JP2002322082A - Prophylactic and therapeutic agent for nephritis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new prophylactic and therapeutic agent for nephritis, a prophylactic and therapeutic agent for glomerular diseases, an inhibitor for infiltration of leukocyte into the glomerulus, an inhibitor for infiltration of CD8 positive cell into the glomerulus and an apoptosis inducer of CD8 positive cell. SOLUTION: This prophylactic and therapeutic agent for nephritis comprises galectin-1, galectin-3 or galectin-9 derived from a mammal such as mouse, rat, human as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an agent for preventing or treating nephritis, an agent for preventing or treating glomerular diseases, an agent for inhibiting infiltration of leukocytes or CD8-positive cells into the glomerulus, and an agent for inducing apoptosis of CD8-positive cells.

[0002]

2. Description of the Related Art The kidney is an organ that plays an important role in removing waste products in the living body and controlling water content. Various types of nephritis caused by bacteria, viruses, chemical substances, and the like are known as diseases related to the kidney. Nephritis, for example, depending on the type of glomerular lesions, membranous nephritis, membranous proliferative nephritis,
Intraproliferative nephritis, mesangial proliferative nephritis, crescent-forming nephritis, IgA nephritis, purpura nephritis, lupus nephritis, etc., and, according to clinical characteristics, acute nephritis syndrome, rapidly progressive nephritis syndrome, It is classified into chronic nephritis syndrome, nephrotic syndrome and the like. Many of these nephritis show symptoms such as proteinuria and hematuria. At present, corticosteroids, immunosuppressants and the like are used for the treatment of nephritis.

[0003] Among glomerular diseases including glomerulonephritis, crescentic nephritis progresses rapidly and leads to renal failure unless strong immunosuppressive therapy is started (Yang RY et al., Bioche).
mistry 37: 4086-4092, 1998; Bolton WK, Semin. Neph.
rol. 16: 517-526, 1996). Lesions resembling human crescentic nephritis are identified by anti-glomerular basement membrane serum (anti-GBM antibody)
Is induced by a single dose of WKY to WKY rats.
As glomerular lesions, in addition to crescent formation, infiltration of CD8-positive cells and macrophages into the glomeruli is observed (Kawasaki K et al., Kidney Int. 41: 1517-1526, 199).
2). In addition, glomerular lesions are exacerbated by the production of antibodies to the administered heterologous IgG antibodies and the deposition of immunoglobulins and complement in the glomeruli. This crescent-forming nephritis is suppressed by administration of dexamethasone.

[0004] On the other hand, mammalian lectins are sugar chain-binding proteins and recognize specific sugar chain structures. Animal lectins are classified into four groups: C-type lectins, P-type lectins, pentraxins, and galectins. Sugar chains are important as components of many cell membrane proteins and extracellular matrix,
Interactions between lectins and their ligands are involved in various biological functions.

[0005] Galectin, a kind of animal lectin,
It specifically recognizes and binds specifically to β-galactoside. To date, at least ten galectins (galectins-1 to 10) have been identified and cloned (Perilo NL et al., J. Mol. Med. 76: 402-412, 1998; Wada).
J et al., J. Biol. Chem. 272: 6078-6086, 1997; Wada J et al.
J. Clin. Invest. 99: 2452-2461, 1997). These galectins have structural homology and common ligand recognition, but are involved in different physiological or pathological functions. The diversity of these functions is related to the diversity of sugar chains as ligands on cells and the distribution of sugar chains in various tissues. Among the various functions of galectins, there are many reports on their role in inducing apoptosis and immune responses. For example, galectin-1 ameliorates experimental autoimmune diseases (Levi G et al., Eur. J. Immu
nol. 13: 500-507, 1983; Offner H et al., Neuroimmnology.
28: 177-184, 1990), which induces apoptosis of activated human peripheral T cells and isolated human thymocytes (Pe
rillo NL et al., Nature 378: 736-739, 1995; PerilloNL
J. Exp. Med 185: 1851-1858, 1997). Galectin-9, which is strongly expressed in the thymus, also induces thymocyte apoptosis (Wada J et al., J. Clin. Invest. 99: 2452-2461,
1997). Conversely, galectin-3 acts to protect T cells and blocks apoptosis by forming a heterodimer with bcl-2 in the cytoplasm (Yang RY et al., Proc. N
atl.Acad.Sci.USA 93: 6737-6742, 1996).

[0006]

The present invention relates to a novel agent for preventing or treating nephritis, an agent for preventing or treating glomerular diseases, an agent for inhibiting infiltration of leukocytes into the glomerulus, and an agent for inhibiting infiltration of CD8-positive cells into the glomerulus. And an agent for inducing apoptosis of CD8-positive cells.

[0007]

In order to achieve the above object, the present invention provides a preventive / therapeutic agent for nephritis, a preventive / therapeutic agent for glomerular disease, and an inhibitor for leukocyte infiltration into the glomerulus provided by the present invention. (A) or (b) as an active ingredient. (A) a polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 3; (b) one or more amino acids are deleted, substituted or added in the amino acid sequence of any of SEQ ID NOs: 1 to 3 Having a specific amino acid sequence and a binding activity specific to β-galactoside

[0008] The agent for suppressing glomerular infiltration of CD8-positive cells and the agent for inducing apoptosis of CD8-positive cells provided by the present invention contain the following polypeptide (c) or (d) as an active ingredient. It is characterized by the following. (C) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3; (d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acids have been deleted, substituted or added, and Polypeptide having specific binding activity to galactoside

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The agent for preventing / treating nephritis, the agent for preventing / treating glomerular diseases and the agent for inhibiting infiltration of leukocytes into the glomerulus of the present invention comprise the polypeptide shown in the following (a) or (b): The agent for inhibiting infiltration of CD8-positive cells into the glomerulus and the agent for inducing apoptosis of CD8-positive cells of the present invention contain the polypeptide shown in the following (c) or (d) as an active ingredient.

(A) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 3 (hereinafter sometimes referred to as “polypeptide (a)”); A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the described amino acid sequence and having a specific binding activity to β-galactoside (hereinafter referred to as “polypeptide (b)”). (C) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (hereinafter sometimes referred to as “polypeptide (c)”); (d) one or more amino acids in the amino acid sequence of SEQ ID NO: 3 A polypeptide comprising an amino acid sequence in which amino acids have been deleted, substituted or added and having a specific binding activity to β-galactoside (hereinafter referred to as “polypeptide”). (D)).

The polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is mouse galectin-1, and the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 is
The polypeptide which is mouse-derived galectin-3 and has the amino acid sequence of SEQ ID NO: 3 is mouse-derived galectin-9.

Galectin is a general term for proteins and glycoproteins having specific binding activity to β-galactoside. Among them, galectin-1 exists as a homodimer (prototype) of a sugar chain binding domain (subunit of about 14 kD) composed of one polypeptide chain (see FIG. 1), and as another galectin belonging to this type, Examples include galectin-2, galectin-5, galectin-7, galectin-10 and the like. Galectin-3 exists as a chimeric type having one sugar chain binding domain at the C-terminus and an hnRNP-like domain at the N-terminus (see FIG. 1). Galectin-9 also exists as a tandem repeat type (tandem repeat type) having two sugar chain binding domains in the same peptide chain (see FIG. 1), and other galectins belonging to this type include galectin. -4, galectin-6, galectin-
8, galectin-9 and the like. The two sugar chain binding domains in tandem repeat type galectins such as galectin-9 are not the same,
It has 38% homology.

In each galectin, the amino acid sequence of the sugar chain binding domain is conserved over its entire length. The sugar chain binding domain is, for example, a portion consisting of the amino acid residues at positions 1 to 135 in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (that is, the entire amino acid sequence shown in SEQ ID NO: 1); In the polypeptide consisting of the amino acid sequence, a portion consisting of the 130th to 262nd amino acid residues, and in the polypeptide consisting of the amino acid sequence of SEQ ID NO: 3, 15th to 146th or 1st
It is thought to be a portion consisting of the 92th to 322nd amino acid residues. Particularly, in the amino acid sequence of SEQ ID NO: 1, 30
It is thought that a portion consisting of the amino acid residues at positions 90 to 90 is involved in the binding activity to β-galactoside, and this portion is well conserved in each galectin.

The number of amino acids deleted, substituted or added in the polypeptide (a) or (c) is not particularly limited as long as the polypeptide has a specific binding activity to β-galactoside. The number is one or more, preferably one or several, and the specific range is preferably 1 to 25, and more preferably 1 to 10. At this time, the amino acid sequence of polypeptide (b) is at least 25 times the amino acid sequence of polypeptide (a).
%, Preferably at least 50%, more preferably at least 80%, and the amino acid sequence of polypeptide (d) is at least 25% the amino acid sequence of polypeptide (c).
% Or more, preferably 50% or more, more preferably 80% or more.

In the polypeptide (a) or (c), the positions of amino acids to be deleted, substituted or added are as follows:
There is no particular limitation as long as the polypeptide has specific binding activity to β-galactoside. The position of the amino acid to be deleted, substituted or added in the polypeptide (a) or (c) may be, for example, a portion other than the sugar chain binding domain.

Here, "having a specific binding activity to β-galactoside" means that the compound has a binding activity to β-galactoside (for example, lactose (glucose-β-D-galactoside)), but has a binding activity other than β-galactoside. It means that it has no binding activity to the sugar chain structure. Examples of the sugar chain structure other than β-galactoside include glucose-α1,4-glucose and glucose-α1,2-fructose. In addition, "galactoside"
Is a general term for those in which the hemiacetal hydroxyl group of galactose is ether-bonded to another compound. Specific examples thereof include oligosaccharides such as lactose and melibiose, as well as glycosides and glycolipids containing galactose. Galactoside is classified into α-form and β-form according to the bonding coordination, and “β-galactoside” is of the β-form.

In the amino acid sequence of SEQ ID NO: 1, 1
Or a polypeptide comprising an amino acid sequence in which a plurality of amino acid sequences have been deleted, substituted or added, while having specific binding activity to β-galactoside, and having the same structure as galectin-1, that is, It is preferable that a homodimer of a sugar chain binding domain consisting of one polypeptide chain can be formed, and it is more preferable that it has the same biological function as galectin-1. The biological functions of galectin-1 include, for example, control of cell adhesion, cell proliferation, apoptosis of thymocytes and peripherally activated T cells, and the like.

In the amino acid sequence of SEQ ID NO: 2,
Or a polypeptide comprising an amino acid sequence in which a plurality of amino acid sequences have been deleted, substituted or added, has specific binding activity to β-galactoside and can have a structure similar to that of galectin-3, It preferably has one sugar chain binding domain at the C-terminus and an hnRNP-like domain at the N-terminus, and more preferably has the same biological function as galectin-3. The biological functions of galectin-3 include, for example, cell adhesion, inflammation (promoting IL-1 production in monocytes and production of active oxygen from neutrophils), mRNA splicing, human T-cell leukemia cell Apoptosis-suppressing action of the line (Jurkat E6-1).

In the amino acid sequence of SEQ ID NO: 3, 1
Or a polypeptide comprising an amino acid sequence in which a plurality of amino acid sequences have been deleted, substituted or added, has specific binding activity to β-galactoside, and can have a structure similar to that of galectin-9, that is, The same peptide chain preferably has two sugar chain binding domains, and more preferably has the same biological function as galectin-9. Examples of the biological function of galectin-9 include eosinophil chemotaxis, differentiation of thymocytes and induction of thymocyte apoptosis, and induction of apoptosis of peripherally activated T cells.

A polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2 or 3 wherein one or more amino acids have been deleted, substituted or added, and having a β-galactoside-specific binding activity In
In addition to polypeptides into which mutations such as deletion, substitution, or addition have been introduced into mouse-derived galectin-1, galectin-3, or galectin-9, they naturally exist in a deleted, substituted, or added state. Polypeptides, for example, galectin-1, galectin-3, or galectin-9 derived from mammals such as humans and rats, and polypeptides into which mutations such as deletion, substitution, and addition are artificially introduced into these galectins. Peptides are also included. Human-derived galectin-1,
The amino acid sequences of 3, 9 are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In addition, the amino acid sequences of rat-derived galectins-1, 3, and 9 are shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

In the present invention, “polypeptide” includes:
Both polypeptides to which a sugar chain is added and polypeptides to which no sugar chain is added are included. The type, position, and the like of the sugar chain added to the polypeptide vary depending on the type of host cell used in the production of the polypeptide. The polypeptide obtained by the above is also included. Also,
"Polypeptide" also includes pharmaceutically acceptable salts thereof.

The polypeptides (a), (b), (c) and (d) correspond to the DNs encoding the respective polypeptides.
It can be produced according to a conventional method using A. For example, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 can be produced using a DNA consisting of the base sequence shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

The DNA encoding the polypeptide (a) or (c) can be obtained, for example, according to the following (1) to (3).

(1) Preparation of cDNA Library Mouse-derived mRNA is prepared according to a conventional method. For example,
A mouse fetal kidney tissue or cell is treated with a guanidine reagent, a phenol reagent or the like to obtain total RNA, and then an affinity column method using oligo dT-cellulose or poly U-sepharose using Sepharose 2B as a carrier, Poly (A +) RNA (mRNA) by batch method
Get. Using the resulting mRNA as a template, a single-stranded cDNA is synthesized using an oligo dT primer and a reverse transcriptase, and then a double-stranded cDNA is synthesized from the single-stranded cDNA. The double-stranded cDNA thus obtained is inserted into an appropriate cloning vector to prepare a recombinant vector. By transforming a host cell such as Escherichia coli using the obtained recombinant vector, tetracycline resistance, by selecting a transformant with ampicillin resistance as an index,
A library of cDNAs is obtained.

Here, the cloning vector for preparing the cDNA library may be any one capable of autonomous replication in a host cell, such as a phage vector,
A plasmid vector or the like can be used. As the host cell, for example, Escherichia coli or the like can be used.

Transformation of host cells such as E. coli
n method (Hanahan, D., J. Mol. Biol. 166: 557-580 (1
983)), that is, a method of adding a recombinant vector to competent cells prepared in the presence of calcium chloride, magnesium chloride or rubidium chloride. When a plasmid is used as a vector, a drug resistance gene such as tetracycline or ampicillin is contained.

The preparation of the cDNA library is performed by using a commercially available kit, for example, SuperScript Plasmid System for cDNA.
Synthesis and Plasmid Cloning (Gibco BRL), ZA
It can be performed using a P-cDNA Synthesis Kit (Stratagene) or the like.

(2) Screening of Clones Containing the Target DNA Primers are synthesized based on the known amino acid sequence of galectin, and the primers are used for polymerase chain reaction (PC).
R) to obtain a PCR amplified fragment. The PCR amplified fragment may be subcloned using an appropriate plasmid vector.

As a primer used for PCR, for a DNA consisting of the nucleotide sequence of SEQ ID NO: 10,
For example, 5′-gcttcaatcatggcctgt-3 ′ (sense primer), 5′-ggctggcttcactcaaaggc-3 ′ (antisense primer), DNA consisting of the nucleotide sequence of SEQ ID NO: 11
For example, 5′-gcacagagagcatacccagg-3 ′ (sense primer), 5′-cttctggcttagatcatg-3 ′ (antisense primer), and DNA consisting of the nucleotide sequence of SEQ ID NO: 12, 5 '-gcattggttcccctgag
atag-3 '(sense primer), 5'-cgttccagagaccggatc
c-3 '(antisense primer) can be used.

The target DNA can be obtained by performing colony hybridization or plaque hybridization on the cDNA library using the PCR amplified fragment as a probe. As a probe, a PCR amplified fragment is an isotope (for example, ³² P,
Those labeled with ³⁵ S), biotin, digoxigenin or the like can be used.

A clone containing the target DNA can also be obtained by expression screening such as immunoscreening using an antibody.

(3) Determination of Nucleotide Sequence The nucleotide sequence of the obtained DNA is obtained by digesting the DNA fragment as it is or after cutting it with an appropriate restriction enzyme or the like, incorporating the DNA fragment into a vector by a conventional method, and using a commonly used nucleotide sequence analysis method. For example, using the Maxam-Gilbert chemical modification method, dideoxynucleotide chain termination method,
Usually, a 373A DNA sequencer (PerkinElmer)
The sequence is determined using a base sequence analyzer such as

As described above, the DNA encoding the polypeptide (a) (specifically, the DNA comprising the nucleotide sequence represented by SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12)
NA) and DNA encoding polypeptide (c)
(Specifically, a DNA comprising the base sequence of SEQ ID NO: 12) can be obtained.

DNA encoding polypeptide (a)
Is not limited to DNA consisting of the nucleotide sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and includes DNA consisting of other nucleotide sequences as long as it encodes the polypeptide (a). Polypeptide (c)
The same applies to the DNA encoding Such DNA can be obtained by chemical synthesis according to its base sequence. For chemical synthesis of DNA, a commercially available DNA synthesizer, for example, a DNA synthesizer using the thiophosphite method (manufactured by Shimadzu Corporation) and a DNA synthesizer using the phosphoramidite method (manufactured by Perkin Elmer) are used. You can do it.

The DNA encoding the polypeptide (b) or (d) may be, for example, a DNA encoding the polypeptide (a) or (c) from a cDNA library derived from a mammal such as mouse, rat or human. And DNA that hybridizes under stringent conditions.

The term "DNA that hybridizes with the DNA encoding the polypeptide (a) or (c) under stringent conditions" refers to a colony using the DNA encoding the polypeptide (a) or (c) as a probe.・
A hybridization method, a plaque hybridization method, a DNA obtained by a Southern hybridization method or the like.For example, after performing hybridization using a filter in which DNA derived from colonies or plaques is immobilized, 2 × SSC, 0.1% SDS, 4
DN obtained by washing the filter under the conditions of 2 ° C., preferably 1 × SSC, 0.1% SDS, 42 ° C., more preferably 0.1 × SSC, 0.1% SDS, 42 ° C.
A is included.

Specific examples of the DNA which hybridizes with the DNA encoding the polypeptide (a) or (c) under stringent conditions include a DNA encoding the polypeptide (a) or (c) and at least a DNA encoding the polypeptide (a) or (c), respectively.
DNA having homology of at least 60%, preferably DNA having homology of at least 60%, more preferably DNA having homology of at least 80%.

The DNA encoding the polypeptide (b) or (d) may be artificially introduced into the DNA encoding the polypeptide (a) or (c) by, for example, site-directed mutagenesis. Can be obtained. Mutagenesis can be introduced using, for example, a mutagenesis kit, for example, Mutant-K (manufactured by TAKARA), Mutant-G (manufactured by TAKARA), or TAKARA's LA PCR in vitro Mutagenesis series kit. . In addition, DNA whose base sequence has already been determined can also be obtained by chemical synthesis as described above.

The polypeptides (a), (b), (c) and (d) can be prepared, for example, according to the following (1) to (3)
It can be produced by expressing DNAs encoding the respective polypeptides in host cells.

(1) Preparation of Recombinant Vector and Transformant A DNA fragment of an appropriate length containing the coding region of the desired polypeptide is prepared. In addition, a DNA is prepared in which bases have been substituted so that the base sequence of the coding region of the polypeptide of interest becomes an optimal codon for expression in a host cell.

A recombinant vector is prepared by inserting the above DNA fragment downstream of the promoter of an appropriate expression vector, and the recombinant polypeptide is introduced into an appropriate host cell to produce a polypeptide of interest. The resulting transformant can be obtained.

As host cells, prokaryotic cells, yeast, animal cells, insect cells,
Any of plant cells and the like may be used. Moreover, an animal individual or a plant individual can also be used.

The expression vector is not particularly limited as long as it is capable of autonomous replication in a host cell. For example, a plasmid vector, a phage vector and the like can be used. Specific examples of the expression vector include pBTrp2, pBTac
1, pBTac2 (Boehringer Mannheim), pKK233-2
(Pharmacia), pSE280 (Invitrogen), pG
EMEX-1 (Promega), pQE-8 (QIAGEN), pBlues
cript II SK + (Stratagene), pBluescript II
SK (-) (Stratagene), pGEX (Pharmacia), pET system (Novagen), pSupex, pUB11
0, pTP5, pC194, pTrxFus (Invitrogen), pMAL-c2
(Manufactured by New England Biolabs), pSTV28 (manufactured by Takara Shuzo), pUC118 (manufactured by Takara Shuzo) and the like.

When a bacterium is used as a host cell, for example, the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, or Pseudomonas putida is used.
putida) and bacteria belonging to the genus Rhizobium such as Rhizobium meliloti can be used as host cells. Specifically, Escheric
hia coli XL1-Blue, Escherichia coli XL2-Blue, Esch
erichia coli DH1, Escherichia coli K12, Escherichi
E. coli such as a coli JM109, Escherichia coli HB101,
Bacillus subtilis MI 114, Bacillus subtilis 207-21
And the like can be used as host cells. The promoter of the case is not particularly limited as long as it can be expressed in bacteria such as Escherichia coli, for example, using a promoter derived from the trp promoter, lac promoter, P _L promoter, P _R promoter of Escherichia coli or phage, etc. it can. In addition, tac promoter, lacT7 promoter, let
It is also possible to use artificially designed and modified promoters such as the I promoter.

The method for introducing the recombinant vector into bacteria is not particularly limited as long as it is a method capable of introducing DNA into bacteria. For example, a method using calcium ions (Cohen,
SN et al., Proc. Natl. Acad. Sci., USA, 69: 211.
0-2114 (1972)), electroporation and the like.

When yeast is used as a host cell, Saccharomyces cerevisiae, Schizosaccharomyces pomb
e), Pichia pastoris and the like can be used as host cells. The promoter in this case is not particularly limited as long as it can be expressed in yeast, for example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, P
HO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like can be used.

The method for introducing a recombinant vector into yeast is not particularly limited as long as it can introduce DNA into yeast.
For example, electroporation (Becker, DM et
al., Methods. Enzymol., 194: 182-187 (1990)), spheroplast method (Hinnen, A. et al., Proc. Natl. Aca.
d. Sci., USA, 75: 1929-1933 (1978)), lithium acetate method (Itoh, H., J. Bacteriol., 153: 163-168 (198
3)) etc. can be used.

When animal cells are used as host cells, monkey cells
COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells and the like can be used as host cells. The promoter in this case is not particularly limited as long as it can be expressed in animal cells, and examples thereof include SRα promoter, SV40 promoter, LTR (Long T
erminal Repeat) promoter, CMV promoter, human cytomegalovirus early gene promoter, and the like.

The method for introducing the recombinant vector into animal cells is not particularly limited as long as it can introduce DNA into animal cells, and examples thereof include an electroporation method, a calcium phosphate method, and a lipofection method.

When an insect cell is used as a host, Spodopte
Ra frugiperda ovary cells, Trichoplusia ni ovary cells, silkworm ovary-derived cultured cells, and the like can be used as host cells. Sf is the ovarian cell of Spodoptera frugiperda
9, High for ovarian cells of Trichoplusia ni such as Sf21
5, Bombyx mori N4 and the like are examples of cultured cells derived from silkworm ovaries such as BTI-TN-5B1-4 (manufactured by Invitrogen).

The method for introducing the recombinant vector into the insect cells is not particularly limited as long as the DNA can be introduced into the insect cells. For example, a calcium phosphate method, a lipofection method, an electroporation method and the like can be used.

(2) Culturing of a transformant A transformant into which a recombinant vector incorporating a DNA encoding a polypeptide of interest has been introduced is cultured according to a conventional culture method. Culture of the transformant can be performed according to a usual method used for culture of host cells.

A culture medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host cell contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the microorganism, and cultivates the transformant. Any of a natural medium and a synthetic medium may be used as long as the medium can efficiently carry out the above.

As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used. As a nitrogen source, ammonia,
Ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate and the like can be used.
As the inorganic salt, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.

The transformant is cultured under aerobic conditions such as shaking culture or aeration and stirring culture. Culture temperature is usually 37
At 培養 39 ° C., the culture time is usually 8 to 12 hours, and the pH is maintained at 7.2 to 7.4 during the culture period. Adjustment of the pH can be achieved with inorganic acids,
The reaction can be performed using an organic acid, an alkali solution, urea, calcium carbonate, ammonia or the like. In addition, at the time of culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as needed.

When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used.When culturing a microorganism transformed with an expression vector using the trp promoter, indole acryl is used. An acid or the like may be added to the medium.

As a medium for culturing a transformant obtained by using an animal cell as a host cell, generally used RP
MI1640 medium, Eagle's MEM medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums can be used. Culture of the transformant is usually performed at 37 to 42 ° C in the presence of 5% CO ₂ for 2 hours.
Perform for ~ 4 days. In addition, at the time of culture, an antibiotic such as kanamycin, penicillin, streptomycin, etc. may be added to the medium as needed.

As a medium for culturing a transformant obtained by using an insect cell as a host cell, generally used TN is used.
Transformants that can use M-FH medium (Pharmingen), Sf-900 II SFM medium (Gibco BRL), ExCell400, ExCell405 (JRH Biosciences), etc., are usually cultured at 27 ° C. For 12 to 24 hours. When culturing,
If necessary, an antibiotic such as gentamicin may be added to the medium.

The polypeptide of interest can be expressed as a secreted protein or a fusion protein. Examples of the protein to be fused include β-galactosidase, protein A, an IgG binding region of protein A, chloramphenicol acetyltransferase, poly (Arg), poly (Glu), protein G, maltose binding protein, and glutathione S-protein. Transferase, polyhistidine chain (His-tag), S peptide, DNA binding protein domain, Tac antigen,
Thioredoxin, green fluorescent protein, and the like.

(3) Isolation and Purification of Polypeptide The desired polypeptide is obtained by collecting the desired polypeptide from the culture of the transformant.
Here, the “culture” includes any of culture supernatants, cultured cells, cultured cells, and crushed cells or cells.

When the polypeptide of interest is accumulated in the cells of the transformant, the culture is centrifuged to collect the cells in the culture, and after washing the cells, the cells are disrupted. To extract the desired polypeptide.

When the target polypeptide is secreted out of the cells of the transformant, the culture supernatant is used as it is, or cells or cells are removed from the culture supernatant by centrifugation or the like.

The polypeptide (a) thus obtained,
(B), (c) or (d) is a solvent extraction method, a salting out method using ammonium sulfate or the like, a desalting method using an organic solvent, a diethylaminoethyl (DEAE) -Sepharose, an ion exchange chromatography method, a hydrophobic chromatography. It can be purified by a chromatography method, a gel filtration method, an affinity chromatography method, or the like.

The polypeptides (a), (b),
(C) or (d) is based on the amino acid sequence,
Fmoc method (fluorenylmethyloxycarbonyl method), tB
It can also be produced by a chemical synthesis method such as the oc method (t-butyloxycarbonyl method). In this case, a commercially available peptide synthesizer can be used.

The polypeptide (a) or (b) can be administered alone as a prophylactic / therapeutic agent for nephritis, a prophylactic / therapeutic agent for glomerular disease, or an inhibitor of leukocyte infiltration into the glomerulus. Usually, it is formulated and used according to a conventional method together with one or more pharmaceutically acceptable carriers and / or additives. Further, the polypeptide (c) or (d)
Similarly, it can be administered alone as an agent for inhibiting infiltration of CD8-positive cells into the glomerulus or an agent for inducing apoptosis of CD8-positive cells, but usually, one or more carriers that are pharmaceutically acceptable and / or Formulated and used together with additives according to a conventional method. When formulated, the amount of the active ingredient in the formulation is usually 0.1 to 5% by weight, preferably 1 to 5% by weight.
It is about 5% by weight.

Pharmaceutically acceptable carriers include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran,
Examples include sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose and the like.

The additives used in the preparation include, for example, fillers, extenders, binders, humectants, disintegrants, surfactants, lubricants, excipients, stabilizers, bactericides. ,
Examples include a buffer, an isotonic agent, a chelating agent, a pH adjuster, and a surfactant. These additives are appropriately selected according to the dosage unit form of the preparation and the like. Among these, components used in ordinary protein preparations and the like, for example, stabilizers, bactericides,
Buffers, isotonic agents, chelating agents, pH adjusters, surfactants and the like are preferably selected.

Specific examples of the additives are shown below. Stabilizer: human serum albumin; L-amino acids such as glycine, cysteine and glutamic acid; monosaccharides such as glucose, mannose, galactose and fructose; sugar alcohols such as mannitol, inositol, xylitol, sucrose, maltose and lactose; Sugars such as saccharides, polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, and hyaluronic acid and derivatives thereof; cellulose derivatives such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and sodium carboxymethyl cellulose;

Surfactants: polyoxyethylene glycol sorbitan alkyl ester, polyoxyethylene alkyl ether type, sorbitan monoacyl ester type,
Surfactants such as fatty acid glycerides.

Buffers: boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and salts thereof (for example, alkali metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, etc., and alkaline earth salts) Metal salts).

Isotonizing agents: sodium chloride, potassium chloride, sugars, glycerin and the like.

Chelating agents: sodium edetate, citric acid and the like.

The agent for preventing or treating nephritis, the agent for preventing or treating glomerular diseases or the agent for inhibiting infiltration of leukocytes into the glomerulus according to the present invention include:
Either polypeptide (a) or (b) may be contained as an active ingredient, or both may be contained as an active ingredient. Similarly, the agent for suppressing infiltration of CD8-positive cells into the glomerulus or the agent for inducing apoptosis of CD8-positive cells of the present invention is also the polypeptide (c) or (d).
May be contained as an active ingredient, or both may be contained as an active ingredient.

The polypeptide (a), (b), (c) or (d) may be a pharmaceutically acceptable salt thereof, and specific examples thereof include sodium, potassium, lithium, calcium, Non-toxic alkali metal salts such as magnesium, barium and ammonium, alkaline earth metal salts,
Ammonium salts and the like. The salts also include non-toxic acid addition salts resulting from the reaction of a polypeptide with a suitable organic or inorganic acid. Representative non-toxic acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide,
Sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate,
Phosphate, p-toluenesulfonate (tosylate),
Citrate, maleate, fumarate, succinate,
Tartrate, sulfonate, glycolate, maleate, ascorbate, benzenesulfonate and the like.

It is desirable to use the most effective route for administration and the dosage form in the treatment. Administration routes include, for example, oral administration, oral cavity, respiratory tract, rectum,
Parenteral administration such as subcutaneous, intramuscular and intravenous,
Dosage forms include, for example, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments,
Tape agents and the like are included.

Formulations suitable for oral administration include, for example,
Examples include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid. It can be produced using preservatives such as esters and flavors such as strawberry flavor and peppermint as additives. Capsules, tablets, powders, granules and the like are lactose, glucose, sucrose, excipients such as mannitol, starch, disintegrants such as sodium alginate,
It can be produced using lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin as additives.

Formulations suitable for parenteral administration include, for example, injections, suppositories, sprays and the like. For example, an injection can be produced using a salt solution, a glucose solution or a mixture thereof as a carrier. Suppositories can be prepared using cocoa butter, hydrogenated fat, carboxylic acid or the like as a carrier. Sprays can be produced using lactose, glycerin, etc. as a carrier that does not irritate the oral and respiratory tract mucosa of the recipient and facilitates absorption by dispersing the active ingredient as fine particles, aerosol, dry powder. Etc. can be formulated.

The dosage and the number of administrations vary depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but the dosage can be appropriately selected usually from 0.01 to 1 mg / kg per day for an adult. The frequency of administration can be appropriately selected from the range of once to several times a day.

The agent for preventing or treating nephritis of the present invention can prevent and / or treat nephritis. The type of nephritis to be prevented or treated is not particularly limited, but includes symptoms such as proteinuria, glomerular hypertrophy, excessive cells in the glomerulus, crescent formation, infiltration of leukocytes into the glomerulus, and CD8 positivity. It can be suitably used for nephritis associated with glomerular lesions such as infiltration of cells into the glomerulus and deposition of fibrinogen in the glomerulus. Such nephritis includes glomerulonephritis, for example, crescentic nephritis, lupus nephritis, IgA nephropathy, membranous proliferative glomerulonephritis and the like.

The agent for preventing or treating glomerular diseases of the present invention can prevent and / or treat glomerular diseases. The types of glomerular diseases to be prevented or treated are not particularly limited, but include glomerular hypertrophy, hypertrophy of glomeruli, crescent formation, infiltration of leukocytes into glomeruli, glomeruli of CD8-positive cells It can be suitably used for glomerular diseases showing lesions such as infiltration in the body and deposition of fibrinogen in the glomerulus. Such glomerular diseases include glomerulonephritis, for example, crescentic nephritis, lupus nephritis, IgA nephropathy, and membranous proliferative glomerulonephritis.

The leukocyte infiltration inhibitor in the glomerulus of the present invention is
It can suppress or inhibit infiltration of leukocytes into the glomerulus. Examples of leukocytes to be targeted for suppression of glomerular infiltration, for example,
Monocytes, macrophages, T cells, natural killer cells and the like can be mentioned, but they can be suitably used particularly for suppressing infiltration of macrophages into the glomerulus. By suppressing or inhibiting leukocyte infiltration into the glomerulus, diseases caused by leukocyte infiltration into the glomerulus, for example, glomerular diseases such as glomerulonephritis (for example, crescentic nephritis) and / or can be prevented. Or can be treated.

The agent for inhibiting infiltration of CD8-positive cells into glomeruli of the present invention can suppress or inhibit the infiltration of CD8-positive cells into glomeruli. The type of CD8-positive cells targeted for suppression of glomerular infiltration is not particularly limited, and examples include CD8-positive cytotoxic T cells. CD
Diseases caused by glomerular infiltration of CD8-positive cells by suppressing or inhibiting the infiltration of 8-positive cells into the glomerulus, such as glomerulonephritis (eg, crescentic nephritis)
And / or other glomerular diseases can be prevented and / or treated.

The agent for inducing apoptosis of CD8-positive cells of the present invention can selectively induce apoptosis in CD8-positive cells without inducing apoptosis in CD4-positive cells. The type of CD8-positive cells for which apoptosis is to be induced is not particularly limited.
Positive cytotoxic T cells and the like. By inducing apoptosis of CD8-positive cells, diseases caused by CD8-positive cells, for example, glomerular diseases such as glomerulonephritis (for example, crescentic nephritis) can be prevented and / or treated.

[0084]

[Examples] 1. METHODS: (1) rabbit anti-glomerular basement membrane serum (nephrotoxic seru
m: NTS) The glomerular basement membrane (GBMs) was isolated from a 4-week-old female WKY rat (body weight: about 100 g) and used as an antigen of an anti-glomerular basement membrane antibody. The WKY rats used were Char
les River Japan (Atsugi, Kanagawa, Japan).

After perfusing the kidneys of WKY rats with saline, they were homogenized and the glomerular basement membrane was isolated. Homogenate and Freund's complete adjuvant (Difco La
boratories, Detroit, MI, USA) to prepare an emulsion.
Rabbits were administered by subcutaneous injection for one month. Three weeks after the last administration, blood was collected from rabbits to separate antisera. The antiserum was immobilized at 56 ° C. for 30 minutes and used to induce nephritis in WKY rats after adsorption with fresh erythrocytes.

(2) Mouse galectin-1, -3, -9
Preparation of Recombinant Protein Recombinant galectin-1, -3, -9 (hereinafter G1, G
3, G9. ) Is a known method (Wada J. et al., J. Bi).
ol. Chem. 272: 6078-6086, 1997; Wada J. et al., J. Clin.I.
nvest 99: 2452-2461, 1997) using the pTrcHis2 vector (Invitrogen, San Diego, CA, USA). These recombinant proteins are produced as fusion proteins having a c-myc epitope and (His) ₆ at the C-terminus.

Vectors containing the G1 gene (SEQ ID NO: 10), G3 gene (SEQ ID NO: 11), and G9 gene (SEQ ID NO: 12) (hereinafter referred to as “pTrcHis2 / G1” and “pTrcHis
2 / G3 "and" pTrcHis2 / G9 ". ) Was transformed into a TOP10 bacterial host (Invitrogen). Transformed bacterial colonies are
Culture in ria-Bertani's medium and add 1 mmol / L isopropyl-
Protein synthesis was induced by adding β-D-tiogalactopyranoside (IPTG). 1% TritonX-100, 10mm
ol / L benzamidine, 10 mmol / L ε-amino-n-caproic aci
d and Tris-dithiothreitol (Tris-DTT) buffer containing 20 mmol / L phenylmethanesulfonyl fluoride (20 mmol / L
L Tris (pH 7.4), 5mmol / L ethylenediaminetetraace
tic acid (EDTA), 150mmol / L sodium chroride, 1mmo
1 / L DTT) to lyse the bacteria. The lysate was centrifuged at 4 ° C. for 30 minutes at 20,000 × g. Centrifuge supernatant in 10 mL of lactosyl-S
It was added to an epharose column (Sigma, ST. Louis, MO, USA). After washing the unbound protein, the fusion protein
Elution was performed with Tris-DTT buffer containing 200 mmol / L Lactose. The eluted fraction was dialyzed against phosphate buffer (PBS) containing 1 mmol / L DTT and stored at -70 ° C. 12.5 samples
% SDS-PAGE (sodium dodecyl sulfate-polyacrylamid
When analyzed by e-gel electrophoresis), a single band was obtained with any of the galectins, and the molecular weights were consistent. That is, the molecular weights of G1 are 17 kDa, G3 is 37 kDa,
9 was 39 kDa, which was consistent with the molecular weight predicted from the number of constituent amino acids.

(3) Induction of NTS Nephritis Anti-glomerular basement membrane serum was intraperitoneally administered (2 mL / kg body weight) to 40 WKY rats on the first day to induce NTS nephritis, followed by DTT (1 mmol / kg). L, n = 8), dexamethasone (DE
X: 0.17 mg / kg body weight, n = 8), G1 (1 mg / kg body weight, n =
8) PBS containing G3 (1 mg / kg body weight, n = 8) and G9 (1 mg / kg body weight, n = 8) were administered every other day for 2 weeks. Urine was collected and measured for 24-hour urine protein excretion.

WKY rats were sacrificed on the 14th day, and their kidney tissues were used for observation by light microscopy, fluorescent antibody method, and in situ TUNEL method for examining apoptosis (i
n situterminal deoxytransferase (TdT) -mediated urid
ine triphosphate (dUTP) nickend labeling reaction)
Was performed.

As another experiment, 20 NTS nephritis (n = 20) and 20 normal kidneys (n = 20) were prepared, and 1 mmol / L DTT, DEX, G1, G3 or G9 was added thereto.
Was administered. The ten groups (n = 4 each) were sacrificed on the eighth day, and CD4 positive cells and CD8 positive cells were detected by enzyme-linked immunosorbent assay (immunoperoxidase staining) using each tissue, and FACS (fluorescence).
-activated cell sorter) analysis was performed.

The CD8-positive cells in the glomerulus were
The animals were sacrificed on day 8 as they emerged from day 1 and then gradually declined until day 11.

(4) Observation by Optical Microscope The tissue was fixed with 10% formaldehyde and embedded in paraffin to prepare a 3 μm-thick section. Section is PA
The cells were stained with S (periodic acid-Schiff). The diameter of 50 glomeruli was measured in each animal, and the total number of glomerular cells,
The number of glomeruli with crescents was counted.

(5) Fluorescent antibody method (observation by immunofluorescence microscopy) Goat anti-rabbit IgG, goat anti-rat IgG, goat anti-rat C3 antibody, goat anti-rat fibrinogen antibody (Capp) in which a 4 μm thick cryostat section was labeled with FITC.
el, Costa Mesa, CA, USA). The fluorescence intensity was evaluated by the following index.

(-) = Negative; (+) = weak positive; (++)
= Moderately positive; (++) = positive; (+++) = strong positive

Further, a continuous cryostat section having a thickness of 2 μm was prepared. For staining for the proliferation marker Ki-67, the sections were fixed in acetone on ice and boiled for another 10 minutes. These sections were stained with a mouse monoclonal antibody against Ki-67 (Dako, Glostrup, Denmark) for 2 hours. Serial sections were further purified with a mouse monoclonal antibody against rat monocytes / macrophages (ED1; Serotec, Oxf).
ord, UK), and α-smooth muscle actin (α-SMA; S
igma) for 1 hour. Finally, anti-mouse IgG labeled with FITC (Cappel)
For 30 minutes. Ki-67, E per glomerulus
The number of D1, α-SMA positive cells was counted. Rat 1
Twenty glomeruli were observed per animal, and the average number of positive cells was determined.

(6) Enzyme antibody method (immune peroxidase staining) Infiltration of leukocytes into glomeruli was examined by the enzyme antibody method. Enzyme antibody method is an immunoperoxidase ABC kit
(Vector laboratories, Burlingame, CA, USA).

First, cryostat sections were reacted with a mouse monoclonal antibody against rat leukocytes (OX1), a mouse monoclonal antibody against rat monocytes / macrophages (ED1), and a mouse monoclonal antibody against rat CD4 or CD8 (Serotec) for 60 minutes. . Next, a horse anti-mouse IgG labeled with bistin was added.
After reacting at 22 ° C. for 30 minutes, it was reacted with 3,3-diamino-benzidine and hydrogen peroxide. By counting the number of cells with brown reaction products, OX in glomeruli
1. The number of ED1, CD4 and CD8 positive cells was counted. Twenty glomeruli were observed per rat, and the average number of positive cells was determined.

(7) In situ TUNEL in kidney tissue
Method Apoptotic cells in kidney tissue were analyzed using FITC-dUPT.
situ TUNEL method (In Situ Cell Death Detection kit; Bo
ehringer Mannheim, Mannheim, Germany).

Prepare a 4 μm thick cryostat section,
The cells were fixed with 4% paraformaldehyde at 22 ° C. for 20 minutes. 0.1% Triton X-100, 0.1% sodi on ice for 2 minutes
Sections were processed with um citrate. The TUNEL reaction mixture was further treated in the dark for 60 minutes under humidified conditions. Each W
Fifty glomeruli were examined for KY rats, and the number of apoptotic cells per glomerulus was counted.

(8) Measurement of anti-rabbit IgG antibody by ELISA The antibody titer of anti-rabbit IgG antibody in rat serum was determined by ELISA.
It examined by A (enzyme-linked immunosorbent assay).

Normal rabbit IgG was added to each of the 96 wells.
To 50 μL (10 mg / ml; Chemicon, Temecula, USA)
Incubated overnight at 2 ° C. The next day, the serum collected on the 14th day was diluted 40-fold, added to each well by 50 μL, and reacted for 2 hours. Thereafter, rabbit anti-rat IgG labeled with alkaline phosphatase (Southern Bio
technology Associates, Birmingham, AL, USA) 2000
The solution was diluted two-fold, added to each well, and reacted for 2 hours. Finally, a p-nitrophenyl phosphate solution (Sigma) was added to each well and reacted for 1 hour. The absorbance at 405 nm was measured using a microplate reader (Japan Bio-Rad, Tokyo,
Japan).

(9) FACS analysis Mononuclear cells isolated from spleen were stained with CD4 and CD8, and TUNEL
It was subjected to the reaction. First, homogenize the spleen and RPMI-1640
(Gibco BRL, Grand Island, NY, USA). Then, Ficoll-Paque (Pharma
cia Biotech, Uppsala, Sweden).
Centrifuged for 0 minutes. Mononuclear cells were collected, washed twice with PBS at 4 ° C., and adjusted to 2 × 10 ⁷ cells / mL. Anti-rat CD4 labeled cells with phycoerythrin
Antibody (Serotec) or anti-rat CD8 antibody labeled with phycoerythrin (Antigenix America, Franklin
Square, NY, USA) and fixed with 100 μL of PBS containing 4% paraformaldehyde. After centrifugation, remove the fixative and remove cells with 0.1% Triton X-100 and 0.1% sodi.
Treated on ice for 2 minutes using um citrate. PB cells
After washing twice with S, 50 μL of the TUNEL reaction mixture (fluore
(including scein-dUTP and TdT) (In Situ C
ell Death Detection Kit). After the reaction, the mixture was reacted at 37 ° C. for 60 minutes in a humidified container in a dark place, washed twice with PBS, and then subjected to two-color flow cytometry (FACS Caribur 3).
A; Becton Dickinson, Mountain View, CA, USA) was used to analyze the percentage of T cell apoptosis.

(10) Administered exogenous galectin-9
Kidney distribution Galectin-9 (1 mg / kg body weight) was administered to normal WKY rats and sacrificed 4 hours after administration. A cryostat section of the kidney was prepared and reacted with an anti-myc antibody (Invitrogen). Furthermore, it was stained with a horse anti-mouse IgG antibody (Vector Laboratories) labeled with rhodamine. In addition, rabbit anti-galectin-9 was used for double staining.
Antibodies (Wada J. et al., J. Biol. Chem. 272: 6078-6086, 199
7; Wada J. et al., J. Clin. Invest 99: 2452-2461, 1997),
Subsequently, mule anti-rabbit IgG labeled with FITC (Chemicon)
Stained. The secondary antibody is already adsorbed by serum proteins from other animals and has no cross-reactivity to other species of IgG. Only administered exogenous fusion protein at the C-terminus
Since it has myc and there is no myc epitope in endogenous galectin-9, the two can be distinguished by an anti-myc antibody.

(11) Statistical processing Data were shown as mean ± standard deviation. P <0.05 was considered significant using the ANOVA test. Fisher analysis was also performed.

[0105] 2. Results (1) Quantitative results of urinary protein excretion FIG. 2 shows the quantification results of urinary protein excretion in NTS nephritis rats. In FIG. 2, (■) of A, B, C, and D indicates NTS
The figure shows the amount of urinary protein excreted when DTT-containing PBS was administered to nephritis rats, and (●) in A, B, C and D indicates NT
Fig. 4 shows urinary protein excretion when PBS containing G1, G3, G9, and DEX was administered to rats with S nephritis.

The control rats to which normal rabbit serum was administered had 24
The amount of urinary protein excreted per hour was as small as 1.9 ± 0.4 mg / day.

On the other hand, in NTS nephritis rats,
As shown in A to D, the amount of proteinuria was small in the first week, but increased gradually in the second week.
On the day, it reached 35.8 ± 7.1 mg / day.

On the other hand, dexamethasone, G1, G
3. In NTS nephritis rats administered G9, FIG.
As shown in A to D, urine protein output per day was significantly suppressed. That is, N administered with dexamethasone
In the TS nephritis group, urine protein excretion was significantly reduced,
The urinary protein excretion on day 14 was 2.3 ± 0.4 mg / day. Similarly, in the group of NTS nephritis rats to which G1, G3, and G9 were administered, urinary protein excretion was significantly reduced, and the urinary protein excretion on day 14 was 5.6 ± 1.8, 11.6 ± 2.3, and 9.5 ± 4.5 mg, respectively. / da
was y.

From the above results, G1, G3 and G9
Has been shown to have an effect of suppressing proteinuria caused by nephritis.

(2) Findings by Light Microscope and Immunofluorescence Microscopy Table 1 shows the results of examination of glomerular hypertrophy and cell hyperplasia in NTS nephritis rats. In addition, Table 2 shows the test results regarding the deposition of rat IgG, C3, and fibrinogen on glomeruli of NTS nephritis rats. FIG. 3 shows the results of examination of a kidney specimen of an NTS nephritis rat by an optical microscope and an immunofluorescence microscope.

TABLE 1 Glomerular diameter Total number of glomerular cells Cellular crescent (μm) (cells / glomerular) (%) PBS 206.3 ± 40.2 167 ± 32 56 ± 10 G1 143.8 ± 32.1 61 ± 24 5 ± 2 G3 150.0 ± 34.8 72 ± 18 8 ± 3 G9 146.6 ± 36.2 66 ± 23 5 ± 2 DEX 131.3 ± 20.4 52 ± 15 3 ± 8

[Table 2] IgG C3 fibrinogen PBS (++++) (++) (++++) G1 (++) (++) (-) G3 (++) (++) (-) G9 (++) (++) (-) DEX (-) (-) (-)

In Tables 1 and 2 and FIG. 3, "PB
"S" represents the results obtained when PBS containing DTT was administered to rats with NTS nephritis, as "G1,""G3,""G9,"
"DEX" is G1 and G in rats with NTS nephritis, respectively.
3 shows the results when PBS containing G9 and DEX was administered (the same applies to the other tables and figures below). Also,
In Table 2, (-) indicates negative, (+) indicates weak positive, (++)
Indicates moderately positive, (+++) indicates positive, and (++++) indicates strong positive. In FIG. 3, “LM” indicates the inspection result by the optical microscope.

As shown in Table 1, in the rat kidney in which NTS nephritis was induced, the result of examination by an optical microscope showed that
Glomerular hypertrophy and excessive cells in the glomerulus were observed. In the group of NTS nephritis rats, Table 1 and FIG.
As shown in Fig. 5, cellular crescent was observed in about 56% of the glomeruli, and strong necrotic lesions were observed in the glomeruli. In the group of rats with NTS nephritis, as a result of examination by immunofluorescence microscopy, as shown in Table 2 and FIGS. 3a to 3c, linear and granular deposition of rat IgG, and granular deposition of rat C3 and fibrinogen were observed. Was found on the glomerular snare wall.

On the other hand, in the group of NTS nephritis rats to which dexamethasone was administered, as shown in Table 1 and FIG. 3t, less than 3% of glomeruli showed crescent formation.
No significant cell proliferation or glomerular hypertrophy was observed. In addition, as shown in Table 2 and FIG.
No deposition of G, C3 and fibrinogen into the glomeruli was observed.

On the other hand, NTS to which G1, G3 and G9 were administered
In the nephritis rat administration group, the results of examination by immunofluorescence microscopy and light microscopy showed that dexamethasone-administered NTS
Almost the same morphological changes as in the nephritis rat group were observed.
That is, as shown in Table 1 and FIGS. 3h, 1 and 3p, crescents were recognized in 5 to 8% of glomeruli, and no increase in glomerular cells was observed. Table 2 and FIGS.
As shown in g, ik, and mo, no deposition of fibrinogen into the glomeruli was observed.
And C3 glomerular deposition was not inhibited.

In all the control and experimental groups, exogenously administered rabbit IgG was stained linearly and no change was observed.

From the above results, G1, G3, and G9 suppress glomerular lesions such as glomerular hypertrophy caused by nephritis, excessive cells and crescent formation in glomeruli, and fibrinogen deposition in glomeruli. It has been shown to have an effect.

(3) Quantitative Results of Cells Infiltrating into the Glomerular Body and Cell Proliferation in the Glomerular Body FIG. 4 shows quantitative results of cells infiltrating into the glomerular body of the NTS nephritis rat and cell proliferation in the glomerular body. In FIG. 4, A
(□) indicates the number of CD8-positive cells that infiltrated into the glomerulus on the eighth day (cells / glomerular), and (A) in A indicates the number of CD4-positive cells that infiltrated into the glomerulus on the eighth day. (□) in B indicates the number of leukocytes infiltrated into the glomerulus on the 14th day (pieces / glomerus), and (■) in B indicates the number of leukocytes infiltrated on the 14th day. Number of monocytes / macrophages infiltrating into the sphere (pieces / glomerular)
Is shown.

Enzyme antibody method (immune peroxidase reaction)
As a result, as shown in FIG. 4A, on day 8, the number of CD8-positive cells and the number of CD4-positive cells infiltrated into the glomerular body were 3.5 ± as shown in FIG. 4A. 0.8 / glomerular, 0.9 ± 0.2 /
It was a glomerulus. As shown in FIG. 4B, on the 14th day, the number of leukocytes infiltrating into the glomerular body increased remarkably, reaching 28.3 ± 3.4 cells / glomerular, and most of the cells (25.0 ± 2.6 cells) / Glomeruli) were macrophages.

On the other hand, in the group of NTS nephritis rats to which dexamethasone was administered, as shown in FIG. 4A, the number of CD8-positive cells infiltrating into the glomeruli was 0.
It decreased to 6 ± 0.2 / glomerular. Further, as shown in FIG. 4B, on the 14th day, the number of macrophages infiltrating into the glomeruli was reduced to 2.3 ± 0.1 / glomeruli.

On the other hand, in the group of NTS nephritis rats to which G1 and G3 were administered, as shown in FIG. 4A, the infiltration of CD8-positive cells could not be suppressed. In the group, as shown in FIG. 4A,
The infiltration of CD8-positive cells was significantly suppressed, and the number of CD8-positive cells that infiltrated into the glomerulus was reduced to 1.2 ± 0.2 / glomerular. In addition, in the group of NTS nephritis rats to which G1, G3 and G9 were administered, as shown in FIG. 4B, the infiltration of macrophages into the glomeruli was significantly suppressed.

FIGS. 5A and 5B show the results of immunofluorescence microscopy of kidney specimens from NTS nephritis rats and the results of quantification of proliferating cells in glomeruli, respectively.
In FIG. 5A, (a) is ED1, (b) is Ki-6.
7, (c) shows the results for α-SMA, and the white line in the figure indicates the length of 100 μm. In FIG. 5B, (■) indicates E
The number of D1-positive cells (cells / glomerular) and (□) indicate the number of Ki-67 positive cells (cells / glomerular).

As a result of detecting Ki-67 as a proliferation marker and ED-1 as a monocyte / macrophage cell type marker on serial sections of renal tissue, NTS to which PBS was administered was used.
In the nephritis rat group, Ki-67 immunoreactivity was observed in the glomerular nucleus as shown in FIGS. 5A (a) and (b).
Most of them were also positive for the monocyte / macrophage marker ED-1. In the glomeruli of the NTS nephritis rat group to which PBS was administered, a large amount of ED-1 positive cells (24 ± 8 / glomerular) and Ki-67 positive cells (22 ± 7 / glomerular) were observed. However, in the NTS nephritis rat group to which G1, G3, G9 and dexamethasone were administered, the generation of ED-1 positive cells and Ki-67 positive cells was significantly suppressed. On the other hand, the distribution of α-SMA is E
The distribution differs from that of D-1 and Ki-67, and in the group of NTS nephritis rats to which PBS was administered, only a part of the mesangial cells was α-SMA positive (FIG. 5A).
(C)). The results showed that the proliferation activity of mesangial cells was low, and the accumulation of glomerular cells in NTS nephritis was due to the influx and proliferation of macrophages into the glomeruli.

From the above results, it was shown that G1, G3, and G9 have an effect of suppressing infiltration of macrophages into glomeruli, which causes glomerular diseases such as glomerulonephritis.
Furthermore, G9 was shown to have an effect of suppressing infiltration of CD8-positive cells into glomeruli, which causes glomerular diseases such as glomerulonephritis.

(4) Peripheral blood anti-rabbit I by ELISA
Measurement of gG antibody (circulating anti-rabbit IgG) level The measurement results of the peripheral blood anti-rabbit IgG antibody level by ELISA are shown in FIG. As shown in FIG. 6, administration of dexamethasone completely inhibited the production of anti-rabbit IgG antibodies, but administration of G1, G3, and G9 did not affect antibody production. These results indicate that dexamethasone, a steroid hormone, inhibits / suppresses functions involved in antibody production of immunocompetent cells such as antigen presenting cells, helper T cells (CD4-positive T cells), and B cells.
3. G9 does not inhibit or suppress the function of these immunocompetent cells involved in antibody production, that is, G1, G3, and G9 have fewer side effects than the steroid hormone dexamethasone. became.

(5) In Situ TUNEL Assay of Kidney Tissue FIG. 7 shows the results of the in situ TUNEL assay of kidney tissue. Arrows in FIG. 7A indicate TUNEL-positive apoptotic cells observed in the group of NTS nephritis rats to which PBS was administered. In FIG. 7A, a white line indicates a length of 50 μm. FIG. 7B shows the number of apoptotic cells (cells / glomerular) present in the glomeruli of NTS nephritis rats.

As shown in FIG. 7A, N was administered with PBS.
In the group of TS nephritis rats, almost no apoptotic cells were found in the glomeruli on day 14. FIG. 7B
As shown in Table 2, no increase in apoptotic cells was observed in the renal tissue of the NTS nephritis rat group to which any of dexamethasone, G1, G3, and G9 was administered.

(6) Apoptosis assay of T cells isolated from spleen Table 3 and FIG. 8 show the results of an apoptosis assay (FACS analysis) of T cells isolated from spleens of a normal rat group. The numerical values in Table 3 and FIG. 8 indicate the percentage (%) of apoptotic cells among CD4-positive cells and CD8-positive cells.
Represents

[Table 3] Normal rat NTS nephritis rat CD4 ⁺ CD8 ⁺ CD4 ⁺ CD8 ⁺ PBS 6.8 ± 1.8 7.0 ± 2.1 5.7 ± 1.8 4.8 ± 2.0 G1 9.1 ± 2.0 8.9 ± 2.2 9.1 ± 2.3 8.6 ± 1.8 G3 8.1 ± 1.6 6.4 ± 1.8 5.9 ± 2.1 7.0 ± 1.6 G9 8.6 ± 1.9 8.4 ± 2.1 9.2 ± 1.6 16.8 ± 2.2 DEX 24.9 ± 2.8 28.3 ± 2.4 20.1 ± 2.6 25.7 ± 2.9

As shown in Table 3 and FIG. 8, about 7% of the CD4-positive cells and CD8-positive cells isolated from the spleen of the normal rat group to which PBS was administered spontaneously reached apoptosis. Apoptosis in normal rats receiving dexamethasone was approximately 25% of CD4 positive cells, CD
Approximately 28% of the 8 positive cells showed G1, G3, G9
No significant increase in apoptosis was observed in the group of normal rats treated with.

Further, as shown in Table 3 and FIG.
In the S nephritis rat group, about 6% of CD4 positive cells and about 5% of CD8 cells isolated from the spleen spontaneously reached apoptosis, and the proportion of apoptotic cells was similar to that of the normal rat group to which PBS was administered. .

On the other hand, in the group of NTS nephritis rats to which dexamethasone was administered, about 20% of CD4-positive cells
Approximately 26% of D8-positive cells have reached apoptosis,
Apoptosis was increased by dexamethasane administration as in the normal rat group to which BS was administered. On the other hand, G1, G3
In the group of NTS nephritis rats administered with, 5 to 9% of CD4-positive cells and CD8-positive cells reached apoptosis, and the apoptosis induced by G1 and G3 was similar to the basal level. However, G9 administered NT
In the group of S nephritis rats, about 9% of CD4 positive cells and CD8
Approximately 17% of the positive cells reached apoptosis, and the apoptosis of CD4-positive cells induced by administration of G9 remained at a basal level, whereas the apoptosis of CD8-positive cells was promoted.

From the above results, dexamethasone induces apoptosis in both resting T cells and activated T cells, but G9 selectively induces apoptosis in activated NT8-positive T cells in the group of NTS nephritis rats. It was shown to. That is, G9 has an action of selectively suppressing infiltration of CD8-positive cells into glomeruli, which causes glomerular diseases such as glomerulonephritis. It was shown to have no side effects of induction.

(7) Distribution of administered exogenous galectin in kidney tissue FIG. 9 shows the distribution of administered exogenous galectin in kidney tissue. When the administered exogenous G9 was detected by an anti-c-myc antibody, as shown in FIG. 9, the exogenous G9 was distributed to the glomerular endothelial cells and capillaries around the tubule (red portion in FIG. 9). In addition, not all of the glomerular loops and endothelial cells in the capillaries around the tubules were positive, but some were positive. Endogenous G9 was detected in glomerular cells as previously reported and was distributed in both endothelial cells and mesangial cells (Wada J. et al., J. Clin. Invest. 99:24
52-2461, 1997). Exogenous G administered by double staining
9 was mainly distributed in glomerular loop endothelial cells, and its distribution pattern was different from endogenous G9.

The effect of G1 and G3 on the infiltration of macrophages into the glomerulus is exerted by the administration of exogenous G1 and G3 by crosslinking the sugar chains expressed on endothelial cells and macrophages to suppress the adhesion between the two. It is considered to be.
The confirmation of the immunohistological localization of exogenous galectins in glomerular endothelial cells supports this idea.

[0137]

Industrial Applicability According to the present invention, a novel agent for preventing and treating nephritis, an agent for preventing and treating glomerular diseases, an agent for inhibiting infiltration of leukocytes into the glomerulus, an agent for inhibiting infiltration of CD8-positive cells into the glomerulus and C
An agent for inducing apoptosis of D8-positive cells is provided.

[0138]

[Sequence List] SEQUENCE LISTING <110> Jun Wada, Akira Morooka <120> A pharmaceutical composition for treating or preventing nephritis <130> P01-1009 <160> 12 <210> 1 <211> 135 <212> PRT <213> Mus musculus <220> <223> mouse galectin-1 <400> 1 Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu 1 5 10 15 Cys Leu Lys Val Arg Gly Glu Val Ala Ser Asp Ala Lys Ser Phe Val 20 25 30 Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro 35 40 45 Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Thr Lys 50 55 60 Glu Asp Gly Thr Trp Gly Thr Glu His Arg Glu Pro Ala Phe Pro Phe 65 70 75 80 Gln Pro Gly Ser Ile Thr Glu Val Cys Ile Thr Phe Asp Gln Ala Asp 85 90 95 Leu Thr Ile Lys Leu Pro Asp Gly His Glu Phe Lys Phe Pro Asn Arg 100 105 110 Leu Asn Met Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys 115 120 125 Ile Lys Cys Val Ala Phe Glu 130 135 <210> 2 <211> 264 <212> PRT <213> Mus musculus <220> < 223> mouse galectin-3 <400> 2 Met Ala Asp Ser Phe Ser Leu Asn Asp Ala Leu Ala Gly Ser Gly Asn 1 5 10 15 Pro Asn Pro Gln Gly Tyr Pro Gly Ala Trp Gly Asn Gln Pro Gly Ala 20 25 30 Gly Gly Tly Pro Gly Ala Ala Ala Tyr Pro Gly Ala Tyr Pro Gly Gln Ala 35 40 45 Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Ala Tyr Pro Gly 50 55 60 Gln Ala Pro Pro Ser Ala Tyr Pro Gly Pro Thr Ala Pro Gly Ala Tyr 65 70 75 80 Pro Gly Pro Thr Ala Pro Gly Ala Tyr Pro Gly Gln Pro Ala Pro Gly 85 90 95 Ala Phe Pro Gly Gln Pro Gly Ala Pro Gly Ala Tyr Pro Gln Cys Ser 100 105 110 Gly Gly Tyr Pro Ala Ala Gly Pro Tyr Gly Val Pro Ala Gly Pro Leu 115 120 125 Thr Val Pro Tyr Asp Leu Pro Leu Pro Gly Gly Val Met Pro Arg Met 130 135 140 Leu Ile Thr Ile Met Gly Thr Val Lys Pro Asn Ala Asn Arg Ile Val 145 150 155 160 Leu Asp Phe Arg Arg Gly Asn Asp Val Ala Phe His Phe Asn Pro Arg 165 170 175 Phe Asn Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys Gln Asp 180 185 190 Asn Asn Trp Gly Lys Glu Glu Arg Gln Ser Ala Phe Pro Phe Glu Ser 195 200 205 Gly Lys Pro Phe Lys Ile Gln Val Leu Val Glu Ala Asp His Phe Lys 210 215 220 Val Ala Val Asn Asp Ala His Leu Leu Gln Tyr Asn His Arg Met Lys 225 230 235 240 Asn Leu Arg Glu Ile Ser Gln Leu Gly Ile Ser Gly Asp Ile Thr Leu 245 250 255 Thr Ser Ala Asn His Ala Met Ile 260 <210> 3 <211> 322 <212> PRT <213> Mus musculus <220> <223> mouse galectin-9 <400> 3 Met Ala Leu Phe Ser Ala Gln Ser Pro Tyr Ile Asn Pro Ile Ile Pro 1 5 10 15 Phe Thr Gly Pro Ile Gln Gly Gly Leu Gln Glu Gly Leu Gln Val Thr 20 25 30 Leu Gln Gly Thr Thr Lys Ser Phe Ala Gln Arg Phe Val Val Asn Phe 35 40 45 Gln Asn Ser Phe Asn Gly Asn Asp Ile Ala Phe His Phe Asn Pro Arg 50 55 60 Phe Glu Glu Gly Gly Tyr Val Val Cys Asn Thr Lys Gln Asn Gly Gln 65 70 75 80 Trp Gly Pro Glu Glu Glu Arg Lys Met Gln Met Pro Phe Gln Lys Gly Met 85 90 95 Pro Phe Glu Leu Cys Phe Leu Val Gln Arg Ser Glu Phe Lys Val Met 100 105 110 Val Asn Lys Lys Phe Phe Val Gln Tyr Gln His Arg Val Pro Tyr His 115 120 125 Leu Val Asp Thr Ile Ala Val Ser Gly Cys Leu Lys Leu Ser Phe Ile 130 135 140 Thr Phe Gln Thr Gln Asn Phe Arg Pro Ala H is Gln Ala Pro Met Ala 145 150 155 160 Gln Thr Thr Ile His Met Val His Ser Thr Pro Gly Gln Met Phe Ser 165 170 175 Thr Pro Gly Ile Pro Pro Val Val Tyr Pro Thr Pro Ala Tyr Thr Ile 180 185 190 Pro Phe Tyr Thr Pro Ile Pro Asn Gly Leu Tyr Pro Ser Lys Ser Ile 195 200 205 Met Ile Ser Gly Asn Val Leu Pro Asp Ala Thr Arg Phe His Ile Asn 210 215 220 Leu Arg Cys Gly Gly Asp Ile Ala Phe His Leu Asn Pro Arg Phe Asn 225 230 235 240 Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asn Asn Ser Trp Gly Gln 245 250 255 Glu Glu Arg Ser Leu Leu Gly Arg Met Pro Phe Ser Arg Gly Gln Ser 260 265 270 Phe Ser Val Trp Ile Ile Cys Glu Gly His Cys Phe Lys Val Ala Val 275 280 285 Asn Gly Gln His Met Cys Glu Tyr Tyr His Arg Leu Lys Asn Leu Gln 290 295 300 Asp Ile Asn Thr Leu Glu Val Ala Gly Asp Ile Gln Leu Thr His Val 305 310 315 320 Gln Thr <210> 4 <211> 135 <212> PRT <213> Homo sapiens <220> <223> human galectin-1 <400> 4 Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu 1 5 10 15 Cys Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val 20 25 30 Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro 35 40 45 Arg Phe Asn Ala His Gly Asp Ala Asn Thrle Ile Val Cys Asn Ser Lys 50 55 60 Asp Gly Gly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe 65 70 75 80 Gln Pro Gly Ser Val Ala Glu Val Cys Ile Thr Phe Asp Gln Ala Asn 85 90 95 Leu Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg 100 105 110 Leu Asn Leu Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys 115 120 125 Ile Lys Cys Val Ala Phe Asp 130 135 <210> 5 <211> 250 <212> PRT <213 > Homo sapiens <220> <223> human galectin-3 <400> 5 Met Ala Asp Asn Phe Ser Leu His Asp Ala Leu Ser Gly Ser Gly Asn 1 5 10 15 Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn Gln Pro Ala Gly 20 25 30 Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln 35 40 45 Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Ala Tyr Pro 50 55 60 Gly Ala Pro Gly Ala Tyr Pro Gly Ala Pro Ala Pro Gly Val Tyr Pro 65 70 75 80 Gly Pro Pro Ser G ly Pro Gly Ala Tyr Pro Ser Ser Gly Gln Pro Ser 85 90 95 Ala Pro Gly Ala Tyr Pro Ala Thr Gly Pro Tyr Gly Ala Pro Ala Gly 100 105 110 Pro Leu Ile Val Pro Tyr Asn Leu Pro Leu Pro Gly Gly Val Val Pro 115 120 125 Arg Met Leu Ile Thr Ile Leu Gly Thr Val Lys Pro Asn Ala Asn Arg 130 135 140 Ile Ala Leu Asp Phe Gln Arg Gly Asn Asp Val Ala Phe His Phe Asn 145 150 155 160 Pro Arg Phe Asn Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys 165 170 175 Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg Gln Ser Val Phe Pro Phe 180 185 190 Glu Ser Gly Lys Pro Phe Lys Ile Gln Val Leu Val Glu Pro Asp His 195 200 205 Phe Lys Val Ala Val Asn Asp Ala His Leu Leu Gln Tyr Asn His Arg 210 215 220 Val Lys Lys Leu Asn Glu Ile Ser Lys Leu Gly Ile Ser Gly Asp Ile 225 230 235 240 Asp Leu Thr Ser Ala Ser Tyr Thr Met Ile 245 250 <210> 6 <211> 355 <212> PRT <213> Homo sapiens <220> <223> human galectin-9 (Long isoform) <400> 6 Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser Pro Ala Val Pro 1 5 10 15 Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln Ile Thr 20 25 30 Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn 35 40 45 Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe His Phe Asn Pro 50 55 60 Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly 65 70 75 80 Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro Phe Gln Lys Gly 85 90 95 Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val 100 105 110 Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe 115 120 125 His Arg Val Asp Thrile Ile Ser Val Asn Gly Ser Val Gln Leu Ser Tyr 130 135 140 Ile Ser Phe Gln Asn Pro Arg Thr Val Pro Val Gln Pro Ala Phe Ser 145 150 155 160 Thr Val Pro Phe Ser Gln Pro Val Cys Phe Pro Pro Arg Pro Arg Gly 165 170 175 Arg Arg Gln Lys Pro Pro Gly Val Trp Pro Ala Asn Pro Ala Pro Ile 180 185 190 Thr Gln Thr Val Ile His Thr Val Gln Ser Ala Pro Gly Gln Met Phe 195 200 205 Ser Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His Pro Ala Tyr Pro 210 215 220 Met Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr Pro Ser Lys Ser 225 230 235 240 Ile Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln Arg Phe His Ile 245 250 255 Asn Leu Cys Ser Gly Asn His Ile Ala Phe His Leu Asn Pro Arg Phe 260 265 270 Asp Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asp Asn Ser Trp Gly 275 280 285 Ser Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe Val Arg Gly Gln 290 295 300 Ser Phe Ser Val Trp Ile Leu Cys Glu Ala His Cys Leu Lys Val Ala 305 310 315 320 Val Asp Gly Gln His Leu Phe Glu Tyr Tyr His Arg Leu Arg Asn Leu 325 330 335 Pro Thr Ile Asn Arg Leu Glu Val Gly Gly Asp Ile Gln Leu Thr His 340 345 350 Val Gln Thr 355 <210> 7 <211> 135 <212> PRT <213> Ruttus norvegicus <220> <223> rat galectin-1 <400> 7 Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu 1 5 10 15 Cys Leu Lys Val Arg Gly Glu Leu Ala Pro Asp Ala Lys Ser Phe Val 20 25 30 Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro 35 40 45 Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys 50 55 60 Asp Asp Gly Thr Trp Gly Thr Glu Gln Arg Glu Thr Ala Phe Pro Phe 65 70 75 80 Gln Pro Gly Ser Ile Thr Glu Val Cys Ile Thr Phe Asp Gln Ala Asp 85 90 95 Leu Thr Ile Lys Leu Pro Asp Gly His Glu Phe Lys Phe Pro Asn Arg 100 105 110 Leu Asn Met Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys 115 120 125 Ile Lys Cys Val Ala Phe Glu 130 135 <210> 8 <211> 262 <212> PRT <213> Ruttus norvegicus <220> <223> rat galectin-3 <400> 8 Met Ala Asp Gly Phe Ser Leu Asn Asp Ala Leu Ala Gly Ser Gly Asn 1 5 10 15 Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn Gln Pro Gly Ala 20 25 30 Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln Ala 35 40 45 Pro Pro Gly Gly Tyr Pro Gly Gln Ala Pro Pro Ser Ala Tyr Pro Gly 50 55 60 Pro Thr Gly Pro Ser Ala Tyr Pro Gly Pro Thr Ala Pro Gly Ala Tyr 65 70 75 80 Pro Gly Pro Thr Ala Pro Gly Ala Phe Pro Gly Gln Pro Gly Gly Pro 85 90 95 Gly Ala Tyr Pro Ser Ala Pro Gly Ala Tyr Pro Ser Ala Pro Gly Ala 100 105 110 Tyr Pro Ala Thr Gly Pro Phe Gly Ala Pro Thr Gly Pro Leu Thr Val 115 120 125 Pro Tyr Asp Met Pr o Leu Pro Gly Gly Val Met Pro Arg Met Leu Ile 130 135 140 Thr Ile Ile Gly Thr Val Lys Pro Asn Ala Asn Ser Ile Thr Leu Asn 145 150 155 160 Phe Lys Lys Gly Asn Asp Ile Ala Phe His Phe Asn Pro Arg Phe Asn 165 170 175 Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys Gln Asp Asn Asn 180 185 190 Trp Gly Arg Glu Glu Arg Gln Ser Ala Phe Pro Phe Glu Ser Gly Lys 195 200 205 Pro Phe Lys Ile Gln Val Leu Val Glu Ala Asp His Phe Lys Val Ala 210 215 220 Val Asn Asp Val His Leu Leu Gln Tyr Asn His Arg Met Lys Asn Leu 225 230 235 240 Arg Glu Ile Ser Gln Leu Gly Ile Ile Gly Asp Ile Thr Leu Thr Ser 245 250 255 Ala Ser His Ala Met Ile 260 <210> 9 <211> 354 <212> PRT <213> Ruttus norvegicus <220> <223> rat galectin-9 (Long isoform) <400> 9 Met Ala Phe Phe Ser Thr Gln Pro Pro Tyr Met Asn Pro Val Ile Pro 1 5 10 15 Phe Thr Gly Ile Ile Gln Gly Gly Leu Gln Asn Gly Leu Gln Ile Thr 20 25 30 Leu Gln Gly Thr Val His Pro Phe Pro Asn Arg Ile Ala Val Asn Phe 35 40 45 Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe His Phe Asn Pro Arg 50 55 60 Phe Glu Glu Gly Gly Tyr Val Val Cys Asn Thr Lys Gln Asn Gly Lys 65 70 75 80 Trp Gly Pro Glu Glu Arg Lys Met Gln Met Pro Phe Gln Lys Gly Met 85 90 95 Pro Phe Glu Leu Cys Phe Leu Val Gln Arg Ser Glu Phe Lys Val Met 100 105 110 Val Asn Lys Asn Phe Phe Val Gln Tyr Ser His Arg Val Pro Tyr His 115 120 125 Leu Val Asp Thr Ile Ser Val Ser Gly Cys Leu His Leu Ser Phe Ile 130 135 140 Asn Phe Gln Asn Ser Thr Ala Ala Pro Val Gln Pro Val Phe Ser Thr 145 150 155 160 Met Gln Phe Ser Gln Pro Val Gln Phe Pro Arg Met Pro Lys Gly Arg 165 170 175 Lys Gln Arg Thr Gln Gly Phe Gln Pro Ala Leu Gln Ala Pro Val Ala 180 185 190 Gln Thr Ile Ile His Thr Val His Ser Ile Pro Gly Gln Met Leu Ser 195 200 205 Thr Pro Gly Ile Pro Pro Met Ala Tyr Pro Thr Pro Ala Tyr Thr Ile 210 215 220 Pro Phe Phe Thr Ser Ile Pro Asn Gly Phe Tyr Pro Ser Lys Ser Ile 225 230 235 240 Asn Ile Ser Gly Val Val Leu Pro Asp Ala Lys Arg Phe His Ile Asn 245 250 255 Leu Arg Cys Gly Gly Asp Ile Ala Phe His Leu Asn Pro Arg P he Asn 260 265 270 Glu Lys Val Val Val Arg Asn Thr Gln Ile Asn Asn Ser Trp Gly Pro 275 280 285 Glu Glu Arg Ser Leu Pro Gly Arg Met Pro Phe Asn Arg Gly Gln Ser 290 295 300 Phe Ser Val Trp Ile Leu Cys Glu Gly His Cys Phe Lys Val Ala Val 305 310 315 320 Asp Gly Gln His Ile Cys Glu Tyr Tyr His Arg Leu Lys Asn Leu Pro 325 330 335 Asp Ile Asn Thr Leu Glu Val Ala Gly Asp Ile Gln Leu Thr His Val 340 345 350 Gln Thr <210> 10 <211> 545 <212> DNA <213> Mus musculus <220> <221> CDS <222> (72) .. (479) <220> <223> mouse galectin-1 gene < 400> 10 cgtctctcgg gtggagtctt ctgactgctg gtggagcagg tctcaggaat ctcttcgctt 60 cagcttcaat c atg gcc tgt ggt ctg gtc gcc agc aac ctg aat ctc aaa 110 Met Ala Cys Gly Leu Val Ac gt As Lec gga gag gtg gcc tcg gac gcc aag 158 Pro Gly Glu Cys Leu Lys Val Arg Gly Glu Val Ala Ser Asp Ala Lys 15 20 25 agc ttt gtg ctg aac ctg gga aaa gac agc aac aac ctg tgc cta cac 206 Ser Phe Val Leu Leu Gly Lys Asp Ser Asn Asn Leu Cys Le u His 30 35 40 45 ttc aat cct cgc ttc aat gcc cat gga gac gcc aac acc att gtg tgt 254 Phe Asn Pro Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys 50 55 60 aac acc aag gaa gat ggg acc tgg gga acc gaa cac cgg gaa cct gcc 302 Asn Thr Lys Glu Asp Gly Thr Trp Gly Thr Glu His Arg Glu Pro Ala 65 70 75 ttc ccc ttc cag ccc ggg agc atc aca gag gtg tgc atc acc ttt gac 350 Phe Pro Phe Gln Pro Gly Ser Ile Thr Glu Val Cys Ile Thr Phe Asp 80 85 90 cag gct gac ctg acc atc aag ctg cca gac gga cat gaa ttc aag ttc 398 Gln Ala Asp Leu Thr Ile Lys Leu Pro Asp Gly His Glu Phe Lys Phe 95 100 105 ccc aac cgc ctc aac atg gag gcc atc aac tac atg gcg gcg gat gga 446 Pro Asn Arg Leu Asn Met Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly 110 115 120 125 gac ttc aag att aag tgc gtg gcc tttgag tcc 499 Asp Phe Lys Ile Lys Cys Val Ala Phe Glu 130 135 tcaataaagg cagctgcctc tgctccccat ataaaaaaaa aaaaaa 545 <210> 11 <211> 955 <212> DNA <213> Mus musculus <220> <221> CDS <222> (44). . (838) <220> <223> mouse galectin-3 gene <400> 11 agcactaatc aggtgagcgg cacagagagc actacccagg aaa atg gca gac agc 55 Met Ala Asp Ser 1 ttt tcg ctt aac gat gcc tta gct ggc tct gga aac cca aac cct caa 103 Phe Ser Leu Asu Asp Ser Gly Asn Pro Asn Pro Gln 5 10 15 20 gga tat ccg ggt gca tgg ggg aac cag cct ggg gca ggg ggc tac cca 151 Gly Tyr Pro Gly Ala Trp Gly Asn Gln Pro Gly Ala Gly Gly Tyr Pro 25 30 35 ggg gct gcc tat cct ggg gcc tat cca gga cag gct cct cca ggg gcc 199 Gly Ala Ala Tyr Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Ala 40 45 50 tac cca gga cag gct cct cca ggg gcc tat cca gga cag gct cct cct 247 Tyr Pro Gly Gln Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro 55 60 65 agt gcc tac ccc ggc cca act gcc cct gga gct tat cct ggc cca act 295 Ser Ala Tyr Pro Gly Pro Thralla Pro Gly Ala Tyr Pro Gly Pro Thr 70 75 80 gcc cct gga gct tat cct ggt caa cct gcc cct gga gcc ttc cca ggg 343 Ala Pro Gly Ala Tyr Pro Gly Gln Pro Ala Pro Gly Ala Phe Pro Gly 85 90 95 100 caa cct ggg gca cct ggg gcc tacccc cag tgc tct gga ggc tat cct 391 Gln Pro Gly Ala Pro Gly Ala Tyr Pro Gln Cys Ser Gly Gly Tyr Pro 105 110 115 gct gct ggc cct tat ggt gtc ccc gct gga cca ctg acg gtg ccc tat 439 Ala Ala Gly Pro Tyr Gly Val Pro Ala Gly Pro Leu Thr Val Pro Tyr 120 125 130 gac ctg ccc ttg cct gga gga gtc atg ccc cgc atg ctg atc aca atc 487 Asp Leu Pro Leu Pro Gly Gly Val Met Pro Arg Met Leu Ile Thr Ile 135 140 145 atg ggc aca gtg aaa ccc aac gca aac agg att gtt cta gat ttc agg 535 Met Gly Thr Val Lys Pro Asn Ala Asn Arg Ile Val Leu Asp Phe Arg 150 155 160 aga ggg aat gat gtt gcc ttc cac ttt aac ccc cgc ttc gag aac 583 Arg Gly Asn Asp Val Ala Phe His Phe Asn Pro Arg Phe Asn Glu Asn 165 170 175 180 aac aga aga gtc att gtg tgt aac acg aag cag gac aat aac tgg gga 631 Asn Arg Arg Val Ile Val Cys Asn Thr Lys Gln Asp Asn Asn Trp Gly 185 190 195 aag gaa gaa aga cag tca gcc ttc ccc ttt gag agt ggc aaa cca ttc 679 Lys Glu Glu Glu Arg Gln Ser Ala Phe Pro Phe Glu Ser Gly Lys Pro Phe 200 205 210 aaa ata caa gtc ctcgtt gaa gct gac cac ttc aag gtt gcg gtc aac 727 Lys Ile Gln Val Leu Val Glu Ala Asp His Phe Lys Val Ala Val Asn 215 220 225 gat gct cac cta ctg cag tac aac cat cgg atg aag aac ctc cgg Aga 775 Asa His Leu Leu Gln Tyr Asn His Arg Met Lys Asn Leu Arg Glu 230 235 240 atc agc caa ctg ggg atc agt ggt gac ata acc ctc acc agc gct aac 823 Ile Ser Gln Leu Gly Ile Ser Gly Asp Ile Thr Leu Thr Ser Ala Asn 245 250 255 260 cac gcc atg atc taa gccagaaggg gcggcaccga aacgccctgt gtgccttagg 878 His Ala Met Ile 265 agtgggaaac tttgcatttc tctctcctta tccttcttgt aagacatccc atttaataaa 938 gtctcatgct <again> <a> 221> CDS <222> (35) .. (1000) <220> <223> mouse galectin-9 gene <400> 12 ggaagagagc attggttccc ctgagataga agag atg gct ctc ttc agt gcc cag 55 Met Ala Leu Phe Ser Ala Gln 15 tct cca tac att aac ccg atc atc ccc ttt act gga cca atc caa gga 103 Ser Pro Tyr Ile Asn Pro Ile Ile Pro Phe Thr Gly Pro Ile Gln Gly 10 15 20 ggg ctg cag gag gga ctt cag gtg ac c ctc cag ggg act acc aag agt 151 Gly Leu Gln Glu Gly Leu Gln Val Thr Leu Gln Gly Thr Thr Lys Ser 25 30 35 ttt gca caa agg ttt gtg gtg aac ttt cag aac agc ttc aat gga aat 199 Phe Ala Gln Arg Phe Val Val Asn Phe Gln Asn Ser Phe Asn Gly Asn 40 45 50 55 gac att gcc ttc cac ttc aac ccc cgg ttt gag gaa gga ggg tat gtg 247 Asp Ile Ala Phe His Phe Asn Pro Arg Phe Glu Glu Gly Gly Gly Tyr Val 60 65 70 gtt tgc aac acg aag cag aac gga cag tgg ggt cct gag gag aga aag 295 Val Cys Asn Thr Lys Gln Asn Gly Gln Trp Gly Pro Glu Glu Arg Lys 75 80 85 atg cag atg ccc ttc cag aag ggg atg ccc ttt gag ctt tgc ttc ctg 343 Met Gln Met Pro Phe Gln Lys Gly Met Pro Phe Glu Leu Cys Phe Leu 90 95 100 gtg cag agg tca gag ttc aag gtg atg gtg aac aag aaa ttc ttt gtg 391 Val Gln Arg Ser Glu Val Hes Val Asn Lys Lys Phe Phe Val 105 110 115 cag tac caa cac cgc gta ccc tac cac ctc gtg gac acc atc gct gtc 439 Gln Tyr Gln His Arg Val Pro Tyr His Leu Val Asp Thr Ile Ala Val 120 125 130 135 tcc ggc tgc ttg aag ctg tcc ttt atc acc ttc cag act cag aac ttt 487 Ser Gly Cys Leu Lys Leu Ser Phe Ile Thr Phe Gln Thr Gln Asn Phe 140 145 150 cgt cct gcc cac cag gca ccc atg gct caa act acc atc cat atg gtt 535 Arg Pro Ala His Gln Ala Pro Met Ala Gln Thr Thr Ile His Met Val 155 160 165 cac agc acc cct gga cag atg ttc tct act cct gga atc cct cct gtg 583 His Ser Thr Pro Gly Gln Met Phe Ser Thr Pro Gly Ile Pro Pro Val 170 175 180 gtg tac ccc acc cca gcc tat acc ata cct ttc tac acc ccc att cca 631 Val Tyr Pro Thr Pro Ala Tyr Thr Ile Pro Phe Tyr Thr Pro Ile Pro 185 190 195 aat ggg ctt tac ccg tcc aag tcc atc atg ata tca ggc aat gtc ttg 679 Asn Gly Leu Tyr Pro Ser Lys Ser Ile Met Ile Ser Gly Asn Val Leu 200 205 210 215 cca gat gct acg agg ttc cat atc aac ctt cgc tgt gga ggt gac att 727 Pro Asp Ala Thr Arg Phe His Ile Asn Leu Arg Cys Gly Gly Asp Ile 220 225 230 gct ttc cac ctg aac ccc cgt ttc aat gag aat gct gtt gtc cga aac 775 Ala Phe His Leu Asn Pro Arg Phe Asn Glu Asn Ala Val Val Arg Asn 235 240 245 act cag atc aac aactcc tgg ggg cag gaa gag cga agt ctg ctt ggg 823 Thr Gln Ile Asn Asn Ser Trp Gly Gln Glu Glu Arg Ser Leu Leu Gly 250 255 260 agg atg ccc ttc agt cga ggc cag agc ttc tcg gtg tgg atc Atgat1 87 Pro Phe Ser Arg Gly Gln Ser Phe Ser Val Trp Ile Ile Cys 265 270 275 gaa ggt cac tgc ttc aag gta gct gtg aat ggt caa cac atg tgt gaa 919 Glu Gly His Cys Phe Lys Val Ala Val Asn Gly Gln His Met Cys Glu 280 285 290 295 tat tac cac cgc ctg aag aac ttg cag gat atc aac act cta gaa gtg 967 Tyr Tyr His Arg Leu Lys Asn Leu Gln Asp Ile Asn Thr Leu Glu Val 300 305 310 gcg ggt gat atc cag ctg acccac aca taggcaaggt ctctggccta 1020 Ala Gly Asp Ile Gln Leu Thr His Val Gln Thr 315 320 gggataaggg ctggagcact ctgcctgtgt cttatctttc ccctgtctca gccctggcac 1080 catcagaaga gatcatcgct tataggaatt ccaggaaggt gaaattccca attgactccc 1140 tccacaaagg gggttttcta ggctgtgtgg cacatgctgt cagcccatag tctgagccat 1200 tgcccccaag ctagctatat actgagggaa gtgaccctcc tgggtttgct cagatctctg 1260 atcgttcccc cctctgtggc ccttttcttt cacccctcca ggagagccgc cctgatatca 1320 tcccactggc ctccaactga cccacaatgt ccacagtaac tttcccccat tctcacccag 1380 tatccataaa ataaagaaat aatattgctt gtctacac 1418

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing types of galectin gene family.

FIG. 2 is a graph showing urinary protein excretion in NTS nephritis rats.

FIG. 3 shows the results of examination of a kidney specimen of an NTS nephritis rat by an optical microscope and an immunofluorescence microscope.

FIG. 4 shows the results of quantification of cells infiltrating into the glomerulus.

FIG. 5 is a diagram showing an examination result of a kidney specimen of an NTS nephritis rat by an immunofluorescence microscope (A) and a quantitative result of proliferating cells in a glomerulus (B).

FIG. 6 is a diagram showing the results of measuring peripheral blood anti-rabbit IgG antibody levels by ELISA.

FIG. 7 shows the results of an in situ TUNEL assay on kidney tissue.

FIG. 8 shows the results of an apoptosis assay on T cells isolated from spleen.

FIG. 9 shows the distribution of administered exogenous G9 in renal tissue.

 ──────────────────────────────────────────────────続 き Continued on front page F term (reference) 4B024 AA01 BA80 CA04 DA05 EA04 GA11 HA11 4C084 AA02 BA01 BA21 BA22 BA23 CA23 DA31 MA23 MA28 MA31 MA35 MA37 MA52 MA57 MA60 MA66 ZA812 4H045 AA10 AA30 BA10 CA40 DA80 EA26 FA74

Claims (8)

[Claims] 1. A prophylactic / therapeutic agent for nephritis, comprising a polypeptide represented by the following (a) or (b) as an active ingredient: (A) a polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 3; (b) one or more amino acids are deleted, substituted or added in the amino acid sequence of any of SEQ ID NOs: 1 to 3 Having a specific amino acid sequence and a binding activity specific to β-galactoside 2. A prophylactic / therapeutic agent for glomerular disease, comprising a polypeptide represented by the following (a) or (b) as an active ingredient: (A) a polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 3; (b) one or more amino acids are deleted, substituted or added in the amino acid sequence of any of SEQ ID NOs: 1 to 3 Having a specific amino acid sequence and a binding activity specific to β-galactoside 3. The glomerular disease includes glomerular hypertrophy, hypertrophy of glomeruli, crescent formation, infiltration of leukocytes into glomeruli, C
3. The method for preventing glomerular disease according to claim 2, wherein the glomerular disease shows one or more lesions selected from the group consisting of infiltration of D8-positive cells into the glomerulus and deposition of fibrinogen in the glomerulus. Therapeutic agent. 4. The preventive / therapeutic agent for glomerular disease according to claim 2, wherein the glomerular disease is glomerulonephritis. 5. An agent for inhibiting infiltration of leukocytes into a glomerulus, which comprises a polypeptide represented by the following (a) or (b) as an active ingredient: (A) a polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 3; (b) one or more amino acids are deleted, substituted or added in the amino acid sequence of any of SEQ ID NOs: 1 to 3 Having a specific amino acid sequence and a binding activity specific to β-galactoside 6. The method according to claim 5, wherein the leukocytes are monocytes or macrophages. 7. A CD comprising a polypeptide represented by the following (c) or (d) as an active ingredient:
Inhibitor of glomerular infiltration of 8-positive cells. (C) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3; (d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acids have been deleted, substituted or added, and Polypeptide having specific binding activity to galactoside 8. A CD comprising a polypeptide represented by the following (c) or (d) as an active ingredient:
An apoptosis inducer for 8-positive cells. (C) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3; (d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acids have been deleted, substituted or added, and Polypeptide having specific binding activity to galactoside