JP2000354487A - Midkine receptor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new midkine receptor which binds to midkine and pleiotrophin, whose binding with midkine is inhibited by heparin, whose complex with midkine is slowly degraded in a cell, and is useful for developing a therapeutic agent for cancer, etc. SOLUTION: This is a new midkine receptor which binds to midkine and pleiotrophin, whose binding with midkine is inhibited in the presence of heparin, which is incorporated into a cell as a complex with midkine after binding with midkine, which is slowly degraded in the cell, is used for screening antibody, agonist, and antagonist against midkine receptor, etc., and is capable of developing a therapeutic agent for cancer and for a prophylactic for cancer metastasis, an antiinflammatory agent, a therapeutic agent for treating Alzheimer's disease, etc. This receptor is obtained by screening a cDNA library produced from a brain of mouse fetus with a probe coding for a partial amino acid sequence of midkine receptor, followed by integrating the obtained gene into an expression vector to express in a host cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to the identification and isolation of a heparin-binding growth factor midkine receptor, the use of the receptor, and the preparation of the receptor. The invention also relates to the inhibition or activation of the receptor.

[0002]

2. Description of the Related Art Midkine (MK) is a product of a gene that is transiently expressed in the early stage of retinoic acid-induced differentiation of embryonic tumor cells. Cloned the cDNA (Kadomatsu, K. et al., Biochem. Biophys. Res.
Commun., 151: 1312-1318). Midkine is a 13 kDa heparin-binding secretory protein rich in basic amino acids and cysteine (Tomomura, M. et a
l., J. Biol. Chem., 265: 10765-10770, 1990). Two years after the cloning of midkine cDNA, Pleiotroph has high homology with midkine.
in; hereinafter, also referred to as “PTN”) (Li, Y.-S. et al .: Science, 250: 1690-1694; Me)
renmies, J. & Rauvala, H .: J. Biol. Chem., 265: 1672
1-16924, 1990). Pleiotrophin has a molecular weight of 18k
Da is a heparin-binding secretory protein. Midkine and pleiotrophin are about
When they have 50% homology and compare their amino acid sequences, they show that the N-terminal and C-terminal
Due to the abundance of lysine residues and the conserved position of cysteine residues, a midkine gene family consisting of only these two members is formed (Tsutsui J. et al .: Biochem. Biophys. . Res. Commu
n., 176: 792-797, 1991).

[0003] Midkine and pleiotrophin have neurotrophic factor activity (Li, L.-S. et al .: Science, 250: 16).
90-1694. 1990; Merenmies, J. & Rauvala, H .: J. Bi
o. Chem., 265: 16721-16924, 1990; Muramatsu, H. et.
al .: Dev. Biol., 110: 284-296, 1995), induction of acetylcholine receptor clustering at the neuromuscular junction,
Enhanced fibrinolytic system (Kojima, S. et al., J. Biol. Chem., 27
0: 9590-9596, 1995), transforming NIH3T3 cells (Chauhan, AK et al .: Proc. Natl. Acad. Sci. USA,
90: 679-682, 1993) and angiogenesis.

[0004] Midkine is associated with various diseases. That is, in many human cancers, the expression of midkine is enhanced as compared to surrounding normal tissues (Tsutsui, J. et.
al., Cancer Res., 53: 1281-1285, 1993; Aridome, K.
et al .: Cancer Res., 86: 655-661, 1995). Midkine is strongly deposited in senile plaques of Alzheimer's disease (Ya
suhara, O. et al .: Biochem. Biophys. Res. Commun., 1
92: 246-251, 1993), and a high concentration of midkine protein is present in the synovial fluid of rheumatoid arthritis (Taka
da, T. et al .: J. Biochem., 122: 453-458, 1997). On the other hand, the expression of pleiotrophin is rather suppressed in advanced cancer in many cases. Midkine and pleiotrophin's angiogenesis ability and fibrinolytic enhancement ability also seem to play important roles in cancer progression.

[0005] The various biological activities of midkine probably occur through signal transduction through the interaction of midkine with high affinity receptors.
In both neurite outgrowth and plasminogen activator activation of vascular endothelial cells, treatment of target cells with hepatilinase reduces sensitivity (Kaneda, N. eta).
l., J. Biochem., 119: 1150-1156, 1996; Akhter, S. et.
al .: J. Biochem., 123: 1127-1136,1998) Therefore, for the action of midkine, the binding of midkine to the heparin-like domain present in the heparan sulfate chain on heparan sulfate priteoglycan is essential. (Takamura Muramatsu: Protein Nucleic Acid Enzyme 43: 2288-2294, 1998).

[0006] Pleiotrophin is a chondroitin sulfate proteoglycan that binds strongly to PTPζ, one of the receptor-type phosphotyrosine phosphatases (Maeda, N. et al.
etal .: J. Biol. Chem., 271: 21446-21452, 1996), midkine also binds strongly to PTPζ. On the other hand, tyrosine phosphorylation as a receptor for midkine, JAK (JanusKi
nase) A protein with a molecular weight of 200 kDa + that binds tyrosine kinase has also been reported (Patovitski, E. et al .:
J. Biol. Chem., 273: 3654-3660, 1998). Midkine also antagonizes the action of the TGF-β superfamily (Kojima, S. et al., J. Biol. Chem., 270: 9590-95).
96, 1995; Mitsiadis, T. et al .: J. Cell Biol., 129: 26
7-281, 1995). However, the full extent of the receptor on midkine target cells has not been determined to date. Some of the data reported so far are not reproducible. Midkine and pleiotrophin have unusually high nonspecific binding, making isolation and purification of their receptors difficult.

[0007] The diverse biological activities of midkine are defined by receptors in target cells and signal transduction pathways. Searching for receptors at the tissue or cellular level is the first step in elucidating the mechanism of midkine's biological activity. It is also important for elucidation of diseases related to the receptor abnormality and for clinical application.

[0008]

Accordingly, an object of the present invention is to isolate and identify a midkine receptor. Another object of the present invention is to provide antibodies, agonists, and antagonists to the receptor.

[0009]

SUMMARY OF THE INVENTION In the absence of antibodies to the receptor, crosslinking experiments are often performed to identify membrane protein molecules that interact with the ligand. That is, after binding a radiolabeled ligand to a receptor, disuccinimidyl suberate (disuccinimidyl suberate:
The two are covalently cross-linked with a cross-linking agent such as DSS), and the resulting radiolabeled ligand-receptor complex is analyzed by autoradiography after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The number and molecular weight of the types of molecules that react with the ligand, which cannot be obtained in the ligand binding experiment, are estimated by the crosslinking experiment. Through this experiment, another IL-2 binding protein, β chain, which differs from the previously known α chain (Tac antigen), was identified, and the starting point for elucidating the molecular mechanism of affinity regulation of the IL-2 receptor was identified. (Tsudo,
M. et al .: Proc. Natl. Acad. Sci. USA ,: 83:96
94, 1986; Teshigawara, H. et al .: J. Exp. Med., 16
5: 223, 1987).

[0010] As described above, the receptor for midkine has not been identified so far, and there is no antibody against it. Therefore, the present inventors have proposed that midkine or a half molecule of the C-terminal side of the midkine molecule (MK C-ha
lf; Asp at position 121 from Ala at position 60 in the midkine molecule
(Muramatsu, H. et al .: Biochem. Bioph
ys. Rea. Commun., 203: 1131-1139, 1994) to identify cell membrane protein molecules that specifically bind. Here, MK C-ha-lf was used because strong heparin binding ability was in the MK C-half region (Muramatsu, H. et a
l .: Biochem. Biophys. Rea. Commun., 203: 1131-113
9, 1994), and a site exhibiting a neurite outgrowth ability and an action of enhancing placminogen activator activity is also in this region (Kojima, S. et al .: Biochem. Biophys. R
ea. Commun., 206: 468-473, 1995). Furthermore, a cross-linking experiment was also performed on pleiotrophin to identify membrane protein molecules on the cell membrane.

As a result, as shown in FIG. 1 (a), ¹²⁵ I midkine or ¹²⁵ I pleiotrophin and an affinity cross-linked product of a membrane protein were subjected to auto-crosslinking after 4-20% density gradient gel SDS-PAGE. When analyzed by radiography, in each case, a band having a molecular weight of 400 kDa + indicating the crosslinked product was observed (lanes 1 and 2). On the other hand, ¹²⁵ I midkine, ¹²⁵ I MK C-half, or ¹²⁵ I pleiotrophin and affinity cross-linked membrane protein
After immunoprecipitation with an anti-midkine antibody, 4-20% density gradient gel SDS-PAGE was performed and analyzed by autoradiography. As shown in FIG. 1 (b), the crosslinked product of midkine and MK C-half was: In each case, a band having a molecular weight of 400 kDa + was observed (lanes 2 and 3), but a band of a crosslinked product of pleiotrophin was not observed. ^{In 125} I midkine alone (lane 1), a band was observed at a molecular weight of 13 kDa. These results revealed that the binding of midkine to the membrane protein molecule was specific, and that this protein molecule also bound to pleiotrophin.

[0012] It is known that a neurotransmitter such as a hormone, after binding to a receptor on a cell membrane, is taken up inside the cell as a complex of the hormone and the receptor. Such a phenomenon is called receptor-dependent endocytosis (Biochemical Dictionary 3rd Edition, 1998, Tokyo Chemical Doujin).
Therefore, the present inventors investigated from two aspects whether or not ¹²⁵ I midkine and the membrane protein were transported as a complex after binding to the cells. After binding of ¹²⁵ I midkine to the membrane protein molecule, transfer to 37 ° C. (the ligand bound to the receptor is rapidly transferred into the cell at 37 ° C.), then 0, 10, 30, 60, and 120 After a lapse time of minutes, it is cross-linked and immediately 4-20%
The density gradient gel was subjected to SDS-PAGE and analyzed by autoradiography. As a result, as shown in FIG. 2, it was observed that the band of 400 kDa + peaked at 10 minutes, gradually diminished at 30 and 60 minutes, and disappeared at 120 minutes.
That is, the complex of midkine and membrane protein is
It was presumed that it was taken into cells.

On the other hand, in order to observe this phenomenon from the inside of the cells, ¹²⁵ I midkine is incubated with the cells, and after the cells are treated with trypsin, the midkine on the cell surface is digested and the cells are lysed. According to the analysis of midkine incorporated into the cells, as shown in FIG. 3, the complex of midkine and the membrane protein was immediately incorporated into the cells (within 30 minutes) after the incubation, and then gradually It was observed that it decomposed within. From the above results, it has been clarified that midkine is formed into a complex with a cell membrane protein molecule and then transferred into a cell depending on the cell membrane protein molecule.

Heparin-binding growth factor binds to heparan sulfate, which is a sugar chain portion of heparan sulfate proteoglycan on the cell surface or extracellular matrix.
Such binding is not only involved in growth factor storage and release, but also in activation of growth factor receptors (Schlessinger, J. et al .: Cell, 83: 357-36).
0, 1995). In fact, midkine is syndecan-1 (Mits
iadis, TA et al .: Development, 121: 37-51, 199
5; Nakanishi, T. et al: J. Biochem., 121: 197-20
5, 1997), Syndecan-3 (Nakanishi, T. et al: J.
Biochem., 121: 197-205, 1997), Rudokan-4 (Ko
jima, T. et al: J. Biol. Chem., 271: 5914-5920,
1996), has been found to bind strongly to heparan sulfate proteoglycans belonging to the syndecan family.

Therefore, the present inventors determined whether the binding of midkine to a membrane protein molecule was inhibited by heparin, in the presence and absence of heparin.
After reacting the cells with ¹²⁵ I midkine and cross-linking,
0% density gradient gel was subjected to SDS-PAGE and examined by autoradiography. As a result, in the absence of heparin, a band of a molecular weight of 400 kDa +, which is observed as a complex of midkine and a membrane protein molecule (1 and 2 in FIG. 4)
However, it was not observed in the presence of heparin (lane 4 in FIG. 4). The complex of midkine and membrane protein was not inhibited by the subsequent addition of heparin (lane 3). On the cell membrane, it is thought that there are two types of receptors, a high-affinity receptor for midkine (midkine receptor with a molecular weight of 400 kDa +) and a low-affinity receptor, and heparin binds to the first-stage low-affinity receptor. It is thought to inhibit binding.

A midkine affinity column in which an extract of cultured cells labeled with ³⁵ S cysteine-methionine is immobilized on a support such as a particulate hydrophilic gel using midkine as a ligand and used as a carrier for affinity chromatography. , The eluate of the protein adsorbed on the column was collected, and a 4 to 20% density gradient SDS-PA
When analyzed by autoradiography after GE, as shown in FIG. 5, a band having a molecular weight of 400 kDa +, which is considered to be a membrane protein binding to midkine, was observed. The fraction of this band and the midkine / membrane protein complex were analyzed by autoradiography after 4-20% density gradient SDS-PAGE, and as shown in FIG. 7, the bands (molecular weight 400 kDa +) were found at almost the same position as shown in FIG. Was observed.

That is, the present inventors searched for a cell membrane protein molecule that specifically binds to radioactive iodine-labeled midkine by a cross-linking experiment. The present inventors have found a cell membrane protein molecule (midkine receptor) having a molecular weight of 400 kDa + and exhibiting a phenomenon (receptor-dependent endocytosis) of being taken into cells as a complex of the present invention, thereby completing the present invention.

As described above, midkine has been reported to be associated with diseases such as cancer, and the midkine receptor protein discovered by the present inventors is a drug candidate for these diseases. It can be used as a tool for screening compounds. Also,
Agonists and antagonists for the midkine receptor are expected to be used for treating these diseases.

Therefore, the present invention more specifically
(1) a midkine receptor having the following characteristics: (a)
Binds to midkine and pleiotrophin;
(B) binding to midkine is inhibited in the presence of heparin; (c) after binding to midkine, it is taken up inside the cell as a complex with midkine and then gradually degraded; ( 2) a DNA encoding the midkine receptor according to (1), (3) a vector containing the DNA according to (2), (4) a transformant carrying the vector according to (3), 5) the method for producing a midkine receptor according to (1), which comprises a step of culturing the transformant according to (4); (6) an isolated antibody that specifically binds to a midkine receptor; (7) A method for identifying a compound that inhibits the binding of midkine or pleiotrophin to a midkine receptor, comprising: (a) in the presence of a test compound, the midkine receptor according to (1) or The partial peptide and midka Down or contacting the pleiotrophin, measured (b) said midkine receptor protein or its partial peptide midkine or binding activity to pleiotrophin, then
(C) a method characterized by selecting a compound that reduces the binding activity of the midkine receptor protein or a partial peptide thereof to midkine or pleiotrophin; (8) identified by the method of (7) A compound that inhibits the binding of midkine or pleiotrophin to a midkine receptor, (9) a method for identifying an agonist of a midkine receptor, wherein (a) a cell that produces a midkine receptor and a candidate substance And contact
(B) determining whether the candidate substance generates a signal generated by activation of the midkine receptor, and then (c) selecting a compound that generates a signal generated by activation of the midkine receptor. (10) A method for identifying a midkine receptor agonist identified by the method of (9), (11) a method for identifying a midkine receptor antagonist, wherein (a) producing a midkine receptor Contacting the cells with midkine and the candidate substance; (b) determining whether the signal generated by activation of the midkine receptor is reduced in the presence of the candidate substance; (12) (11) a method characterized by selecting a compound that reduces a signal generated by activation.
(13) A method for treating a subject requiring enhancement of the activity or expression of a midkine receptor, which is identified by the method of (1), wherein a therapeutically effective amount of an agonist for the receptor is provided. (14) A method for treating a subject in need of inhibition of the activity or expression of a midkine receptor, which comprises administering to the subject a therapeutically effective amount of an antagonist against the receptor. (15)
An agonist of the midkine receptor as an active ingredient,
(16) An agent for treating a subject requiring enhancement of the activity or expression of a midkine receptor, (16) a method for inhibiting the activity or expression of a midkine receptor comprising an antagonist of the midkine receptor as an active ingredient. For treating a subject to be treated.

The following definitions are provided to facilitate understanding of terms frequently used in this specification. “Agonist” refers to a compound that binds to the midkine receptor of the present invention and enhances or prolongs the duration of various biological activities possessed by midkine. Agonists include proteins, peptides, nucleic acids, carbohydrates, or any other compounds that bind to the midkine receptor of the invention and modify the biological activity of midkine. An "antagonist" binds to the midkine receptor of the invention,
A compound that reduces the duration or amount of various biological activities possessed by midkine. Antagonists include proteins, peptides, nucleic acids, carbohydrates, or any other compounds that bind to the midkine receptor of the invention and modify the biological activity of midkine. "Midkine receptor" refers to a protein that binds to midkine and has receptor activity, or an allelic variant thereof.
"Receptor activity" or "biological activity of a receptor"
Refers to the metabolic or physiological function of the midkine receptor, and includes activities that have similar, or improved, or reduced undesirable side effects. Also included are the antigenic and immunogenic activities of the midkine receptor. "Antibodies" encompasses polyclonal or monoclonal antibodies, chimeric, Fv, and humanized antibodies, as well as Fab fragments, and also encompasses the products of Fab or other immunoglobulin expression libraries.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTIONPreparation of midkine receptor  The midkine receptor protein of the present invention comprises the receptor
Expressing cells, for example, Wilms' t
umor) cells G401, rat osteosarcoma cells UMR-106, rat
Extraction of vascular smooth muscle cells, mouse fetal nerve cells, L cells, etc.
Add the effluent to the Midkine affinity column,
The protein adsorbed on the column is eluted and the molecular weight is 400 kDa +
Can be obtained by further purification.
Wear. Affinityca with midkine as ligand
Lamb is a method described in the literature (eg, Hideyoshi Yokozawa, Shinichi Ishii "
Protein I "(Shinsei Kagaku Laboratory 1), Japan Biochemistry, p.21
4, Tokyo Chemical Dojin (1990)). Sand
For example, mature human midkine (see, for example, JP-A-9-65454)
The recombinant midkine obtained by the method described in the gazette)
Supports activated with cyanogen bromide, such as CNBr-Sepharo
Supports midkine by mixing with se4B gel
Create a midkine affinity column immobilized on the body
To make. This column expresses the midkine receptor
Cells, such as an extract of cultured L cells,
Biological material paired with immobilized midkine
Only certain membrane proteins can do this through specific interactions.
Adsorb to. Then, take appropriate action to eliminate this interaction.
Appropriate conditions, e.g., elution buffer (10 mM
Buffer (pH 4.6), 150 mM NaCl, 5 mM EDTA, 0.6% CHAPS)
Eluted from the column and eluted from the column, indicating a molecular weight of 400 kDa +.
The fraction of the deposited protein is purified by HPLC or the like.

A molecular weight of 400 kDa + as measured on a 4-20% density gradient SDS-polyacrylamide gel indicates that a protein band is detected around a position slightly larger than 400 kDa when electrophoresed with a molecular weight marker. The molecular weight of the protein is 400 kDa or more and 800 kDa or less.

The midkine receptor protein of the present invention can be prepared not only as a natural protein but also as a recombinant protein utilizing a genetic recombination technique. As described below, the recombinant protein is obtained by isolating a DNA encoding the midkine receptor protein of the present invention, incorporating the DNA into an appropriate expression vector, culturing cells transformed with the vector, and expressing the DNA. It can be prepared by recovering the protein that has been made.

For the cDNA encoding the midkine receptor protein of the present invention, for example, a partial amino acid sequence (eg, N-terminal sequence) of the purified midkine receptor protein of the present invention is obtained by the phenylisothiocyanate method (PITC method, Ed method).
one determined from the codon table,
Or more synthetic oligonucleotides, ³²
CDNA prepared from fetal mouse brain with P-labeled probe
It can be prepared by screening the A library. Once the cDNA is obtained, a genomic DN encoding the midkine receptor protein of the present invention is screened by screening the genomic library in the same manner.
A can also be prepared.

To prepare a recombinant protein, a DNA encoding the midkine receptor protein of the present invention is inserted into an appropriate expression vector, and the transformant obtained by introducing the vector into appropriate cells is obtained. And the expressed protein is purified. Escherichia coli, yeast, insect cells, animal cells, and the like can be considered as hosts in a host-vector system used for production of a recombinant protein, and the vector to be used is specified. Examples of the vector include pHIL-D1 and pHIL-D4 in yeast. Methods for introducing a vector into a host include biological methods, physical methods, and chemical methods. As a biological method, for example, a method using a viral vector,
A method using a specific receptor, a cell fusion method (HVJ (Sendai virus), polyethylene glycol (PEG), an electric cell fusion method, a micronucleus fusion method (chromosome transfer)) are exemplified. Examples of the physical method include a microinjection method, an electroporation method, and a method using a gene particle gun. Examples of the chemical method include a calcium phosphate precipitation method, a liposome method, a DEAE dextran method, a protoplast method, an erythrocyte ghost method, an erythrocyte membrane ghost method, and a microcapsule method. As a method for purifying the recombinant protein, a known method, for example, a method using an ion exchange column, an affinity column, or the like is used. Finally, it is preferable to purify through an affinity column on which the purified midkine antigen is immobilized.

[0026]Preparation of Antibody for Midkine Receptor  As a specific antibody against the midkine receptor of the present invention
Is a polyclonal or monoclonal antibody, human
Antibodies, and human antibodies. Midkine
Receptor, or a variant thereof, or expresses them
Cells as antigens, preferably adjuvanted to non-human animals.
By administering together with the antigen,
Can give rise to the body. Monoclonal antibodies are
Can be prepared using techniques well known to those skilled in the art.
(Kohler, et al., Nature 256: 495, 1975; Curret
Protocols in Molecular Biology, Ausubel, et al., E
d., 1989). Polymorphs for the midkine receptor of the invention
Clonal antibodies can also be prepared using methods known to those of skill in the art.
Kiru (Antibodies: A Laboratory Manual ed. ByHarlow
 and Lane (Cold Spring Harbor Laboratory Press: 198
8)). Continuous cells for preparation of monoclonal antibodies
Techniques for providing antibodies produced by line culture include:
Either can be used. Such techniques are, for example,
Hybridoma technology (Kohler, et al., Nature 256)
 : 495, 1975), Trioma technology, human B cell hybrid
Doma technology (Kozbor, et al., 1983, Immunology Today
 4:72), and EBV hybridoma technology (Cole, et.
 al., 1985, inMonoclonal Antibodies and Cancer The
rapy, Alan R. Liss, Inc., pp. 77-96).
You.

Recently, a phage display vector (Smith GP, Science, 228: 13) designed by Smith et al.
15, 1985), a peptide library or an antibody library expressed in the coat protein is screened with a ligand of interest, and a phage clone that binds to the ligand is selected using a purification method such as an affinity column. Collecting proteins displayed on the surface, or cloning genes encoding the proteins using the properties (phenotype) of the proteins as an index has been performed (Burt).
on DR et al .: Adv. Immunol., 57: 191, 1994; Winter
G. et al .: Ann. Rev. Immunol., 12: 433, 1994; O'N
eil KT et al .: Curr. Opin. Struct. Biol., 5:44
3, 1995). For example, a 12 amino acid long linear peptide (Ma) that binds to E-selectin and inhibits binding to neutrophils
rtens CL et al .: J. Biol. Chem., 270: 21129, 199
5), a 20-amino acid peptide having a loop structure of 8 amino acids in length that functions as an agonist by binding to the EPO receptor (Wrighton NC et al .: Science, 273: 458,
1996) or alternatively, a peptide that binds to the TPO receptor c-MPL was identified and chemically dimerized, and an agonist showing the same level of activity as natural TPO (Cwirla, S .;
E. et al .: Science, 276: 1696, 1997).

This method (phage display screening) can be used to obtain a humanized antibody, human antibody, agonist, antagonist or the like against the midkine receptor of the present invention or to modify their functions. ) Can be used. For example, a phage display library (Grif
fiths, AD et al .: EMBO J., 13: 3245, 1994)
If you want to obtain a human antibody against the midkine receptor, use ELIS with immobilized midkine receptor protein.
Contact the A plate, wash and elute. In the binding process, for example, a method of adsorbing a midkine receptor protein on a plastic surface such as an ELISA plate can be used. In addition to this method, various ideas will be helpful. For example, a method of recovering phage bound to a biotinylated target protein using a micromagnet linked to avidin (Hawkins, R. et al.
E. et al .: J. Bol. Biol., 226: 889, 1992) or targeting proteins expressed on the cell surface,
A method using cells themselves for binding to phage (Kruif,
J. et al .: Proc. Natl. Acad. Sci. USA., 92: 3938,
1995) can be applied to the present invention. In the elution process,
Since treatments with acids or alkalis are generally used, these can be tried in the present invention. If the desired phage is present only in a few clones in the library, it is very difficult to completely remove the phage having no binding ability in the washing step. for that purpose,
Infect E. coli with the phage obtained after the elution operation,
It is necessary to grow the cells and repeat the selection operation with the midkine receptor protein. The phages thus selected are individually grown in E. coli, and clones that specifically bind to the midkine receptor protein are identified by ELISA using an anti-M13 phage antibody. By repeating the above-mentioned operations such as binding, washing, elution, and proliferation, a human antibody that specifically binds to the midkine receptor protein can be separated from the antibody library.

[0029]Midkine receptor agonists and
Identification of antagonists  The midkine receptor protein of the present invention
Use to identify compounds that inhibit docaine binding
You. The identification is performed by (a) the method of the present invention in the presence of the test compound.
Docaine receptor protein or its partial peptide
And (b) the midkine of the present invention.
Receptor protein or its partial peptide and Midkai
The binding activity to the protein is measured, and then (c)
Cain receptor protein or its partial peptide
Those who select compounds that reduce the binding activity to docaine
Method. Also, to Midkine
Perform the same experiment using pleiotrophin instead
For example, the midkine receptor protein of the present invention
To identify compounds that inhibit binding to trophin
it can.

The binding experiments (b
The inding assay is performed by those skilled in the art according to methods described in the literature, for example, “" Receptor properties and measurement "growth factor and its receptor (Neochemical Experiment Lecture 7), edited by The Japanese Biochemical Society, p236-26.
7, Tokyo Chemical Dojin, (1991) '', `` Schumacher, R. et al .:
J. Biol. Chem. 266: 19288-19295, 1991 "and the like. The principle is to use drugs labeled with radioisotopes (radioisotope) to elucidate the mode of binding to receptors contained in single cells, homogenates, or membrane fractions obtained from tissues. . As another technique, a technique described in WO 84/03564 can be used. The midkine receptor protein or its partial peptide used may be free in solution, bound to a solid support, present on a cell surface, or present in a cell. Good. The test compound includes, but is not limited to, a protein as a cell extract, a purified protein or peptide, an artificially synthesized low molecular compound, and the like. Compounds isolated by this include, for example, agonists and antagonists that bind to receptors. In binding experiments, these are often collectively referred to as ligands. Further, the midkine receptor protein of the present invention can be used for identifying an agonist or antagonist binding thereto.

The agonist of the present invention can be identified by (a) contacting a cell producing a midkine receptor with a candidate substance, and (b) generating a signal generated by activation of the midkine receptor. And then (c) selecting a compound that generates a signal generated by activation of the midkine receptor. The identification of the antagonist of the present invention includes (a) contacting a cell producing a midkine receptor with a midkine and a candidate substance, and (b) a signal generated by activation of the midkine receptor generates a signal of the candidate substance. It can be carried out by determining whether it is reduced in the presence and then (c) selecting a compound that reduces the signal generated by activation of the midkine receptor. Examples of the candidate compound include a protein as a cell extract, a purified protein, or a peptide, an artificially synthesized low-molecular compound, and the like.
Not limited to these. As the candidate compound, a compound identified by the above-described method for identifying a compound that inhibits the binding between midkine receptor and midkine can also be used. In yet another embodiment, neutralizing antibodies capable of binding to the midkine receptor can be used in competitive drug screening assays. According to this method, the antibody can detect the presence of any peptide that shares one or more antigenic determinants with the midkine receptor.

[0032]Application to treatment  Midkine is derived from mammals, including humans,
It has been found all the way down to the frog
Protein is highly preserved in humans and mice
In Cain, there is 87% (amino acid level) homology (T
sutsui, J. etal .: Biochem. Biophys. Res. Commun.,
 176: 792-797, 1991). In addition, midkine is
As mentioned above, lesions of various diseases (cancer,
Expression is enhanced in Zheimer's disease, rheumatoid arthritis, etc.)
Has no reparative effect, but promotes malignancy of the disease image
it seems to do.

Accordingly, the present invention provides antagonists to the midkine receptor for reducing the activity of midkine. The antagonist can be administered to a patient to prevent or treat the above-mentioned diseases associated with midkine, especially cancer. Such cancers include colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, etc., in which the expression of midkine is observed in 80 to 90% of cases (Aridome, K. et al .: Jpn. J. Cancer Res. ., 655-661,
1995), lung cancer (Garver, RI et al .: Am. J. Respi
r. Cell Mol. Biol., 9: 463-466, 1993), breast cancer (Gar
ver, RI et al .: Cancer, 74: 1574-1590, 199
4) In addition, patients with high expression of midkine have a worse prognosis than patients with weaker expression of neuroblastoma, glioma, and bladder cancer (Nakagawara, A. et al .: Cancer Res., 5).
5: 1792-1797, 1995; Mishima, K. etal .: Neurosci,
Lett., 233: 29-32, 1997; O'Brien, D. et al .: Ca
ncer Res., 56: 2515-2518, 1996), etc., but these are only examples and do not limit the present invention.

From another viewpoint, an antibody that specifically binds to the midkine receptor can be used as an antagonist to cells and tissues expressing the midkine receptor directly as an antagonist.
Alternatively, it can be used indirectly as a delivery system for delivering drugs. Also, agonists that modify the activity of the midkine receptor in the present invention,
It can be administered to a patient for the prevention or treatment of a neurological disease, in particular, the neurological disease described above.

From another viewpoint, the midkine receptor of the present invention can be administered in combination with a therapeutic protein, antagonist, antibody, agonist or other appropriate therapeutic agent. Selection of the appropriate agent for use in combination therapy is possible according to normal pharmaceutical principles.
The combination of therapeutic agents can be expected to work synergistically (enhancing) for the treatment or prevention of the various diseases described above. Using this approach, it is possible to achieve therapeutic effects with lower dosages of each drug, and hopefully reduce side effects.

Midkine antagonists or inhibitors can be prepared using methods known to those skilled in the art. In particular, purified midkine can be used to produce antibodies or to screen a library of drugs to identify substances that specifically bind to midkine.

When a pharmaceutical composition is prepared using the agonist or antagonist of the midkine receptor protein of the present invention as an active ingredient and used as a preventive or therapeutic agent for a disease associated with the receptor protein, a known pharmaceutical production method is used. Thus, it can be formulated and administered. For example, a suitable carrier or vehicle commonly used as a drug,
For example, sterile water or saline, vegetable oils (eg, sesame oil, olive oil, etc.), coloring agents, emulsifiers (eg, cholesterol),
Suspension (eg, gum arabic), surfactant (eg, polyoxyethylene hydrogenated castor oil-based surfactant), solubilizer (eg, sodium phosphate), stabilizer (eg, sugar, sugar alcohol, albumin) ) Or preservatives (parabens) as appropriate, and pharmaceutical preparations suitable for effective administration to living bodies, such as injections, nasal absorbents, transdermal absorbents, and oral preparations, preferably injections Can be prepared. For example, injection preparations can be provided in the form of a lyophilized product, a liquid preparation for injection, or a form enclosed in an osmotic pump.

[0038]Application to diagnosis  In another embodiment, it specifically binds to the midkine receptor.
Antibodies that are characterized by expression of the receptor
Condition or disease, or
Monitors patients treated with agonists and antagonists.
Can be used in assays for monitoring. Diagnosis
Antibodies useful for the purpose are produced in the same manner as described above.
Can be Antibodies may be used modified or unmodified
Can be covalently or non-covalently linked with the reporter molecule.
Label by linking them with any of the covalent bonds
May be. ELIS for measuring midkine receptors
A, RIA, and FACS are known to those skilled in the art and
Diagnose mutated or abnormal expression of in receptors
Provide a way to: Normal or standard mid
The expression level of the cytokine receptor is determined under conditions suitable for complex formation.
In addition, body fluid obtained from normal mammals, or cell extraction
The antibody to the midkine receptor.
And is established by. Patients, controls, and biopsies
-Amount of midkine receptor expressed in tissue samples
Is compared to a standard value.

[0039]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.

[Example 1] Identification of midkine receptor (1) ^{125 of} midkine and pleiotrophin
I Labeling preparation Midkine and pleiotrophin are chloramine T
(Chloramine T) -labeled with ¹²⁵ I. For midkine, recombinant mature human midkine obtained by the method described in JP-A-9-65454 was used (the same applies to midkine in the following examples). 4.7 μg midkine, 90 μL 50 mM
Sodium phosphate buffer solution (pH 7.5), and 10 μL of Na
¹²⁵ I (1 mCi) was placed in a 1.5 mL Eppendorf tube, 50 μL of chloramine T prepared at a concentration of 3 mg / mL in a 50 mM sodium phosphate buffer solution was added, and the mixture was reacted at 4 ° C. for 1 minute. Further, 50 μL of sodium pyrosulfite adjusted to a concentration of 7.5 mg / mL with a 50 mM sodium phosphate buffer solution was added to the tube, and the mixture was reacted at room temperature for 15 seconds. Finally, 100 μL of a 150 mM KI solution was added. PD-10 (Pharmacia)
Using a column, ¹²⁵ I midkine and free ¹²⁵ I were fractionated. Elution was performed with a 75 mM NaCl solution containing 1 mg / mL BSA. Fractionation was performed for each 500 μL, and fractions near the peak were stored. The specific activity of ¹²⁵ I midkine is 2-4 x 10 ⁷ c
pm / μg. Pleiotrophin was labeled with ¹²⁵ I in the same manner as for midkine.

(2) Cross-linking experiment L cells or C were placed in a dish (10 cmφ) (FALCON, 3003).
OS cells were cultured until they reached approximately confluency (5 × 10 ⁶ -1 × 10 ⁷ cells / dish). Transfer cells to PBS / B
Washed three times with BSA [PBS / B 500 mL, BSA 0.5 g]. Subsequently, 10% FBS / DMEM 5mL, 125 I midkine 50 to each cell
μg / dish (10 cmφ) was added. Incubated at 4 ° C for 2 hours. Thereafter, the operation was continued at 4 ° C. P
Cells were washed three times with BS / B BSA and once with PBS / B. 5 mL of PBS / B was added to the dish. Next, as a crosslinker
68 μL of DSS (20 mM) (final concentration: 0.27 mM) was added and reacted at 4 ° C. for 15 minutes. Reaction stop solution (10 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 10% glyserol, 0.3 mM PMSF (prepared for use)]. Further, 1 mL of a reaction stop solution was added. The cells stuck to the culture dish were peeled off with a scraper and collected in an Eppendorf tube. Centrifuge at 12000-15000 rpm for 2 minutes,
Cells were collected. Lysis buffer in the collected cell pellet
[1% Triton X-100, 1% deoxycholate, 20mM Tris-HCl (p
H7.5), 150mM NaCl, 1mM EDTA (pH7.5), 0.1% aprotini
n, 1 mM PMSF (business preparation)] was added. The cells were solubilized by pipetting well. It was left on ice for 20 minutes. After the reaction, the mixture was centrifuged at 12,000 to 15000 rpm for 10 minutes. After centrifugation, transfer 40 μL of the supernatant to a new tube, and add 2.5 mL of sample buffer [0.5 M Tris-HCl (pH6.8), 4.0 mL of 10% SDS,
0.1% bromophenol blue 0.5mL, glycerol 1
2.0 mL, β-mercaptoethanol 0.5 mL, H ₂ O 10 mL] 40
μl was added. 4-20% density gradient gel After SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie staining was performed. ^A similar experiment was performed for pleiotrophin labeled with ¹²⁵ I. The result detected by autoradiography is shown in FIG. In each of the pleiotrophin in lane 1 and the midkine in lane 2, a band that appears to be a complex with a membrane protein molecule is present.
It is observed at a position where the molecular weight is larger than 400 kDa.

Example 2 Crosslinking Experiment Using MK C-half and Pleiotrophin (PTN) According to the method of Example 1, midkine and midkine were synthesized.
C-terminal partial peptide (MK C-half) (PEPTIDE INSTITUT
E, INC.) And pleiotrophin (PTN, R & D) were labeled with radioactive iodine, and crosslinking experiments were performed. Thereafter, an anti-midkine antibody and protein A Sepharose CL-4B were added, and immunoprecipitation was performed. The sediment is subjected to a 4-20% density gradient SDS-
PAGE and detection by autoradiography. The results are shown in FIG. In both midkine in lane 2 and MK C-half in lane 3, a band that appears to be a complex with a membrane protein molecule is observed at a position larger than 400 kDa in molecular weight. In the pleiotrophin in lane 4, the band observed when the anti-midkine antibody shown in Example 1 was not used disappeared. Lane 1 shows the case where only ¹²⁵ I midkine was subjected to SDS-PAGE. From these results, it is clear that both MK C-half and pleiotrophin, like midkine, bind to the same membrane protein molecule.

Example 3 Internalization (Inwardization) Experiment After binding of midkine to a membrane protein molecule, it was examined whether or not it was taken into cells as a complex of midkine and the membrane protein molecule. . For a 10cm dish,
L cells cultured to near confluency (5 × 10 ⁶ -1 × 10 ⁷ cells / dish) were added to ¹²⁵ I midkine
50 μg / dish was added and reacted at 4 ° C. for 2 hours. After the reaction, the cells were washed with PBS / B BSA. DMEM containing 10% FBS was added, the cells were transferred to 37 ° C, and then 0 minutes, 10 minutes, 30 minutes, 60 minutes.
After a lapse of 120 minutes, the same crosslinking experiment as in Example 2 was performed. 4-20% density gradient After SDS-PAGE, detection was performed by autoradiography. The results are shown in FIG. From the peak at 10 minutes, it is observed that the band of the membrane protein molecule (400 kDa +) detected in Examples 1 and 2 becomes thinner as time passes. From this result, it is assumed that the complex of midkine and the membrane protein molecule is taken up into cells. Furthermore, in order to observe this phenomenon from within the cells, ¹²⁵ I midkine and L cells were incubated at 4 ° C.
The reaction was performed for 4 hours. After washing the cells with PBS, the cells are
Moved to After 0, 30, 60 and 120 minutes, the cells were trypsinized to digest midkine on the cell surface. The cells were lysed, and midkine incorporated into the cells was analyzed by autoradiography after 15% SDS-PAGE. The result is shown in FIG. From this result, it is observed that the complex of midkine and the membrane protein molecule is taken up into the cell immediately after the reaction (within 30 minutes), and then gradually degraded. From the above results, it is clear that after binding to the membrane protein molecule, midkine is taken into the cell as a complex of midkine and the membrane protein molecule, and then degraded.

Example 4 Experiment of Inhibition of Binding by Heparin Whether or not the binding between midkine and a membrane protein molecule was inhibited by heparin was examined. Add ¹²⁵ I midkine and 10 μg / mL sodium heparin (Nacalai) to L cells cultured in a 10 cm dish until they are almost confluent (5 × 10 ⁶ to 1 × 10 ⁷ cells / dish). After reacting for an hour, crosslinking was carried out in the same manner as described above (lane 4 in FIG. 4). Also, for comparison, L cells
^After adding ¹²⁵ I midkine and reacting at 4 ° C for 2 hours, washing was performed, and 10 μg / mL sodium heparin was added.
After reacting at 2 ° C. for 2 hours, crosslinking was carried out in the same manner as described above (FIG. 4).
Lane 3). As a control, a system to which heparin was not added (lanes 1 and 2 in FIG. 4) was set. Lanes 1 and 2
In the figure, a band which appears to be a complex of midkine and a membrane protein molecule is observed at a position larger than the molecular weight of 400 kDa. A similar band is observed in lane 3. No corresponding band is observed in lane 4. From these results, it is clear that the binding of midkine to membrane protein molecules is inhibited by the presence of heparin.

Example 5 Preparation of Midkine Affinity Column Midkine 5 mg was added to 170 μL of a coupling buffer (0.
0.1 M NaHCO ₃ containing 5M NaCl, dissolved in pH 8.3), the 0.5mL
Add CNBr-Sepharose 4B gel (Pharmacia) and add 4
Mix by rotator at ° C. After 12 hours, the gel was washed with 2.5 mL of coupling buffer. 0.1M gel
It was added to a Tris-HCl buffer (pH 8.0) and reacted at 4 ° C. for 2 hours. Further, the gel was washed three times alternately with 2.5 mL of 0.1 M acetate buffer (pH 4.0) containing 0.5 M NaCl and 2.5 m of 0.1 M Tris-HCl buffer (pH 8) containing 0.5 M NaCl, A midkine affinity column was prepared. Similarly, a BSA affinity column was prepared.

Example 6 Isolation of Membrane Protein Molecules
Purified 10cm dish (FALCON, 3003)³⁵S cysteine-meth
L-cells labeled with ionine (metabolic label)
Confluent (5x10 per dish⁶~ Lx10⁷ cell)
Until it reached. Paste on culture dish with scraper
Remove the cells and place in an Eppendorf tube.
Was. Centrifugation was performed at 4 ° C and 12000-15000 rpm for 2 minutes. Discard the supernatant
And 1 mL of equilibration buffer (20 mM Tris-HCl (pH 7.5), 150
mM NaCl, 2mH CaCl_Two, 1mM MgCl_Two, 1mM PMSF, 1mM sodium
 vanadate, 0.1% aprotinin, 10μg / ml leupeptin)
In addition, pipette 10 times with a 26G syringe
Destroyed. Centrifugation was performed at 4 ° C. and 12000 rpm for 20 minutes. Supernatant
Discard and add 0.5 mL of the above equilibration buffer containing 1% Triton.
And suspended at 4 ° C. for 20 minutes. 20 at 4 ℃, 12000rpm
Centrifuge for minutes. Equilibration with 1% Triton in advance
Midkine-affinity equilibrated with buffer
Injected into the column (0.5 mL). Inject the eluate into the column
Repeat twice to allow the protein to adsorb to the column
Was. 10 mL of buffer (10 mM sodium phosphate buffer (pH
7.4), 150mM NaCl, 2mM CaCl_Two, 0.6% CHAPS)
Was washed. Subsequently, the elution buffer (10 mM sodium
Acid buffer (pH 4.6), 150 mM NaCl, 5 mM EDTA, 0.6% CH
APS) elutes the protein adsorbed on the column
Was. Similarly, experiments using BSA affinity columns
went. Aliquot the eluate by 0.5 mL and perform SDS-PAGE.
The analysis of the adsorbed protein was performed. The results are shown in FIG.
6 and FIG. Fractions 3 and 4 and Example 1
Of midkine and membrane protein molecule obtained in step
And the position of the band are almost the same, but the cross link is
Slightly high molecular weight. This is due to the cross link
It is thought that the molecular weight increased by the amount of midkine
(FIG. 7).

[0047]

According to the present invention, a midkine receptor has been identified and isolated. Further, using the receptor protein,
Methods for screening for antibodies, agonists, and antagonists to the receptor have been provided. This will enable the development of drugs useful for the treatment and prevention of diseases associated with midkine receptor abnormalities, such as new cancer drugs, cancer metastasis preventive drugs, anti-inflammatory drugs, and Alzheimer's disease drugs. .

[Brief description of the drawings]

1 (a) is a diagram showing the ¹²⁵ I midkine and ¹²⁵ I play Override by neurotrophin after 4-20% density gradient gel SDS-PAGE of L cells or affinity cross-linking of COS cells autoradiographic galley fees is there. Lane 1 is pleiotrophin-midkine receptor complex, lane 2
Indicates a midkine / midkine receptor complex. (B) ¹²⁵ I midkine, ¹²⁵ I MK C-half,
FIG. 4 is a diagram showing autoradiography of an affinity cross-linked product of L cells with ¹²⁵ I pleiotrophin after immunoprecipitation with an anti-midkine antibody and 4-20% density gradient gel SDS-PAGE of the precipitate. Lane 1 is ¹²⁵ I midkine alone, lane 2 is midkine / midkine receptor complex, lane 3 is MK C-half / midkine receptor complex, lane 4 is pleiotrophin / midkine receptor complex Is shown.

FIG. 2 is an autoradiograph showing one aspect of midkine receptor-dependent endocytosis. Lanes 1-5 show ¹²⁵ I midkine and cultured L cells at 4 ° C.
After reacting for 2 hours, the cells were transferred to 37 ° C, and then 0, 10,
Autoradiography after cross-linking at 30, 60, and 120 minutes and 4-20% density gradient gel SDS-PAGE.

FIG. 3 is also an autoradiography showing one aspect of midkine receptor-dependent endocytosis. After reacting ¹²⁵ I midkine with cultured L cells at 4 ° C for 4 hours, the cells were transferred to 37 ° C, and then the midkine on the cell surface was treated with trypsin, analyzed by SDS-PAGE and analyzed by autoradiography. FIG. Lane 1
4 shows the amount of the midkine / midkine receptor complex present in the cells after 0, 30, 60, and 120 minutes, respectively.

FIG. 4. ¹²⁵ I in the presence or absence of heparin
4 shows the autoradiography of affinity cross-linked products of cultured L cells by midkine after 4 to 20% density gradient gel SDS-PAGE. Lanes 1 and 2 are in the absence of heparin, lane 3 is L-cells, first added ¹²⁵ I midkine, allowed to react at 4 ° C for 2 hours, washed, added 10 μg / mL sodium heparin, and further added at 4 ° C. Reaction, followed by crosslinking (adding heparin on the way).
The results of crosslinking (in the presence of heparin) after adding ¹²⁵ I midkine and 10 μg / mL sodium heparin and reacting at 4 ° C. for 2 hours are shown.

FIG. 5. Labeling of L cells with ³⁵ S cysteine-methionine (me
tabolic label) and dissolve the resulting protein,
Midkine column (MK column) or BSA column (0.5
of the eluate, 0.25 mL of the eluate was collected, and this was autoradiography after 4-20% density gradient gel SDS-PAGE. A band likely to be a midkine receptor is observed only in the eluate of the midkine column.

FIG. 6: A protein obtained by lysing L cells is injected into a midkine column in the same manner as in FIG. 5 except that the L cells are not labeled with ³⁵ S cysteine-methionine, and
The figure shows that 0.25 mL of the gel was collected and subjected to 4% SDS-PAGE, followed by silver staining of the gel.

FIG. 7 shows autoradiography after midkine / midkine receptor complex and the eluate of the midkine affinity column after 4% SDS-PAGE of fractions 3 and 4 (FIG. 5). Lane 1 is the midkine / midkine receptor complex, and lane 2 is the fractions 3 and 4 of the midkine column in FIG.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 45/00 A61K 45/00 4H045 C07K 14/705 C07K 14/705 16/30 16/30 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 5/10 C12P 21/00 C 15/02 21/08 C12P 21/00 G01N 33/15 Z 21/08 33/50 Z G01N 33/15 33/566 33/50 C12N 5/00 A 33/566 B 15/00 C (72) Inventor Takashi Muramatsu 2845-161 Black Stone, Daijiro-cho, Tenpaku-cho, Tenpaku-ku, Nagoya-shi, Aichi Prefecture 540 Narita, Odawara-shi, Meiji Meiji Dairies Co., Ltd.Cell Engineering Center Co., Ltd. (72) Inventor Munehiro Oda 540, Narita, Odawara-shi, Kanagawa Prefecture Meiji Dairies Co., Ltd. address Meiji Dairies Co., Ltd. Cell Engineering Center F-term (reference) 2G045 AA34 AA35 AA40 CB01 CB21 DA12 DA13 DA14 DA36 FB03 4B024 AA11 BA63 CA04 DA02 DA03 DA05 EA04 GA11 4B064 AG20 AG26 CA19 CC24 DA13 4B065 AA26CA24 4C084 AA17 ZB261 4H045 AA10 AA11 BA10 CA40 DA50 DA75 EA20 EA50 FA72 FA74

Claims (16)

[Claims] 1. A midkine receptor having the following characteristics. (A) binds to midkine and pleiotrophin; (b) binds to midkine in the presence of heparin; (c) binds to midkine and then binds to midkine as a complex with midkine inside the cell Is taken up in water and then gradually decomposed; 2. A DNA encoding the midkine receptor according to claim 1. 3. A vector containing the DNA according to claim 2. A transformant carrying the vector according to claim 3. 5. The method for producing a midkine receptor according to claim 1, comprising a step of culturing the transformant according to claim 4. 6. An isolated antibody that specifically binds to the midkine receptor. 7. A method for identifying a compound that inhibits the binding of midkine or pleiotrophin to a midkine receptor, wherein (a) in the presence of a test compound,
Contacting midkine or a partial peptide thereof according to the above with midkine or pleiotrophin,
(B) measuring the binding activity between the midkine receptor protein or its partial peptide and midkine or pleiotrophin; and then (c) measuring the binding activity of the midkine receptor protein or its partial peptide with midkine or pleiotrophin A method comprising selecting a compound that reduces the binding activity to a compound. 8. A compound identified by the method of claim 7, which inhibits binding of midkine or pleiotrophin to a midkine receptor. 9. A method for identifying an agonist of a midkine receptor, comprising: (a) bringing a cell that produces a midkine receptor into contact with a candidate substance; and (b) the activity of the midkine receptor as a candidate substance. Determining whether a signal generated by the activation is generated, and then (c) selecting a compound that generates a signal generated by activation of the midkine receptor. 10. An agonist of the midkine receptor identified by the method of claim 9. 11. A method for identifying a midkine receptor antagonist, comprising: (a) contacting a cell that produces a midkine receptor with midkine and a candidate substance;
(B) measuring whether the signal generated by activation of the midkine receptor is reduced in the presence of the candidate substance, and then (c) selecting a compound that reduces the signal generated by activation of the midkine receptor. A method comprising: 12. Identified by the method of claim 11,
Midkine receptor antagonist. 13. A method of treating a subject in need of enhanced midkine receptor activity or expression, comprising administering to the subject a therapeutically effective amount of an agonist for the receptor. 14. A method of treating a subject in need of inhibition of midkine receptor activity or expression, comprising administering to the subject a therapeutically effective amount of an antagonist to said receptor. 15. An agent for treating a subject in need of enhanced midkine receptor activity or expression, comprising a midkine receptor agonist as an active ingredient. 16. An agent for treating a subject in need of inhibition of midkine receptor activity or expression, comprising an antagonist of midkine receptor as an active ingredient.