host cell is E. coli, examples of the host cell include those derived from the Escherichia coli K12 strain, and examples thereof include NM533, XL1-Blue, C600, DH1, DH5, DH11.
S, DH12S, DH5α, DH10B, HB101, MC1061, JM109
Examples of the STBL2 and B834 strains include BL21 (DE3) pLysS. When the host cell is an animal cell, for example, African green monkey fibroblast-derived COS-7 cells, COS-1 cells, CV-1 cells, mouse fibroblast-derived COP cells, MOP
C derived from cells, WOP cells, Chinese hamster cells
HO cells, CHO DHFR ⁻ cells, human HeLa cells, mouse cell-derived C127 cells, mouse cell-derived NIH 3T3 cells and the like can be mentioned. Insect cells include silkworm nuclear polyhedrosis virus
(Bombyx mori nuclear polyhedrosis virus) or one derived therefrom can be used as a vector, and silkworm larvae or silkworm cultured cells such as BM-N cells can be used. It is also possible to use plant cells as host cells, together with suitable vectors, which are well known in the art.

In the genetic engineering method of the present invention, in order to modify or convert a restriction enzyme, reverse transcriptase, or DNA fragment known or used in the art into a structure suitable for cloning. The enzymes such as DNA modifying / degrading enzyme, DNA polymerase, terminal nucleotidyl transferase, and DNA ligase can be used. Examples of restriction enzymes include RJ Roberts, Nuc
leic Acids Res., 13: r165, 1985; S. Linn et al. ed.
Nucleases, p. 109, Cold Spring Harbor Lab., Cold
Spring Harbor, New York, 1982; RJ Roberts, D. M
acelis, Nucleic Acids Res., 19: Suppl. 2077, 1991
And the like. As reverse transcriptase,
For example, the mouse Moloney leukemia virus (mouse Moloney
leukemia virus; MMLV-derived reverse transcriptase (reverse tra
nscriptase), chicken myeloblastosis virus (avian mye
and a reverse transcriptase derived from loblastosis virus (AMV). Reverse transcriptases such as RNase H deficient can be preferably used, and in particular, modified MM lacking RNase H activity.
LV RT can be preferably used, and one having high thermal stability is preferable. Suitable reverse transcriptases include MMLV RT (G
ibco-BRL), Superscript RT plus (Life Technologie
s) etc.

Examples of the DNA polymerase include E. coli
DNA polymerase, its derivative Klenow Flag
Ment, coliphage T4 DNA polymerase, coliphine
Image T7 DNA polymerase, thermostable bacterium DNA polymerase
Which can be mentioned. Terminal nucleotidyl transferer
For example, R. Wu et al. Ed., "Methods in Enz
ymology ", Vol. 100, p. 96, Academic Press, New Yor
k (1983) at the 3'-OH end with deoxynucleotides.
Examples include TdTase that adds (dNMP). DNA modification
Decomposing enzymes include exonuclease and endonuclease.
And snake venom phosphodiestera.
, Spleen phosphodiesterase, E. coli DNA exonu
Clearase I, E. coli DNA exonuclease III, Large
Enterobacterial DNA exonuclease VII, λ exonuclear
, DNase I, Nuclease S1, Micrococcus (Micr
ococcus) nuclease and the like. DNA rigger
Examples of the enzyme include E. coli DNA ligase and T4 DNA ligase
Ze and the like. DNA gene cloning and DN
As a suitable vector for constructing A libraries
Is a plasmid, lambda phage, cosmid, P1 phage,
F factor, YAC, etc., and preferably λ phage origin
There are conventional vectors such as Charon 4A and Charon 21.
A, λgt10, λgt11, λDASHII, λFIXII, λEMBL3,
λZAPII ^TM (Stratagene), etc.

The transformant transformed with the expression vector containing the nucleic acid encoding the protein of the present invention stabilizes its high expression ability by carrying out repeated cloning using an appropriate selectable marker if 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 protein of the present invention is encoded by gradually increasing the MTX concentration and culturing and selecting a resistant strain. It is possible to amplify the DNA and obtain a cell line with higher expression. The transformant of the present invention can be cultured under conditions in which the nucleic acid encoding the protein of the present invention can be expressed to produce and accumulate the desired product. The transformant can be cultured in a medium commonly used in the art. For example, a liquid medium can be preferably used for a transformant using a prokaryotic host such as Escherichia coli or Bacillus subtilis or a yeast host. The medium contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein,
Examples of inorganic or organic substances such as meat extract, malt extract, soybean meal, and potato extract, and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, calcium carbonate, and the like. In addition, yeast, vitamins, casamino acids, growth promoting factors and the like may be added. Further, if necessary, a drug such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. The pH of the medium is preferably about 5-8.

The culture is generally about 15 to about 45 for E. coli.
It is carried out at a temperature of about 3 to about 75 hours, and if necessary, aeration and stirring can be added. When culturing a transformant whose host is an animal cell, as the medium, for example, MEM medium containing about 5 to about 20% fetal bovine serum, PRMI1640 medium, DMEM medium and the like are used. The pH is preferably about 6 to about 8. Culturing is usually performed at about 30 ° C to about 40 ° C for about 15 to about 72 hours, and aeration and agitation are added as necessary. When extracting from the cultured cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and disrupted by ultrasonic waves, lysozyme and / or freeze-thawing. After that, a method of obtaining a crude extract by centrifugation or filtration can be appropriately used. The buffer contains protein denaturants such as urea and guanidine hydrochloride, and Triton X.
A surfactant such as -100 (trade name) or Tween-80 (trade name) may be added. When the desired product is secreted into the culture solution, after the completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.
The target product contained in the thus obtained culture supernatant or the extract can be purified by appropriately combining separation / purification methods known per se, for example, salts such as ammonium sulfate precipitation method. Gel filtration by Sephadex or Sephadex, for example, ion exchange chromatography using a carrier having a diethylaminoethyl group or carboxymethyl group, for example, a carrier having a hydrophobic group such as a butyl group, an octyl group or a phenyl group. It can be obtained by purification by the used hydrophobic chromatography method, dye gel chromatography method, electrophoresis method, dialysis, ultrafiltration method, affinity chromatography method, high performance liquid chromatography method and the like. Preferably, it can be purified and separated by polyacrylamide gel electrophoresis, affinity chromatography with immobilized ligand and the like. For example, gelatin-agarose affinity chromatography, heparin-agarose chromatography and the like can be mentioned.

In the present invention, a method commonly used in genetic engineering based on the gene base sequence of galectin-9 is used to appropriately substitute or delete one or more amino acids in a given amino acid sequence. Corresponding proteins with mutations such as loss, insertion, transfer or addition can be produced. Examples of such mutation / conversion / modification methods are edited by The Biochemical Society of Japan, “Sequel Biochemistry Experimental Course 1, Gene Research Method II”, p105 (Susumu Hirose), Tokyo Kagaku Dojin (1986); Experimental Course 2, Nucleic Acid III (Recombinant DNA Technology) ", p233 (Susumu Hirose), Tokyo Kagaku Dojin (199
2); R. Wu, L. Grossman, ed., "Methods in Enzymolog
y ", Vol. 154, p. 350 & p. 367, AcademicPress, New
York (1987); R. Wu, L. Grossman, ed., "Methods in
Enzymology ", Vol. 100, p. 457 & p. 468, Academic P
ress, New York (1983); JA Wells et al., Gene,
34: 315, 1985; T. Grundstroem et al., Nucleic Acid
s 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: 17
7, 1986 and the like. For example, site-directed mutagenesis using synthetic oligonucleotides (site-directed mutagenesis) (Zoller et al., Nucl. Acid
s Res., 10: 6487, 1987; Carter et al., Nucl. Acids
Res., 13: 4331, 1986), cassette mutagenesis method (casset
te mutagenesis: Wells et al., Gene, 34: 315, 198
5), restriction site selection mutagenesis
mutagenesis: Wells et al., Philos.Trans.R.Soc.L
ondon Ser A, 317: 415, 1986), alanine scanning method (Cunningham & Wells, Science, 244: 1081-108)
5, 1989), PCR mutagenesis method, PCR megaprimer method, Ku
Examples include the nkel method, dNTP [αS] method (Eckstein), and region-directed mutagenesis method using sulfite or nitrite.

Further, the obtained protein of the present invention can be modified in its amino acid residue by a chemical method, and peptidases such as pepsin, chymotrypsin, papain, bromelain, endopeptidase, exopeptidase, etc. The enzyme can be used for modification or partial decomposition to give a derivative or the like. Proteins of the present invention, C-terminal, usually a carboxyl group (-COOH) or carboxylate (-COO ^-) is a, C-terminal, may be an amide (-CONH ₂₎ or ester (-COOR). Here, R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl or cyclohexyl, for example, phenyl. , A C _6-12 aryl group such as α-naphthyl, for example, a phenyl-C _1-2 alkyl group such as benzyl and phenethyl, or an α such as α-naphthylmethyl.
In addition to C _7-14 aralkyl group such as -naphthyl-C _1-2 alkyl group, pivaloyloxymethyl group widely used as an oral ester is used. The protein of the present invention
When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminal, the carboxyl group of which is amidated or esterified is also included in the protein of the present invention. Examples of the ester in this case include those described above.
A C-terminal ester or the like is used.

Furthermore, in the protein of the present invention, in the above-mentioned protein, the amino group of the N-terminal methionine residue is a protecting group (for example, formyl group, C such as acetyl, etc.).
_(1-5 alkyl-carbonyl group, etc., C _1-6 acyl group, etc.)
Protected with, the N-terminal side is cleaved in vivo glutamyl group produced by pyroglutamyl, substituents on the side chain of amino acids in the molecule (for example, -OH, -COOH,
Amino group, imidazole group, indole group, guanidino group, etc.) protected by a suitable protecting group (eg, formyl group, C _1-6 acyl group such as acetyl group, etc.),
Alternatively, a complex protein such as a so-called glycoprotein having a sugar chain bound thereto is also included. Alternatively, it may be expressed as a fusion protein when it is produced by a gene recombination method, and may be converted / processed into one having a biological activity substantially equivalent to that of natural CRD in vivo or in vitro. Although a fusion production method commonly used in genetic engineering can be used, such a fusion protein can be purified by affinity chromatography or the like using the fusion portion. Such fusion proteins include those fused to a histidine tag, or β-galactosidase (β-gal), maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX) or Cre Recombinase. Examples include those fused to an amino acid sequence. Similarly, the polypeptide can be tagged with a heterogeneous epitope so that it can be purified by immunoaffinity chromatography using an antibody that specifically binds to the epitope.

In a more preferred embodiment, the epitope tag is, for example, AU5, c-Myc, CruzTag 09, C
ruzTag 22, CruzTag 41, Glu-Glu, HA, Ha.11, KT3, FL
AG (registered trademark, Sigma-Aldrich), Omni-pro
be, S-probe, T7, Lex A, V5, VP16, GAL4, VSV-G, etc. (Field et al., Molecular and Cellula
r Biology, 8: pp.2159-2165 (1988); Evan et al., Mo
lecular and CellularBiology, 5: pp.3610-3616 (198
5); Paborsky et al., Protein Engineering, 3 (6): pp.
547-553 (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., 26.
6: pp.15163-15166 (1991); Lutz-Freyermuth et al.,
Proc. Natl. Acad. Sci. USA, 87: pp.6393-6397 (199
0) etc.). A two-hybrid method using yeast can also be used. Further, the fusion protein may be one with a marker attached so that it becomes a detectable protein. In a more preferred embodiment, the detectable marker is a biotin / streptavidin system of Biotin.
It may be an Avi Tag, a fluorescent substance or the like. The substance that emits the fluorescence is Aequorea victo.
rea) and other green jellyfish-derived green fluorescent proteins (green
fluorescent protein: GFP) and its modified variants (G
FP variant), for example EGFP (Enhanced-humanized GF
P), rsGFP (red-shift GFP), yellow fluorescent protein (yel
low fluorescent protein: YFP), green fluorescent protein
(green fluorescent protein: GFP), indigo fluorescent protein
(cyan fluorescent protein: CFP), blue fluorescent protein (BFP), Renilla
Examples include GFP derived from (Renilla reniformis) (Miyawaki Atsushi, Ed., Experimental Medicine, Separate volume, Post-genomic era experimental course 3-GFP and bio-easing, Yodosha (2000)). Detection can also be performed using an antibody (including a monoclonal antibody and a fragment thereof) that specifically recognizes the above fusion tag.

In a preferred embodiment of the present invention, a marker sequence, such as hexa-
Those in which a histidine peptide is fused can be used.
Expression and purification of such a fusion protein can be carried out using a commercially available kit suitable therefor, and can also be carried out according to the protocol disclosed by the kit manufacturer or kit vendor. Modifications and alterations in protein structure were referred to, for example, the method described therein or cited therein by referring to "Shinsei Chemistry Laboratory Lecture 1, Protein VII, Protein Engineering", edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin (1993). It can be carried out by the method described in the literature or a method substantially similar thereto. Further, as described below, the biological activity may include immunological activity, for example, having an antigenicity. Among the modifications and alterations, deamination, hydroxylation, phosphorylation, methylation, acetylation, ring opening, ring closing,
For example, the type of contained sugar chain may be changed to a different type, the number of contained sugar chains may be increased or decreased, and substitution with a D-form amino acid residue may be performed. Those methods are known in the art (eg TE Creighton, Proteins: Structure and
Molecular Properties, pp.79-86 WH Freeman & C
o., San Francisco, USA (1983), etc.).

Thus, the mutant protein of the present invention has 1
One or more amino acid residues may differ from the naturally occurring form in terms of identity, and one or more amino acid residue positions may differ from the naturally occurring form. The mutant protein of the present invention has one or more amino acid residues specific to CRD of galectin-9 (for example, 1 to 80, preferably 1 to 60,
More preferably 1 to 40, more preferably 1 to 20
Deletion analogues lacking one, in particular 1 to 10, etc., one or more (eg 1 to 80, preferably 1 to 60, more preferably 1 to 40) of unique amino acid residues, More preferably 1 to 20, particularly 1 to 10 etc.) substituted analogs substituted with other residues, 1 or more (eg 1 to 80)
Preferably 1 to 60, more preferably 1 to 40,
More preferably, 1-20, especially 1-10 etc.) amino acid residues are added. All the above mutants are included in the present invention as long as the domain structure or active center structure characteristic of CRD in the naturally-derived form of galectin-9 is maintained. It is also considered that the mutants of the present invention may include those having a primary structure conformation or a part thereof that is substantially equivalent to CRD in a naturally-derived form of galectin-9, and further galectin-9. It is considered that those having a biological activity substantially equivalent to that of the naturally-derived form of CRD may be included. It can also be one of the naturally occurring variants.

The polypeptide of the present invention is, for example, 60% of the CRD amino acid sequence of the naturally occurring form of galectin-9.
In some cases, those having a homology higher than 70% can be mentioned, and more preferably, those having a homologous amino acid sequence of 80% or 90% or more can be mentioned. A part of the polypeptide of the present invention means a part of the peptide of the galectin-9 CRD protein (i.e.,
Galectin-9, which is a partial peptide of the protein)
Any one may be used as long as it has an activity substantially equivalent to that of CRD. For example, the partial peptide of the protein of the present invention is at least 5 or more, preferably 20 or more, more preferably 50 or more, more preferably 70 or more, and still more preferably the constituent amino acid sequence of CRD of galectin-9. Is a peptide having an amino acid sequence of 100 or more, and in some cases 120 or more, preferably those corresponding to consecutive amino acid residues, or, for example, the amino acid of CRD of galectin-9. Regarding homology to the corresponding region of the sequence,
Those having the same homology as described above can be mentioned.

In the present specification, "substantially equivalent" means protein activity, for example, sugar binding activity, eosinophil migration activity,
It means that physiological activity and biological activity are substantially the same. Furthermore, within the meaning of the term,
It may include the case of having substantially the same activity,
Examples of the substantially same activity include a binding activity to a sugar chain having an N-acetyllactosamine structure, an eosinophil migration activity, and the like. The substantially same activity means that those activities are the same in nature. For example, activities such as binding activity to a sugar chain having an N-acetyllactosamine structure are equivalent (for example, about 0.0
01 to about 1000 times, preferably about 0.01 to about 100 times, more preferably about 0.1 to about 20 times, still more preferably about 0.5 to about 2 times.
However, quantitative factors such as the degree of activity and the molecular weight of the protein may be different. Second, amino acid substitutions, deletions, or insertions often do not result in a significant change in the physiological or chemical properties of the polypeptide, in which case the substitution,
Polypeptides with deletions or insertions will be substantially identical to those without such substitutions, deletions or insertions. 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 acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, phenylalanine, leucine,
Isoleucine, valine, proline, tryptophan, methionine, etc. are mentioned, and polar (neutral) amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, etc., and amino acids having a positive charge (basic amino acid Examples of) include arginine, lysine, histidine, and the like, and examples of the amino acid having a negative charge (acidic amino acid) include aspartic acid and glutamic acid.

For the synthesis of the protein of the present invention and its partial peptide, a method known in the peptide synthesis field,
For example, a chemical synthesis method such as a liquid phase synthesis method or a solid phase synthesis method can be used. In such a method, for example, a resin for protein or peptide synthesis is used, and appropriately protected amino acids are sequentially bound to the desired amino acid sequence on the resin by various condensation methods known per se. For the condensation reaction, various activation 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 appropriately removing the protecting group.
When the protein of the present invention and a part of the peptide thereof are obtained in a free form, they can be converted into a salt by a method known per se or a method analogous thereto, and they are converted into a salt. When obtained, it can be converted into a free form or another salt by a method known per se or a method analogous thereto. The salt of the protein of the present invention and a part of the peptide thereof is preferably physiologically acceptable or pharmaceutically acceptable, but is not limited thereto. As such salt,
Salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., such as acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid, malic acid, benzoic acid, methanesulfonic acid , P-toluenesulfonic acid, salts with organic acids such as benzenesulfonic acid, and the like. Further, examples of the salt include ammonium salts, for example, salts with organic bases such as ethylamine, dimethylamine, trimethylamine and hydroxyethylamine.

The mutants, modified products, derivatives and the like of the present invention can be subjected to the separation / purification treatment as described above. In the present invention, the terms “fragment”, “derivative” and “analog” refer to a galectin-9 CRD-related polypeptide, transcribed from the corresponding nucleotide sequence and unspliced or specifically spliced.
A polypeptide encoded by hnRNA or mRNA, or in the context of a polypeptide encoded by genomic DNA is referred to as a "fragment,""derivative," or "analog" of that polypeptide and is essentially Means a polypeptide having the same biological function or activity. Thus, analogs include activatable proproteins, etc., such that the proprotein portion is cleaved to produce an active mature polypeptide. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments, this is a recombinant polypeptide.

The mutant proteins related to CRD, fragments thereof, and nucleic acids including DNA (including mRNA and oligonucleotide) disclosed herein may be used alone or organically, and Sense method,
It can be applied to the genomics and proteomics techniques by appropriately combining with antibodies including monoclonal antibodies and transgenic animals. For example, CRD mutants can also be used for functional analysis using the dominant negative effect. In addition, RNAi using double-stranded RNA (dsRNA)
(RNA interference) There is a potential application for technology. Thus, single nucleotide polymorphism (SNP)
polymorphism analysis centered on (s), gene expression analysis using nucleic acid array, protein array, gene function analysis,
It becomes possible to analyze protein-protein interactions, related diseases, and therapeutic drugs for diseases. For example, in the nucleic acid array technology, a cDNA library was used or PCR technology was used.
DNA is placed at high density on a substrate with a spotting device, and a sample is analyzed using hybridization. The array is formed by using a needle or a pin, or by an ink jet printing technique,
This can be done by attaching DNA to each unique position of a substrate such as a slide glass, a silicon plate, and a plastic plate. Data is acquired by observing the signal obtained as a result of hybridization on the nucleic acid array. The signal is a label such as a fluorescent dye (for example, Cy3, Cy5, BODIPY, FITC, Alex
a Fluordyes (trade name), Texas red (trade name), etc.). A laser scanner or the like may be used for detection, and the obtained data may be processed by a computer system equipped with a program according to an appropriate algorithm. Also, protein array technology may utilize tagged recombinantly expressed protein products for two-dimensional electrophoresis (2-DE), mass spectrometry (MS) including enzymatic digestion fragments (electrophoresis). Spray ionization (ESI),
Matrix-assisted laser desorption / ionization (matrix-assi
technology such as sted laser desorption / ionization: MALDI) is included, MALDI-TOF analyzer, ESI-3 quadrupole analyzer, E
SI-ion trap analyzer may be used), staining technology, isotope labeling and analysis, image processing technology, etc. can be used. Therefore, the present invention may also include software, databases and the like related to the CRD mutants obtained or utilized above.

When transferring the DNA obtained in the present invention (eg, a DNA encoding a CRD-related mutant such as a CRD-containing mutant) to a target animal, it is used as a DNA fragment or the DNA is used in an animal cell. It is generally advantageous to use it linked downstream of an expressible promoter. For example, when introducing galectin-9 CRD DNA into mice,
Highly homologous animal-derived galectin-9 CRD DNA gene constructs linked to the downstream of various promoters that can be expressed in animal cells are microinjected into fertilized eggs of target animals, such as mouse fertilized eggs. It is possible to create a transgenic mouse that highly produces The mouse is not particularly limited to a pure line mouse, but for example, C57BL / 6, Bal
b / C, C3H, (C57BL / 6 × DBA / 2) F ₁ (BDF ₁ ) and the like. As this promoter, for example, a virus-derived promoter, a ubiquitous expression promoter such as metallothionein, etc. can be preferably used. When the galectin-9 CRD DNA is introduced, it can be recombined into a recombinant retrovirus and used. Fertilized mouse eggs into which the DNA of interest is preferably introduced can be grown using, for example, a foster mouse such as ICR. Transfer of the DNA obtained by the present invention (for example, a DNA encoding a CRD-related mutant such as a CRD-containing mutant) at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target animal. To be done. The presence of DNA encoding a CRD-related mutant, such as a CRD-containing mutant, in the germ cells of the producing animal after DNA transfer means that all the offspring of the producing animal have the galectin-9 in all of the germ cells and somatic cells. It is meant to have the DNA encoding the CRD.
The offspring of this type of animal that inherited the gene have the potential to express the galectin-9 CRD in all of its germ cells and somatic cells.

It was confirmed that the galectin-9 CRD DNA-introduced animal stably retains the gene by mating, and
As a holding animal, breeding passage can be performed in a normal breeding environment. Furthermore, a male and female animal carrying the target DNA is mated to obtain a homozygous animal having the transgene on both homologous chromosomes, and by mating the male and female animals, all offspring will have the DNA. Can be bred and passaged. Since the mutant protein associated with the CRD is highly expressed in the animal into which the galectin-9 CRD DNA has been introduced, an animal for screening an inhibitor (inhibitor) for the mutant protein associated with the CRD, etc. Is useful as Also galectin-9 C
Antisense capable of inhibiting RD gene expression
It is useful as an animal for screening oligonucleotides such as antisense DNA. This transgenic animal can also be used as a cell source for tissue culture. For example, in the tissues of transgenic mice
Proteins associated with inhibition of galectin-9 activity can be analyzed by direct analysis of DNA or RNA or by analysis of protein tissues expressed by the gene. The cells of the tissue having the CRD are cultured by a standard tissue culture technique, and by using these, the function of cells or blood cells derived from, for example, brain, thymus, testis, intestine, kidney, liver, bone marrow and other tissues, etc. Can be researched. Further, by using the cells, it is possible to contribute to, for example, drug development for enhancing the functions of various tissues. In addition, if there is a highly expressing cell line, CRD can be isolated and purified from it.
Techniques related to transgenic mice and the like are described in, for example, Brinster, RL, et al.,; Proc. Natl. Acad.
Sci. USA, 82: 4438, 1985; Costantini, F. & Jaenisc
h, R. (eds): Genetic manipulation of the early mam
malian embryo, Cold Spring Harbor Laboratory, 1985
Can be carried out by the method described in the literature such as the above, the method described in the literature cited therein, and a modification thereof.

A mutant mouse (knockout mouse) having the nucleic acid (eg, a DNA encoding a CRD-related mutant such as a CRD-containing mutant) obtained in the present invention and not expressing galectin-9 at all can be produced. it can. For example, targeting with a mutant gene in which a gene cassette consisting of neo resistance gene-polyA addition signal was inserted into an exon located near the center of genomic DNA of approximately 8 kb including 4 kb before and after the translation start codon of the gene and close to the translation start codon. Vectors can be constructed. In addition to the neo resistance gene cassette, the DT-A cassette,
Examples include the tk cassette and the lacZ cassette. The targeting vector was opened linearly and introduced into established mouse embryonic stem cells (ES cells) by electroporation and further cultured to acquire neo resistance.
Select ES cells. ES cells have 129, C57BL / 6, F1 (C57BL / 6
It can be prepared by selecting from mouse strains such as (/ 6 × CBA) mouse. It is assumed that the ES cells that have acquired neo-resistance undergo homologous recombination with the targeting vector in which the gene cassette has been inserted in the galectin-9 gene region, and at least one of the galectin-9 gene alleles is destroyed and galectin- 9 cannot be expressed normally.
For selection, an appropriate method is selected depending on the inserted gene cassette, and the introduction of mutation can be confirmed using a method such as PCR, Southern hybridization or Northern hybridization.

ES cells into which the mutation was introduced were C57BL / 6 and BALB.
/ c, injection into 8-cell stage embryos removed from ICR mice, etc.
Individuals can be grown by culturing for one day and developing the blastocysts into a foster mother such as ICR. The offspring mouse is a chimeric mouse derived from a mutant ES cell and a normal host embryo, and the color of the individual's coat determines how much ES cell-derived cells are contained. Therefore, it is desirable that the ES cells and the host embryo have a combination of lines with different coat colors. The mutation of the obtained chimeric mouse is hetero, and a homozygous mouse can be obtained by appropriately mating these.
The homozygous mutant mice thus obtained had the same gene expression system in all germ cells and somatic cells, in which only the galectin-9 gene was disrupted and no galectin-9 was expressed. With. This knockout mouse shows development, growth, reproduction, and
It is useful for analyzing the role of galectin-9 in the life cycle of individuals such as aging and death, and the function of galectin-9 in each organ and tissue. It can also be applied to drug development related to galectin-9 inhibition. The knockout mouse can be used not only as these model animals but also as a cell source for tissue culture, and can be used for functional analysis of galectin-9 at the cell level. Technologies related to knockout mice, for example,
Mansour, SL, et al.,; Nature, 336: 348-352, 198.
8; Joyner, AL, ed .; Gene targeting, IRL Press,
1993; Shinichi Aizawa, Generation of mutant mice using gene-targeting ES cells, the method described in the literature such as Yodosha, 1995 or the method cited therein, and the modification thereof You can

According to the present invention, an antisense oligonucleotide (nucleic acid) capable of inhibiting the expression of the CRD site of galectin-9 was cloned or determined, and the nucleotide sequence of DNA encoding galectin-9 was determined. It can be designed and synthesized based on information. Such an oligonucleotide (nucleic acid) can hybridize with the CRD site mRNA of galectin-9 and inhibit the function of the mRNA, or through the interaction of galectin-9 with the CRD-related mRNA, etc. Can regulate and control the expression of the CRD site. An oligonucleotide complementary to a selected sequence of galectin-9 CRD-related gene and an oligonucleotide capable of specifically hybridizing with a CRD-related gene regulates and controls the expression of the CRD portion in vivo and in vitro. It is also useful for treating or diagnosing diseases related to galectin-9. The term "corresponding" means having homology with or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponds" between nucleotides, base sequences or nucleic acids and peptides (proteins)
The term usually refers to the amino acid of a peptide (protein) in a command derived from the sequence of nucleotides (nucleic acid) or its complement.

The relationship between the target nucleic acid and the oligonucleotide complementary to at least a part of the target region means the relationship between the oligonucleotide capable of hybridizing with the target and it is "antisense". Can be said. Antisense oligonucleotides are 2-
Deoxy-D-ribose-containing polydeoxynucleotides, D-ribose-containing polydeoxynucleotides, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or other with non-nucleotide backbones Polymers (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages (provided the base pairing or bases are found in DNA or RNA). Containing a nucleotide having a configuration that allows attachment) and the like. They are double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA.
It may be a hybrid and may further be an unmodified polynucleotide or an unmodified oligonucleotide, as well as those with known modifications, such as those labeled in the art, capped, methylated. And one or more naturally occurring nucleotides substituted with analogs, intramolecular nucleotide modifications such as uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) What it has, charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.)
Those having side chain groups such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine) and sugars (eg monosaccharides), intercurrents Compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals, radioactive metals, boron, oxidizable metals, etc.), alkylating agents, modified It may have a bond (for example, an α-anomeric nucleic acid). Where "nucleoside" and "nucleotide"
The term "nucleic acid" includes not only known purine and pyrimidine bases, but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. The modified nucleoside and modified nucleotide may also have a modified sugar moiety, for example, one or more hydroxyl groups are substituted with halogen or an aliphatic group, or converted into a functional group such as ether or amine. It can be done.

The antisense nucleic acid of the present invention includes RNA and DNA.
, Or a modified nucleic acid. Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is,
Make the antisense nucleic acid more stable in the cell,
Make the antisense nucleic acid more cell permeable, have a greater affinity for the target sense strand, and, if toxic, make the antisense nucleic acid less toxic. Many such modifications are known in the art, for example J. Kawakami et al., Pharm Tech Ja.
pan, 8: 247, 1992; 8: 395, 1992; ST Crooke et a
l. ed., Antisense Research and Applications, CRC Pr
It is disclosed in ess, 1993, etc. The antisense nucleic acid of the present invention may contain altered or modified sugars, bases, linkages, provided in a special form such as liposomes and microspheres, or applied by gene therapy, It can be provided in added form. Examples of such added forms include polycationic substances such as polylysine that act to neutralize the charge of the phosphate skeleton, lipids that enhance interaction with cell membranes and increase uptake of nucleic acids ( Examples thereof include hydrophobic ones such as phospholipids and cholesterol). Preferred lipids to add include cholesterol and its derivatives (eg cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'-end or 5'-end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Examples of the other group include a capping group specifically arranged at the 3'-end or 5'-end of a nucleic acid, which is for preventing decomposition by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl group-protecting groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory activity of antisense nucleic acid can be examined by using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of galectin-9 CRD portion. . The nucleic acid can be applied to cells by various methods known per se.

Based on the above-mentioned research results of the present inventors, a mutant gene and recombination focused on the galectin-9 CRD region.
There is provided a method of introducing a DNA molecule into a host and expressing the mutant to obtain a mutant of interest. Thus, according to the present invention, a recombinant or transfectant that substantially expresses a mutant gene focused on the galectin-9 CRD portion, a method for producing the same, and uses thereof are provided. In another aspect, the present invention is a mutant focused on the galectin-9 CRD part and is a kind of a unique polypeptide having a sugar chain-binding activity and has an activity substantially equivalent to that of natural human galectin-9CRD. Or a salt thereof, more preferably a galectin-9 CRD-containing variant or a salt thereof, which has substantially the same activity or substantially the same primary structure conformation. DN that enables expression of a polypeptide having at least a part or all of a protein in a prokaryote such as Escherichia coli or a eukaryote such as a mammalian cell
It can be related to nucleic acids such as A and RNA. In addition, such a nucleic acid, particularly DNA, should hybridize with (a) a sequence capable of encoding a mutant focused on galectin-9 CRD or a sequence complementary thereto, (b) a DNA sequence of (a) or a fragment thereof. And (c) the sequence (a) or
It may be a sequence having a degenerate code capable of hybridizing to the sequence of (b). Here, the hybridization conditions may be stringent conditions. A prokaryote such as E. coli or a eukaryote such as a mammalian cell transformed with such a nucleic acid and capable of expressing the polypeptide of the present invention is also a feature of the present invention.

Typically, it is an object of the present invention to use a mutant galectin-9 CRD protein to screen a galectin-9 binding substance in a test sample, for example a galectin-9 activity inhibitor. The present invention provides an excellent method for detection / separation / quantification and a reagent kit therefor. A technique using an N-terminal CRD or a C-terminal CRD of human galectin-9, particularly, a solid-phased species thereof is provided. The present invention uses such galectin-9 CRD protein or its gene, and further, detection / utilization using a unique substance including producing cells.
It is understood that all of the reagents of the reagent kit capable of differential quantification are included in that embodiment. Also,
A substance having a binding affinity to the N-terminal CRD or C-terminal CRD of human galectin-9, for example, a substance having a specific sugar chain, has some biological or physiological activity against human galectin-9 activity. And in relation to human galectin-9,
Provided are methods, reagents, diagnostic agents, and therapeutic agents capable of monitoring many diseases and the like by participating in many normal cell or abnormal cell processes such as intracellular protein metabolism in the living body. Therefore, the use of the above-mentioned reagents for the purpose of various uses of the above-mentioned reagents in the medical / physiological fields, research / analysis / measurement of cells / tissues of animals such as humans such as tumors and cancers is not limited to the practice of the present invention. It is understood to be included in the aspects.

Human galectin-9 immobilized CRD of the present invention,
For example, immobilized N-terminal CRD or C-terminal CRD is typically prepared by immobilizing a recombinant polypeptide on at least a water-insoluble carrier. Examples of the solid-phase immobilization method include a carrier binding method, a cross-linking method, an encapsulation method, and a composite method combining them. A method generally used in the art can be used for the immobilization. The carrier binding method can be carried out by, for example, a method utilizing ionic interaction, hydrophobic interaction, physical adsorption, etc., or a chemical bond such as covalent bond. When utilizing physical adsorption, for example, activated carbon, acid clay, bleaching earth, kaolinite, alumina, silica gel, bentonite, metal oxides, hydroxyapatite, inorganic substances such as calcium phosphate, starch, chitin, gluten, cellulose, agarose, Examples of the carrier include natural polymers such as tannin, synthetic polymers such as polystyrene, and agarose derivatives having a hydrophobic group. When ionic bond is used, dextran, cellulose, agarose, ion exchangers of polysaccharides such as starch, for example, DEAE group, TEAE group,
It is possible to use a derivative having a CM group, an alkyl sulfonate group or the like, an ion exchange resin or the like as a carrier.

As the covalent bond method, a peptide method, a diazo method, an alkylation method, a cyanogen bromide activation method, a binding method using a crosslinking reagent, an immobilization method utilizing a Ugi reaction, and a thiol / disulfide exchange reaction are used. Immobilization method used, Schiff base formation method, chelate binding method, tosyl chloride method, biochemical specific binding method and the like can be mentioned, but for more stable binding such as covalent binding, preferably a thiol group and a maleimide group are used. The reaction can be carried out by utilizing a reaction, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, etc., and a known method or a method that can be easily performed by a person skilled in the art, or a modification thereof The method can be appropriately selected and applied. Preferably, a chemical binder, a cross-linking agent or the like that can form a more stable bond such as a covalent bond is used.

These include carbodiimide,
Examples thereof include isocyanates, diazo compounds, benzoquinone, aldehydes, periodic acid, maleimide compounds, and pyridyl disulfide compounds. Preferred reagents include, for example, formaldehyde, glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, N'-polymethylenebisiodoacetamide, N, N'-ethylene bismaleimide, ethylene glycol bissuccinimidyl succinate. , Bisdiazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (N
-Maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-
Succinimidyl (4-iodoacetyl) aminobenzoate, N-succinimidyl 4- (1-maleimidophenyl)
Butyrate, N- (ε-maleimidocaproyloxy) succinimide (EMCS), iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4'-dithiopyridyl) propionimidate, methyl-4-mercapto Examples include butyrylimidate, methyl-3-mercaptopropionimidate, N-succinimidyl-S-acetylmercaptoacetate and the like.

In the peptide method, a peptide bond is formed between a carrier and a desired polypeptide to immobilize it. For example, a carrier having a carboxyl group is used as a derivative of azide, chloride, isocyanate or the like to form a peptide bond with a free amino group in the polypeptide. Reagents used for peptide synthesis, such as carbodiimide reagent, Woodward reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate), etc. are used. It is also possible to form peptide bonds between the amino and carboxyl groups of the carrier and the amino and carboxyl groups of the desired polypeptide. The diazo method is a method in which a carrier having an aromatic amino group is a diazonium compound, and this and a required polypeptide are diazo-coupled and immobilized. Free amino group, imidazole group of histidine,
It can be suitably applied to the polypeptide having a phenolic hydroxyl group of tyrosine. As the carrier, polysaccharides, amino acid copolymers, polyacrylamides, styrene resins,
Examples thereof include ethylene / maleic acid copolymers and porous glass / aromatic amino derivatives.

The alkylation method is a method in which a free amino group, a phenolic hydroxyl group, and a sulfhydryl group in the polypeptide are alkylated with a carrier having a reactive functional group such as halogen to immobilize them. Examples of the carrier include halogenated acetyl derivatives, triazinyl derivatives, halogenated methacryl derivatives and the like. The cyanogen bromide activation method is a method in which dextran, cellulose, agarose, polysaccharides such as starch, porous glass, etc. are activated with cyanogen bromide, and then the polypeptide is immobilized.
Among the binding methods using a cross-linking reagent, particularly when a bifunctional reagent such as glutaraldehyde is used, a natural polymer or synthetic polymer having or introducing an amino group such as cellulose, agarose, albumin, gelatin, and chitosan.
Examples thereof include aminosilane derivatives of inorganic carriers such as porous glass and porous ceramics. The Yugi reaction utilizes the fact that a condensation reaction occurs when a carboxyl group, an amino group, an aldehyde group and an isonitrile group coexist and are reacted. An example is one in which acetaldehyde and 3-dimethylaminopropyl isocyanide are added to a mixture of a carrier having a carboxyl group or an amino group and the polypeptide, and the mixture is reacted. Examples of the carrier include polysaccharides, amino derivatives of polyacrylamide, and isonitrile derivatives of nylon.

The biochemical specific binding method utilizes the biochemical specific binding reaction of specific binding reaction pairs, and includes, for example, an antigen and an antibody thereto, an antibody and a hapten,
Effectors and receptors, enzymes and enzyme inhibitors,
Enzyme substrates, coenzymes, prosthetic groups in complex proteins,
Examples include lectin and sugar chain-containing substance, enzyme and enzyme substrate, nucleic acid and nucleic acid complementary thereto, and these may be selected from known ones. The encapsulation method is mainly for enclosing the polypeptide in a thin gel matrix of natural or synthetic polymers such as polysaccharides and proteins and for enclosing the polypeptide in a space separated by a membrane. Can be separated. Examples of the membrane encapsulation include a microcapsule type encased in a semipermeable solid membrane, an encapsulating type in a space formed by a semipermeable membrane hollow fiber or an ultrafiltration membrane, and a liposome type encased in a liquid membrane. The method using a polymer gel is to enclose the polypeptide in a matrix of polymer gel with a network structure and immobilize it, and the gel can be spherical, film-shaped, tube-shaped or membrane-shaped when fixed. Can be molded into The gel is prepared by polymerizing a monomer and a crosslinking agent to form a polymer gel, polymerizing a prepolymer or an oligomer, and changing the polymer from a soluble state to an insoluble state to form a gel. The method of making it etc. is mentioned. As a polymer,
Synthetic polymers such as polyacrylamide, polyvinyl alcohol, photocurable resin, urethane polymer, κ-carrageenan, alginic acid, pectin, chitosan, starch,
Examples include natural polymers such as collagen. In the case of polyacrylamide, acrylamide monomer, cross-linking agent
N, N'-methylenebisacrylamide, polymerization accelerator N, N, N ',
It is possible to use N'-tetramethylethylenediamine, a polymerization initiator potassium persulfate for gelation, or to use radiation such as γ-rays or X-rays. In the case of using calcium alginate, sodium alginate is soluble in water, but its calcium salt and aluminum salt are insoluble in water. First, an aqueous sodium alginate solution and the polypeptide are mixed and brought into contact with an aqueous calcium chloride solution.

In the case of κ-carrageenan, κ-carrageenan dissolves in water when heated, but ammonium ion,
Since gelation occurs in the presence of potassium ions, calcium ions, aliphatic amines, etc., the gel thus obtained is crosslinked and stabilized with glutaraldehyde, hexamethylenediamine or the like. As a method of using a photo-crosslinkable resin polymer, polyethylene glycol (P
For example, a prepolymer having EG) or polypropylene glycol (PPG) as a main chain and a photosensitive group such as an acryloyl group, a methacryloyl group, or a cinnamoyl group incorporated at its end is used. Such a prepolymer can be mixed with a solution containing the polypeptide in the presence of the photosensitizer benzoin ethyl ether or benzoin isobutyl ether and irradiated with ultraviolet rays to cause gelation.
The urethane prepolymer can be gelled simply by mixing it with an aqueous solution containing the polypeptide.

The microcapsule type membrane encapsulation method is, for example, when the hydrophilic monomer and the hydrophobic monomer are polymerized at the interface, the polypeptide is coated and solid-phased, or in-liquid drying method such as benzene, hexane, A polymer is dissolved in a highly volatile organic solvent such as chloroform, and an aqueous solution containing the polypeptide is dispersed therein to form a primary emulsion, which is then used as a protective colloid such as gelatin, polyvinyl, or a surfactant. A capsule is formed by dispersing in an aqueous solution containing a substance and removing an organic solvent from the obtained secondary emulsion. In the method of immobilizing the polypeptide on a hollow fiber or an ultrafiltration membrane, it is possible to immobilize a plurality of the polypeptides, and further immobilize in a free state without binding to the membrane. You can References include, for example, US Pat. No. 4,003,988, BK Van Weemen and AHA S.
chuurs, Febs Letters, Vol. 15, No. 15, pp.232-235,
(1971, 6); P. Leinikki and Suvi Passila, J. Clin.
Path., 29, pp.116-120, (1976); BR Brodeur, F.
E. Ashton and BB Diena, The Journal of MedicalM
icrobiology, Vol. 15, No. 1, pp.1-9, (1981); J. Cl
in. Path., 29, pp.150-153, (1976); Eiji Ishikawa, et al., "Enzyme Immunoassay", Medical Shoin Co., Ltd., 1978.

The water-insoluble carrier refers to a carrier which is substantially insoluble in a liquid medium used for immobilization, storage, measurement and the like. As these carriers, various carriers that are used in a specific binding reaction are known, and in the present invention, these carriers can be selected and used. Particularly preferably used are those mentioned above, for example, cross-linked albumin, collagen, gelatin, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, cellulose acetate, cross-linked dextran, polyacrylamide,
Cross-linked polyacrylamide, polyethylene, polypropylene, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, polystyrene, styrene-
Polybutadiene such as butadiene copolymer, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, polyamide such as nylon, polyurethane,
Organic polymeric substances such as those obtained by emulsion polymerizing organic polymeric substances such as polyepoxy resin, glass, such as activated glass, silica gel, silica-alumina, inorganic materials such as alumina, etc. In some cases, a functional group is introduced with a silane coupling agent or the like.

In a preferred embodiment, the human galectin-9
The N-terminal CRD or C-terminal CRD-immobilized carrier is packed in a column or the like, and affinity chromatography technology, particularly frontal affinity chromatography technology (H
irabayashi et al., J. Chromatogr. A, 890, 261-271
(2000); Arata et al., J. Biol. Chem., 276: 3068-30.
77 (2001)) can be used for screening using the binding affinity of CRD for the test sample. For example, a substance having a binding affinity for the N-terminal CRD or C-terminal CRD of human galectin-9 has a longer residence time in the column than a substance having a lower binding affinity. Examples of the test sample include those having a sugar chain. Glycoproteins, glycopeptides, glycolipids, non-peptidic sugar chain-containing compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts of animals, etc. ,
Cell extracts and the like can be mentioned. Examples of the test compound used in the test sample preferably have an N-acetyllactosamine structure, galectin-9 inhibitor, galectin-9.
It may include compounds having inhibitory activity against, especially synthetic compounds. These compounds may be novel compounds or known compounds. The sugar chains recognized by galectin-9 (which may include analogs thereof) typically include, but are not limited to, glycans. Examples include glycoprotein-derived N-glycans and glycolipid-derived glycans, which have a 3-branched or 4-branched sugar chain structure, and more preferably an N-bond having an N-acetyllactosamine structure. Examples thereof include 3-branched and 4-branched complex type glycans, forssman pentasaccharide, and the like, which have a part of such a characteristic structure or are derived therefrom and have the binding ability. Examples thereof include glycopeptides, glycoproteins, glycolipids, sugar chain-containing receptors, and the like.

Thus, the N-terminal CRD of human galectin-9
Alternatively, a substance having a binding affinity to the C-terminal CRD, for example, a substance having a specific sugar chain structure (which may include an analog thereof) or a salt thereof is a galectin-9 (galectin-9, -9M
, And -9L) are useful as agents (agonists or promoters) that promote functions such as biological activity (eg, eosinophil chemotactic activity), such as human galectin-9 dysfunction symptoms. It can be used as a medicament useful as a therapeutic and / or preventive agent for various diseases. Further, a substance having a binding affinity to the N-terminal CRD or C-terminal CRD of human galectin-9, for example, a substance having a specific sugar chain structure (which may include an analog thereof) or a salt thereof is It is useful as a compound (antagonist or inhibitor) that inhibits functions such as biological activity of 9 (eg, eosinophil migration activity), and treats various diseases such as hyperhuman galectin-9 dysfunction and the like. It can be used as a medicine such as a prophylactic agent. Polypeptides such as mutants of the present invention promote functions such as biological activity of galectin-9 (including galectin-9, -9M and -9L) (eg, eosinophil migration activity). It is useful as a reagent for screening compounds (agonists), compounds that inhibit (antagonists) or salts thereof, and biological activities such as galectin-9-related proteins, some peptides thereof or salts thereof. There is also provided a method for screening a compound (agonist) or a compound (antagonist) that promotes a function such as (for example, eosinophil migration activity, hemagglutination activity, etc.) or a salt thereof.

The compound or its salt obtained by using the screening method or screening kit of the present invention is a test compound described above, for example, glycopeptide, glycoprotein, glycolipid, sugar-containing non-peptidic compound, synthetic compound,
It is a compound selected from fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like, and compounds that promote or inhibit the functions of galectin-9 protein and the like. Examples of the salt of the compound include pharmaceutically acceptable salts. Examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and aluminum salt and ammonium salt. Suitable examples of salts with organic bases include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N'-
Examples thereof include salts with dibenzylethylenediamine and the like.
Preferable examples of the salt with an inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Suitable examples of salts with organic acids include, for example, formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid,
Examples thereof include salts with citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid and the like.
Suitable examples of salts with basic amino acids include, for example, salts with arginine, lysine, ornithine,
Preferable examples of the salt with acidic amino acid include salts with aspartic acid, glutamic acid and the like.

In the present specification, the term "antibody" may be used in a broad sense, and it has a binding affinity to the desired N-terminal CRD or C-terminal CRD of human galectin-9. A composition of a single monoclonal antibody against a substance such as a sugar chain or an analog thereof (including those having an N-acetyllactosamine structure and related peptide fragments) or an antibody composition having specificity for various epitopes. Often, it also includes monovalent or polyvalent antibodies, polyclonal antibodies and monoclonal antibodies, and further represents natural molecules (intact molecules) and fragments and derivatives thereof, F (ab ′) ₂ , Fab ′. as well as
Chimeric antibody or hybrid antibody having a fragment such as Fab and further having at least two antigens or epitopes (epitope) binding sites, or, for example, a bispecific recombinant antibody such as quadrome, triome, etc., Interspecific hybrid antibodies, anti-idiotype antibodies, and further those chemically modified or processed to be considered as derivatives thereof, known cell fusion or hybridoma technology or antibody engineering, or synthetic or semisynthetic technology Antibodies obtained by use, antibodies to which conventional techniques known from the viewpoint of antibody production are applied, or antibodies prepared using DNA recombination techniques, target antigen substances or target epitopes described and defined in this specification May have antibodies that have neutralizing properties or antibodies that have binding properties.

Monoclonal antibodies made against the antigenic material are produced using any method that can provide for the production of antibody molecules by a series of cell lines in culture. The modifier "monoclonal" refers to the character of the antibody as being obtained from a population of substantially homogeneous antibodies, which is not considered necessary for the antibody to be produced by any particular method. I won't. Individual monoclonal antibodies, except that there may be small amounts of naturally occurring variants,
It contains a population of antibodies that are identical.
Monoclonal antibodies have a high degree of specificity, which is directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is a single antigen on that antigen. It is aimed at determinants. In addition to its specificity, the monoclonal antibody is also excellent in that it is synthesized by hybridoma culture and is free from or less contaminated with other immunoglobulins. Monoclonal antibodies include hybrid antibodies and recombinant antibodies. They may replace the variable region domain with a constant region domain (e.g., humanized antibody), or replace the light chain with a heavy chain, as long as it exhibits the desired biological activity, regardless of its origin or immunoglobulin class or subclass. It can be obtained by replacement, replacement of one type of chain with another type of chain, or fusion with a heterogeneous protein (eg, US Pat. No. 4,816,567).
Issue; Monoclonal Antibody Production Techniques and
Applications, pp.79-97, Marcel Dekker, Inc., New Y
ork, 1987, etc.).

Examples of suitable methods for producing monoclonal antibodies include the hybridoma method (G. Kohler and C. Milst
ein, Nature, 256, pp.495-497 (1975)); human B cell hybridoma method (Kozbor et al., Immunology Today,
4, pp.72-79 (1983); Kozbor, J. Immunol., 133, pp.30.
01 (1984); Brodeur et al., Monoclonal Antibody Pro
duction Techniques and Applications, pp.51-63, Mar
cel Dekker, Inc., New York (1987); Trioma Method; EB
V-hybridoma method (Cole et al., Monoclonal Antibo
dies and Cancer Therapy, Alan R. Liss, Inc., pp.77
-96 (1985)) (Method for producing human monoclonal antibody); U.S. Pat. Biocca et al., EMBO J, 9, pp.101-108 (19
90); RE Bird et al., Science, 242, pp.423-426 (1
988); MA Boss et al., Nucl. Acids Res., 12, pp.3
791-3806 (1984); J. Bukovsky et al., Hybridoma, 6,
pp.219-228 (1987); M.DAINO et al., Anal. Bioche
m., 166, pp.223-229 (1987); JS Huston et al., Pr
oc. Natl. Acad. Sci. USA, 85, pp.5879-5883 (1988);
PT Jones et al., Nature, 321, pp.522-525 (198
6); JJ Langone et al. (Ed.), "Methods in Enzymolo
gy ", Vol. 121 (Immunochemical Techniques, Part I:
Hybridoma Technology and Monoclonal Antibodies), A
academic Press, New York (1986); S. Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81, pp.6851-6855 (198
4); VT Oi et al., BioTechniques, 4, pp.214-221
(1986); L. Riechmann et al., Nature, 332, pp.323-32.
7 (1988); A. Tramontano et al., Proc. Natl. Acad.S.
ci. USA, 83, pp.6736-6740 (1986); C. Wood et al.,
Nature, 314, pp.446-449 (1985); Nature, 314, pp.45
2-454 (1985) or references cited therein (the descriptions therein are incorporated by reference into the disclosure herein).

Monoclonal antibodies according to the present invention are antibodies of which part of the heavy and / or light chains is derived from a particular species or belongs to a particular antibody class or subclass, as long as they exhibit the desired biological activity. A "chimeric" antibody (immune) that is identical or homologous to the corresponding sequence, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass. Globulin) (US Pat. No. 4,816,567; Morrison et al., Proc. Natl.
Acad. Sci. USA, 81, pp.6851-6855 (1984)). Less than,
The production of the antibody will be described in detail by taking a monoclonal antibody as an example. The monoclonal antibody of the present invention is a cell fusion technology (G. Kohler and C. Mil) using myeloma cells.
Stein, Nature, 256, pp.495-497 (1975), etc.), and may be prepared by the following steps, for example. (1) Preparation of immunogenic antigen, (2) Immunization of animal with immunogenic antigen, (3) Preparation of myeloma cells (myeloma cells),
(4) Cell fusion between antibody-producing cells and myeloma cells, (5)
Selection of hybridoma (fused cell) and monocloning, and (6) production of monoclonal antibody

(1) The immunogenic antigen can be prepared as follows. As an antigen, the N-terminal CRD or C- of human galectin-9 identified as described above is used.
Substances such as sugar chains or analogs thereof having binding affinity to the terminal CRD (including those having an N-acetyllactosamine structure and related fragments thereof, etc., more preferably sugar chains having binding affinity to human galectin-9 Etc.) or fragments derived therefrom. The antigen may be one isolated from a natural product or one obtained by an appropriate chemical synthesis method based on the determined sugar chain sequence information. The antigen can be used as it is by mixing with an appropriate adjuvant to immunize an animal, but may be an immunogenic conjugate or the like. Further, a sugar chain fragment is bound to various carrier proteins through an appropriate condensing agent to give an immunogenic conjugate such as a hapten-protein, which can be used to react with only a specific sequence (or a specific sequence). It can also be used to design monoclonal antibodies (which can only recognize). It is possible to add a binding residue or the like to the designed sugar chain fragment in advance so that the immunogenic conjugate can be easily prepared. In binding the carrier proteins, the carrier proteins can first be activated. For such activation, introduction of an activating linking group can be mentioned. As the activated linking group, (1) activated ester or activated carboxyl group, such as nitrophenyl ester group, pentafluorophenyl ester group, 1-benzotriazole ester group, N-succinimide ester group, (2)
An activated dithio group, such as a 2-pyridyldithio group, can be mentioned. Carrier proteins include keyhole limpet hemocyanin (KLH), bovine serum albumin (BS
A), ovalbumin, globulin, polypeptides such as polylysine, and bacterial cell components such as BCG.

(2) Immunization of animals with an immunogenic antigen can be carried out as follows. Immunization can be performed by a method known to those skilled in the art, for example, Shigeru Muramatsu, et al., Laboratory of Experimental Biology 14, Immunobiology, Maruzen Co., Ltd., 1985, edited by The Biochemical Society of Japan, sequel biochemistry experiment. Lecture 5, Immunobiochemistry Research Method, Tokyo Kagaku Dojin, 1986, Japan Society for Biochemistry, Newborn Chemistry Experimental Course 12, Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Kagaku Dojin, 1992, etc. It can be performed according to. Immunizing agent (with adjuvant if necessary)
The mammal is immunized by one or more injections. Typically, the immunizing agent and / or adjuvant is administered to a mammal by multiple subcutaneous injections or intraperitoneal injections. Examples of the immunizing agent include those containing the above antigen sugar chain or a related fragment thereof. The immunizing agent may be used by forming a conjugate with a protein known to be immunogenic in the mammal to be immunized (for example, the above-mentioned carrier proteins). Examples of the adjuvant include Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, lipid A, liposome, aluminum hydroxide, silica and the like. Immunization is performed using, for example, a mouse such as BALB / c, a hamster, and other appropriate animals. The dose of the antigen is, for example, about 1 to 400 μg / animal per mouse, and it is generally injected intraperitoneally or subcutaneously into a host animal, and thereafter every 1 to 4 weeks, preferably 1 to 2
Each week, booster immunization is repeated intraperitoneally, subcutaneously, intravenously or intramuscularly about 2 to 10 times. As the mouse for immunization, in addition to BALB / c mouse, F1 mouse of BALB / c mouse and other mouse can be used. As needed,
The degree of animal immunity can be confirmed by preparing an antibody titer measuring system and measuring the antibody titer. The antibody of the present invention may be obtained from the animal thus obtained and immunized, and includes, for example, antiserum, polyclonal antibody and the like.

(3) Myeloma cells (myeloma cells) can be prepared as follows. The infinitely proliferative strain (tumor cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulin, for example, P3-NS-1.
-Ag4-1 (NS-1, Eur. J. Immunol., 6: 511-519, 1976)
, SP-2 / 0-Ag14 (SP-2, Nature, 276: 269 ~ 270,197
8), P3-X63 derived from mouse myeloma MOPC-21 cell line
-Ag8-U1 (P3U1, Curr. Topics Microbiol. Immunol., 8
1: 1-7, 1978), P3-X63-Ag8 (X63, Nature, 256: 495-
497, 1975), P3-X63-Ag8-653 (653, J. Immunol., 12
3: 1548-1550, 1979) etc. can be used. 8-Azaguanine-resistant mouse myeloma cell line is Dulbecco
To cell culture medium such as MEM medium (DMEM medium) and RPMI-1640 medium, for example, antibiotics such as penicillin and amikacin, fetal calf serum (FCS), etc. are added, and 8-azaguanine (eg 5-45 μg / ml) is further added. It is subcultured in
The required number of cell lines can be prepared by subculturing in normal medium 2 to 5 days before cell fusion. The cell lines used are those prepared by thawing the cryopreserved strain completely at about 37 ° C, washing it with normal medium such as RPMI-1640 medium three times or more, and then culturing in the normal medium to prepare the required number of cell lines. It may be.

(4) Cell fusion between antibody-producing cells and myeloma cells can be carried out as follows. Animals, such as mice, immunized according to the above step (2) are
The spleen is removed after 5 days and a splenocyte suspension is obtained therefrom. In addition to splenocytes, lymph node cells in various parts of the body can be obtained and used for cell fusion. More specifically, cell fusion is carried out in an ordinary nutrient medium in the presence of a cell fusion promoter, for example. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and other common culture medium used for this type of cell culture can be used. , Serum supplement such as fetal calf serum (FCS) can also be used together. Thus, the splenocyte suspension thus obtained and the myeloma cell line obtained according to the step (3) above, for example, a minimal essential medium (MEM medium), DMEM medium, RPMI-1
It is placed in a cell culture medium such as 640 medium and a cell fusion promoter such as polyethylene glycol is added. As the cell fusion promoter, other known ones in the relevant field can be used, and as such, inactivated Sendai virus (HVJ: Hemagglutinating Virus of Japan).
And so on. Preferably, for example, 0.5 to 2 ml of 30 to 60% polyethylene glycol can be added, polyethylene glycol having a molecular weight of 1,000 to 8,000 can be used, and polyethylene glycol having a molecular weight of 1,000 to 4,000 can be more preferably used.

The concentration of polyethylene glycol in the fusion medium is preferably 30 to 60%, for example. If necessary, a small amount of an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency. The ratio of immune cells to myeloma cells used, that is, the ratio of splenocytes (lymphocytes) to myeloma cells used for fusion: myeloma cell line, can be set arbitrarily, for example, 1: 1 to 20: 1. However, it is more preferably 4: 1 to 10: 1. For cell fusion, a predetermined amount of immune cells and myeloma cells are thoroughly mixed in a culture solution and pre-heated to about 37 ° C in a PEG solution (for example, an average molecular weight of about 1000-6000).
Is usually added at a concentration of 30-60% (w / v) and mixed to form a target fused cell (hybridoma). Then, an appropriate culture solution is sequentially added, and the procedure of centrifuging and removing the supernatant is repeated to remove the cell fusion agent and the like that are not preferable for the growth of the hybridoma. The fusion reaction is allowed to proceed for 1-10 minutes and then a cell culture medium such as RPMI-1640 medium is added. The fusion reaction treatment can be performed multiple times.
After the fusion reaction, the cells are separated by centrifugation or the like and then transferred to a selection medium.

(5) Hybridoma (fused cell) selection and monocloning can be carried out as follows. As the selective medium, an ordinary selective culture medium, for example, hypoxanthine, aminopterin and thymidine-containing FCS-containing MEM
Examples of the medium include so-called HAT medium and medium such as RPMI-1640 medium. Culturing in the HAT medium is continued for a sufficient time (usually several days to several weeks) to kill cells (non-fused cells) other than the target hybridoma. The selection medium can be replaced generally by adding the same volume as the volume dispensed to the culture plate on the next day, and then replacing it by half with the HAT medium every 1 to 3 days. This can be modified and performed. After fusion 8
On the -16th day, the so-called HT medium without aminopterin can be replaced every 1 to 4 days. Mouse thymocytes, for example, can also be used as a feeder, which may be preferred.

The culture supernatant of a culture well in which the hybridoma was proliferated was subjected to, for example, radioimmunoassay (RIA), enzyme immunoassay (ELISA), fluorescence immunoassay (FIA), luminescence immunoassay (LI).
A), using a measurement system such as Western blotting, or a fluorescence-induced cell separation device (FACS), using a predetermined sugar chain fragment as an antigen, or measuring a target antibody using a labeled anti-mouse antibody, To screen. The hybridoma producing the desired antibody is cloned. Cloning can be done by picking up colonies in agar or by limiting dilution. The limiting dilution method can be used more preferably. Cloning is preferably performed multiple times. The hybridoma producing the monoclonal antibody thus produced can be subcultured in an ordinary culture medium and can be stored in liquid nitrogen for a long period of time.

(6) The production of a monoclonal antibody can be carried out as follows. To obtain a monoclonal antibody from the hybridoma, a method of culturing the hybridoma according to a usual method and obtaining the culture supernatant,
Alternatively, a method in which the hybridoma is administered to a mammal compatible with the hybridoma to grow it and obtain it as ascites is adopted. The former method is suitable for obtaining high-purity antibody, while the latter method is suitable for mass production of antibody. Thus, the obtained hybridoma strain can be cultured in an appropriate growth medium such as FCS-containing MEM medium and RPMI-1640 medium, and the desired monoclonal antibody can be obtained from the medium supernatant. In order to obtain a large amount of antibody, ascites of the hybridoma may be mentioned. In this case, each hybridoma is transplanted into the abdominal cavity of a histocompatibility animal of the same strain as the myeloma cell-derived animal and proliferated, or, for example, each hybridoma is transplanted and proliferated in a nude mouse etc. It can be obtained by recovering the produced monoclonal antibody.

Prior to the transplantation of the hybridoma, the animal can be intraperitoneally administered with mineral oil such as pristane (2,6,10,14-tetramethylpentadecane). Can also be collected. Ascites fluid as it is, or a conventionally known method,
For example, salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, high performance liquid chromatography, etc. It can be used as a monoclonal antibody. Preferably, the ascitic fluid containing the monoclonal antibody can be purified and separated by ammonium sulfate fractionation, followed by treatment with an anion exchange gel such as DEAE-Sepharose and an affinity column such as a protein A column.
Particularly preferred is an affinity-immobilized antigen or antigen fragment (eg, synthetic peptide, recombinant antigenic protein or peptide, site specifically recognized by antibody).
Chromatography, affinity chromatography with protein A immobilized, hydroxyapatite
Examples include chromatography.

Also, transgenic mice or other organisms, such as other mammals, can be used to express antibodies such as humanized antibodies against the immunogenic sugar chain products of the invention. It is also possible to determine the sequence of the antibody thus obtained in large quantities, or to prepare the antibody by gene recombination technology using the nucleic acid sequence encoding the antibody obtained from the hybridoma strain. The nucleic acid encoding the monoclonal antibody can be isolated and sequenced by a conventional method such as using an oligonucleotide probe capable of specifically binding to a gene encoding a heavy chain or a light chain of a mouse antibody. . Once isolated
The DNA can be put into an expression vector and put into a host cell such as CHO or COS. The DNA can be modified, for example, by substituting a sequence encoding the constant region domain of human heavy chain or light chain in place of the homogenous mouse sequence (Morrison et al., Proc. . Natl. A
cad. Sci. USA, 81: 6581, 1984). Thus, it is possible to prepare a chimeric antibody or a hybrid antibody having a desired binding specificity. Further, the antibody can be modified by preparing a chimeric antibody or a hybrid antibody by applying a chemical protein synthesis technique including the use of a condensing agent as described below.

Humanized antibodies can be made by techniques known in the art (eg Jones et al., Na.
ture, 321: pp.522-525 (1986); Riechmann et al., Na
ture, 332: pp.323-327 (1988); Verhoeyen et al., Sc
ience, 239: pp.1534-1536 (1988)). Human monoclonal antibodies can also be carried out by techniques known in the art, and human myeloma cells and human / mouse heteromyeloma cells for producing human monoclonal antibodies are known in the art (Kozbor, J. Immunol., 133, pp.
3001 (1984); Brodeur et al., Monoclonal Antibody P
roduction Techniques and Applications, pp.51-63, M
arcel Dekker, Inc., New York (1987)). Methods for producing bispecific antibodies are also known in the art (Millstein et al., Nature, 305: pp.537-539 (198
3); WO93 / 08829; Traunecker et al., EMBO J., 10: pp.
3655-3659 (1991); Suresh et al., "Methods in Enzym
ology ", Vol. 121, pp. 210 (1986)). Since the antibody is directed to the sugar chain structure recognized by galectin-9, a mimetic molecule can be designed based on it, and the mimetic is galectin.
-9-like sugar chain recognition activity can be expected. That is, it can be used to obtain a substance having galectin-9 activity by carrying out molecular design based on the antibody. The substances thus obtained are also within the scope of the idea of the present invention and can be treated as the active ingredient of the present invention. Whether a particular characteristic portion is selected from the sequence and (i) the pharmacophore among them is replaced with an isostere, or (ii) at least one of the constituent amino acid residues is replaced with a D-amino acid residue , (Iii) modify the side chain of the amino acid residue, (iv) arrange and link an amino acid residue different from the amino acid residue present in the sequence, or (v) analyze the three-dimensional structure and analyze mimic. It can be done by making full use of the technology adopted in the field, such as designing the body (for example, by Koichi Suto
Drug Development Volume 7 (Molecular Design), issued June 25, 1990, Hirokawa Shoten Co., Ltd. and references and articles cited therein). Some of these techniques include those described above. Further, these antibodies may be treated with an enzyme such as trypsin, papain, pepsin, etc. and optionally reduced to obtain antibody fragments such as Fab, Fab ′ and F (ab ′) ₂ which may be used.

The antibodies can be used in any of the known assays, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal A
ntibodies: A Manual of Techniques, pp.147-158 (CRC
Press, Inc., 1987). Any method known in the art can be used to conjugate the antibody to each detectable moiety, eg, David et al., Bi
ochemistry, 13: 1014-1021 (1974); Pain et al,
J. Immunol. Meth., 40: pp.219-231 (1981); and "Me.
thods in Enzymology ", Vol. 184, pp.138-163 (1990)
The method described by As the antibody that imparts the labeled substance, an IgG fraction, and further, a specific binding site Fab ′ obtained by digestion with pepsin and reduction can be used. Examples of labeled substances in these cases include enzymes (peroxidase, alkaline phosphatase or β-D
-Galactosidase, etc.), chemicals, fluorescent substances or radioisotopes.

The detection / measurement in the present invention can be carried out by immunostaining, for example, tissue or cell staining, immunoelectron microscopy, immunoassay, for example, competitive immunoassay or non-competitive immunoassay, and radioimmunoassay (RIA), F
IA, LIA, EIA, ELISA, etc. can be used, and the BF separation may or may not be performed. RIA, EIA, FIA, LIA are preferable, and a sandwich type assay is also included. For example, in a sandwich type assay, one is used as an antibody against the galectin-9-binding affinity sugar chain of the present invention or a related fragment, the other is used as an antibody against another fragment portion of the galectin-9-binding affinity sugar chain, and one is detected. Label as much as possible (of course, other combinations are possible and can be appropriately designed according to the purpose). Another antibody capable of recognizing the same antigen is immobilized on the solid phase. Incubation treatment is performed in order to sequentially react the sample with the labeled antibody and the immobilized antibody, if necessary, and after separating the unbound antibody, the labeled substance is measured.

The amount of the measured label is proportional to the amount of the antigen, that is, the galectin-9-binding affinity sugar chain antigen. In this assay, depending on the order of addition of insolubilized antibody and labeled antibody, a simultaneous sandwich type assay, forward (forwar
d) It is called a sandwich type assay or an inverse sandwich type assay. For example, washing, stirring, shaking, filtration, pre-extraction of antigen, etc. are appropriately adopted in those measuring steps under specific circumstances. Other measurement conditions such as the concentration of a specific reagent or buffer solution, temperature or incubation treatment time can be changed according to factors such as the concentration of the antigen in the sample and the properties of the sample. Those skilled in the art can appropriately select the optimum conditions effective for each measurement and perform the measurement while using a normal experimental method. Many carriers are known which can solidify an antigen or an antibody, and in the present invention, they can be appropriately selected and used. As the carrier, various carriers used for antigen-antibody reaction and the like are known, and in the present invention, it is needless to say that these carriers can be selected and used.

Particularly preferably used are, for example, glass, activated glass such as aminoalkylsilyl glass, porous glass, silica gel, silica-alumina, alumina, inorganic materials such as magnetized iron and magnetized alloy, polyethylene. , Polypropylene, polyvinyl chloride, polyvinylidene fluoride, polyvinyl, polyvinyl acetate, polycarbonate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, crosslinked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein -Ethylene glycol dimethacrylate copolymer, etc., cross-linked albumin, collagen, gelatin, dextran,
Agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, natural or modified cellulose such as cellulose acetate, cross-linked dextran, polyamide such as nylon, polyurethane, organic polymer substances such as polyepoxy resin, further emulsion polymerization of them The obtained product, silicon gum, etc., cells, erythrocytes, etc., which have a functional group introduced by a silane coupling agent etc., if necessary, can be mentioned.

Furthermore, filter paper, beads, tubes, cuvettes, inner walls of test vessels such as test tubes, titer plates, titer wells, microplates, glass cells,
Cells made of synthetic material such as synthetic resin cells, glass rods, rods made of synthetic material, rods with thickened or thinned ends, rods with rounded or flattened ends, thin plates Examples include the surface of a solid substance (object) such as a bar and the like. An antibody can be bound to these carriers, and preferably a sugar chain antibody having anti-galectin-9 binding activity (including antiserum or purified antibody) or an anti-galectin-9-binding activity that specifically reacts with the sugar chain antigen that can be identified in the present invention. It is possible to bind a sugar chain monoclonal antibody having a galectin-9 binding activity. The binding between the carrier and those involved in these antigen-antibody reactions may be carried out by a physical method such as adsorption, a chemical method using a condensing agent or the like, a chemical method using an activated one, or the like. It can be performed by a method utilizing a chemical bonding reaction.

As the label, an enzyme, an enzyme substrate, an enzyme inhibitor, a prosthetic group, a coenzyme, an enzyme precursor, an apoenzyme, a fluorescent substance, a pigment substance, a chemiluminescent compound,
Examples thereof include a luminescent substance, a coloring substance, a magnetic substance, a metal particle such as a gold colloid, a non-metal element particle such as a selenium colloid, and a radioactive substance. As the enzyme, dehydrogenase, reductase, oxidoreductase such as oxidase, for example, transferase which catalyzes transfer of amino group, carboxyl group, methyl group, acyl group, phosphate group, and the like,
For example, ester bond, glycoside bond, ether bond,
Examples include hydrolases that hydrolyze peptide bonds and the like, lyases, isomerases, and ligases. Enzymes can also be used for detection by using multiple enzymes in combination. For example, enzymatic cycling can be utilized. Typical labeling isotopes of radioactive substances include [ ³² P], [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C], and [ ³⁵ S]. As a typical enzyme label, peroxidase such as horseradish peroxidase, E. coli β
Examples include galactosidases such as -D-galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, acetylcholinesterase, catalase, bovine intestinal alkaline phosphatase, and alkaline phosphatase such as Escherichia coli alkaline phosphatase.

When alkaline phosphatase is used, 4-
Fluorescence produced by using umbelliferone derivatives such as methyl umbelliferyl phosphate, phosphorylated phenol derivatives such as nitrophenyl phosphate, enzymatic cycling systems using NADP, luciferin derivatives, and substrates such as dioxetane derivatives, It can be measured by luminescence. The luciferin and luciferase system can also be used. When catalase is used, it reacts with hydrogen peroxide to generate oxygen, and the oxygen can be detected by an electrode or the like. The electrode may be a glass electrode, an ion electrode using a poorly soluble salt film, a liquid film type electrode, a polymer film electrode, or the like. The enzyme label can be replaced with a biotin label and an enzyme-labeled avidin (streptavidin). Thus, biotin-
A sensitivity enhancement method known in the art, such as using an avidin system or using a secondary antibody such as an antibody against an anti-galectin antibody, can be appropriately adopted. The label can also use a plurality of different types of labels. In such cases, it may be possible to make multiple measurements continuously, or discontinuously, and simultaneously or separately.

In the present invention, 4-hydro is used for signal formation.
Xyphenylacetic acid, o-phenylenediamine (OPD), tet
Lamethylbenzidine (TMB), 5-aminosalicylic acid, 3,3-
Diaminobenzidine tetrahydrochloride (DAB), 3-
Amino-9-ethylcarbazole (AEC), tyramine, lumi
Nol, lucigenin luciferin and its derivatives, Phol
ad luciferin etc. and horseradish peroxidase etc.
Peroxidase, Lumigen PPD, (4-methyl)
Umbelliferyl-phosphoric acid, p-nitrophenol-phosphorus
Acid, phenol-phosphoric acid, bromochloroindolyl phosphorus
Acid (BCIP), AMPAK ^TM(DAKO), AmpliQ^TM(DAKO) etc.
Caliphosphatase, 4-methylumbelliferyl-β
-D- Umbelliferyl galactosides such as galactoside
Such as o-nitrophenol-β-D-galactoside
Nitrophenyl galactoside and β-D-galactosida
, Glucose-6-phosphate dehydrogenase, ABTS
And enzyme reagents such as glucose oxidase
Sese can also be used, hydroquinone, hydroxybenzoquino
, Quinol compounds such as hydroxyanthraquinone,
Lipoic acid, thiol compounds such as glutathione, pheno
A derivative such as a phenol derivative or ferrocene derivative by the action of an enzyme.
What can be made can be used.

As the fluorescent substance or chemiluminescent compound, fluorescein isothiocyanate (FIT
C), for example rhodamine B isothiocyanate, tetramethylrhodamine isothiocyanate (RITC), tetramethylrhodamine isothiocyanate isomer R (TRITC)
Rhodamine derivatives such as, 7-amino-4-coumarin-3-acetic acid, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salt,
Examples thereof include luminol such as lumiferin, luciferase, and equalin, imidazole, oxalate ester, rare earth chelate compound, coumarin derivative, and the like. Color development,
In order to detect the generated signal including the fluorescence and the like, it is possible to use the visual sense, but it is also possible to use a known device, for example, a fluorometer, a plate reader and the like. Further, in order to detect a signal emitted by a radioisotope (isotope) or the like, a known device can be used, for example, a gamma counter, scintillation or the like can be used. The labeling can be carried out by utilizing a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, etc. It can be applied by appropriately selecting from among the methods that can be applied to the above, and methods that have modified them. Further, a condensing agent that can be used for preparing the immunogenic complex, a condensing agent that can be used for binding to a carrier, and the like can be used. Examples of the condensing agent include those used for CRD immobilization.

According to the assay method of the present invention, a labeled antibody reagent such as an antiserum in which a substance to be assayed is labeled with an enzyme or the like, a purified antibody or a monoclonal antibody, and an antibody bound to a carrier can be sequentially reacted. It is possible to react at the same time. The order of adding the reagents depends on the type of carrier system chosen. When sensitized beads such as plastics are used, labeled antibody reagents such as labeled antiserum, purified antibody or monoclonal antibody are first placed together with a sample sample containing the substance to be measured in an appropriate test tube. Then, the measurement can be performed by adding beads such as the sensitized plastic. In the measuring method of the present invention, an immunological measuring method is used, and as the solid phase carrier in that case, polystyrene, polycarbonate, polypropylene or polyvinyl balls, which are well adsorbed to proteins such as antibodies, and microplates are used. Various materials and forms such as, sticks, fine particles, or test tubes can be arbitrarily selected and used.
The measurement can be carried out in an appropriate buffer system so as to maintain the optimum pH, for example, about pH 4 to about 9. Particularly suitable buffers include, for example, acetate buffer, citrate buffer, phosphate buffer, Tris buffer, triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer, Tris-hydrochloric acid. Examples thereof include buffers and veronal buffers. The buffering agents can be used as a mixture with each other in an arbitrary ratio. Antigen-antibody reaction is about 0 ℃ ~ about
Preference is given to working at temperatures between 60 ° C.

Antiserum labeled with enzyme, purified antibody,
Alternatively, an antibody reagent such as a monoclonal antibody and an antibody reagent bound to a carrier, and further an incubation treatment of a substance to be measured can be carried out until equilibrium is reached, but much more than the equilibrium of the antigen-antibody reaction is achieved. The reaction can be stopped after a limited incubation treatment by separating the solid phase and the liquid phase at an early point and the extent of the presence of a label such as an enzyme in either the liquid phase or the solid phase can be measured. The measurement operation can be performed using an automated measuring device,
It is also possible to detect and measure a display signal generated by converting a substrate by the action of an enzyme using a detector, a photo detector or the like. In the antigen-antibody reaction, appropriate means can be taken to stabilize the reagents, substances to be measured, labels such as enzymes, or the antigen-antibody reaction itself. further,
In order to remove non-specific reactions, reduce inhibitory effects, or activate measurement reactions, proteins, stabilizers, surfactants such as those listed below, and chelating agents should be added to the incubation solution. Can also be added to. Ethylenediaminetetraacetic acid salt (EDTA) is more preferable as the chelating agent. Blocking treatment for preventing non-specific binding reaction commonly used in the art or known to those skilled in the art may be performed, and for example,
It can be treated with normal serum or serum protein of mammals, albumin, hemoglobin, ovalbumin (OVA), skim milk, milk fermented substance, collagen, gelatin and the like. As long as the purpose is to prevent non-specific binding reaction, those methods can be used without particular limitation.

Further, for washing the sample or solid phase, an appropriate liquid can be selected from the above-mentioned buffer system and saline solution and used, and Tween 20 (trade name), Tween
80 (Product Name), NP-40 (Product Name), Triton X100 (Product Name)
, Non-ionic surfactants such as Briji (trade name), CHAPS
In addition to the amphoteric surfactants such as the above, those selected from the group consisting of cationic surfactants, anionic surfactants and the like can be added and used. The sample to be measured by the measuring method of the present invention may be a solution or colloidal solution of any form, a non-fluid sample, etc., but preferably a biological sample such as thymus, testis, intestine, kidney, brain, breast. All organs and tissues such as tissues, ovaries, uterus, prostate, colorectal, stomach, lungs, bronchi, pancreatic cancer, liver, malignant tumors of these organs / tissues, leukemia cells, blood, serum,
Plasma, joint fluid, cerebrospinal fluid, pancreatic fluid, bile fluid, saliva, amniotic fluid,
Examples include urine, other body fluids, cell culture medium, tissue culture medium, tissue homogenate, biopsy sample, tissue, cells and the like.
When 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, operations and the like. The measurement conditions related to the target substance of the present invention or a substance having substantially the same activity as the target substance of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary conditions and operating methods in each method. .

Epitope mapping can also be carried out using the anti-galectin-9-binding sugar chain antibody of the present invention, particularly a monoclonal antibody. If an antibody that recognizes each epitope is used, detection of each galectin-9-binding substance and its related analogs can be performed.・ Measurement can be performed. In the EIA assay system, for example, in the competitive method, an antibody against a substance having an anti-galectin-9-binding sugar chain or the like is used as a solid-phased antibody,
Labeled antigen and unlabeled antigen (galectin 9 is used as the antigen
(For example, those having a sugar chain having a binding affinity with the antibody) are used, and non-competitive methods, such as the sandwich method, can use immobilized antibodies and labeled antibodies, or directly label the antibodies. Alternatively, the antibody can be labeled with an antibody against a substance having an anti-galectin-9-binding sugar chain or the like without being immobilized, or immobilized. As a sensitivity amplification method, for example, a combination of a non-enzyme-labeled primary antibody, a polymer polymer, an enzyme and a primary antibody (envision reagent is applied; Enhanced polymer one
-step staining (EPOS), and when combined with a non-enzyme labeled secondary antibody, for example, PAP (peroxidase-antiperox)
SABC with a combination of an enzyme such as the (idase) method and an anti-enzyme antibody complex
ABC (streptavidin-biotin complex), which is a combination of biotin-labeled secondary antibody such as (avidin-biotinylated peroxidase complex) method and biotin-labeled enzyme-avidin complex
Method, a combination of biotin-labeled secondary antibody such as LSAB (labeled streptavidin-biotin) method and a biotin-labeled enzyme-streptavidin complex, CSA (catalyzed signal amplific
ation) and the like, a combination of SABC, biotin-labeled tyramide and enzyme-labeled streptavidin, and those in which a secondary antibody and an enzyme are labeled with a high-molecular polymer.

For details of these general technical means, reference can be made to reviews, textbooks, etc. [eg,
Hiroshi Irie, “Radioimmunoassay”, Kodansha, Showa
Published in 1948; edited by Hiroshi Irie, "Sequel Radioimmunoassay"
Kodansha, published in 1979; edited by Eiji Ishikawa et al., “Enzyme Immunoassay”, Medical Shoin, 1983 published by: Eiji Ishikawa et al., “Enzyme Immunoassay” (second edition), Medical Shoin, 1982 Published: Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition), Medical Institute, 1987; HV Vunakis et al. (Ed.), "Met
hods in Enzymology ", Vol. 70 (Immunochemical Techn
iques, Part A), Academic Press, New York (1980); J.
J. Langone et al. (Ed.), "Methods in Enzymology",
Vol. 73 (Immunochemical Techniques, Part B), Acade
mic Press, New York (1981); JJ Langone et al.
(ed.), "Methods in Enzymology", Vol. 74 (Immunochem
ical Techniques, Part C), Academic Press, New York
(1981); JJ Langone et al. (Ed.), "Methods in E
nzymology ", Vol. 84 (Immunochemical Techniques, Pa
rt D: Selected Immunoassays), Academic Press, New Y
ork (1982); JJ Langone et al. (ed.), "Methods i
n Enzymology ", Vol. 92 (Immunochemical Techniques,
Part E: Monoclonal Antibodies and General Immunoa
ssay Methods), Academic Press, New York (1983); J.
J. Langoneet al. (Ed.), "Methods in Enzymology",
Vol. 121 (Immunochemical Techniques, Part I: Hybri
doma Technology and Monoclonal Antibodies), Academ
ic Press, New York (1986); JJ Langone et al. (e
d.), "Methods in Enzymology", Vol. 178 (Antibodie
s, Antigens, and Molecular Mimicry), Academic Pres
s, New York (1989); M. Wilchek et al. (ed.), "Meth
ods in Enzymology ", Vol. 184 (Avidin-Biotin Techno
logy), Academic Press, New York (1990); JJ Lango
ne et al. (ed.), "Methods in Enzymology", Vol. 203
(Molecular Design and Modeling: Concepts and Appl
ications, Part B: Anibodies and Antigens, Nucleic
Acids, Polysaccharides, and Drugs), Academic Pres
S., New York (1991), etc., or references cited therein (the descriptions therein are incorporated by reference herein).

The antibody of the present invention (antibodies against sugar chains having a binding affinity for galectin-9 and related fragments thereof) is
It is useful for detecting and / or measuring a phenomenon such as inhibition of galectin-9 activity by the sugar chain or its analog, and further for detecting and / or measuring various physiologically active substances caused by excess galectin-9 activity. The antibody, particularly the monoclonal antibody, is used for (i) detection of tissue- or protein-related disorders, abnormalities and / or diseases caused by galectin-9, (ii) tumor formation of cells associated with galectin-9, migration of cells. , Invasion, migration and / or metastasis or the possibility thereof, and / or (iii) tumorigenesis of cells, migration of cells such as tumor cells and blood cells, invasion, migration and / or
Alternatively, it is useful for detecting metastasis or the possibility thereof. It is expected that it can be used to know the degree of motility, invasiveness, chemotaxis and / or metastasis of cancer. The antibody of the present invention is a compound (antagonist or inhibitor) that inhibits functions such as biological activity of galectin-9 (including galectin-9, -9M and -9L) (eg, eosinophil migration activity). It is useful as an agent) and can be used as a medicine such as a therapeutic and / or prophylactic agent for various diseases such as hyperhuman galectin-9 dysfunction. Furthermore, an antibody mimetic designed based on the antibody of the present invention is used as an agent (agonist or promoter) that promotes functions such as the biological activity of galectin-9 (eg, eosinophil migration activity). It is useful and can be used as a medicament useful as a therapeutic and / or preventive agent for various diseases such as human galectin-9 dysfunction symptoms. Polypeptides such as mutants of the present invention promote functions such as biological activity of galectin-9 (including galectin-9, -9M and -9L) (eg, eosinophil migration activity). It is useful as a reagent for screening compounds (agonists), compounds that inhibit (antagonists) or salts thereof, and biological activities such as galectin-9-related proteins, some peptides thereof or salts thereof. There is also provided a method for screening a compound (agonist) or a compound (antagonist) that promotes a function such as (for example, eosinophil migration activity, hemagglutination activity, etc.) or a salt thereof. According to the present invention, the activity inhibition of galectin-9 by a sugar chain having a specific structure or an analog thereof is detected and / or measured and used as a monitor for determining the effect of an anticancer agent, a cancer metastasis inhibitor and / or an immunosuppressant. It becomes possible to do.

The active ingredient of the present invention [eg, (a) a mutant polypeptide of galectin-9, a part of the peptide or a salt thereof, a peptide related thereto, etc., (b) a DNA encoding the mutant, etc. Nucleic acid, etc., (c) the antibody of the present invention, a partial fragment thereof (including a monoclonal antibody) or a derivative thereof (including a mimetic), (d) a compound having a sugar chain having a binding affinity to galectin-9, or Its analog,
Furthermore, a compound or a salt thereof that suppresses and / or inhibits the biological activity of galectin-9, (e) a compound having a sugar chain having a binding affinity to galectin-9 or an analog thereof, and the biological activity of galectin-9 When a compound or a salt thereof that promotes and / or enhances the activity, (f) an antisense oligonucleotide against a nucleic acid such as the DNA of the present invention] is used as a medicine, for example, an agonist or antagonist to galectin-9 or a salt thereof, etc. Can be administered alone or in admixture with various pharmaceutically acceptable formulation auxiliaries, and can be administered as a pharmaceutical composition or pharmaceutical preparation. Preferably, oral administration, topical administration,
Alternatively, it may be administered in the form of a pharmaceutical preparation suitable for use such as parenteral administration, and may be in any administration form (including inhalation method and rectal administration) depending on the purpose.

In addition, the active ingredient of the present invention is combined with an antitumor agent (anticancer agent), tumor metastasis inhibitor, angiogenesis inhibitor, Alzheimer's therapeutic agent, joint destruction therapeutic agent, anti-inflammatory agent and / or immunosuppressive agent. It can also be used. As an antitumor agent (anticancer agent), tumor transfer inhibitor, angiogenesis inhibitor, Alzheimer's treatment agent, joint destruction treatment agent, anti-inflammatory agent or immunosuppressant agent, any agent that has an advantageous action can be used without limitation,
For example, it can be selected from those known in the art. And, as a parenteral administration form, topical,
Transdermal, intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal administration may be included, although direct administration to the affected area is also possible and in some cases also suitable. It is preferably orally administered to mammals including humans, or parenterally (eg, intracellular, tissue, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathoracic, spinal cavity, infusion method, infusion). Intestine, transrectal, ear drop, eye drop or nose drop,
It can be applied to the teeth, skin, mucous membranes, etc.). Specific forms of the formulation preparation include solution formulation, dispersion formulation, semi-solid formulation, powder formulation, molded formulation, leaching formulation, etc. Agents, troches, hard capsules, soft capsules, microcapsules, implants, powders, powders, granules, fine granules, injections, solutions, elixirs, emulsions, irrigation agents, syrups, Water solution, emulsion, suspension, liniment, lotion, aerosol, spray,
Inhalants, sprays, ointments, plasters, patches, pasta, poultices, creams, oils, suppositories (for example, rectal suppositories), tinctures, water solutions for skin, eye drops, nasal drops, Powders for ear drops, coatings, infusions, injectables, etc.
Lyophilized preparations, gel preparations and the like can be mentioned.

Pharmaceutical compositions can be formulated according to conventional methods. For example, physiologically acceptable carriers, pharmaceutically acceptable carriers, adjuvants, excipients, excipients, diluents, flavors, flavors, sweeteners, vehicles, preservatives, stabilizers, etc. Agent, binder, p
H regulator, buffer, surfactant, base, solvent, filler,
Extenders, solubilizers, solubilizers, tonicity agents, emulsifiers, suspending agents, dispersants, thickeners, gelling agents, curing agents, absorbents,
Adhesives, elastic agents, plasticizers, disintegrants, propellants, preservatives, antioxidants, light-shielding agents, moisturizers, emollients, antistatic agents, soothing agents, etc. are used alone or in combination, and together with this book By admixing the protein of the invention and the like, it can be produced in a unit dose form required for generally accepted formulation practice. Formulations suitable for parenteral use include sterile solutions of the active ingredient and water or other pharmaceutically acceptable medium, or suspensions, such as injections. Generally, water, saline, aqueous dextrose solution, other related sugar solutions, and glycols such as ethanol, propylene glycol, and polyethylene glycol are mentioned as preferred liquid carriers for injection. When preparing an injection, the solution is prepared by a method known in the art using a carrier such as distilled water, Ringer's solution, physiological saline, an appropriate dispersing agent or wetting agent and suspending agent. , Suspensions, emulsions, and injectable forms.

Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg D-sorbitol, D-mannitol, sodium chloride etc.). A suitable acceptable solubilizing agent, eg alcohol (eg ethanol),
You may use together with polyalcohol (for example, propylene glycol, polyethylene glycol, etc.), a nonionic surfactant (for example, Polysorbate 80 ^™ , HCO-50, etc.) and the like. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.) or reagents for adjusting osmotic pressure, soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human) Serum albumin, polyethylene glycol, etc.), a preservative (eg, benzyl alcohol, phenol, etc.), an antioxidant such as ascorbic acid, an absorption promoter and the like may be added. The prepared injection solution is usually filled in a suitable ampoule.

For parenteral administration, in a sterile pharmaceutically acceptable liquid such as water, ethanol or oil, with or without surfactants and other pharmaceutically acceptable auxiliaries. It is formulated in the form of a solution or suspension. Examples of the oily vehicle or solvent used in the preparation include natural or synthetic or semi-synthetic mono-, di- or triglycerides, natural, semi-synthetic or synthetic fats and oils, such as peanut oil, corn oil, Examples include vegetable oils such as soybean oil and sesame oil. For example, this injection can be usually prepared so as to contain the compound of the present invention in an amount of about 0.1 to 10% by weight. Formulations suitable for topical, eg buccal, or rectal use include, eg, mouth washes, dentifrices, mouth sprays,
Inhalants, ointments, dental fillers, dental coating agents, dental paste agents, suppositories and the like can be mentioned. The mouthwash and other dental agents are prepared by a conventional method using a pharmacologically acceptable carrier. As an oral spray and an inhalant, the compound of the present invention itself or a pharmacologically acceptable inert carrier can be dissolved in a solution for an aerosol or a nebulizer, or can be administered as fine powder for inhalation to teeth and the like. The ointment is prepared by a conventional method by adding a commonly used base such as an ointment base (white petrolatum, paraffin, olive oil, Macrogol 400, Macrogol ointment, etc.).

Medicaments for topical application to the teeth, skin may be formulated in solutions or suspensions of suitably sterile water or non-aqueous vehicles. Additives include, for example, buffering agents such as sodium bisulfite or disodium edetate; preservatives such as acetic acid or phenylmercuric nitrate, benzalkonium chloride or chlorohexidine, and preservatives such as hypromelrose. Examples include thickeners. Suppositories are carriers well known in the art, preferably suitable non-irritating excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc., preferably solid at room temperature but at intestinal temperature. It is prepared by a conventional method using a liquid that melts in the rectum and releases the drug, and is usually prepared so as to contain the compound of the present invention in an amount of about 0.1 to 95% by weight. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Adjuvants such as local anesthetics, preservatives and buffers can be dissolved in the vehicle. Examples of formulations suitable for oral use include solid compositions such as tablets, pills, capsules, powders, granules, and troches, and liquid compositions such as solutions, syrups, and suspensions. Can be mentioned. When preparing the formulation,
A formulation auxiliary agent or the like known in the art is used. Tablets and pills can also be manufactured with enteric coating. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type.

Furthermore, when the nucleic acid such as the DNA of the present invention is used as the therapeutic and / or prophylactic agent as described above, the nucleic acid can be used alone or by the gene recombination technique as described above. It can be used by ligating it to a suitable vector used, for example, a virus-derived vector such as a retrovirus-derived vector. The nucleic acid such as the DNA of the present invention can be administered by an ordinary known method, and as it is, or together with an appropriate auxiliary agent or a physiologically acceptable carrier or the like so that the uptake into cells can be promoted. , Can be formulated and used, and can be administered as a pharmaceutical composition or a pharmaceutical preparation as described above. Also, a method known as gene therapy can be applied. The dose of the active ingredient of the present invention can be selected over a wide range, and the dose and the number of doses can be determined by the sex of the treated patient,
Age and weight, general health condition, diet, administration time, administration method, excretion rate, drug combination, and the degree of medical condition being treated by the patient at that time are determined in consideration of these factors and other factors. . In manufacturing pharmaceutical products, additives, preparation methods, etc. are, for example, edited by the Japanese Pharmacopoeia Editorial Committee, 13th revision Japanese Pharmacopoeia, published on July 10, 1996, Hirokawa Shoten Co., Ltd .; Ichibankase Takashi et al. Development of Pharmaceuticals Volume 12 (Pharmaceutical Formulation [I]), Heisei 2
Published October 15, 2015, Hirokawa Shoten Co., Ltd .; Development of pharmaceuticals
Volume 12 (Pharmaceutical Material [II]) Issued on October 28, 1990, refer to the description of Hirokawa Shoten Co., Ltd., etc., and can be appropriately selected and applied from among them.

In the present specification, the activity of galectin-9 (including galectin-9S, -9M and -9L) may mean the following. Galectin-9 can be tested for cytokine activity, cell proliferative activity (inducing or inhibiting activity) or cell differentiating activity (inducing or inhibiting activity), and the activity of inducing the production of other cytokines in a particular cell population. Many factor-dependent cell proliferation assays are known, for example 32D, DA2, DA1G, T10, B9, B9 / 11, BaF3, M.
C9 / G, M + (preB M +), 2E8, RB5, DA1, 123, T1165, HT
2, Assays for cell lines such as CTLL2, TF-1, Mo7e, CMK are known. Assays for T cell or thymocyte proliferative activity include, but are not limited to, eg John E. Coligan, Ada M. Kruisbeek, Davi.
d H. Margulies, Ethan M. Shevach, Warren Strober
(Ed.), Current Protocols in Immunology (hereinafter, simply "JE Coligan et al. (Ed.), Current Protocols i
n Immunology, John Wiley & Sons, Inc. "(Cha
pter 3: "In Vitro assays for Mouse Lymphocyte Func
tion (3.1-3.19) "; Chapter 7:" Immunologic studies
in Humans "), John Wiley & Sons, Inc; Takai et al.,
J. Immunol., 137: 3494-3500 (1986); Bertagnolli et
al., J. Immunol., 145: 1706-1712 (1990); Bertagnoll
i et al., Cellular Immunology, 133: 327-341 (1991);
Bertagnolli et al., J. Immunol., 149: 3778-3783 (1
992); Bowman et al., J. Immunol., 152: 1756-1761.
(1994). Assays for cytokine production and / or proliferative activity of spleen cells, lymph node cells and thymocytes include, but are not limited to, eg, JE Coligan et al. (Ed.), C
urrent Protocols in Immunology, John Wiley & Sons,
Inc. (Chapter 3 "In Vitro assays for Mouse Lymphoc
yteFunction--Proliferative Assays for T Cell Funct
ion "and Chapter 6" Cytokines and Their Cellular R
eceptors--Measurement of mouse and human Interfero
n γ "). Assays for proliferation and differentiation activity of hematopoietic and lymphopoietic cells include, but are not limited to, eg, JE Coligan et al. (Ed. ), Current Protocols
in Immunology, John Wiley & Sons, Inc. (Chapter 6
"Cytokines and Their Cellular Receptors--Measurem
ent of Human and Murine Interleukin 2 and Interleu
kin 4;-Measurement of mouse and human interleukin
6; --Measurement of human Interleukin 11; --Measu
rement of mouse and human Interleukin 9); deVriese
t al., J. Exp. Med., 173: 1205-1211 (1991); Moreau
et al., Nature, 336: 690-692 (1988); Greenberger et
al., Proc. Natl. Acad. Sci. USA, 80: 2931-2938
(1983); Smith et al., Proc. Natl. Acad. Sci. US
A., 83: 1857-1861 (1986).

Assays for T cell clone responsiveness to antigens, including, among other things, measuring its proliferation and cytokine production to identify APC-T cell interactions and proteins that act directly on T cells. Are not limited to, for example, JE Co
ligan et al. (Ed.), Current Protocols in Immunolog
y, John Wiley & Sons, Inc. (Chapter 3 "In Vitro as
says for Mouse Lymphocyte Function "; Chapter 6" Cy
tokines and Their Cellular Receptors "; Chapter 7"
Immunologic studies in Humans "); Weinberger et a
L., Proc. Natl. Acad. Sci. USA, 77: 6091-6095 (19
80); Weinberger et al., Eur. J. Immun., 11: 405-411.
(1981); Takai et al., J. Immunol., 137: 3494-3500.
(1986); Takai et al., J. Immunol., 140: 508-512 (19
88). Galectin-9
It can be tested for immunostimulatory or suppressive activity. Activity in the treatment of various immunodeficiencies and disorders, including severe combined immunodeficiency (SCID), eg,
It is possible to test the activity in regulating the proliferation (up-regulation or down-regulation) of T and / or B lymphocytes as well as whether they are active when NK cells and other cell populations exert cytolytic activity. The immunodeficiency may be genetic or may be caused by a viral (eg HIV) as well as a bacterial or fungal infection and may even result from an autoimmune disorder. More specifically, infectious diseases caused by viruses, bacteria, fungi or other infectious diseases (HIV, hepatitis virus, herpes virus, mycobacteria, infection by malaria and various fungal infections such as candidiasis. May be included). In addition, when it is required to stimulate the immune system, a place for treating cancer, for example, may be included. Examples of the autoimmune disorder include connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pneumonia, Guian-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis. , Graft-versus-host disease, autoimmune inflammatory eye disease and the like. It may also include testing for activity in treating allergic reactions and conditions such as asthma (particularly allergic asthma) and other respiratory disorders. Testing for activity against other conditions in which immunosuppression is desired, including, for example, organ transplantation, may be included.

Whether galectin-9 enables an immune response can be tested in a number of ways. For example, down-regulation may be in the form of inhibiting or blocking an already ongoing immune response, or may involve preventing induction of an immune response. The function of activated T cells may be inhibited by suppressing the T cell response, or by inducing specific tolerance in the T cell, or both. In general, immunosuppression of T cell responses is an active and non-antigen specific process that requires continuous contact of T cells with their suppressors. Tolerance, including non-responsiveness or induction of anergy in T cells, is immunosuppressive in that it is generally antigen-specific and persists after contact with the tolerizing factor is stopped. Distinguished from. Whether it is tolerable or not can be confirmed by actual manipulation by checking whether or not there is a T cell response when re-exposed to a specific antigen in the absence of a tolerizing factor. . One or more antigenic functions (including but not limited to B lymphocytes (eg,
Down-regulation or inhibition of (including B7) antigen functions), eg, inhibition of high levels of lymphokine synthesis by activated T cells, is associated with tissue, skin and organ transplant conditions and graft versus host disease. (GVHD) is useful. For example, inhibiting T cell function should reduce tissue destruction in tissue transplants. Typically, when a tissue is transplanted, rejection of the graft is initiated through recognition of the tissue piece as foreign by T cells, followed by an immune reaction that destroys the graft. A molecule that inhibits or blocks the interaction of the B7 lymphocyte antigen with its natural ligand (s) on immune cells (a soluble, monomeric peptide alone with B7-2 activity, or another B lymphocyte Antigen (eg B7
-1, B7-3) active monomeric forms of peptides or combinations with blocking antibodies, etc.) prior to transplantation do not transduce the corresponding secondary stimulatory signals, and Binding of the molecule to the ligand (s) of Thus, blocking B lymphocyte antigen function blocks cytokine synthesis by immune cells such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of a secondary stimulus renders it sufficient to inactivate the T cells, thereby inducing tolerance in the subject. Long-term tolerance induction with B lymphocyte antigen blocking agents could avoid the need for repeated administration of the blocking agents. It may also be necessary to block B lymphocyte antigen combination function in order for the target to be fully immunosuppressed or tolerated.

The efficacy of certain blocking agents in preventing organ transplant rejection or GVHD can be performed using animal models useful in predicting efficacy in humans. Suitable usable systems include, for example, rat allograft heart transplants, mouse xenogeneic islet cell transplants, and the like. Lenschow et al., Science, 257: 789-792 (1992)
And Turka et al., Proc. Natl. Acad. Sci. USA, 8
Both have been used to study the immunosuppressive effects of CTLA4Ig fusion proteins in vivo, as described in 9: 11102-11105 (1992). Furthermore, GVHD mouse model (Paul (Ed.), Fundamental Immunology, Raven
Press, New York, pp.846-847 (1989))
The effect of blocking B lymphocyte antigen function in vivo on disease progression can also be measured. It may also be useful to block antigen function in the therapeutic treatment of autoimmune diseases. Many autoimmune disorders are the result of a response to self tissues, resulting from the inappropriate activation of T cells that promote the production of cytokines and autoantibodies involved in the disease. By preventing activation of self-reactive T cells, symptoms of the disease can be reduced or eliminated. By administering a drug that blocks the co-stimulation of T cells through disrupting the B lymphocyte antigen receptor: ligand interaction, T
It can inhibit cell activation and prevent the production of autoantibodies or T cell-derived cytokines that are involved in the disease process. Furthermore, the blocking agent makes it possible to induce antigen-specific tolerance of autoreactive T cells which makes it possible to induce long-term relief from the disease. Many well-characterized animal models for human autoimmune disease could be used to test the efficacy of blocking agents in preventing or ameliorating autoimmune disorders. Examples include mouse experimental autoimmune encephalitis, systemic lupus erythematosus in MRL / lpr / lpr mice or NZB hybrid mice, mouse autoimmune collagen arthritis, diabetes in NOD mice and BB rats, and mouse experimental myasthenia gravis. (Paul (Ed.), Fundamental Immunology, Rave
n Press, New York, 840-856 (1989). Upregulation of antigen function (preferably B lymphocyte antigen function) may also be therapeutically useful as a means of upregulating immune responses. Upregulation of an immune response may be in the form of enhancing an existing immune response or inducing an initial immune response. For example, enhancing the immune response via stimulating B lymphocyte antigen function may be useful in the case of viral infection. Furthermore, by systemically administering a stimulatory form of B lymphocyte antigen,
It can ameliorate systemic viral diseases such as influenza, common cold, and encephalitis. Upregulation or enhancement of antigen function, preferably B lymphocyte antigen function, may also be useful in inducing tumor immunity. For example, tumor cells (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, transfected with a nucleic acid encoding at least one of galectin-9,
(Carcinoma) can be administered to the target to see if it achieves overcoming tumor-specific tolerance in the target.

Galectin-9 can be tested for hematopoietic regulatory activity and further tested for its usefulness in treating myeloid or lymphoid cell deficiencies.
Suitable assay methods for examining the proliferation and differentiation activity of various hematopoietic cell lines are widely known in the art and can be used. The assay for the differentiation of embryonic stem cells (in particular, the effect on embryonic differentiation and hematopoiesis) is not limited to, for example, Johansson et al.,
Cellular Biology, 15: 141-151 (1995); Keller et a
l., Molecular and Cellular Biology, 13: 473-486 (1
993); McClanahan et al., Blood 81: 2903-2915 (199
Examples include those described in 3). The survival and differentiation of stem cells (in particular, the effect on lympho-hematopoietic regulation) is not limited to, for example, Methyl
cellulose colony forming assays, Freshney, MG Cu
lture of Hematopoietic Cells. RI Freshney et al. Vo
l 265-268, Wiley-Liss, Inc., New York, NY. 1994;
Hirayama et al., Proc. Natl. Acad. Sci. USA 89: 5907-59.
11, 1992; Primitive hematopoietic colonyforming ce
lls with high proliferative potential, McNiece, I.
K. and Briddell, RA Culture of Hematopoietic C
ells. RI Freshney et al. Vol. 23-39, Wiley-Liss, I
nc., New York, NY. 1994; Neben et al., Experimental He
matology 22: 353-359, 1994; Cobblestone area formi
ng cell assay, Ploemacher, RE Culture of Hematopo
ietic Cells. RI Freshney et al. Vol. 1-2 page, Wiley-
Liss, Inc., New York, NY. 1994; Long term bone mar
row cultures in the presence of stromal cells, Spoo
ncer, E., Dexter, M. and Allen, T. Culture of He
matopoietic Cells. RI Freshney et al. Vol. 163-179
Page, Wiley-Liss, Inc., New York, NY. 1994; Long ter
m culture initiating cell assay, Sutherland, HJ
Culture of Hematopoietic Cells. RI Freshney et al.
Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 19
And those described in 94.

Galectin-9 is a pharmaceutical composition used for the growth or regeneration of bone, cartilage, tendon, ligament and / or nerve tissue, as well as for wound healing and tissue repair and replacement, and for the treatment of burns, lacerations and ulcers. It can also be examined for its activity as an agent. The activity of inducing cartilage and / or bone growth under conditions in which bone is not normally formed is useful for healing bone fractures and cartilage damage or defects in humans and other animals. The activity of galectin-9 on the treatment of periodontal disease and other tooth repair processes can also be tested. Activities such as attracting osteogenic cells, stimulating proliferation of osteogenic cells, or inducing differentiation of precursors of osteogenic cells can also be investigated. Galectin-9 is, for example, bone and / or
Or it may be active in the treatment of osteoporosis or osteoarthritis, either through stimulation of cartilage repair or by blocking the processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammation or inflammatory processes There is a nature. In addition, activity for healing tendon or ligament lacerations, deformities and other tendon or ligament defects in humans and other animals may also be tested. Galectin-9
Is active in the proliferation of nerve cells involved in degeneration, death or trauma to nerve cells or tissue and regeneration of nerve and brain tissue, ie in the treatment of central and peripheral nervous system diseases and neuropathy and mechanical and traumatic disorders. And more specifically, peripheral nervous system disorders such as peripheral nerve injury, peripheral neuropathy and localized neuropathy, and Alzheimer's disease, Parkinson's disease, Huntington's disease,
The activity against central nervous system diseases such as amyotrophic lateral sclerosis and Shy-Drager syndrome is investigated.

Galectin-9 includes activin- or inhibin-related activities, mammalian cells (eg monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells). Chemotaxis / migratory activity of
Tumors that also have hemostatic or thrombolytic activity, activity as inhibitors or agonists of receptors, receptor ligands or receptor / ligand interactions, anti-inflammatory activity (including cell-stimulating activity involved in inflammatory response), anti-tumor activity It may be tested for one or more of the following activities, additional activities or effects: inhibition of growth, infection or function of infectious agents, including but not limited to bacteria, viruses, fungi and other parasites. Or its killing;
Height, weight, hair color, eye color, skin, fat-to-weight ratio (fat to
lean ratio) or pigmentation of other tissues, or body characteristics including (but not limited to) size or morphology of organs or body parts (eg, breast augmentation or vice versa, changes in bone morphology or shape). Effects (suppression or enhancement); effects on biorhythm or cardiac cycle or rhythm; effects on fertility of male or female subjects; ingested fats, lipids, proteins, carbohydrates, vitamins, minerals, cofactors or other nutritional factors Or metabolism, catabolism, assimilation, processing, utilization of component (s),
Effects on storage or elimination; effects on behavioral characteristics including appetite, libido, stress, cognition (including cognitive impairment), depression (including depressive disorder) and violent behavior (including but not limited to); Providing analgesic or other pain-relieving effects; promoting differentiation and proliferation of embryonic stem cells in non-hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correction of enzyme defects and treatment of defect-related diseases;
Treatment of hyperproliferative disorders (eg, psoriasis); immunoglobulin-like activities (eg, ability to bind antigen or complement); as well as such proteins acting as antigens in vaccine compositions Or the ability to elicit an immune response against another substance or entity that is cross-reactive with such proteins. In the description and drawings, the terms are IUPAC-IUB Commission on Biochemical
Nomenclature or based on the meaning of terms commonly used in the art.

[0112]

EXAMPLES The present invention will be described in detail below with reference to examples, but these examples are provided merely for the purpose of explaining the present invention and for reference to specific modes thereof. .
These exemplifications are intended to illustrate certain specific embodiments of the invention, but are not meant to limit or limit the scope of the invention disclosed herein. It should be understood that the present invention is capable of various embodiments based on the ideas of the present specification. All examples, unless otherwise indicated in detail, are carried out or can be carried out using standard techniques, which are well known and routine to those skilled in the art. ..

Commonly used molecular biology techniques in the following examples include standard laboratory manuals such as J. Sambrook, EF Fritsch & T. Maniatis (ed.),
"Molecular Cloning: A Laboratory Manual (2nd edit
ion) ", Cold Spring Harbor Laboratory Press, Cold Sp
ring Harbor, New York (1989) and DM Gloveret a
l. (ed.), "DNA Cloning", 2nd ed., Vol. 1 to 4, (Th
e Practical Approach Series), IRL Press, Oxford Un
iversity Press (1995), and especially for PCR, R. Sai
ki et al., Science, 230: 1350, 1985; R. Saiki et a
l., Science, 239: 487, 1988; HA Erlich (ed.), PC
R Technology, Stockton Press, 1989; DM Glover
et al. (ed.), "DNA Cloning", 2nd ed., Vol. 1, (The
Practical Approach Series), IRL Press, Oxford Uni
versity Press (1995); MAInnis et al. (ed.), "PC
R Protocols: a guide to methods and applications ",
Academic Press, New York (1990)); MJ McPherso
n, P. Quirke and GR Taylor (ed.), PCR: a practi
cal approach, IRL Press, Oxford (1991); MA Frohm
an et al., Proc. Natl. Acad. Sci. USA, 85, 8998-90
02 (1988) or the methods described in the literature cited therein, or methods and modifications substantially similar to them, and when commercially available reagents or kits are used, those methods may be used. Instructions attached to (p
rotocols) and attached chemicals, etc. (the descriptions therein are included in the disclosure of the present specification by reference thereto).

Example 1 (1) Galectin-9, Galectin-9NN and Galectin-9
Construction of CC expression vector To investigate the possible morphology of the interaction between galectin-9 and glycoconjugates on the surface of eosinophils, the N-terminal CRD and C-terminal CRD of galectin-9 were sequenced. A recombinant protein in which the two are bound to
Galectin-9NN and galectin-9CC (see FIG. 1) were prepared. Three isoforms of galectin-9, galectin-9S, galectin-9M and galectin-9
For L, eosinophil chemoattra
(ctant: ECA) was prepared to see the effect of the structure of the linker peptide on the activity. The primers used to amplify the cDNAs of galectin-9 isoforms and galectin-9 variants are summarized below:

[0115] G9F1: CGTCCTCTCGAGAATGGCCTTCAGCGGTTCCCAG (SEQ ID NO: 1) G9R1: CGACCGGAATTCCTATGTCTGCACATGGGTCAG (SEQ ID NO: 2) G9NNF1: CGTCCTGGTACCATGGCCTTCAGCGGTTCCCAG (SEQ ID NO: 3) G9NNR1: CGACCGGAATTCCTACTGGAAGCTGATGTAGGACAG (SEQ ID NO: 4) G9NNR2: CGACCGGGTACCCTGTCCAGGGGCGCTCTGCAC (SEQ ID NO: 5) G9CCF1: CGTCCGGGTACCCCTCCCGGCGTGTGGCCTGCC (SEQ ID NO: 6) G9CCF2: CGTCCTCTCGAGAATGTTCTCTACTCCCGCCATC (SEQ ID NO: 7) G9CCR1: CGACCGGGTACCTGTCTGCACATGGGTCAGCTG (SEQ ID NO: 8)

FIG. 1 conceptually shows the positions of the primer sites used to amplify the galectin-9 cDNAs and the structure of the galectin-9 mutant protein. Since it is not easy to reliably determine the C-terminal end portion of the linker peptide without data on the three-dimensional structure, the length of the linker peptide is assumed to be just assumed. Should be. Human galectin-9 / urate transporter
(rpk) genomic structure has been reported (Lipkow
its et al., J. Clin. Invest., 107: 1103-1115 (200
1)), but in the case of galectin-9M, the C-terminal tail site of the linker peptide seems to be Ser ¹⁷⁷ . Except for the production of N-terminal CRD and C-terminal CRD of galectin-9, pTrcHisB prokaryotic expression vector (Invi
trogen) was used (see below).

Wild-type galectin-9 (Galectin-9S,
The expression vectors for Galectin-9M and Galectin-9L) are pT cDNAs amplified with primers G9F1 + G9R1.
It was constructed by incorporating it into the XhoI / EcoRI site of rcHisB. To construct the galectin-9NN expression vector, cDNA (primer) corresponding to N-terminal CRD + linker peptide
G9F1 + G9NNR2) and cDNA corresponding to N-terminal CRD with additional stop codon at 3'end (primer G9NNF1)
+ Amplification with G9NNR1) XhoI of the same vector pTrcHisB
Incorporated into the / KpnI site and KpnI / EcoRI site. To construct the galectin-9CC expression vector, the cDNA corresponding to the C-terminal CRD without the stop codon (primer G9CCF2
+ Amplification with G9CCR1) and linker peptide + C-terminal side
The cDNA corresponding to the CRD (amplified with the primers G9CCF1 + G9R1) was used for the XhoI / KpnI site and K
Incorporated into the pnI / EcoRI site. Galectin-9 N-terminal C
The GST-fusion protein expression vector of RD and C-terminal CRD is described by Matsushita et al., J Biol. Chem., 275: 8355-83.
60 (2000).

(2) Expression and Purification of Recombinant Protein All recombinant proteins were expressed in E. coli. Escherichia coli harboring each expression plasmid
BL-21 strain was cultivated in 2xYT medium containing 2% (w / v) glucose and 100 μg / ml ampicillin, and the optical density at 600 nm was
Grow to 0.7. Expression of the fusion protein was induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside, and galectin-9S, galectin-9M, galectin-9NN and galectin-9CC were incubated at 37 ° C for 2 hours. 30 ° C for Galectin-9L
Cultivated for 3 hours. Cells from a 500 ml culture
0.5 M NaCl, 1 mM dithiothreitol and 10 mM Tris-H
It was suspended in 90 ml of Cl (pH 7.5) solution and then sonicated for 10 minutes. 10% (w / v) Trito on sonicated product
Add 10 ml of nX-100 solution, stir at 4 ° C for 30 min, then
It was centrifuged at 5,000 xg for 30 minutes. The obtained supernatant was subjected to affinity chromatography on lactose-agarose (Seikagaku Corporation, Tokyo). The protein concentration was determined by the BCA protein assay reagent (Pierce) using bovine serum albumin as a standard substance.

FIG. 2 shows the results of SDS-polyacrylamide gel electrophoresis analysis of the purified recombinant proteins. As shown in FIG. 2, all the recombinant proteins were subjected to lactose affinity chromatography. It was purified with high efficiency and was of uniform quality. The recombinant protein purified by lactose-agarose affinity chromatography was electrophoretically separated in an SDS / 12% polyacrylamide gel under reducing conditions. Recombinant protein galectin
-9S (38.7 kDa), Galectin-9M (39.9 kDa), Galectin-9
L (43.5 kDa), galectin-9NN (39.5 kDa) and galectin
-9CC (40.6 kDa) with Coomassie Brilliant Blue (Coomass
ie brilliant blue) G-250 was used for visualization (the molecular weight of the recombinant protein contains a tag sequence of approximately 4 kDa). Galectin-9L, an isoform containing the longest linker peptide
In this case, the yield was lower than that of the other isoforms, probably due to the low solubility of galectin-9L.

(3) In vitro migration activity (chemotaxi
s) Matsumoto et al., J Biol. Chem., 273: 16975-16984
(1978) and Hirashima et al., Lymphokine Cytokine
Res., 11: 331-338 (1992), and in vitro eosinophil chemoattractant.
The activity was measured. That is, human peripheral blood leukocytes from a healthy donor were percolated (Percoll, Amersham Pharmaci
a Biotech) and then treating the cells with anti-CD16 immunoglobulin (DAKO S., Glostrup, Denmark) to obtain a CD16-negative eosinophil-rich sample. Purified eosinophils were> 97% pure and their viable cell ratio was> 95%.

48-well chamber with a polyvinylpyrrolidone-free membrane with a pore size of 5 μm (Neuro Pro
be Inc.) was used to measure eosinophil migration activity. Human eosinophils (0.5-1 × 10 ⁶ / ml) were placed on top of the chamber, while various concentrations of the test chemoattractant were placed on the bottom of the chamber. Each assay was performed in triplicate. After incubation at 37 ° C for 1-2 hours in humidified air containing 5% CO ₂ , the membrane separating the two chambers was removed and the Diff-Quick stain (B
axter Healthcare Corp.). Eosinophils that migrated through the membrane and were stained were counted under the microscope.
As a control, human eotaxin-1 (human eo
taxin-1, Seikagaku Corporation, Tokyo) was used. When recombinant galectins are produced as a glutathione S-transferase (GST) fusion protein and used in an eosinophil migration activity assay, the presence of the GST moiety does not affect the eosinophil migration activity of galectin-9. It is confirmed that it will not be given. It has also been confirmed that the recombinant protein having a tag sequence having a molecular weight of about 4,000 containing a hexahistidine sequence at the N-terminal used in the present invention is indistinguishable from the GST-fused recombinant protein in eosinophil migration activity.

The eosinophil chemotactic activity of 0.1 μM galectin-9CC was found to be a typical eosinophil chemotactic factor, eotaxin-1.
It was equivalent to (100 ng / ml). Galectin-9NN showed some weaker eosinophil migration activity at that concentration than galectin-9CC. However, as shown in FIG. 3, overall, the dose responsiveness was similar between the wild-type galectin (Galectin-9M) and the galectin mutant. Figure 3
Among them, wild-type galectin-9M, galectin-9NN and galectin-9CC were used after being expressed by E. coli and then purified by lactose-agarose affinity chromatography. The results are shown as the average value ± SD, which is the average of two tests performed with three samples as a set. Separately, the results of comparing the eosinophil migration activity of galectin-9 isoforms are shown in FIG. The galectin-9 isoforms that differ only in the linker peptide used were those produced in E. coli and purified by lactose-agarose affinity chromatography. Galectin-9 isoform with the longest linker peptide, i.e., 0 for galectin-9L.
Its eosinophil migration activity was measured at concentrations below 14 μM.
The results are shown as the average value ± SD, which is the average of two tests performed with three samples as a set. Galectin-9 isoform with the shortest linker peptide,
That is, galectin-9S showed a dose-response curve similar to that of galectin-9M. Galectin-9L showed eosinophil migration activity similar to other galectin-9 isoforms in the range of concentrations tested.

(4) Hemagglutination assay Hemagglutination activity was determined by the method of Nowak et al. (Nowak et al., B
iochem. Biophys. Res. Commun., 68: 650-657 (1976))
I looked it up. That is, a recombinant protein was prepared in a 2-fold dilution series on a 96-well microtiter plate to prepare an assay sample. Final concentration 0.25%
bovine serum albumin to (w / v) and trypsin-treated rabbit red blood cells immobilized with glutaraldehyde to a final concentration of 1% (v / v) were added, and the reaction mixture was allowed to stand at room temperature for 1 hour. Incubated for hours. The minimum concentration required for aggregation was determined visually. Hemagglutination activity is one of the typical characteristics of multivalent lectins. The minimum concentration of galectin-9 required for hemagglutination is the monomer-dimer equilibrium (Cho and Cummings, Biochemistry, 35: 13
081-13088 (1996)) is a prototype galectin known to exist, Galectin-1 (Matsushi
ta et al., J. Biol. Chem., 275: 8355-8360 (2000))
, Which was equivalent to the above concentration. To investigate the CRD functionality of both galectin-9NN and galectin-9CC, the hemagglutination activity of the mutant proteins was compared with wild-type galectin-9. The results are shown in Table 1.

[0124]

[Table 1]

Recombinant proteins including galectin-9S and galectin-9L all showed comparable hemagglutination activity. It was shown that both CRDs, galectin-9NN and galectin-9CC, can interact with erythrocyte cell surface glycoconjugates.

(5) Frontal Affinity Chromatography Each of N-terminal CRD and C-terminal CRD of galectin-9,
The interaction with oligosaccharides was investigated by frontal affinity chromatography. Recombinant protein was dissolved in 0.1 M NaCO ₃ solution (pH 8.3) containing 0.25 M NaCl and 0.1 M lactose, and HiTrap N
HS-activated column (Amersham Pharmacia Biotech KK)
(The operation was performed according to the operating method instructed by the column vendor). The agarose beads with immobilized galectin were taken out from the cartridge and packed in a stainless steel column (4 x 10 mm). Methods known in the art (eg, Kasi et al., J. Chromatogr., 376:
33-47 (1986); Hirabayashi et al., J. Chromatogr.
A, 890: 261-271 (2000); Arata et al., J. Biol. Che
m., 276: 3068-3077 (2001)), the elution volume of the analyte (pyridylamino oligosaccharides) was determined, and the Kd value was calculated. Frontal affinity chromatography technology is described by Hirabayashi et al., J.
Chromatogr. A, 890, 261-271 (2000) and Arata et a
l., J. Biol. Chem., 276: 3068-3077 (2001) was performed with reference to the method described in, and pyridylamino of oligosaccharides to immobilized GST-galectin-9NT and immobilized GST-galectin-9CT ( The binding affinity of the PA) derivative was measured and determined. Twelve glycoprotein-derived N-glycans and 21 glycolipid-derived glycans were tested. As a result, Table 2
As shown in FIG. 4 and FIG. 5, both galectin-9NT and galectin-9CT have N-acetyllactosamine structure 3
It showed relatively high affinity for branched or 4-branched N-link type glycans (Kd <1 μM).

[0127]

[Table 2]

[0128]

[Table 3]

[0129]

[Table 4]

[0130]

[Table 5]

In Tables 2 to 5, the following symbols have the following meanings.

[Table 6] In Tables 2 to 5 above, Biantennary: Bifurcated type, Triant
ennary: 3-branch type, Tetraantennary: 4-branch type, core-f
ucosylated: core-fucosylated, monosialylated: monosialized, disialylated: disialylated, fucosyla
ted at N-acetyllactosamine unit: Fucosylated in the N-acetyllactosamine structure, Lactose:
Lactose, asialo: asialo, trisaccharide: trisaccharide, tetrasaccharide: tetrasaccharide, pe
ntasaccharide: pentasaccharide, hexasaccharide:
Hexasaccharide, Globotoriose: Globotriose, Globo-N-tetraose: Globo-N-tetraose, Forss
man pentasaccharide: forssman pentasaccharide, Lacto: lacto, neotetraose: neotetraose, tetraose: tetraose, fucopentaose: fucopentaose, Fucosyllactose: fucosyl lactose. The eosinophil migration activity of galectin-9 was inhibited by 2% of 4-branched glycan (sugar chain) at 87%. On the other hand, the oligosaccharide showed only a negligible effect (lower than 10% inhibition rate) on the eosinophil migration activity of eotaxin. Due to fucosylation or sialylation of the N-acetyllactosamine structure, the affinity between the oligosaccharide and galectin-9 CRD was decreased (6 in Tables 2 to 5).
Compare 9, 10 and 12, 1 and 3-5).

[Discussion] Using a mutant galectin-9 protein (galectin-9NN) that binds two N-terminal CRDs and a mutant galectin-9 protein (galectin-9CC) that binds two C-terminal CRDs By doing so, each CRD
It is possible to screen for a substance that specifically binds to, and the substance obtained as a result of the screening is useful as a substance capable of controlling the activity of galectin-9 and has applicability to medicines and the like. There is. Tandem repeat type galectin artificially produced in the present invention, that is,
The results that galectin-9NN and galectin-9CC show eosinophil migration activity indistinguishable from wild-type galectin-9 are considered to be consistent with the results for galectin-9NT and galectin-9CT.

Galectin-8 has a weak eosinophil chemotactic activity, but the tandem repeat structure itself is not a sufficient condition for obtaining eosinophil chemotactic activity. This is because the molecule having two galectin-1 linked by a peptide was completely inactive in the eosinophil migration activity assay. Therefore, the eosinophil migration activity depends on the sugar-binding properties unique to each CRD, and when acting as eosinophil chemoattractants, the two CRDs of galectin-9 are the same or very similar. It is thought that it is bound to a sugar chain. In the present invention, frontal affinity chromatography and various oligosaccharide compounds are used to use galectin-9, which is an affinity carrier having the N-terminal CRD of galectin-9 as a ligand.
Detailed analysis was carried out on the sugar chain binding specificity of galectin-9CT, which is an affinity carrier using NT and C-terminal CRD as ligands. As a result, galectin-9NT and galectin-9CT show similar binding affinity to most glycoprotein-derived N-glycans, and have N-linked 4-branched complex with N-acetyllactosamine structure. It had the highest affinity for body-type glycans. The fucosylated and sialylated oligosaccharide compounds had reduced affinity for both CRDs. On the other hand, the binding affinities of almost half of the glycolipid-derived glycans were about 10-fold different between the two CRDs. In addition, 4-branched glycans strongly inhibited the eosinophil migration activity of galectin-9, but not the activity of eotaxin.

Taken together, these show that galectin-9 binds to eosinophil surface glycoproteins via N-linked 3- and / or 4-branched glycans having an N-acetyllactosamine structure. It is suggested to do. These interactions may lead to dimerization / multimerization of cell surface glycoproteins / receptors (receptors), thereby activating intracellular signaling pathways linked to chemotactic responses. Galectin-9NT is one of the glycolipid-derived glycans, forsman pentasaccharide (Forss
It showed exceptionally high affinity for man pentasaccharide, whereas galectin-9CT did not. The Forssmann antigen is speculated to be involved in the pathogenesis of autoimmune diseases such as Graves' disease and Hashimoto's thyroiditis (Ariga, T. et.
al., Clin. Exp. Immunol., 86: 483-488 (1991)). The recognition of the Forssmann antigen by the N-terminal CRD of galectin-9 may also be involved in some unknown immunomodulatory activity of galectin-9.

By the way, a mouse galectin having 31 amino acid residues inserted in the linker peptide region
-9 isoform (small intestine isoform) has been reported to exist (Wada et al., J. Biol. Che
m., 272: 6078-6086 (1997)). As a result of analyzing a clone having a galectin-9 cDNA expressed in Jurkat cells, human galectin-9 has three isoforms,
That is, it was found that there are galectin-9S, -9M and -9L, but galectin-9M is an authentic galectin-9.
It is equivalent to (authentic galectin-9), galectin-9L is a type in which 32 amino acid residues are inserted in the linker peptide, and galectin-9S lacks 12 amino acid residues in the linker peptide part. It is the lost type. The expression of these isoforms in Jurkat cells confirms it at the protein level.

The linker peptide of galectin-9L is approximately four times longer than that of galectin-9S, but all three isoforms show similar eosinophil migration activity. Compared to galectin-9S, galectin-9L and -9M both have a linker peptide with an inserted proline-rich amino acid residue, that is, in galectin-9L, of the 44 amino acid residues, 13 are proline residues, galectin-9M
In, 5 out of 12 amino acid residues are proline residues. Glycine residue and proline residue are amino acid residues common to β-folding structure, and it is unlikely that a structure in which a linker peptide of galectin-9M or galectin-9L is extended can be formed. This is also supported by the Chou-Fasman prediction algorithm. Despite the large differences in the size of the linker peptides, there may be no significant difference in the three-dimensional configuration of N-terminal CRD and C-terminal CRD among these isoforms. It is undeniable that these three isoforms may play different roles with respect to previously unknown functions of galectin-9 other than eosinophil migration activity.

[0137]

INDUSTRIAL APPLICABILITY According to the present invention, it was found that binding of sugar chains to CRD of galectin-9 plays an important role in the expression of galectin-9 activity. Therefore, utilizing the CRD of galectin-9, by applying a frontal affinity chromatography technique and the like, means for identifying the sugar chain recognized by galectin-9 is provided, and information on the identified sugar chain is provided. By utilizing this, the biological activity of galectin-9 can be elucidated, and further compounds that promote and suppress the biological activity of galectin-9 can be screened. Further, the antibody against the identified sugar chain is galectin.
It can be expected to study the biological activity of -9 and also as a medicine. Thus, it is useful for the development of medicines and treatments for diseases related to galectin-9 such as cancer, immunity and allergy. Obviously, the present invention may be carried out other than as specifically described in the above description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and are therefore within the scope of the claims appended hereto.

[0138]

[Sequence list]                                SEQUENCE LISTING        <110> GALPHARMA Co., Ltd. <120> Galectin-9 modulators <130> P-01GL365 <140> <141> <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 1 cgtcctctcg agaatggcct tcagcggttc ccag 34 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 2 cgaccggaat tcctatgtct gcacatgggt cag 33 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 3 cgtcctggta ccatggcctt cagcggttcc cag 33 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 4 cgaccggaat tcctactgga agctgatgta ggacag 36 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 5 cgaccgggta ccctgtccag gggcgctctg cac 33 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 6 cgtccgggta cccctcccgg cgtgtggcct gcc 33 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 7 cgtcctctcg agaatgttct ctactcccgc catc 34 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 8 cgaccgggta cctgtctgca catgggtcag ctg 33

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the sites of a primer for amplifying galectin-9 cDNAs and the structure of a recombinant protein.

FIG. 2 shows the results of SDS-polyacrylamide gel electrophoresis analysis of the purified recombinant protein.

FIG. 3 shows the results of eosinophil migration activity assay for galectin-9M, -9NN and -9CC.

FIG. 4 shows the results of eosinophil migration activity assay for galectin-9S, -9M and -9L.

FIG. 5 shows galectin-9 against sugar chains listed in Tables 2-5.
The result of measurement of the binding affinity (1 / Kd) of N-terminal CRD (GST-G9NT) and C-terminal CRD (GST-G9CT) is shown.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 37/02 A61P 37/04 4C085 37/04 37/06 4C086 37/06 43/00 111 4H045 43/00 111 C07K 14 / 47 C07K 14/47 16/18 16/18 17/10 17/10 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 G01N 33/15 Z C12P 21/02 33/50 Z G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A (72) Inventor Atsushi Hirabayashi 1-43-10 112, Mukaihara, Shiroyama Town, Tsukui District, Kanagawa Prefecture Takanaka Nakamura 2284, Takamatsu-cho, Takamatsu-shi, Kagawa 1 Medical School A-304 (72) Inventor Nozomi Nishi 605 No. 605, Kabushi, Miki-cho, Kida-gun, Kagawa Prefecture (72) Mitsuomi Hirashima 505 Maeda-cho, Takamatsu-shi, Kagawa 2 Medical college accommodation B-404 F term (reference) 2G045 AA40 4B024 AA01 AA11 BA80 CA04 DA06 EA04 GA11 HA03 HA15 4B064 AG01 CA02 CA19 CC24 CE13 DA01 DA13 4B065 AA26X AA93Y AB01 AC14 BA0 2 CA24 CA44 CA46 4C084 AA17 ZB072 ZB082 ZB092 ZC022 4C085 AA13 AA14 BB16 DD63 DD88 GG01 GG08 4C086 AA01 AA02 EA20 MA01 MA04 NA14 ZB07 ZB08 FA80 FA80 FA60 FAA FAA FAA FAA FAA FAA FAA DAABA AA FAB AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA 7

Claims (13)

[Claims] 1. An N-terminal sugar chain recognition domain (CRD) of galectin-9.
Alternatively, a carrier characterized by immobilizing a C-terminal CRD. 2. A screening method comprising using the carrier according to claim 1 and screening a sugar chain recognized by galectin-9 or an analog thereof. 3. A galectin-9 activity inhibitor or regulator comprising a substance obtained by the screening method according to claim 2 and having a galectin-9 binding activity. 4. A screening method and a screening kit for a compound which uses the carrier according to claim 1 and promotes or inhibits the biological activity of galectin-9. 5. A medicine, which is obtained by the screening method according to claim 2 and contains a substance having a binding activity to galectin-9. 6. A mutant protein having (a) an N-terminal CRD of galectin-9 and not having a C-terminal CRD, and (b) an N-terminal having a C-terminal CRD of galectin-9. A polypeptide or a salt thereof, which is selected from the group consisting of mutant proteins having no CRD. 7. A nucleic acid having a base sequence encoding the polypeptide according to claim 6. 8. A vector containing the nucleic acid according to claim 7. 9. A host cell, which is transformed with the nucleic acid according to claim 7 or the vector according to claim 8. 10. The host cell according to claim 9 is cultured in a nutrient medium, the polypeptide according to claim 6 is expressed in the host cell, and the expressed polypeptide is recovered. Item 7. A method for producing the polypeptide according to Item 6. 11. An antibody against a sugar chain or an analog thereof, which is obtained by the screening method according to claim 2 and is recognized by galectin-9. 12. Galectin-9 containing a sugar chain or an analog thereof recognized by galectin-9.
An agent that promotes or inhibits the biological activity of. 13. An antibody containing an antibody against a sugar chain or an analog thereof recognized by galectin-9 or a mimetic thereof and having a biological activity equivalent to that of galectin-9, or having a part of the activity, or An agent which lacks a part of the activity or inhibits a part or all of the activity.