Novel polypeptide and its DNA

Abstract

It is intended to construct a screening method, etc. for searching for compounds which are capable of binding to apolipoprotein A-I and thus promote or inhibit the binding of a cubulin fragment to apolipoprotein A-I, and provide preventives or remedies containing compounds obtained by the above screening method, etc. These compounds, etc. are usable in preventives and remedies for various diseases in which cubulin participates and the like.

Description

TECHNICAL FIELD

The present invention relates to partial peptides of cubilin and their functions.

BACKGROUND ART

The level of HDL-cholesterol [cholesterol contained in HDL (high density lipoprotein); hereinafter, sometimes abbreviated to “HDL-C”] is known as a negative risk factor in coronary artery diseases. Various drugs has been developed for the purpose of raising that level, but thus developed drugs, such as fibrate type drugs, are not sufficient though they raise HDL-C levels significantly. Besides, such rise in HDL-C levels represents a consequential factor. Recent studies using transgenic mice, etc. have revealed that, as long as the true purpose is to extract cholesterol from foci of arteriosclerosis, what is needed is technology to raise the level of apolipoprotein A-I (hereinafter, sometimes abbreviated to “apo A-I”) that plays a major role in the cholesterol extraction.

Apo A-I in plasma exists for the most part as HDL in the form of complexes with phospholipid, cholesterol, cholesterol ester and so on. These complexes are catabolized mainly in the liver. However, it is known that in some diseases such as hypertriglyceridemia, HDL particles with increased triglyceride (hereinafter, sometimes abbreviated to “TG”) contents are converted into smaller apo A-I particles by various lipases and then catabolized mainly in the kidney. The catabolism in the kidney is achieved as follows: small particles of apo A-I that have been filtered through the uriniferous tubule are re-absorbed in the proximal tubule and then degraded. Kozyraki et al. have been reported that cubilin (GenBank Accession No. AF034611) molecules expressed on the uriniferous tubule epithelium and known to bind to vitamin B₁₂-intrinsic factor complex and LDL receptor-related protein (LRP)-associated protein (hereinafter, sometimes abbreviated to “RAP”) also bind to apo A-I (see, for example, Nature Medicine Vol. 5, No. 6, 656-661 (1999); Proc. Natl. Acad. Sci. USA, Vol. 96, 10158-10163 (1999)). However, relations between the binding of cubilin to apo A-I and plasma apo A-I or HDL concentration, or relations between this binding and arteriosclerosis have not yet been elucidated.

The binding of cubilin to apo A-I may be an important finding for the development of therapeutics for hypo-high density lipoproteinemia, which is a problem in diseases such as high hypertriglyceridemia, and for the development of drugs for treating arteriosclerosis at the root. To specify the apo A-I-binding domain in cubilin molecules is extremely useful, for example, for developing anti-cubilin-apo A-I-binding domain antibody and screening for certain low molecular weight compounds.

Thus, the finding that the apo A-I-binding site is located in a specific region in cubilin molecules is extremely important in view of clinical application, and the identification of this binding site has been eagerly awaited. Furthermore, the raising of serum apo A-I concentration and HDL concentration by administering a substance that inhibits the binding of apo A-I to cubilin partial fragments, and therapeutics for arteriosclerosis comprising such a substance have been eagerly awaited in order to treat hypertriglyceridemia, hypercholesterolemia and arteriosclerotic diseases.

DISCLOSURE OF THE INVENTION

The present invention provides the identification of the apo A-I-binding site on cubulin; the construction of a screening method for a compound that inhibits the binding of a cubilin fragment comprising the identified binding site to apo A-I; and a medicine comprising a compound obtained by the constructed screening method.

To specify the apo A-I-binding site in cubilin molecules leads to clinical application of apo A-I-binding fragments of cubilin per se, antibodies thereto, and low molecular weight compounds that inhibit the binding of cubilin figments to apo A-I.

As a result of intensive and extensive researches, the present inventors have found apo A-I-binding activity in the CUB7-CUB14 fragment peptide (Blood 91, 3593-3600 (1998)) and the CUB9-CUB14 fragment peptide of cubilin. Then, the inventors have developed an experimental system for measuring apo A-I-binding using these fragments. Based on these findings, the inventors have made further investigations. Thus, the present invention has been achieved.

The present invention relates to:

• • (1) A polypeptide having the ability to bind to apolipoprotein A-I which is a partial fragment of cubilin, an amide or ester of said polypeptide, or a salt of said polypeptide,
  • (2) The polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein the polypeptide is characterized by having an amino acid sequence identical or substantially identical with the amino acid sequence as shown in SEQ ID NO: 10,
  • (3) The polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein the polypeptide has the amino acid sequence as shown in SEQ ID NO: 10,
  • (4) The polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein the polypeptide is characterized by having an amino acid sequence identical or substantially identical with the amino acid sequence as shown in SEQ ID NO: 19,
  • (5) The polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein the polypeptide has the amino acid sequence as shown in SEQ ID NO: 19,
  • (6) A DNA comprising a DNA encoding the polypeptide of (1) above,
  • (7) The DNA of (6) above, wherein the DNA comprises the base sequence as shown in SEQ ID NO: 9,
  • (8) The DNA of (6) above, wherein the DNA comprises the base sequence as shown in SEQ ID NO: 22,
  • (9) A recombinant vector comprising the DNA of (6) above,
  • (10) A transformant transformed with the recombinant vector of (9) above,
  • (11) A method of producing the polypeptide, amide or ester thereof, or salt thereof of (1) above, which is characterized by culturing the transformant of (10) above and allowing the transformant to produce the polypeptide,
  • (12) An antibody to the polypeptide, amide or ester thereof, or salt thereof of (1) above,
  • (13) A method of screening for compounds or salts thereof that promote or inhibit the activity of the polypeptide or salt thereof of (1) above, wherein the method is characterized by using the polypeptide, amide or ester thereof, or salt thereof of (1) above,
  • (14) A screening kit for compounds or salts thereof that promote or inhibit the activity of the polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein the kit comprises the polypeptide or salt thereof of (1) above,
  • (15) A compound or salt thereof that promotes or inhibits the activity of the polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein said compound or salt thereof is obtainable by using the screening method of (13) above or the screening kit of (14) above,
  • (16) A medicine comprising a compound or salt thereof that promotes or inhibits the activity of the polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein said compound or salt thereof is obtainable by using the screening method of (13) above or the screening kit of (14) above,
  • (17) A prophylactic and/or therapeutic agent for renal disorders, nephritis, nephropathy, proteinuria, nervous disorders or vitamin B₁₂ deficiency, comprising a compound or salt thereof that promotes or inhibits the activity of the polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein said compound or salt thereof is obtainable by using the screening method of (13) above or the screening kit of (14) above,
  • (18) A prophylactic and/or therapeutic agent for hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardial infarction or angina, comprising a compound or salt thereof that promotes or inhibits the activity of the polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein said compound or salt thereof is obtainable by using the screening method of (13) above or the screening kit of (14) above,
  • (19) A medicine comprising the polypeptide, amide or ester thereof, or salt thereof of (1) above,
  • (20) A prophylactic and/or therapeutic agent for diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders, comprising the polypeptide, amide or ester thereof, or salt thereof of (1) above,
  • (21) A diagnostic agent that is characterized by the use of the DNA of (6) above,
  • (22) A diagnostic agent comprising the antibody of (12) above,
  • (23) A method for preventing and/or treating renal disorders, nephritis, nephropathy, proteinuria, nervous disorders or vitamin B₁₂ deficiency in a mammal, comprising a compound or salt thereof that promotes or inhibits the activity of the polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein said compound or salt thereof is obtainable by using the screening method of (13) above or the screening kit of (14) abvove,
  • (24) A method for preventing and/or treating hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardial infarction or angina, characterized by administering to a mammal an effective amount of a compound or salt thereof that promotes or inhibits the activity of the polypeptide, amide or ester thereof, or salt thereof of (1) above, wherein said compound or salt thereof is obtainable by using the screening method of (13) above or the screening kit of (14) above,
  • (25) Use of a compound or salt thereof that promotes or inhibits the activity of the polypeptide, amide or ester thereof, or salt thereof of (1) above, for manufacturing a prophylactic and/or therapeutic agent for hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardial infarction or angina, wherein said compound or salt thereof is obtainable by using the screening method of (13) above or the screening kit of (14) above,
  • (26) A method for preventing and/or treating diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders, characterized by administering to a mammal an effective amount of the polypeptide, amide or ester thereof, or salt thereof of (1) above, and
  • (27) Use of the polypeptide, amide or ester thereof, or salt thereof of (1) above for manufacturing a prophylactic and/or therapeutic agent for diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders.

Further, the present invention provides:

• • (28) The polypeptide, amide or ester thereof, or salt thereof of (2) or (4) above, wherein the amino acid sequence substantially identical with the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19 is an amino acid sequence having about 50% or more (preferably about 60% or more, more preferably about 70% or more, still more preferably about 80% or more, especially preferably about 90% or more, and most preferably about 95% or more) homology to the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19, and
  • (29) The polypeptide, amide or ester thereof, or salt thereof of (2) or (4) above, wherein the amino acid sequence substantially identical with the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19 is (i) an amino acid sequence wherein one or more amino acids (preferably about 1-30 amino acids) are deleted from the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; (ii) an amino acid sequence wherein one or more amino acids (preferably about 1-30 amino acids) are added to the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; (iii) an amino acid sequence wherein one or more amino acids (preferably about 1-30 amino acids) amino acids are substituted with other amino acids in the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; or (iv) an amino acid sequence which is a combination of these sequences.

Further, the DNA of the invention and the polypeptide, amide or ester thereof, or salt thereof of the invention may be utilized in basic researches such as molecular markers, tissue markers, chromosome mapping, identification of genetic diseases or designing of primers, probes, and so on.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of a human cubilin mature protein (to be continued to FIG. 2).

FIG. 2 shows the amino acid sequence of a human cubilin mature protein (continued from FIG. 1).

FIG. 3 shows the results of Western blotting carried out in Example 2. In this Figure, lane C shows pSecTag2-transfected COS7 cells; lanes I and II show COS7 cells transfected with expression vectors comprising pSecTag2 plasmids into which cDNAs encoding human cubilin partial fragments I and II are incorporated, respectively; lane III shows the culture supernatant from pTB2116-transfected COS cells; and lanes IV and V show COS7 cells transfected with expression vectors comprising pSecTag2 plasmids into which cDNAs encoding human cubilin partial fragments IV and V are incorporated, respectively. “Intracellular” denotes PVDF membranes blotted from those gels on which disrupted materials from individual cells were electrophoresed, and “Culture Supernatant” denotes PVDF membranes blotted from those gels on which culture supernatants from individual cells were electrophoresed.

FIG. 4 shows the results of Western blotting carried out in Example 3. In this Figure, lanes I to V show culture supernatants containing partial fragments I to V, respectively, and lane M shows molecular markers. “Elution” denotes PVDF membranes blotted from those gels on which EDTA-selectively eluted fractions were electrophoresed, and “Pass” denotes PVDF membranes blotted from those gels on which non-binding fractions were electrophoresed.

FIG. 5 shows the results of Western blotting carried out in Examples 4 and 5.

The utmost left lane in each blotting shows the migration of molecular markers.

Mock, Cub7-12, Cub9-14, Cub7-14 and Cub13-20 represent vectors transfected into COS7 cells. Mock means a vector alone. Cub7-12 means a vector expressing a cDNA encoding the CUB7-CUB12 fragment peptide, and Cub9-14 means a vector expressing a cDNA encoding the CUB9-CUB14 fragment peptide. Cub7-14 corresponds to the fragment III described in Example 3, and Cub13-20 corresponds to the fragment IV described in Example 3.

In this Figure, “Sample” denotes sample solutions applied to human apolipoprotein A-I-binding resin; “Column” denotes fractions passed through columns; “Wash” denotes fractions eluted at the time of washing; and “Elution” denotes fractions eluted by EDTA. In the “Pass” panel, the bands appearing at approx. 70 kDa are considered to be non-specific recognition bands attributable to high concentration albumin.

FIG. 6 shows the results of Western blotting (A) and CBB staining (B) carried out in Example 6. The numbers of individual lanes correspond to the Nos. of fractions eluted with 10 mM-200 mM imidazole gradient in Example 6.

FIG. 7 shows the biotin-added human apolipoprotein A-I-binding curve obtained in Example 7. In this Figure, “FIU” denotes values quantitatively determined with a fluorescence measuring apparatus.

BEST MODE FOR CARRYING OUT THE INVENTION

The polypeptide of the invention that is a partial fragment of cubilin having the ability to bind to apolipoprotein A-I (hereinafter, sometimes simply referred to as the “polypeptide of the invention”) may be a polypeptide derived from cells of human or other warm-blooded animals (e.g. guinea pig, rat, mouse, chicken, rabbit, pig, sheep, bovine, or monkey), such as hepatocyte, splenocyte, nerve cell, glia cell, pancreatic β cell, bone marrow cell, mesangial cell, Langerhans' cell, epidermic cell, epithelial cell, endothelial cell, fibroblast, fibrocyte, myocyte, fat cell, immune cell (e.g. macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synovial cell, cartilage cell, bone cell, osteoblast, osteoclast, mammary gland cell or interstitial cell; or precursor cells, stem cells or cancer cells of these cells; or may be a polypeptide derived from any of the tissues where such cells are present, such as brain, any region thereof (e.g. olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, or cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g. large intestine and small intestine), blood vessel, heart, thymus, spleen, salivary gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, cartilage, joint, skeletal muscle, etc. Alternatively, the polypeptide of the invention may be a recombinant polypeptide or a synthetic polypeptide.

When the polypeptide of the invention has a signal peptide, the polypeptide may be efficiently secreted out of cells.

The partial fragment of cubilin having the ability to bind to apolipoprotein A-I may be any partial peptide as long as it is a partial peptide of cubilin and has the ability to bind to apolipoprotein A-I. Preferably, the partial peptide is a polypeptide having an amino acid sequence identical or substantially identical with the amino acid sequence as shown in SEQ ID NO: 10 or a polypeptide having an amino acid sequence identical or substantially identical with the amino acid sequence as shown in SEQ ID NO: 19.

As an amino acid sequence substantially identical with the amino acid sequence as shown in SEQ ID NO: 10, an amino acid sequence having about 50% or more, preferably about 60% or more, more preferably about 70% or more, still more preferably about 80% or more, especially preferably about 90% or more, and most preferably about 95% or more homology to the amino acid sequence as shown in SEQ ID NO: 10 may be given.

As an amino acid sequence substantially identical with the amino acid sequence as shown in SEQ ID NO: 19, an amino acid sequence having about 50% or more, preferably about 60% or more, more preferably about 70% or more, still more preferably about 80% or more, especially preferably about 90% or more, and most preferably about 95% or more homology to the amino acid sequence as shown in SEQ ID NO: 19 may be given.

As a polypeptide having an amino acid sequence substantially identical with the amino acid sequence as shown in SEQ ID NO: 10, such a polypeptide is preferable that has the above-described amino acid sequence substantially identical with the amino acid sequence as shown in SEQ ID NO: 10 and yet has a substantially identical nature with a nature of a polypeptide having the amino acid sequence as shown in SEQ ID NO: 10.

As a polypeptide having an amino acid sequence substantially identical with the amino acid sequence as shown in SEQ ID NO: 19, such a polypeptide is preferable that has the above-described amino acid sequence substantially identical with the amino acid sequence as shown in SEQ ID NO: 19 and yet has a substantially identical nature with a nature of a polypeptide having the amino acid sequence as shown in SEQ ID NO: 19.

As a substantially identical nature, a nature to bind to apolipoprotein A-I may be given, for example. The term “substantially identical” means that such a nature is qualitatively identical. Therefore, it is preferable that a nature such as apolipoprotein A-I-binding ability is identical (e.g. about 0.1-to about 100-fold, preferably about 0.5-to about 10-fold, more preferably about 0.5- to about 2-fold), but quantitative factors such as the extent of that nature, the molecular weight of the polypeptide, etc. may be different.

More specifically, examples of the polypeptide comprising an amino acid sequence substantially identical with the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19 include polypeptides (the so-called muteins) comprising (i) an amino acid sequence wherein one or more amino acids (preferably about 1-30, more preferably about 1-10, still more preferably several (1-5) amino acids) are deleted from the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; (ii) an amino acid sequence wherein one or more amino acids (preferably about 1-30, more preferably about 1-10, still more preferably several (1-5) amino acids) are added to the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; (iii) an amino acid sequence wherein one or more amino acids (preferably about 1-30, more preferably about 1-10, still more preferably several (1-5) amino acids) are inserted into the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; (iv) an amino acid sequence wherein one or more amino acids (preferably about 1-30, more preferably about 1-10, still more preferably several (1-5) amino acids) amino acids are substituted with other amino acids in the amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; or (v) an amino acid sequence which is a combination of these sequences.

When the amino acid sequence has insertion(s), deletion(s) or substitution(s) as described above, the positions of such insertion(s), deletion(s) or substitution(s) are not particularly limited. However, such positions are preferably those which are not essential for the apo A-I-binding in the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 19.

Throughout this specification, the left end of each polypeptide is its N-terminus, and the right end thereof is its C-terminus according to the convention in peptide description. The C-terminus of the polypeptide of the invention (such as a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 10) is usually a carboxyl group (—COOH) or a carboxylate (—COO), but it may be an amide (—CONH₂) or an ester (—COOR).

Examples of R of the above ester group include C_1-6 alkyl groups (e.g. methyl, ethyl, n-propyl, isopropyl or n-butyl), C_3-8 cycloalkyl groups (e.g. cyclopentyl or cyclohexyl), C_6-12 aryl groups (e.g. phenyl or α-naphthyl), C_7-14 aralkyl groups such as phenyl-C_1-2 alkyl groups (e.g. benzyl or phenethyl) and α-naphthyl-C_1-2 alkyl groups (e.g. α-naphthylmethyl). In addition, the ester group also includes pivaloyloxymethyl esters that are universally used as oral esters.

When the polypeptide of the invention has a carboxyl group (or carboxylate) at any position other than its C-terminus, such a polypeptide that the carboxyl group may be amidated or esterified is also included in the polypeptide of the invention. The ester in this case may be, for example, any of the esters mentioned above for the C-terminal ester.

Furthermore, the polypeptide of the present invention includes those polypeptides in which the N-terminal amino acid residue (e.g. Met) is protected by a protective group (e.g. C_1-6 acyl group such as C_1-6 alkanoyl group (e.g. formyl group or acetyl group)); those polypeptides in which the N-terminal Glu generated through in vivo cleavage is pyroglutaminated; those polypeptides in which a substituent on a side chain of an amino acid (e.g. OH, SH, amino group, imidazole group, indole group, or guannidino group) is protected by an appropriate protective group (e.g. C_1-6 acyl group such as C_1-6 alkanoyl group (e.g. formyl group or acetyl group)); and conjugated polypeptides such as the so-called glycopolypeptides to which sugar chains are linked.

As the salt of the polypeptide of the invention, salts formed with physiologically acceptable acids (e.g. organic or inorganic acids) or bases (e.g. alkali metals) are used. Especially preferable are physiologically acceptable acid addition salts. Examples of such salts include salts formed with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid) and salts formed with organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid).

The polypeptide or salt thereof of the present invention can be produced from the afore-mentioned cells or tissues of human or other warm-blooded animals by publicly known purification methods for polypeptides (proteins). Alternatively, the polypeptide of the invention can be produced by culturing a transformant described later comprising a DNA encoding the polypeptide. It can also be produced in accordance with the procedures for peptide synthesis which are described later.

When the polypeptide of the invention is produced from tissues or cells of human or other warm-blooded animals, the relevant tissue or cell is homogenized and then the polypeptide of the present invention is extracted with acids, etc. The polypeptide can be purified and isolated from the resultant extract by a combination of chromatography, such as reversed phase chromatography, ion exchange chromatography and so on.

For the synthesis of the polypeptide of the invention or salt or amide thereof, any of the commercially available resins for polypeptide synthesis may be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4-(2′,4′-dimethoxyphenylhydroxymethyl)phenoxy resin, and 4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin. Using such a resin, amino acids appropriately protected at their α-amino groups and side chain functional groups are condensed on the resin according to the amino acid sequence of the polypeptide of interest by publicly known condensation methods. At the final stage of the reaction, all protective groups are removed simultaneously with the cleavage of the polypeptide from the resin. Then, in a highly diluted solution, intramolecular disulfide bond formation reaction is carried out to obtain the polypeptide of interest or amide thereof.

With respect to the above-described condensation of protected amino acids, various activators useful for polypeptide synthesis may be utilized. Among all, carbodiimide reagents are especially preferred. Examples of carbodiimide reagents include DCC, N,N′-diisopropylcarbodiimide, and N-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide. For activation by these reagents, protected amino acids and a recemization inhibitor (e.g. HOBt or HOOBt) may be directly added to the resin, or protected amino acids may be activated in advance in the form of symmetric acid anhydride, HOBt ester or HOOBt ester and, then, added to the resin.

The solvent used for the above-mentioned activation of protected amino acids or the condensation thereof with a resin may be appropriately selected from those solvents known to be useful for polypeptide (protein) condensation reactions. Examples of useful solvents include acid amides (e.g. N,N-dimethylformamide, N,N-dimethylacetamide, of N-methylpyrrolidone), halogenated hydrocarbons (e.g. methylene chloride, or chloroform), alcohols (e.g. trifluoroethanol), sulfoxides (e.g. dimethyl sulfoxide), ethers (e.g. pyridine, dioxane, tetrahydrofuran), nitriles (e.g. acetonitrile or propionitrile), esters (e.g. methyl acetate or ethyl acetate), and suitable mixtures of these solvents. The reaction temperature may be appropriately selected from the range known to be useful for polypeptide-forming reactions; usually, the temperature is selected from the range from about −20° C. to about 50° C. The activated amino acid derivative is usually used in 1.5- to 4-fold excess. When the condensation is found insufficient as a result of test using the ninhydrin reaction, sufficient condensation can be achieved by repeating reactions without removing protective groups. When sufficient condensation cannot be achieved even by repeating reactions, unreacted amino acids may be acetylated with acetic anhydride or acetylimidazole so that they do not affect subsequent reactions.

Examples of useful protective groups for the amino group of raw materials include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, and Fmoc.

The carboxyl group can be protected, for example, in the form of an alkyl ester (e.g. straight-chain, branched, or cyclic alkyl esters such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, and so on), aralkyl ester (e.g. benzyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, benzhydryl, and so on), phenacyl ester, benzyloxycarbonylhydrazide, t-butoxycarbonylhydrazide or tritylhydrazide.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of suitable groups for this esterification include lower (C_1-6) alkanoyl groups such as acetyl, aroyl groups such as benzoyl, and carbonic acid-derived groups such as benzyloxycarbonyl and ethyloxycarbonyl. Examples of groups suitable for the etherification include benzyl, tetrahydropyranyl and t-butyl.

Examples of protective groups for the phenolic hydroxyl group of tyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, BrZ, and t-butyl.

Examples of protective groups for the imidazole ring of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt and Fmoc.

Examples of raw materials with activated carboxyl groups include the corresponding acid anhydrides, azides and active esters (esters of alcohols such as pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide and HOBt). Examples of raw materials with activated amino groups include the corresponding phosphoric acid amides.

Methods for removing (eliminating) protective groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd-carbon, acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or mixtures thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, or the like, and reduction with sodium in liquid ammonia. The elimination reaction by the above-mentioned acid treatment is generally conducted at temperatures of about −20° C. to about 40° C. In the acid treatment, it is effective to add a cation scavenger such as anisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide, 1,4-butanedithiol or 1,2-ethanedithiol. The 2,4-dinitrophenyl group used as the protective group for the imidazole of histidine is removed by thiophenol treatment. The formyl group used as the protective group for the indole of tryptophan may be removed by the above-mentioned deprotection by the acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol or the like, or by alkali treatment using dilute sodium hydroxide, dilute ammonia or the like.

The protection of functional groups in raw materials that should not be involved in the reaction, protective groups therefor, the removal of these protective groups and the activation of functional groups involved in the reaction can be appropriately selected from groups or methods publicly known.

An alternative method for obtaining amides of a polypeptide of interest comprises, for example, protecting the α-carboxyl group of the C-terminal amino acid by amidation, extending the peptide (polypeptide) chain to a desired length on the side of the amino group, preparing a polypeptide with its N-terminal α-amino group selectively deprotected, preparing a polypeptide with its C-terminal carboxyl group selectively deprotected, and condensing these two polypeptides in a mixed solvent such as described above. Details of this condensation reaction are the same as described above. After purification of the protected polypeptide thus obtained by condensation, all the protective groups are removed by the method described above to thereby provide a crude polypeptide of interest. This crude polypeptide is purified by various known purification techniques and the major fractions are lyophilized to provide the desired polypeptide in an amide form.

A method for obtaining esters of a polypeptide of interest comprises, for example, condensing the α-carboxyl group of the C-terminal amino acid with a desired alcohol to prepare the corresponding amino acid ester, and subjecting this ester to the same procedures as described above in the preparation of amides to thereby provide the desired polypeptide in an ester form.

The polypeptide of the invention or salt thereof can be produced by publicly known methods for peptide synthesis. The method for peptide synthesis may be solid-phase synthesis or liquid-phase synthesis. Briefly, a peptide of interest can be produced by condensing a partial peptide or amino acid capable of constituting the partial peptide of the invention with the residual part thereof and, if the product has protective groups, removing the protective groups. Examples of condensation methods and methods for removal of protective groups publicly known include those described in the following references (1) to (5).

• (1) M. Bodanszky & M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York, 1966
• (2) Schroeder & Luebke, The Peptide, Academic Press, New York, 1965
• (3) Nobuo izumiya et al., Fundamentals and Experiments in Peptide Synthesis, Maruzen, 1975
• (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment Series 1, Polypeptide Chemistry IV, 205, 1977
• (5) Haruaki Yajima (ed.), Development of Drugs (Continued), Vol. 14, Peptide Synthesis, Hirokawa Shoten

After the reaction, the polypeptide of the invention can be isolated and purified by a combination of conventional purification techniques such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the polypeptide thus obtained is a free form, it can be converted to a suitable salt by publicly known methods or methods based thereon. On the contrary, when the polypeptide is obtained in a salt form, it can be converted to a free form or another salt by publicly known methods or methods based thereon.

The DNA encoding the polypeptide of the invention may be any DNA as long as it comprises a base sequence encoding the above-described polypeptide of the invention. The DNA may be genomic DNA, cDNA derived from the above-mentioned cells or tissues, or synthetic DNA.

Vectors used for library may be any vectors such as bacteriophage, plasmid, cosmid, phagemid, and so on. Alternatively, total RNA or mRNA fraction may be prepared from the above-mentioned cells or tissues, followed by direct amplification by reverse transcriptase polymerase chain reaction (hereinafter, referred to as “RT-PCR”).

Examples of DNA encoding the polypeptide of the invention include a DNA comprising the base sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 22; or a DNA which has a base sequence hybridizing to the base sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 22 under high stringent conditions and encodes a polypeptide having a property (e.g. immunogenicity) substantially identical with the corresponding property of the polypeptide of the invention. Any of such DNA may be used.

As a DNA hybridizable to the base sequence as shown in SEQ ID NO: 9 under high stringent conditions, a DNA comprising a base sequence having about 60% or more, preferably about 70% or more, and more preferably about 80% or more homology to the base sequence as shown in SEQ ID NO: 9 may be used, for example.

As a DNA hybridizable to the base sequence as shown in SEQ ID NO: 22 under high stringent conditions, a DNA comprising a base sequence having about 60% or more, preferably about 70% or more, and more preferably about 80% or more homology to the base sequence as shown in SEQ ID NO: 22 may be used, for example.

Hybridization can be carried out according to publicly known methods or methods based thereon, e.g. those methods described in Molecular Cloning, 2nd Ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) and the like. When commercial libraries are used, hybridization can be carried out in accordance with the methods described in the instructions attached to the kits; preferably, hybridization is carried out under high stringent conditions.

“High stringent conditions” refers to, for example, conditions where sodium concentration is about 19-40 mM, preferably about 19-20 mM, and temperature is about 50-70° C., preferably about 60-65° C.

As a DNA encoding the polypeptide of the invention having the amino acid sequence as shown in SEQ ID NO: 10, a DNA having the base sequence as shown in SEQ ID NO: 9 may be used, for example. As a DNA encoding the polypeptide of the invention having the amino acid sequence as shown in SEQ ID NO: 19, a DNA having the base sequence as shown in SEQ ID NO: 22 may be used, for example.

A DNA encoding the entire polypeptide of the invention can be cloned either by PCR amplification using synthetic DNA primers each having a partial base sequence encoding the polypeptide of the invention, or by hybridization of DNA fragments inserted into a suitable vector to a labeled DNA fragment encoding a part or full length of the polypeptide of the invention or a labeled synthetic DNA fragment. The hybridization can be carried out, for example, according to the method described in Molecular Cloning, 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When commercial libraries are used, the hybridization can be carried out according to the instructions attached thereto.

Substitution of the base sequence of a DNA can be performed by publicly known methods such as ODA-LA PCR, gapped duplex method, Kunkel method and the like using publicly known kits such as Mutan™-Super Express Km (Takara), Mutan™-K (Takara), etc.

The cloned DNA encoding the polypeptide of the invention may be used as it is or after digestion with restriction enzymes or addition of linkers, if desired. The DNA may have ATG at its 5′ end as the translation initiation codon and TAA, TGA, or TAG at its 3′ end as the translation termination codon. The translation initiation and termination codons may be added by using appropriate synthetic DNA adapters.

Expression vectors for the polypeptide of the invention can be prepared by, for example, (a) cutting out a DNA fragment of interest from a DNA encoding the polypeptide of the invention and (b) ligating the DNA fragment to an appropriate expression vector downstream of its promoter.

Examples of vectors include plasmids derived from Escherichia coli (e.g. pBR322, pBR325, pUC12, and pUC13); plasmids derived from Bacillus subtilis (e.g. pUB110, pTP5 and pC194); plasmids derived from yeast (e.g. pSH19 and pSH15); bacteriophages such as λ-phage; animal viruses such as retrovirus, vaccinia virus, baculovirus; and other vectors such as pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo and so on.

Any promoter may be used in the invention as long as it is appropriate for the host that will be used for expressing a gene of interest. When the host is an animal cell, examples of promoters include SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and β-actin promoter.

Among these promoters, CMV (cytomegalovirus) promoter, SRα promoter or the like is preferably used. When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP_L promoter, Ipp promoter, T7 promoter or the like is preferably used. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter or the like is preferably used. When the host is a yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, or the like is preferably used. When the host is an insect cell, polyhedrin promoter, P10 promoter, or the like is preferably used.

The expression vectors may, if desired, further comprise enhancers, splicing signals, polyadenylation signals, selective markers, SV40 replication origin (hereinafter, sometimes abbreviated to “SV40 ori”) and the like. Examples of selective markers include dihydrofolate reductase (hereinafter, sometimes abbreviated to “dhfr”) gene [methotorexate (MTX) resistance], ampicillin resistance gene (hereinafter, sometimes abbreviated to “Amp^r”), neomycin resistance gene (hereinafter, sometimes abbreviated to “Neo^r”: Geneticin resistance) and the like. When dhfr gene-deficient Chinese hamster cells are used in combination with dhfr gene as a selective marker, recombinant cells may be selected even in a thymidine-free medium.

Furthermore, a signal sequence appropriate for the host may be added, if necessary, to the N-terminal of the polypeptide. When the host is Escherichia, the utilizable signal sequences may include PhoA signal sequence, OmpA signal sequence, or the like may be added. When the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, or the like may be added. When the host is yeast, MFα signal sequence, SUC2 signal sequence, or the like may be added. When the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, or the like may be added.

When the polypeptide of the invention has such a signal sequence as described above, the polypeptide of the invention is secreted out of cells effectively.

Transformants (or transfectants) can be prepared by using the thus constructed vector comprising a DNA encoding the polypeptide of the invention.

Examples of hosts include bacteria belonging to the genus Escherichia, bacteria belonging to the genus Bacillus, yeasts, insect cells, insects, and animal cells.

Specific examples of bacteria belonging to the genus Escherichia include E. coli K12 DH1 (Proc. Natl. Acad. Sci. USA, Vol. 60, 160 (1968)), JM103 (Nucleic Acids Research, Vol. 9, 309 (1981)), JA221 (Journal of Molecular Biology, Vol. 120, 517 (1978)), HB101 (Journal of molecular Biology, Vol, 41, 459 (1969)) and C600 (Genetics, Vol. 39, 440 (1954)).

Specific examples of bacteria belonging to the genus Bacillus include B. subtilis MI114 (Gene, Vol. 24, 255 (1983)) and 207-21 (Journal of Biochemistry, Vol. 95, 76 (1984)).

Specific examples of yeasts include Saccharomyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-SD and 20B-12, Schizosaccharomyces pombe NCYC1913 and NCYC2036, and Pichia pastoris KM71.

Specific examples of insect cells include, when the virus used is AcNPV, a cell line derived from larvae of Spodoptera frugiperda (Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five™ cells derived from eggs of Trichoplusia ni, Mamestra brassicae-derived cells and Estigmena acrea-derived cells. When the virus used is BmNPV, insect cells such as a silkworm-derived cell line (Bombyx mori N cells; BmN cells) may be used. Specific examples of Sf cells useful in the invention include Sf9 cells (ATCC CRL 1711) and Sf21 cells (both disclosed in Vaughn J. L. et al., In Vivo, 13, 213-217 (1977)).

Specific examples of insects include larvae of silkworm (Maeda et al., Nature, 315, 592 (1985)).

Specific examples of animal cells include a simian cell COS-7 (COS7), Vero cells, a Chinese hamster cell CHO(CHO cells), a dhfr gene-deficient Chinese hamster cell CHO (hereinafter, abbreviated to “CHO(dhfr⁻) cells”), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FL cells.

Transformation of bactera belonging to the genus Escherichia can be performed in accordance with methods disclosed, for example, in Proc. Natl. Acad. Sci. USA, Vol. 69, 2110 (1972) and Gene, Vol. 17, 107 (1982).

Transformation of bacteria belonging to the genus: Bacillus can be performed in accordance with methods disclosed, for example, in Molecular & General Genetics, Vol. 168, 111 (1979).

Transformation of yeasts can be performed in accordance with methods disclosed, for example, in Methods in Enzymology, 194, 182-187 (1991) and Proc. Natl. Acad. Sci. USA, Vo. 75, 1929 (1978).

Transformation of insect cells or insects can be performed in accordance with methods disclosed, for example, in Bio/Technology, 6, 47-55 (1988).

Transformation of animal cells can be performed by methods disclosed, for example, in Cell Engineering, Separate Vol. 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (Shujunsha Co.) and Virology, Vol. 52, 456 (1973).

Thus, transformants transformed with an expression vector comprising a DNA encoding the polypeptide of the invention can be obtained.

As a medium to culture transformants obtained from Escherichia or Bacillus bacteria as hosts, a liquid medium is appropriate. The medium is allowed to contain carbon sources, nitrogen sources, minerals, and so on which are necessary for the growth of the transformant. As carbon sources, glucose, dextrin, soluble starch, sucrose or the like may be enumerated. As nitrogen sources, organic or inorganic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, yeast extract, meat extract, bean cake, potato extract, or the like may be enumerated. As minerals, calcium chloride, sodium dihydrogen phosphate, magnesium chloride, or the like may be enumerated. Further, yeast extract, vitamins, growth-promoting factors, etc. may also be added to the medium. Preferable pH of the medium is about 5 to about 8.

As a medium to culture Escherichia bacteria, M9 medium containing glucose and casamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York, (1972)) is preferable, for example. If necessary, drugs such as 3β-indolyl acrylic acid can be added to the medium to improve efficiency of the promoter.

When the host is an Escherichia bacterium, the transformant is cultured usually at about 15-43° C. for about 3-24 hours. If necessary, aeration and stirring may be applied.

When the host is a Bacillus bacterium, the transformant is cultured usually at about 30-40° C. for about 6-24 hours. If necessary, aeration and stirring may also be applied.

As a medium to culture transformants obtained from yeasts as hosts, a medium such as Burkholder minimum medium (Bostian, K. L. et al., Proc. Natl. Acad. Sci. USA, Vol. 77, 4505 (1980)) or SD medium containing 0.5% casamino acid (Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA, Vol. 81, 5330 (1984)) may be used, for example. It is preferable that the pH of the medium is adjusted to about 5 to about 8. The transformant is cultured usually at about 20-35° C. for about 24-72 hours. If necessary, aeration and stirring may be applied.

As a medium to culture transformants obtained from insect cells or insects as hosts, Grace's Insect Medium (Grace, T. C. C., Nature, 195, 788 (1962)) supplemented with additives such as inactivated 10% bovine serum may be used, for example. It is preferable that the pH of the medium is adjusted to about 6.2-6.4. The transformant is cultured usually at about 27° C. for about 3-5 days. If necessary, aeration and stirring may be applied.

As a medium to culture transformants obtained from animal cells as hosts, examples of useful media include MEM medium (Science, Vol. 122, 501 (1952)) containing about 5-20% fetal calf serum, DMEM medium (Virology, Vol. 8, 396 (1959)), RPMI 1640 medium (Journal of the American Medical Association, Vol. 199, 519 (1967)), 199 medium (Proceedings of the Society of the Biological Medicine, Vol. 73, 1 (1950)). Preferable pH of the medium is about 6 to about 8. The transformant is cultured usually at about 30-40° C. for about 15-60 hours. If necessary, aeration and stirring may be applied.

Thus, the polypeptide of the invention can be produced inside of transformant cells, in their cell membranes, or outside of these cells.

Separation and purification of the polypeptide from the resultant culture can be carried out, for example, according to the methods described below.

For extraction of the polypeptide of the invention from cultured microorganisms or cells, the microorganisms or cells are harvested by publicly known methods after the cultivation, suspended in a suitable buffer, and disrupted by sonication or by lysozyme and/or freezing and thawing, etc. Then, a crude extract of the polypeptide is obtained by centrifugation or filtration. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100™. If the polypeptide is secreted into the culture broth, the supernatant is separated from the microorganisms or cells after completion of the cultivation and collected by publicly known methods.

Purification of the polypeptide contained in the thus obtained culture supernatant or extract can be performed by an appropriate combination of publicly known methods for separation and purification. These publicly known methods include methods utilizing solubility such as salting out or sedimentation with solvents, methods mainly utilizing difference in molecular weight such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods utilizing difference in electric charge such as ion-exchange chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing difference in the hydrophobicity such as reversed-phase high-performance liquid chromatography, and methods utilizing difference in isoelectric point such as isoelectric electrophoresis.

When the thus obtained polypeptide is a free form, the polypeptide can be converted into a salt by publicly known methods or methods based thereon. On the contrary, when the polypeptide is obtained in a salt form, the salt can be converted into a free form or another salt according to publicly known methods or methods based thereon.

The polypeptide produced by the transformant can be arbitrarily modified or a part thereof can be removed therefrom by using an appropriate protein modification enzyme or proteolytic enzyme before or after the purification. Examples of such enzymes include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase and glycosidase.

The presence of the polypeptide of the invention thus obtained can be measured by enzyme immunoassay, Western blot analysis or the like using specific antibodies.

Antibodies to the polypeptide of the invention or salt thereof may be either polyclonal or monoclonal antibodies as long as they can recognize the polypeptide of the invention or salt thereof.

Antibodies to the polypeptide of the invention or salt thereof can be prepared using the polypeptide of the invention as antigen and according to publicly known producing methods for antibody or anti-serum preparation.

[Preparation of Monoclonal Antibodies]

(a) Preparation of Monoclonal Antibody-Producing Cells

The polypeptide of the invention or salt thereof is administered to warm-blooded animals either alone or together with a carrier or diluent to a site capable of producing antibodies upon the administration. In order to enhance the ability to produce antibodies, complete Freund's adjuvants or incomplete Freund's adjuvants may also be administered. The administration is usually carried out once in every two to six weeks and two to ten times in the total. Examples of warm-blooded animals utilized include monkey, rabbit, dog, guinea pig, mouse, rat, sheep, goat and chicken. Among them, mouse or rat is used preferably.

In the preparation of monoclonal antibody-producing cells, individuals with detectable antibody titers are selected from warm-blooded animals (e.g. mice) immunized with antigen. Then, the spleen or lymph nodes are collected from them two to five days after the final immunization, and antibody-producing cells contained therein are fused with myeloma cells of a homologous or heterologous animal to thereby obtain monoclonal antibody-producing hybridomas. Measurement of antibody titers in antisera may be carried out, for example, by reacting a labeled polypeptide, which will be described later, with the antiserum followed by measuring the activity of the labeling agent bound to the antibody. The cell fusion may be carried out by a known method, for example, the method of Koehler and Milstein (Nature, 256, 495, (1975)). Examples of fusion promoters include polyethylene glycol (PEG), Sendai virus, etc. Preferably, PEG is used.

Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2/0, AP-1, etc. Preferably, P3U1 is used. A preferable ratio of the number of antibody-producing cells used (spleen cells) to the number of myeloma cells is about 1:1 to 20:1. When PEG (preferably, PEG 1000 to PEG 6000) is added at a concentration of about 10-80% and the resultant cell mixture is incubated at 2040° C. (preferably, at 30-37° C.) for about one to ten minutes, an efficient cell fusion can be performed.

Various methods may be used for screening for monoclonal antibody-producing hybridomas. For example, hybridoma culture supernatant is added to a solid phase (e.g. microplate) on which the polypeptide antigen has been adsorbed either directly or with a carrier. Then, a radioactively or enzymatically labeled anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used when mouse cells are used in the cell fusion) or protein A is added thereto to detect monoclonal antibodies bound to the solid phase. Alternatively, a method may be used in which hybridoma culture supernatant is added to a solid phase on which an anti-immunoglobulin antibody or protein A has been adsorbed; then, a radioactively or enzymatically labeled polypeptide is added thereto to thereby detect monoclonal antibodies bound to the solid phase.

Selection of monoclonal antibodies may be carried out by publicly known methods or methods based on them. Usually, selection can be carried out in a medium for culturing animal cells supplemented with HAT (hypoxanthine, aminopterin and thymidine). As a medium for selection and culturing, any medium may be used as long as hybridomas are capable of growing therein. Examples of media include RPMI 1640 medium containing about 1-20% (preferably about 10-20%) of fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing about 1-20% of fetal calf serum and a serum-free medium for hybridoma cultivation (SFM-101; Nissui Pharmaceutical Co.). The cultivation temperature is usually about 20-40° C., preferably about 37° C. The cultivation period is usually from five days to three weeks, preferably one to two weeks. The cultivation may be carried out usually under 5% carbon dioxide. The antibody titer of hybridoma culture supernatant may be measured in the same manner as in the above-mentioned measurement of the antibody titers in antisera.

(b) Purification of the Monoclonal Antibodies

Separation and purification of monoclonal antibodies may be carried out by publicly known methods, such as methods for separating/purifying immunoglobulin [e.g. salting-out, alcohol precipitation, isoelectric precipitation, electrophoresis, adsorption/desorption using ion exchangers (e.g. DEAE), ultracentrifugation, gel filtration, specific purification methods in which only an antibody is collected by means of an antigen-binding solid phase or active adsorbent such as protein A or protein G, followed by dissociation of the bond].

[Preparation of Polyclonal Antibodies]

The polyclonal antibody of the invention can be produced by publicly known methods or methods based on them. For example, an immunogen (antigen polypeptide) per se or a complex of the immunogen and a carrier protein is prepared. Then, using the immunogen or the complex, warm-blooded animals are immunized in the same manner as described for the production of monoclonal antibodies. Fractions containing the antibody against the polypeptide of the invention or salt thereof are harvested from the immunized animals, followed by separation and purification of the antibody.

With respect to the immunogen-carrier protein conjugate for use in the immunization of warm-blooded animals, the kind of carrier protein and the mixing ratio of the carrier and the hapten are not particularly restricted as long as antibodies are produced efficiently against the hapten cross-linked to the carrier. For example, bovine serum albumin, bovine thyroglobulin, hemocyanine, or the like is coupled to the hapten at a weight ratio of about 0.1-20:1, preferably about 1-5:1.

A variety of condensing agents can be used for the coupling between the hapten and the carrier. For example, glutaraldehyde, carbodiimide, maleimide, or active ester reagents containing a thiol or dithiopyridyl group may be used.

The condensation product is administered to a warm-blooded animal either alone or together with a carrier or diluent at a site capable of producing antibodies upon the administration. In order to enhance the antibody production ability, complete Freund's adjuvant or incomplete Freund's adjuvant may also be administered. Administration is carried out generally once in about every 2-6 weeks and about 3-10 times in the total.

Polyclonal antibodies can be recovered from the blood, abdominal dropsy or other body fluid, preferably from the blood, of the warm-blooded animal immunized as described above.

Polyclonal antibody titers in antisera can be determined in the same manner as described above for the determination of monoclonal antibody titers in antisera. The separation and purification of polyclonal antibodies can be carried by the same methods for separation and purification of immunoglobulin as those described for the separation and purification of monoclonal antibodies.

With respect to the antisense DNA having a base sequence complementary to or substantially complementary to a DNA encoding the polypeptide of the invention (hereinafter, sometimes referred to as the “DNA of the invention”), any antisense DNA may be used as long as it has a base sequence complementary to or substantially complementary to the DNA of the invention and is able to inhibit the expression of the DNA.

A base sequence substantially complementary to the DNA of the invention refers to, for example, a base sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more homology to the full-length or partial base sequence of the complementary base sequence to the DNA of the invention (i.e., the complementary strand to the DNA of the invention). Particularly preferable is an antisense DNA having about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more homology to a partial base sequence of the complementary strand to the DNA of the invention that is complementary to a base sequence encoding an N-terminal portion of the polypeptide of the invention (e.g. base sequence encoding a region adjacent to the initiation codon). These antisense DNAs can be synthesized with DNA synthesizers that are publicly known.

Hereinbelow, uses of the polypeptide of the invention, amide or ester thereof, or salt thereof (sometimes, they are simply referred to as the “polypeptide of the invention”); the DNA encoding the polypeptide of the invention (sometimes referred to as the “DNA of the invention”); the antibody to the polypeptide of the invention, amide or ester thereof, or salt thereof (sometimes referred to as the “antibody of the invention”); and the antisense DNA will be described.

(1) Therapeutic and/or Prophylactic for Various Diseases where Cubilin is Involved

Cubilin exists as a membrane protein in vivo and binds to apolipoprotein A-I. The polypeptide of the invention, which is a partial fragment of cubilin having the ability to bind to apolipoprotein A-I, is able to repress the catabolism of apolipoprotein A-I by binding to apolipoprotein A-I contained in the blood. Therefore, the polypeptide and the DNA of the invention may be used as medicines such as therapeutics and/or prophylactics for diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders. It is possible to allow the role of the polypeptide of the invention to manifest sufficiently in patients by, for example, (a) administering the DNA of the invention to patients and allowing the polypeptide of the invention to express in vivo; (b) inserting the DNA of the invention into cells, allowing the expression of the polypeptide of the invention therein, and transplanting the cells into patients; or (c) administering the polypeptide of the invention to patients.

When the DNA of the invention is used as the above-mentioned therapeutic and/or prophylactic agent, the DNA per se or the DNA inserted into an appropriate vector such as a retrovirus vector, adenovirus vector, adeno-associated virus vector, etc. may be administered to human or other warm-blooded animals using conventional means. The DNA of the invention or a vector into which the DNA of the invention is inserted may be administered as it is or after formulation with physiologically acceptable carriers such as adjuvants to promote uptake. Usually, the DNA or the vector may be administered parenterally by means of, e.g., a gene gun or a catheter such as hydrogel catheter.

When the polypeptide of the invention is used as the above-described therapeutic and/or prophylactic agent, at least 90%, preferably 95% or more, more preferably 98% or more, still preferably 99% or more purified polypeptide of the invention is used.

The polypeptide of the invention may be used, for example, orally in the form of tablets (sugar-coated, if necessary), capsules, elixirs, microcapsules or the like; or parenterally in the form of injections such as aseptic solutions or suspensions in water or other pharmaceutically acceptable liquids. These preparations may be produced, for example, by mixing the polypeptide of the invention with physiologically acceptable carriers, flavoring agents, excipients, vehicles, antiseptics, stabilizers, binders, etc. in unit dosage forms required for preparing generally approved pharmaceutical preparations. The amounts of active ingredients in these formulations are decided so that an appropriate dose within the specified range can be obtained.

Examples of additives miscible with tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid, lubricants such as magnesium stearate, sweetening agents such as sucrose, lactose and saccharin, and flavoring agents such as peppermint, akamono oil and cherry. When the unit dosage form is capsule, liquid carrier such as oils and fats may further be included in addition to the above-mentioned materials. Sterile compositions for injection can be formulated according to conventional practices in pharmaceutical manufacturing, e.g., by dissolving or suspending active ingredients, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. in vehicles such as water for injection.

Examples of aqueous liquids for injection include physiological saline and isotonic solutions containing glucose and other auxiliary agents (e.g. D-sorbitol, D-mannitol, sodium chloride, etc.). They may be used in combination with a suitable auxiliary solubilizer such as alcohol (e.g. ethanol, etc.), polyalcohol (e.g. propylene glycol, polyethylene glycol, etc.), nonionic surfactant (e.g. Polysorbate 80™, HCO-50, etc.). Examples of oily liquids for injection include sesame oil, soybean oil, etc. They may be used in combination with an auxiliary solubilizer such as benzyl benzoate, benzyl alcohol, etc. In addition, buffers (e.g. phosphate buffer, sodium acetate buffer, etc.), soothing agents (e.g. benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g. human serum albumin, polyethylene glycol, etc.), preservatives (e.g. benzyl alcohol, phenol, etc.), antioxidants, etc. may also be admixed therewith. Usually, the prepared injections are filled in appropriate ampoules.

Since the thus obtained preparations are safe and of low toxicity, they can be administered to human or other warm-blooded animals (e.g., rat, mouse, guinea pig, rabbit, avian, sheep, pig, bovine, horse, cat, dog, monkey, etc.).

Dose of the polypeptide of the invention may vary depending upon the target disease, the target to be administered, administration route, and so on. However, when the polypeptide of the invention is administered orally for treating diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders, generally the polypeptide of the invention is administered to adult (60 kg in body weight) at a dose of about 1-1000 mg/day, preferably about 10-500 mg/day, more preferably about 10-200 mg/day. With respect to parenteral administration, when the polypeptide of the invention is administered to adult (60 kg in body weight) in the form of an injection for treating diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders, it is convenient to inject the polypeptide of the invention into the affected part of the body at a dose of about 1-1000 mg/day, preferably about 1-200 mg/day, and more preferably about 10-100 mg/day, though the dose per administration may vary depending on the patient to be treated, the target disease, etc. For other animals, corresponding doses may be administered after conversion of the above-mentioned values per 60 kg based on actual body weights.

(2) Screening for Candidate Compounds for Medicine to Diseases

(i) When the expression level of cubilin is abnormally enhanced, various diseases such as, for example, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardial infarction or angina occur.

Therefore, compounds or salts thereof that inhibit the binding of cubilin to apolipoprotein A-I may be used as medicines such as therapeutics and/or prophylactics for, e.g., hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardial infarction or angina.

(ii) On the other hand, since cubilin exists as a membrane protein in vivo and binds to apolipoprotein A-I, various diseases such as renal disorders, nephritis, nephropathy, proteinuria, nervous disorders and vitamin B₁₂ deficiency occur when cubilin or DNA encoding the same is abnormal or deficient, or cubilin expression is abnormally reduced.

Therefore, compounds or salts thereof that promote the binding of cubilin to apolipoprotein A-I may be used as medicines such as therapeutics and/or prophylactics for various diseases such as renal disorders, nephritis, nephropathy, proteinuria, neurological disorders and vitamin B₁₂ deficiency.

Therefore, use of cubilin for screening for those compounds or salts thereof that inhibit or promote the function of cubilin can be contemplated.

However, since cubilin is a large protein consisting of more than 3,500 amino acids, non-specific adsorption or binding, or degradation of cubilin molecules per se may occur when cubilin molecules are used in the screening for such compounds or salts thereof; due to these drawbacks, it is impossible to carry out highly sensitive and efficient screening.

On the other hand, the screening method using the polypeptide of the invention does not have such drawbacks. By using this screening method, compounds or salts thereof that inhibit or promote the binding between the polypeptide of the invention and apo A-I can be screened with high sensitivity and efficiently. Since such compounds or salts thereof inhibit or promote the binding between cubilin and apo A-I, they can be used as medicines such as therapeutics and/or prophylactics for the above-mentioned various diseases.

That is, the present invention provides:

• 1) {circle over (1)} a method for screening for compounds or salts thereof that inhibit the binding between cubilin and apo A-I (sometimes abbreviated to “inhibitor(s)” in “(2) Screening for Candidate Compounds for Medicine to Treat Diseases”) or compounds or salts thereof that promote the binding between cubilin and apo A-I (sometimes abbreviated to “promoter(s)” in “(2) Screening for Candidate Compounds for Medicine to Treat Diseases”), wherein the method is characterized by using the polypeptide of the invention; and {circle over (2)} a screening kit for inhibitors or promoters comprising the polypeptide of the invention (sometimes referred to as the “screening kit of the invention” in “(2) Screening for Candidate Compounds for Medicine to Treat Diseases”). More specifically, the present invention provides:
• 2) {circle over (1)} a method for screening for promoters or inhibitors, comprising comparing (i) the case where apo A-I is contacted with the polypeptide of the invention and (ii) the case where apo A-I and a test compound are contacted with the polypeptide of the invention; and {circle over (2)} a screening kit for promoters and inhibitors, comprising the polypeptide of the invention and apo A-I.

Specifically, the above-mentioned screening method is characterized by measuring and comparing the amounts of binding of apo A-I to the polypeptide of the invention or the amounts of binding of the polypeptide of the invention to apo A-I in the cases (i) and (ii).

These amounts of binding can be measured by publicly known methods or methods based on them.

Examples of test compounds include, but not limited to, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts. These compounds may be either novel compounds or publicly known compounds.

As the peptide, partial peptides of apo A-I are preferable.

For example, a test compound that reduces the amount of binding in case (ii) above by about 20% or more, preferably 30% or more, more preferably about 50% or more compared to the amount of binding in case (i) above may be selected as a compound that inhibits the binding between cubilin and apo A-I. On the other hand, a test compound that increases the amount of binding in case (ii) above by about 20% or more, preferably 30% or more, more preferably about 50% or more compared to the amount of binding in case (i) above may be selected as a compound that promotes the binding between cubilin and apo A-I.

The compounds of salts thereof obtained by using the screening method or the screening kit of the invention are compounds that are selected from the above-mentioned test compounds, e.g. peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc., and that promote or inhibit the function of cubilin (e.g. binding to apolipoprotein A-I).

As salts of such compounds, salts same as those salts of the polypeptide of the invention described herein earlier may be used.

Hereinbelow, the screening method will be described specifically.

The screening may be performed by SPA (scintillation proximity assay) method (Anal. Biochem. 1987 March; 161 (2): 494-500), fluorescence binding assay, a method using a surface plasmon sensor, or a method based on them.

[I] SPA method may be performed as follows.

The polypeptide of the invention is bound to SPA beads directly or indirectly and then mixed with (i) apo A-I labeled with a radioactive isotope or (ii) apo A-I labeled with a radioactive isotope and a test compound. Subsequently, the fluorescence intensities in case (i) and case (ii) are measured and compared.

More specifically, the method is performed as follows:

{circle over (1)} The polypeptide of the invention is bound to SPA beads. The binding may be either direct or indirect

As a method for direct binding, the method described in the protocol attached to SPA beads may be used. As a method for indirect binding, the following methods may be enumerated, for example:

• • (i) A method in which SPA beads coated with copper are used, and histidine tag-added polypeptide of the invention are bound to these SPA beads,
  • (ii) A method in which SPA beads coated with glutathione are used, and a fusion protein composed of GST and the polypeptide of the invention is bound to these SPA beads,
  • (iii) A method in which SPA beads coated with a secondary antibody are used, and the polypeptide of the invention and these SPA beads are bound via a linker that is an antibody to the polypeptide,
  • (iv) A method in which SPA beads coated with a secondary antibody are used, and the polypeptide of the invention to which a tag such as FLAG or myc has been added is bound to these SPA beads via an antibody to the tag,
  • (v) A method in which SPA beads coated with streptavidin are used, and biotin-labeled polypeptide of the invention is bound to these SPA beads,
  • (vi) A method in which SPA beads coated with streptavidin are used, and the polypeptide of the invention to which a tag such as FLAG or myc has been added is bound to these SPA beads via a linker that is a biotin-labeled antibody to the tag.

When the polypeptide of the invention has sugar chains, the following method may also be used:

• • (vii) A method in which SPA beads coated with WGA (wheat germ agglutinin) are used, and the polypeptide of the invention is bound to these SPA beads.

SPA beads coated with such materials may be prepared by publicly known methods or methods based on them. However, it would be convenient to purchase them from Amersham Pharmacia Biotech.

{circle over (2)} (i) When a tag is added to the polypeptide of the invention, any conventional tag used in the art may be used as long as it can be detected specifically.

Preferable examples include tags consisting of several amino acids, such as FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) or myc (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu), and histidine tag that do not affect the binding between the polypeptide of the invention and apo A-I.

By expressing a fusion composed of such a tag and the polypeptide of the invention, a tag-added polypeptide of the invention can be obtained. The fusion can be obtained by inserting a DNA encoding the polypeptide of the invention into a vector such as pFLAG-CMV-1, pSecTag or pcDNA3.1/His, introducing the vector into an appropriate host cell such as E. coli, COS7 or CHO, and allowing the host cell to produce the polypeptide in cells or cell membranes or outside of cells. The tag may be added to any site of the polypeptide of the invention, but a site that does not affect the binding with apo A-I is preferable. Hereinafter, the polypeptide of the invention to which a tag has been added is sometimes referred to as “Tag-CUBIII”.

(ii) A fusion protein composed of GST and the polypeptide of the invention can be prepared by publicly known methods or methods based on them. The site of fusion may be any site of the polypeptide of the invention, but a site that does not affect the binding with apo A-I is preferable.

(iii) When apo A-I is labeled with a radioactive isotope (e.g. [¹²⁵I], [¹³¹I], [³H], [¹⁴C], [³⁵S] or [³³P]), the labeling may be performed by publicly known methods or methods based on them. Specifically, apo A-I is dissolved in 40 ml of 0.4 M glycine-NaOH buffer (pH 8.5) at 50 μg/ml, followed by addition of 1 mCi of ¹²⁵I for labeling reaction. To the reaction solution, 50 μl of Cloramin T solution (4 mg/ml) is added and maintained at room temperature for 15 min. Then, 100 μl of metabisulfite solution (16 mg/ml) is added thereto and stirred for 10 min. The resultant reaction solution is extracted with 40 ml of acetone/diethyl ether (3:1, v/v). The resultant precipitate is dried, dissolved in a phosphate buffer and subjected to dialysis against the buffer to thereby obtain ¹²⁵I-labeled apo A-I.

(iv) When an anti-tag antibody is modified with biotin, an anti-tag antibody may be prepared by publicly known methods or methods based on them using a tag such as FLAG or myc as an antigen. Such an antibody may be either a monoclonal antibody or a polyclonal antibody. Commercial anti-tag antibodies may also be used. As an anti-myc antibody, Clone 9E10 (Sigma) or the like may be used. As an anti-FLAG antibody, Clone M1 (Sigma) or the like may be used.

The modification of anti-tag antibodies and the polypeptide of the invention with biotin may be performed by publicly known methods or methods based on them. Specifically, an anti-tag antibody or the polypeptide of the invention is dissolved in a carbonate buffer (0.1 M sodium hydrogencarbonate, 0.1 M sodium chloride, pH 8.3) to give a concentration of 1 mg/ml. To this solution, 50 μl of an aqueous solution of Sulfo-NHS-Biotin (Pierce) (1 mg/ml) is added and mixed gently overnight at 4° C. Then, the reaction solution is dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride to perform biotin modification.

(v) After the procedures described in (i) to (iv) have been completed, the fluorescence intensity is measured with a scintillator. If the fluorescence intensity is decreased by the addition of a test compound, the compound can be selected as an inhibitor. If the fluorescence intensity is increased by the addition of a test compound, the compound can be selected as a promoter.

Hereinbelow, operational procedures will be described more specifically. Briefly, 1 mg of streptavidin-yttrium silicate type SPA beads, 0.5 μl of biotin-modified myc monoclonal antibody (9E10; Sigma), 10-200 ng of purified polypeptide of the invention (CUBIII-myc-His fragment), 250,000 cmp of radioactive iodine-labeled apo A-I, and 10 pmol to 100 pmol of unlabeled apo A-I are mixed in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, 1.0 mM calcium chloride and 0.1% sodium azide hereinafter, referred to as the “binding reaction buffer”) to give a reaction solution of 200 μl.

This reaction solution is mixed gently at 25° for 1 hr and centrifuged in a bench type centrifuge at 1,000 rpm for 2 min to precipitate SPA beads. Then, the fluorescence emitted by SPA beads is measured with a scintillation counter. For the purpose of evaluation of substances that inhibit or promote binding, a test substance is dissolved in the above-mentioned binding reaction buffer and included in a 200 μl reaction solution. Substances that change scintillation counts are evaluated taking the scintillation count in the absence of the test substance as 100%.

The screening kit of the invention comprises labeled apo A-I, variously coated SPA beads and the polypeptide of the invention.

[II] Fluorescence apo A-I binding assay is performed using a plate coated with the fragment peptide of the invention.

Specifically, the assay is performed as described below. After the polypeptide of the invention is solidified in a plate, labeled apo A-I and a test compound are mixed and incubated on the plate. After washing, the amount of labeled apo A-I bound to the polypeptide is determined by an appropriate method.

For example, the polypeptide of the invention is diluted to 0.1-10 μg/ml in a buffer and solidified by incubation in a high binding ability-type microplate for fluorescence measurement (e.g. Black Clini plate enhanced binding; Labsystems). As the buffer, a buffer containing 20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 2 mM CaCl₂ and 0.1% sodium azide may be used, for example.

After the polypeptide is solidified by an overnight or longer incubation, non-specific binding is inhibited with an appropriate blocking reagent. For this purpose, SuperBlock TBS (Pierce) may be used, for example.

After this blocking, the microplate is washed with a buffer to be used in the subsequent binding reaction. Then, a mixture of labeled apo A-I and a test compound is incubated on wells of the microplate to carry out competitive binding reaction. As the buffer, a buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl₂, 0.1% sodium azide and 5% Block-Ace (Dainippon Pharmaceutical) may be used, for example. For labeling apo A-I particles, Sulfo-NHS-LC-LC-Biotin may be used, for example. Specifically, apo A-I is dissolved in 50 mM sodium bicarbonate (pH 8.5) at a concentration of 1 mg/ml. To this solution, 1/10 volume of Sulfo-NHS-LC-LC-Biotin dissolved in pure water at 1 mg/ml is added and incubated overnight at 4° to thereby prepare biotin-labeled apo A-I. As the concentration of biotin-labeled apo A-I in the competitive binding reaction solution, a concentration of approximately 0.1 μg/ml may be used, for example.

For quantitative determination of biotin-labeled apo A-I bound to the polypeptide after washing, β-galactosidase-labeled streptavidin may be used, for example. For measuring its activity, 4-methylumbelliferyl-β-D-galactopyranoside or the like may be used. In this case, the amount of biotin-labeled apo A-I specifically bound to the polypeptide of the invention can be calculated and determined by measuring excitation light at around 365 nm and fluorescence emission at around 460 nm.

[III] The method using a surface plasmon sensor may be performed using BIACORE™ 3000 (Biacore) or the like according to the protocol attached thereto. Specifically, this method may be performed as described below.

The polypeptide of the invention is bound to a sensor chip directly or indirectly, and

• • (i) apo A-I solution is applied to the sensor chip; or (ii) apo A-I solution containing a test substance is applied to the sensor chip. Then, the surface plasmon values in the cases of (i) and (ii) are measured and compared.

If the fluorescence intensity is decreased by the addition of a test compound, the compound can be selected as an inhibitor. If the fluorescence intensity is increased by the addition of a test compound, the compound can be selected as a promoter.

More specifically:

{circle over (1)} The polypeptide of the invention is fixed on the sensor chip. This fixing may be either direct fixing or indirect fixing.

Direct fixing may be performed by any binding method. Preferably, the polypeptide is fixed on the sensor chip by covalent bond as NHS-ester. As a method for indirect fixing, a method based on the above-mentioned method may be used.

Alternatively, apo A-I may be fixed on a sensor chip, and a solution of the polypeptide of the invention may be applied to the sensor chip.

{circle over (2)} Subsequently, apo A-I solution is applied to the sensor chip.

{circle over (3)} The surface plasmon is measured.

Specifically, a sensor chip CM5 is treated with a 1:1 mixture of 0.2 M n-ethyl-N′-(3 diethylaminopropyl)-carbodiimide aqueous solution and 0.05 M N-hydroxysuccinimide aqueous solution. To this sensor chip, the polypeptide of the invention (CUBIII-myc-His fragment) dissolved in 10 mM sodium acetate (pH 4.0) at 40 μg/ml is applied to thereby fix the polypeptide of the invention to censor chip CM5. For blocking, 1 M ethanol amine (pH 8.5) is applied. As a buffer for measuring binding, a buffer containing 10 mM sodium dihydrogen phosphate, 150 mM sodium chloride, 1.0 mM calcium chloride and 0.1% sodium azide (pH 7.4) is used. After equilibration with this buffer and stabilization of plasmon changes, apo A-I diluted with this buffer to 50-500 nM is applied to the sensor chip to measure changes in surface plasmon. For the purpose of evaluation of substances that inhibit or promote the binding, a test substance is dissolved in the above-described buffer and mixed with the apo A-I solution. By comparing changes in surface plasmon in the absence of a test compound with changes in the presence of the test compound, binding inhibitors or promoters are evaluated.

The screening kit of the invention comprises a sensor chip on which the polypeptide of the invention or apo A-I is fixed, and apo A-I or the polypeptide.

Compounds or salts thereof that are obtainable by using the screening method or screening kit of the invention are those compounds that are selected from, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and plasma, and that promote or inhibit the function of the polypeptide of the invention.

As salts of such compounds, salts same as those salts of the polypeptide of the invention described herein earlier may be used.

When the compound obtained by using the screening method or screening kit of the invention is used as the above-mentioned therapeutic and/or prophylactic agent, it may be used according to conventional procedures. For example, the compound may be formulated into tablets, capsules, elixirs, microcapsules, aseptic solutions, suspensions, etc. in the same manner as described in the preparation of medicines comprising the polypeptide of the invention.

Since the thus obtained pharmaceutical preparations are safe and of low toxicity, they can be administered to human or other warm-blooded animals (e.g., mouse, rat, rabbit, sheep, pig, bovine, horse, avian, cat, dog, monkey, etc.).

Dose levels of the compound or salt thereof may vary depending upon the target disease, the target to be administered, administration route, and so on. However, when a compound that promotes the binding between the polypeptide of the invention and apo A-I is administered orally for treating nephritis, nephropathy, hyper-high density lipoproteinemia, hyperapolipoproteinemia A-I or proteinuria, generally the compound is administered to adult (60 kg in body weight) at a dose of about 0.1-100 mg/day, preferably about 1.0-50 mg/day, more preferably about 1.0-20 mg/day. With respect to parenteral administration, when a compound that promotes the binding between the polypeptide of the invention and apo A-I is administered to adult (60 kg in body weight) in the form of an injection for treating nephritis, nephropathy, hyper-high density lipoproteinemia, hyperapolipoproteinemia A-I or proteinuria, it is convenient to inject the compound intravenously at a dose of about 0.01-30 mg/day, preferably about 0.1-20 mg/day, and more preferably about 0.1-10 mg/day, though the dose per administration may vary depending on the target to be administered, the target disease, etc. For other animals, corresponding doses may be administered after conversion of the above-mentioned values per 60 kg based on actual body weights.

On the other hand, when a compound that inhibits the binding between the polypeptide of the invention and apo A-I is administered orally for treating diseases such as hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, arteriosclerosis, myocardial infarction or angina, generally the compound is administered to adult (60 kg in body weight) at a dose of about 0.1-100 mg/day, preferably about 1.0-50 mg/day, more preferably about 1.0-20 mg/day. With respect to parenteral administration, when a compound that inhibits the binding between the polypeptide of the invention and apo A-I is administered to adult (60 kg in body weight) in the form of an injection for treating diseases such as hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, arteriosclerosis, myocardial infarction or angina, it is convenient to inject the compound intravenously at a dose of about 0.01-30 mg/day, preferably about 0.1-20 mg/day, and more preferably about 0.1-10 mg/day, though the dose per administration may vary depending on the target to be administered, the target disease, etc. For other animals, corresponding doses may be administered after conversion of the above-mentioned values per 60 kg based on actual body weights.

(3) Quantitative Determination of the Polypeptide of the Invention or Salt Thereof

Since an antibody to the polypeptide of the invention (hereinafter, sometimes referred to as the “antibody of the invention”) can specifically recognize the polypeptide of the invention and cubilin (hereinafter, sometimes referred to as the “polypeptide of the invention” in “(4) Quantitative Determination of the Polypeptide of the Invention of Salt Thereof”), the antibody may be used for quantitative determination of the polypeptide of the invention contained in a sample solution. In particular, the antibody may be used in quantitative determination by sandwich immunoassay technique.

The present invention provides:

• (i) a method of quantitative determination of the polypeptide of the invention in a sample solution, comprising reacting the antibody of the invention with the sample solution and the polypeptide of the invention labeled, competitively and determining the ratio of the labeled polypeptide of the invention bound to the antibody; and (ii) a method of quantitative determination of the polypeptide of the invention in a sample solution, comprising reacting the sample solution with the antibody of the invention insolubilized on a carrier and another antibody of the invention labeled, simultaneously or in succession and determining the activity of the label on the insolubilized carrier.

A monoclonal antibody to the polypeptide of the invention (hereinafter, sometimes referred to as the “monoclonal antibody of the invention”) may be used to quantitatively determine the polypeptide of the invention or may be used for detection of the polypeptide by tissue staining. For these purposes, either antibody molecules per se or the F(ab′)₂, Fab′ or Fab fragment thereof may be used.

Methods of quantitative determination of the polypeptide of the invention using the antibody of the invention are not particularly limited. Any measuring method may be used in which the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of the antigen in a sample solution (e.g. the amount of the polypeptide of the invention) is detected by chemical of physical means, and then calculated from a standard curve prepared with a standard solution containing a known amount of the antigen. For example, nephrometry, competitive method, immunometric method and sandwich method may be used suitably and, in terms of sensitivity and specificity, the sandwich method described later is particularly preferred.

Examples of labeling agents used in measuring methods utilizing labeling substances include radioisotopes, enzymes, fluorescent substances, and luminescent substances. Examples of radioisotopes include [¹²⁵I], [¹³¹I], [³] and [¹⁴C]. Preferred examples of enzymes are those which are stable and with high specific activity, e.g., β-galactosidase, β-glucosidase, alkali phosphatase, peroxidase and malate dehydrogenase. Examples of fluorescent substances include fluorescamine and fluorescein isothiocyanate. Examples of luminescent substances include luminol, luminol derivatives, luciferin, and lucigenin. Further, a biotin-avidin system may also be used for binding an antibody or antigen with a labeling agent

Insolubilization of antigens or antibodies may be performed by physical adsorption or by chemical binding usually used for insolubilizing or immobilizing polypeptides or enzymes. Examples of carriers include insoluble polysaccharides such as agarose, dextran and cellulose; synthetic resins such as polystyrene, polyacrylamide and silicone; and glass.

In sandwich method, a sample solution is reacted with an insolubilized monoclonal antibody of the invention (primary reaction); then, another monoclonal antibody of the invention that is labeled is reacted therewith (secondary reaction); and the activity of the labeling agent on the insolubilized carrier is measured to thereby quantitatively determine the amount of the polypeptide of the invention in the sample solution. The primary reaction and the secondary reaction may be conducted in a reverse order, or they may be conducted simultaneously or with an interval. The type of the labeling agent and the method of insolubilization may be the same as those described herein earlier. In immunoassays using the sandwich technique, the antibody insolubilized on a solid phase or the antibody labeled is not necessarily a single antibody; a mixture of two or more antibodies may be used for the purposes of enhancing the sensitivity of measurement, etc.

In the method of measuring the polypeptide of the invention by the sandwich method of the invention, the monoclonal antibodies of the invention used in the primary and the secondary reactions are preferably those antibodies wherein their sites binding of the polypeptide of the invention are different from each other. For example, if the antibody used in the secondary reaction recognizes the C-terminal region of the polypeptide of the invention, an antibody that recognizes a site other than the C-terminal region, e.g. an N-terminal region, is preferably used in the primary reaction.

The monoclonal antibody of the invention may be used in a measuring system other than the sandwich method, such as competitive methods, immunometric methods and naphrometry.

In competitive methods, an antigen in a sample solution and a labeled antigen are reacted competitively with an antibody; then, unreacted labeled antigen (F) and labeled antigen bound to the antibody (B) are separated (i.e. B/F separation); and the amount of the label of B or F to thereby quantitatively determine the amount of the antigen in the sample solution. With respect to this reaction method, there are a liquid phase method in which a soluble antibody is used and the B/F separation is conducted with polyethylene glycol and a second antibody to the above-mentioned antibody; and a solid phase method in which a solidified antibody is used as the first antibody or a soluble antibody is used as the first antibody while a solidified antibody is used as the second antibody.

In immunometric methods, an antigen in a sample solution and a solidified antigen are reacted competitively with a specific amount of a labeled antibody, followed by separation of the solid phase from the liquid phase; or an antigen in a sample solution is reacted with an excessive amount of a labeled antibody, and then a solidified antigen is added to bind unreacted labeled antibody to the solid phase, followed by separation of the solid phase from the liquid phase. Subsequently, the amount of label in one of the phases is measured to determine the amount of the antigen in the sample solution.

In nephrometry, the amount of insoluble precipitate generated as a result of antigen-antibody reaction in a gel or solution is measured. Even when the amount of the antigen in a sample solution is small and thus only a small amount of such precipitate is obtained, laser nephrometry utilizing the scattering of laser can be used suitably.

In applying each of those immunological measuring methods to the measuring method of the present invention, no special conditions or operations are required. A measuring system for the polypeptide of the present invention may be constructed using the conventional conditions and operational procedures in the relevant measuring method while taking into account usual technical consideration of those skilled in the art. For details of these commonly used technical means, a variety of reviews, reference books, etc. may be referred to.

For example, Hiroshi Irie (ed.): “Radioimmunoassay” (Kodansha, 1974); Hiroshi Irie (ed.): “Radioimmunoassay; Second Series” (Kodansha, 1979); Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay” (Igaku Shoin, Japan, 1978); Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay” (Second Edition) (Igaku Shoin, 1982); Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay” (Third Edition) (Igaku Shoin, 1987); “Methods in Enzymology”, Vol. 70 (Immunochemical Techniques (Part A)); ibid., Vol. 73 (Immunochemical Techniques (Part B)); ibid., Vol. 74 (Immunochemical Techniques (Part C)); ibid., Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)); ibid., Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (Academic Press) and the like may be referred to.

By using the antibody of the invention as described above, the polypeptide of the invention can be quantitatively determined with high sensitivity.

Further, by quantitatively determining the concentration of the polypeptide of the invention using the antibody of the invention, it is possible to diagnose that a subject has a disease such as, e.g., hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, arteriosclerosis, myocardial infarction or angina, or that a subject is very likely to develop such a disease in the future, when an increase of decrease is detected in the concentration of the polypeptide of the invention in the subject.

Further, the antibody of the invention may be used for detecting the polypeptide of the invention present in body fluid, tissue or other samples. The antibody of the invention may also be used in the preparation of antibody columns for use in the purification of the polypeptide of the invention; in the detection of the polypeptide of the invention in individual fractions generated in the course of purification; and in the analysis of the behavior of the polypeptide of the invention in test cells.

(4) Gene Diagnostic Agent

When used as a probe, the DNA of the invention can detect abnormalities (gene abnormalities) in DNA or mRNA encoding the polypeptide of the invention in human or other warm-blooded animals (e.g. rat, mouse, guinea pig, rabbit, avian, sheep, pig, bovine, horse, cat, dog, monkey, etc.). Thus, the DNA of the invention is useful as a gene diagnostic agent for, e.g., damage, mutations or reduced expression of the DNA or mRNA encoding the polypeptide of the invention, or increase or excessive expression of the DNA or mRNA.

Gene diagnosis using the DNA of the invention described above, may be performed by publicly known methods such as Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, 874-879 (1989); Proc. Natl. Acad. Sci. USA 86: 2766-2770 (1989)).

When a reduction in expression is detected by Northern hybridization or when mutations are detected in the DNA by PCR-SSCP method, for example, it is possible to diagnose that the relevant subject is very likely to have a disease such as renal disorders, nephritis, nephropathy, proteinuria, nervous disorders, or vitamin B₁₂ deficiency.

On the other hand, when excessive expression is detected by Northern hybridization, it is possible to diagnose that the relevant subject has a disease such as, e.g., hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, arteriosclerosis, myocardial infarction or angina.

(5) Medicines Containing the Antibody of the Invention

The antibody of the invention that neutralizes the activity of the polypeptide of the invention may be used as a therapeutic and/or prophylactic agent for, e.g., diseases caused by excessive expression of the polypeptide of the invention.

The neutralizing antibody of the invention specifically recognizes the apo A-I-binding site in cubilin or regions in the vicinity thereof. Therefore, like the inhibitor, the antibody may be used as medicines such as therapeutics and/or prophylactics for diseases such as, e.g., hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia hypoapolipoproteinemia A-I, after meal hyperlipemia, arteriosclerosis, myocardial infarction or angina.

For these purposes, either antibody molecules per se or the F(ab′)₂, Fab′ or Fab fragment thereof may be used.

Therapeutics and/or prophylactics for the above-mentioned diseases comprising the antibody of the invention may be administered orally or parenterally to human or other warm-blooded animals (e.g., rat, rabbit, sheep, pig, bovine, cat, dog, monkey, etc.) in the form of a liquid preparation without any processing or in appropriate forms of pharmaceutical compositions. Dose levels may vary depending upon the patient to be treated, the target disease, symptoms, administration route, and so on. However, for the purpose of treating diseases such as hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, arteriosclerosis, myocardial infarction or angina, it is convenient to inject the antibody of the invention intravenously at a dose of about 0.01-20 mg/kg body weight, preferably about 0.1-10 mg/kg body weight, more preferably about 0.1-5 mg/kg body weight per administration about one to five times a day, preferably about one to three times a day. In other parenteral administration and oral administration, similar dose levels may be used. If symptoms are particularly heavy, the dose may be increased accordingly.

The antibody of the invention may be adminstered per se or in the forms of appropriate pharmaceutical compositions. The pharmaceutical compositions for the above administration comprise the antibody or salt thereof, pharmacologically acceptable carriers, and diluents or excipients. Such compositions are provided in forms appropriate for oral or parenteral administration.

For example, compositions for oral administration include solid or liquid preparations such as tablets (including sugar-coated tablets and film-coated tablets), pills, granules, dispersants, capsules (including soft capsules), syrups, emulsions and suspensions. These compositions are prepared according to conventional methods and contain carriers, diluents or excipients conventionally used in the field of medicine manufacture. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers or excipients for tablets.

Compositions for parenteral administration include, for example, injections and suppositories. Injections include intravenous injections, subcutaneous injections, intradermal injections, muscle injections, instilment injections, etc. Such injections may be prepared by dissolving, suspending or emulsifying the above antibody or salt thereof in an aseptic, aqueous or oily liquid. Examples of aqueous liquids for injection include physiological saline and isotonic solutions containing glucose and other auxiliary agents. They may be used in combination with a suitable auxiliary solubilizer such as alcohol (e.g. ethanol), polyalcohol (e.g. propylene glycol, polyethylene glycol), nonionic surfactant [e.g. Polysorbate 80™, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.). Examples of oily liquids for injection include sesame oil and soybean oil. They may be used in combination with an auxiliary solubilizer such as benzyl benzoate, benzyl alcohol, etc. Usually, the prepared injections are filled in appropriate ampoules.

It is convenient to formulate the above-described pharmaceutical compositions for oral or parenteral administration into unit dosage forms that would give an appropriate dose of the active ingredient Examples of such unit dosage forms include tablets, pills, capsules, injections (ampoules), and suppositories. Usually, each unit of these dosage forms contains preferably about 5-500 mg of the above-described antibody. In particular, each unit contains preferably about 5-100 mg in injections, and each unit in other dosage forms contains preferably about 10-250 mg.

The above-described pharmaceutical compositions may contain other active ingredients as long as they do not produce undesirable interaction with the above-described antibody.

In the specification and drawings of the present application, the abbreviations used for bases, amino acids and so forth are those recommended by the IUPAC-IUB Commission on Biochemical Nomenclature or those conventionally used in the art. Examples of such abbreviations are given below. Amino acids, which may have optical isomers are intended to represent their L-isomer unless otherwise specified.

• DNA: Deoxyribonucleic acid
• cDNA: Complementary deoxyribonucleic acid
• A: Adenine
• T: Thymine
• G: Guanine
• C: Cytosine
• RNA: Ribonucleic acid
• mRNA: Messenger ribonucleic acid
• dATP: Deoxyadenosine triphosphate
• dTTP: Deoxythymidine triphosphate
• dGTP: Deoxyguanosine triphosphate
• dCTP: Deoxycytidine triphosphate
• ATP: Adenosine triphosphate
• EDTA: Ethylenediaminetetracetic acid
• SDS: Sodium dodecyl sulfate
• Gly: Glycine
• Ala: Alanine
• Val: Valine
• Leu: Leucine
• Ile: Isoleucine
• Ser: Serine
• Thr: Threonine
• Cys: Cysteine
• Met: Methionine
• Glu: Glutamic acid
• Asp: Aspartic acid
• Lys: Lysine
• Arg: Arginine
• His: Histidine
• Phe: Phenylalanine
• Tyr: Tyrosine
• Trp: Tryptophan
• Pro: Proline
• Asn: Asparagine
• Gln: Glutamine
• pGlu: Pyroglutamic acid

The substituents, protective groups and reagents that are frequently used in the specification are represented by the following abbreviations.

• Me: Methyl group
• Et: Ethyl group
• Bu: Butyl group
• Ph: Phenyl group
• TC: Thiazolidine-4(R)-carboxamide group
• Tos: p-Toluene sulfonyl
• CHO: Formyl
• Bzl: Benzyl
• Cl₂Bzl: 2,6-Dichlorobenzyl
• Bom: Benzyloxymethyl
• Z: Benzyloxycarbonyl
• Cl-Z: 2-Chlorobenzyloxycarbonyl
• Br-Z: 2-Bromobenzyloxycarbonyl
• Boc: t-Butoxycarbonyl
• DNP: Dinitrophenol
• Trt: Trityl
• Bum: t-Butoxymethyl
• Fmoc: N-9-Fluorenylmethyloxycarbonyl
• HOBt: 1-Hydroxybenzotriazole
• HOOBt: 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
• HONB: 1-Hydroxy-5-norbornene-2,3-dicarboximide
• DCC: N,N′-Dicyclohexylcarbodiimide

The SEQ ID NOS of the SEQUENCE LISTING of the present specification represent the sequences as indicated below.

[SEQ ID NO: 1]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 2]

This shows the base sequence of an antisense-strand primer used in Example 1.

[SEQ ID NO: 3]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 4]

This shows the base sequence of an antisense-strand primer used in Example 1.

[SEQ ID NO: 5]

This shows the base sequence of a cDNA encoding a cubilin partial peptide obtained in Example 1.

[SEQ ID NO: 6]

This shows the base sequence of a cDNA encoding a cubilin partial peptide obtained in Example 1.

[SEQ ID NO: 7]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 8]

This shows the base sequence of an antisense-strand primer used in Example 1.

[SEQ ID NO: 9]

This shows the base sequence of a cDNA encoding partial fragment III of human cubilin.

[SEQ ID NO: 10]

This shows the amino acid sequence of partial fragment III of human cubilin.

[SEQ ID NO: 11]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 12]

This shows the base sequence of an antisense-strand primer used in Example 1.

[SEQ ID NO: 13]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 14]

This shows the base sequence of an antisense-strand primer used in Example 1.

[SEQ ID NO: 15]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 16]

This shows the base sequence of an antisense-strand primer used in Example 1.

[SEQ ID NO: 17]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 18]

This shows the base sequence of an antisense-strand primer used in Example 1.

[SEQ ID NO: 19]

This shows the amino acid sequence of fragment peptide CUB9-CUB14 that is a partial fragment of human cubilin.

[SEQ ID NO: 20]

This shows the base sequence of a cDNA encoding fragment peptide CUB7-CUB12 that is a partial fragment of human cubilin.

[SEQ ID NO: 21]

This shows the amino acid sequence of a polypeptide used in Example 5.

[SEQ ID NO: 22]

This shows the base sequence of a cDNA encoding fragment peptide CUB9-CUB14 that is a partial fragment of human cubilin.

A transformant Escherichia coli DH5α/pTB2116 obtained in Example 1 has been deposited at the International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology, located at Central 6, 1-1 Higashi 1-chome, Tsukuba, Ibaraki under the Accession No. FERM BP-7190 since Jun. 19, 2000, and at the Institute for Fermentation, Osaka (IFO), located at 17-85 Jusanboncho 2-chome, Yodogawa-ku, Osaka, Osaka under the Accession No. IFO 16438 since Jun. 1, 2000.

Another transformant Escherichia coli DH5α/pTB2231 obtained in Example 4 has been deposited at the International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology, located at Central 6, 1-1 Higashi 1-chome, Tsukuba, Ibaraki under the Accession No. FERM BP-7607 since May 24, 2001, and at the Institute for Fermentation, Osaka (IFO), located at 17-85 Jusanboncho 2-chome, Yodogawa-ku, Osaka, Osaka under the Accession No. IFO 16626 since May 17, 2001.

EXAMPLES

The present invention will be described in more detail with reference to the following Examples. These Examples are provided only for explanation, and are not intended to limit the scope of the present invention.

Example 1

Cloning of cDNAs Encoding Partial Fragments of Human Cubilin

Using a human small intestine-derived cDNA library, cDNAs encoding partial fragments of human cubilin were cloned by PCR as described below.

A PCR reaction was performed using Marathon Ready cDNA (Clontech) derived from human small intestine, the oligo DNA shown in SEQ ID NO: 1 as a sense strand primer and the oligo DNA shown in SEQ ID NO: 2 as an anti-sense strand primer to thereby obtain a cDNA (SEQ ID NO: 5) encoding a partial peptide of human cubilin. A cDNA (SEQ ID NO: 6) encoding a partial peptide of human cubilin was obtained in the same manner by PCR using the oligo DNA shown in SEQ ID NO: 3 as a sense strand primer and the oligo DNA shown in SEQ ID NO: 4 as an anti-sense strand primer. Then, the cDNA having the base sequence as shown in SEQ ID NO: 5 and the cDNA having the base sequence as shown in SEQ ID NO: 6 were mixed. Using this mixture as a template, a PCR reaction was performed with the oligo DNA shown in SEQ ID NO: 7 as a sense strand primer and the oligo DNA shown in SEQ ID NO: 8 as an anti-sense strand primer. After digestion with restriction enzymes BamHI and NotI, the PCR product was inserted into a vector pSecTag2 (Invitrogen) pre-treated with the same restriction enzymes. As a result, a 2781 bp cDNA shown in SEQ ID NO: 9 encoding partial fragment III of human cubilin [SEQ ID NO: 10; a fragment having the amino acid sequence consisting of the amino acids from position 1165 to position 2091 of the amino acid sequence of human cubilin mature protein (FIGS. 1 and 2). This fragment corresponds to fragment peptide CUB7-CUB14.] was cloned into the vector that is an eukaryotic expression vector where Igκ signal sequence has been added to an upstream region and Myc and His tags have been added to a downstream region.

Similarly, using a cDNA obtained from Caco-2 cell-derived purified mRNA by RT-PCR with random primers, a PCR reaction was performed with the oligo DNA shown in SEQ ID NO: 11 as a sense strand primer and the oligo DNA shown in SEQ ID NO: 12 as an anti-sense strand primer to thereby obtain a cDNA encoding partial fragment I [a fragment having the amino acid sequence consisting of the amino acids from position 25 to position 816 of the amino acid sequence of human cubilin mature protein (FIGS. 1 and 2)]. A cDNA encoding partial fragment II [a fragment having the amino acid sequence consisting of the amino acids from position 474 to position 1390 of the amino acid sequence of human cubilin mature protein (FIGS. 1 and 2)] was obtained in the same manner using the oligo DNA shown in SEQ ID NO: 13 as a sense strand primer and the oligo DNA shown in SEQ ID NO: 14 as an anti-sense strand primer. These cDNAs were individually cloned into the above-described expression vector.

Further, using a human small intestine-derived Marathon Ready cDNA (Clontech), a PCR reaction was performed similarly with the oligo DNA shown in SEQ ID NO: 15 as a sense strand primer and the oligo DNA shown in SEQ ID NO: 16 as an anti-sense strand primer to thereby obtain a cDNA encoding partial fragment IV [a fragment having the amino acid sequence consisting of the amino acids from position 1852 to position 2804 of the amino acid sequence of human cubilin mature protein (FIGS. 1 and 2)]. A cDNA encoding partial fragment V [a fragment having the amino acid sequence consisting of the amino acids from position 2569 to position 3623 of the amino acid sequence of human cubilin mature protein (FIGS. 1 and 2)] was obtained in the same manner using the oligo DNA shown in SEQ ID NO: 17 as a sense strand primer and the oligo DNA shown in SEQ ID NO: 18 as an anti-sense strand primer. These cDNAs were individually cloned into the above-described expression vector.

Further, a plasmid pTB2116 obtained by integrating the cDNA (SEQ ID NO: 9) encoding a partial fragment III of human cubilin into pSecTag2 vector as described above was introduced into Escherichia coli DH5α according to publicly known methods to thereby obtain a transformant designated Escherichia coli DH5α/pTB2116.

Example 2

Expression of Human Cubilin Partial Fragments in COS7 Cells

The expression vector pTB2116 obtained in Example 1 comprising the cDNA having the base sequence as shown in SEQ ID NO: 9 was transiently transfected into COS7 cells using a transfection reagent TransFast (Promega). Subsequently, the cells were transferred to a serum-free medium to allow them to produce a polypeptide partial fragment III) having the amino acid sequence as shown in SEQ ID NO: 10 in the serum-free medium. With respect to partial fragments I, II, IV and V, corresponding expression vectors were separately transfected into COS7 cells in the same manner to allow the resultant cells to produce respective fragments in the serum-free medium. In order to confirm expression, an aliquot of each culture supernatant was subjected to SDS-PAGE (12.5% gel) under non-reducing conditions. After the gel was transferred onto a PVDF membrane by a semi-dry method, the fragment of interest was detected by Western blotting using anti-Myc monoclonal antibody 9E10 (Sigma). As shown in FIG. 3, it was confirmed that overlapping partial fragments I to V of human cubilin were expressed in COS7 cells and secreted into the culture supernatant.

Example 3

Interaction between Human Apohpoprotein A-I-conjugated Resin and Partial Fragments of Human Cubilin

Human apolipoprotein A-I-cross linking Sepharose resin was prepared by mixing human apolipoprotein A-I (Sigma) and swollen CNBr-activated Sepharose (Amersham Pharmacia) at a ratio of 2 mg/l ml in a binding buffer (pH 8.3) containing 0.5 M NaCl and 0.1 M NaHCO₃ overnight at 4° C. The resultant human apolipoprotein A-I-linking resin (50 μl) was mixed with 200 μl each of the culture supernatants obtained in Example 2 containing partial fragments I to V, respectively, overnight at 4° C. After removal of the unbound fraction by centrifugation, the reaction mixture was washed with 400 μl of 0.2% bovine albumin-containing D-MEM medium (Gibco) four times, with D-MEM medium once, and mixed with 20 mM EDTA-containing D-MEM medium. After incubation for 5 min, a fraction that elutes in an EDTA selective manner was obtained by centrifugation. The unbound fraction and the fraction that elutes in an EDTA selective manner were subjected to SDS-PAGE (12.5% gel) separately. The resultant gel was transferred onto a PVDF membrane by a semi-dry method and subjected to detection using anti-Myc monoclonal antibody 9E10 (Sigma). As shown in FIG. 4, it was revealed that only partial fragment III bound to the human apolipoprotein A-I-linking resin and was eluted with EDTA selectively.

From the above, it was made clear that the human apolipoprotein A-I-binding site of human cubilin is in partial fragment III (corresponding to the fragment peptide CUB7-CUB14).

Example 4

The Apo A-I-Binding Action of CUB9-CUB14 Fragment Peptide

In order to further specify the location of the human apolipoprotein A-I-binding site, a cDNA encoding the amino acid sequence as shown in SEQ ID NO: 19 corresponding to fragment peptide CUB9-CUB14 [having the amino acid sequence consisting of the amino acids from position 1391 to position 2091 of the amino acid sequence of human cubilin mature protein (FIGS. 1 and 2)] and a cDNA encoding the amino acid sequence as shown in SEQ ID NO: 20 corresponding to fragment peptide CUB7-CUB12 were sub-cloned separately into the expression vector used in Example 1.

The above-mentioned cDNA (SEQ ID NO: 22) encoding human CUB9-CUB14 fragment peptide was inserted into pSecTag2 vector. The resultant plasmid pTB2231 was introduced into Escherichia coli DH5α by a publicly known method to obtain a transformant designated Escherichia coli DH5α/pTB2231.

These cubilin partial fragments were expressed in culture supernatant of COS7 cells in the same manner as in Example 2, and their interaction with human apolipoprotein A-I-linking resin was examined in the same manner as in Example 3.

The partial protein containing CUB9-CUB14 fragment peptide bound to human apolipoprotein A-I-linking resin (FIG. 5). This result revealed that the human apolipoprotein A-I-binding site of human cubilin is in CUB9-CUB14 fragment peptide.

Example 5

The Apo A-I-Binding Action of CUB9-CUB14 Fragment Peptide

The cDNA (SEQ ID NO: 22) encoding CUB9-CUB14 fragment peptide was inserted into a baculovirus for expressing the peptide in insect cells.

Specifically, a cDNA encoding the amino acid sequence as shown in SEQ ID NO: 21 (Myc tag and His tag are added to the C-terminal of CUB9-CUB14 fragment peptide) was sub-cloned into pFasBac vector. Using this vector, a baculovirus was constructed according to conventional methods to thereby prepare a system for expressing the fragment peptide in culture supernatant of HiFive cell, an insect cell.

It was found in the same manner as in Example 4 that the partial protein having CUB9-CUB14 fragment peptide expressed in the culture supernatant bound to human apolipoprotein A-I-linking resin (FIG. 5). This result revealed that CUB9-CUB14 fragment peptide expressed in an insect cell also binds to human apolipoprotein A-I.

Example 6

Purification of CUB9-CUB14 Fragment Peptide

The CUB9-CUB14 fragment peptide expressed in the insect cell obtained in Example 5 was purified using the His tag added to the C-terminal.

Specifically, the culture supernatant was precipitated with 50% saturated ammonium sulfate, and the precipitate was recovered by centrifugation (12,000 g, 30 min). The resultant precipitate was dissolved in a buffer containing 20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 10 mM imidazole and 0.1% sodium azide. The resultant solution was applied to HiTrap Chelating column (Amersham Pharmacia), charged with 0.1 M NiSO₄ and equilibrated with the same buffer. After washing with the same buffer, a linear gradient from 10 mM imidazole to 200 mM imidazole was formed with the buffer to thereby elute CUB9-CUB14 fragment peptide.

The thus eluted fractions were subjected to SDS-PAGE (12.5% gel). The resultant gels stained with CBB are shown in FIG. 6A. The gels transferred onto PVDF membrane and subjected to detection with anti-Myc monoclonal antibody 9E10 (Sigma) are shown in FIG. 6B.

From these results, it was found that the eluted fractions contain CUB9-CUB14 fragment peptide as a major component.

Example 7

Quantitative Determination of the Binding Between CUB9-CUB14 Fragment Peptide and ApoA-I

The CUB9-CUB14 fragment peptide expressed in the insect cell obtained in Example 5 was dissolved in a buffer containing 20 mM Tris-HCl, 500 mM NaCl, 2 mM CaCl₂ and 0.1% sodium azide to give a final concentration of 1-5 μg/ml, and incubated in a fluoroimmunoassay plate (Clini plate Black enhanced binding; Flow Lab.) containing 100 μl/well of this solution to thereby solidify the peptide. After blocking the non-specific binding sites with 200 μl/well of SuperBlock TBS (Pierce), the peptide was incubated with biotin-added human apolipoprotein A-I and, after washing, reacted with streptavidin β-galactosidase. After washing, β-galactosidase activity was measured by conventional methods to thereby quantitatively determine the binding between solidified CUB9-CUB14 fragment peptide and human apolipoprotein A-I. For the above binding reaction and washing, a buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl₂, 0.1% sodium azide and 5% (v/v) Block-Ace (Dainippon Pharmaceutical Co., Ltd.) was used.

As shown in FIG. 7, binding reaction dependent on the amount of solidified CUB9-CUB14 fragment peptide and dependent on the amount of biotin-added human apolipoprotein A-I was recognized. The binding was so strong that the amount of binding did not change even after washing for 2-3 hours. From the binding curve, the approximate affinity (Kd) of apolipoprotein A-I is judged to be at levels of 20-100 nM.

Example 8

Screening Method

The CUB9-CUB14 fragment peptide expressed in the insect cell obtained in Example 5 was incubated at a concentration of 3 μg/ml in a fluoroimmunoassay plate (Clini plate Black enhanced binding; Flow Lab.) containing 100 μl/well of the peptide solution to thereby solidify the peptide. After blocking the non-specific binding sites with 200 μl/well of SuperBlock TBS (Pierce), the test compound was incubated with biotin-added human apolipoprotein A-I (final concentration: 0.1 μg/ml) overnight. After washing, the test compound (100 μl/well) was reacted with 0.05 U/ml of streptavidin β-galactosidase for 1 hour. After washing, 0.5 mM 4-methylumbelliferyl β-D-galactopyranoside was added to the reaction solution dissolved in a buffer containing 10 mM K₂HPO₄, 150 nM NaCl, 2 mM MgCl₂, 0.1% sodium azide and 0.2% BSA (pH 7.0) and incubated, followed by measurement of excitation at 365 nm and absorbance at 460 nm. For the above binding reaction and washing, a buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl₂, 0.1% sodium azide and 5% (v/v) Block-Ace (Dainippon Pharmaceutical Co., Ltd.) was used.

INDUSTRIAL APPLICABILITY

The polypeptide of the invention (or amide or ester thereof, or salts thereof) and the DNA of the invention may be used as medicines such as therapeutic and/or prophylactic agent for diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, or nervousl disorders.

Further, compounds or salts thereof, which are obtainable by a screening method or kit using the polypeptide of the invention, that inhibit the binding of cubilin to apolipoprotein A-I may be used as medicines such as therapeutics and/or prophylactics for diseases such as hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardial infarction or angina; and compounds or salts thereof, which are obtainable by a screening method or kit using the polypeptide of the invention, that promote the binding of cubilin to apolipoprotein A-I may be used as medicines such as therapeutics and/or prophylactics for diseases such as renal disorders, nephritis, nephropathy, proteinuria, neurological disorders or vitamin B₁₂ deficiency.

Further, since the antibody of the invention can specifically recognize the polypeptide of the invention and cubilin, it may be used in quantitative determination of the polypeptide of the invention in a sample solution. Also, the antibody of the invention may be used as a medicine such as a therapeutic and/or prophylactic agent for diseases caused by excessive expression of the polypeptide of the invention.

The DNA of the invention may be useful as a gene diagnostic agent when used as a probe, for example.

Claims

1. A polypeptide having the ability to bind to apolipoprotein A-I, which is a partial fragment of cubilin, an amide or ester of said polypeptide, or a salt of said polypeptide.

2. The polypeptide, amide or ester thereof, or salt thereof of claim 1, wherein the polypeptide is characterized by having an amino acid sequence identical or substantially identical with the amino acid sequence as shown in SEQ ID NO: 10.

3. The polypeptide, amide or ester thereof, or salt thereof of claim 1, wherein the polypeptide has the amino acid sequence as shown in SEQ ID NO: 10.

4-5. (canceled)

6. A DNA comprising a DNA encoding the polypeptide of claim 1.

7. The DNA of claim 6, wherein the DNA comprises the base sequence as shown in SEQ ID NO: 9.

8. The DNA of claim 6, wherein the DNA comprises the base sequence as shown in SEQ ID NO: 22.

9. A recombinant vector comprising the DNA of claim 6.

10. A transformant transformed with the recombinant vector of claim 9.

11. A method of producing the polypeptide, amide or ester thereof, or salt thereof of claim 1, comprising:

culturing a transformant transformed with a recombinant vector comprising a DNA encoding the polypeptide of claim 1 and

allowing the transformant to produce the polypeptide.

12. An antibody to the polypeptide, amide or ester thereof, or salt thereof of claim 1.

13. A method of screening for compounds or salts thereof that promote or inhibit the activity of the polypeptide or salt thereof of claim 1, wherein the method is characterized by using the polypeptide, amide or ester thereof, or salt thereof of claim 1 in a screening assay.

14. A screening kit for compounds or salts thereof that promote or inhibit the activity of the polypeptide, amide or ester thereof, or salt thereof of claim 1, wherein the kit comprises the polypeptide or salt thereof of claim 1.

15. A compound or salt thereof that promotes or inhibits the activity of a polypeptide having the ability to bind to apolipoprotein A-I, which is a partial fragment of cubilin, amide or ester thereof, or salt thereof wherein said compound or salt thereof is obtainable by using the screening method of claim 13.

16. A medicine comprising a compound or salt thereof that promotes or inhibits the activity of a polypeptide having the ability to bind to apolipoprotein A-I, which is a partial fragment of cubilin, amide or ester thereof, or salt thereof wherein said compound or salt thereof is obtainable by using the screening method of claim 13.

17. A prophylactic and/or therapeutic agent for renal disorders, nephritis, nephropathy, proteinuria, nervous disorders or vitamin B12 deficiency, comprising a compound or salt thereof that promotes or inhibits the activity of a polypeptide having the ability to bind to apolipoprotein A-I, which is a partial fragment of cubilin, amide or ester thereof, or salt thereof wherein said compound or salt thereof is obtainable by using the screening method of claim 13.

18. A prophylactic and/or therapeutic agent for hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardial infarction or angina, comprising a compound or salt thereof that promotes or inhibits the activity of a polypeptide having the ability to bind to apolipoprotein A-I, which is a partial fragment of cubilin, amide or ester thereof, or salt thereof wherein said compound or salt thereof is obtainable by using the screening method of claim 13.

19. A pharmaceutical composition comprising the polypeptide, amide or ester thereof, or salt thereof of claim 1 and a pharmacologically acceptable carrier, excipient or diluent.

20. A prophylactic and/or therapeutic agent for diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders, comprising the polypeptide, amide or ester thereof, or salt thereof of claim 1.

21. A diagnostic agent that is characterized by the use of the DNA of claim 6.

22. A diagnostic agent comprising the antibody of claim 12.

23. A method for preventing and/or treating renal disorders, nephritis, nephropathy, proteinuria, nervous disorders or vitamin B12 deficiency in a mammal, comprising a compound or salt thereof that promotes or inhibits the activity of a polypeptide having the ability to bind to apolipoprotein A-I, which is a partial fragment of cubilin, amide or ester thereof, or salt thereof wherein said compound or salt thereof is obtainable by using the screening method of claim 13.

24. A method for preventing and/or treating hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardial infarction or angina, characterized by administering to a mammal an effective amount of a compound or salt thereof that promotes or inhibits the activity of a polypeptide having the ability to bind to apolipoprotein A-I, which is a partial fragment of cubilin, amide or ester thereof, or salt thereof wherein said compound or salt thereof is obtainable by using the screening method of claim 13.

25. A method for manufacturing a prophylactic and/or therapeutic agent for treating or preventing, in a mammal in need thereof, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardial infarction or angina, comprising

combining a therapeutic amount of a compound or salt thereof that promotes or inhibits the activity of a polypeptide having the ability to bind to apolipoprotein A-I, which is a partial fragment of cubilin, amide or ester thereof, or salt thereof, wherein said compound or salt thereof is obtainable by using the screening method of claim 13 with

a pharmacologically acceptable carrier, excipient or diluent.

26. A method for preventing and/or treating diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders, characterized by administering to a mammal an effective amount of the polypeptide, amide or ester thereof, or salt thereof of claim 1.

27. A method for manufacturing a prophylactic and/or therapeutic agent for treating or preventing, in a mammal in need thereof, diabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders, comprising

combining a therapeutic amount of a compound or salt thereof that promotes or inhibits the activity of a polypeptide having the ability to bind to apolipoprotein A-I, which is a partial fragment of cubilin, amide or ester thereof, or salt thereof with

a pharmacologically acceptable carrier, excipient or diluent.