JP2004275186A - Antibody and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody capable of sensitively, specifically and quantitatively determining TGR (G protein conjugated type receptor) 23-2 ligand, a method for detecting and quantitatively determining the TGR23-2 ligand using the antibody, and a medicine and a diagnostic agent using these. <P>SOLUTION: Provided are an antibody to specifically react to a partial peptide at an N terminal or C terminal of the TGR23-2 ligand, the method for detecting and quantitatively determining the TGR23-2 using the antibody, and a preventing/treating agent for cancer, the diagnostic agent, or the like, using these. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel antibody having binding specificity for a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a salt thereof. More specifically, a method for quantifying the polypeptide or a salt thereof based on an antigen-antibody reaction, and development of a diagnostic and prophylactic / therapeutic agent for a disease (eg, cancer, etc.) involving the polypeptide or a salt thereof utilizing neutralizing activity And the like useful for antibodies.

At present, about half of genes targeted for drug discovery are G protein-coupled receptors (GPCRs) characterized by seven transmembrane structures. The G protein-coupled receptor TGR23 is a receptor whose expression is enhanced in cancer cell lines such as human colon cancer cells LS 174T, LS 180, SW 403 and human gastric cancer cell KATOIII and in colon cancer tissues. A polypeptide having the amino acid sequence represented by (hereinafter sometimes referred to as human TGR23-2 ligand); a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 (hereinafter sometimes referred to as rat TGR23-2 ligand) And a polypeptide having the amino acid sequence represented by SEQ ID NO: 3 (hereinafter sometimes referred to as mouse TGR23-2 ligand) and the like were found as ligand peptides thereof (Patent Document 1 WO 02 / No. 31145).
WO 02/31145 Publication

  To further clarify the physiological function of human TGR23-2 ligand, neutralize human TGR23-2 ligand, rat TGR23-2 ligand or mouse TGR23-2 ligand, and detect these TGR23-2 ligands easily and with high sensitivity -There has been a long-awaited need for a measurement system for quantification.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, specifically recognize the N-terminal side and the C-terminal side of human TGR23-2 ligand, rat TGR23-2 ligand or mouse TGR23-2 ligand. To produce a plurality of monoclonal antibodies, blood, cerebrospinal fluid, using a variety of TGR23-2 ligand in biological components such as urine can be easily detected and quantified with high sensitivity, furthermore, The inventors have also found that these antibodies neutralize the activity of TGR23-2 ligand, and have completed the present invention.

That is, the present invention
(1) an antibody that specifically reacts with a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a partial peptide on the N-terminal side of a salt thereof,
(2) the antibody according to (1), which specifically reacts with a peptide having the first to seventh amino acid sequences of the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;
(3) The first to third, first to fourth, first to fifth, first to sixth, and first amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 7th, 2nd to 4th, 2nd to 5th, 2nd to 6th, 2nd to 7th, 3rd to 5th, 3rd to 6th, 3rd to 7th, 4th to 6th The antibody according to the above (1), which specifically reacts with a peptide having an amino acid sequence of the fourth, seventh to seventh or fifth to seventh amino acids;
(3a) the antibody according to (1), which specifically reacts with a peptide having the first to eighth amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2;
(3b) the antibody according to (1), which specifically reacts with a polypeptide having the amino acid sequence represented by SEQ ID NO: 5, its amide, or a salt thereof;
(4) the antibody according to (1), which does not recognize a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a partial peptide on the C-terminal side of a salt thereof;
(5) the antibody according to (1), which has a neutralizing activity against a peptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;
(6) a labeled antibody according to (1) above,
(7) the antibody according to (1), which is a monoclonal antibody;
(8) The monoclonal antibody according to the above (7), which is represented by 23L-1Na that can be produced from the hybridoma cell represented by 23L-1N (FERM BP-8302),
(9) an antibody that specifically reacts with a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a partial peptide on the C-terminal side of a salt thereof,
(10) the antibody according to (9), which specifically reacts with a peptide having the 15th to 18th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3;
(11) The 12th to 14th, 12th to 15th, 12th to 16th, 12th to 17th, 12th to 18th, and 12th positions of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3. 13th to 15th, 13th to 16th, 13th to 17th, 13th to 18th, 14th to 16th, 14th to 17th, 14th to 18th, 15th to 17th, 15th to 15th The antibody according to the above (9), which specifically reacts with a peptide having the 18th or 16th to 18th amino acid sequence,
(12) The antibody according to the above (9), which does not recognize a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a partial peptide on the N-terminal side of a salt thereof.
(13) the antibody according to (9), which has a neutralizing activity against a peptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3;
(14) a labeled antibody according to (9) above,
(15) the antibody according to (9), which is a monoclonal antibody;
(16) the monoclonal antibody according to the above (15), represented by 23L-2Ca that can be produced from a hybridoma cell represented by 23L-2C (FERM BP-8303);
(17) a medicine comprising the antibody according to (1) or (9),
(18) The medicament according to the above (17), which is a prophylactic / therapeutic agent for cancer or anorexia nervosa.
(19) A diagnostic agent comprising the antibody according to (1) or / and (9) above,
(20) A method for quantifying a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a salt thereof, comprising using the antibody according to (1) above;
(21) The method according to (20), further comprising using the antibody according to (9),
(22) A disease involving a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a salt thereof, wherein the antibody described in (1) above is used. Diagnostic methods,
(23) A method for diagnosing cancer, comprising using the antibody according to (1) above,
(24) The diagnostic method according to (22) or (23), further comprising using the antibody according to (9).
(25) A method for quantifying a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof, comprising using the antibody according to (9) above,
(26) The antibody according to (1) above, a test solution, and a labeled polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a salt thereof. SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: in the test solution, wherein the ratio of the labeled polypeptide or the salt thereof bound to the antibody is measured by reacting competitively. A method for quantifying a polypeptide or a salt thereof containing the amino acid sequence represented by 3,
(27) The antibody of (9) is allowed to competitively react with the test solution and a labeled polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or 3 or a salt thereof. Measuring the ratio of the labeled polypeptide or a salt thereof bound to the antibody, comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 in the test solution. A method for determining a polypeptide or a salt thereof,
(28) (i) After reacting the antibody of (1) insolubilized on a carrier, the labeled antibody of (9) and a test solution, and then measuring the activity of the labeling agent, or
(Ii) reacting the antibody according to (9), the labeled antibody according to (1), and the test solution which are insolubilized on a carrier, and then measuring the activity of the labeling agent. A method for quantifying a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof in a test solution,
(29) a hybridoma cell producing the monoclonal antibody according to (7),
(30) The hybridoma cell according to the above (29), which is denoted by 23L-1N (FERM BP-8302),
(31) The monoclonal antibody according to (7), wherein the hybridoma cell according to (29) is cultured in vivo or in vitro, and the monoclonal antibody according to (7) is collected from the body fluid or culture. Antibody production method,
(32) a hybridoma cell producing the monoclonal antibody according to (15),
(33) The hybridoma cell according to the above (32), which is designated by 23L-2C (FERM BP-8303),
(34) The monoclonal antibody according to (15), wherein the hybridoma cell according to (32) is cultured in vivo or in vitro, and the monoclonal antibody according to (15) is collected from the body fluid or culture. Antibody production method,
(35) a polypeptide having the amino acid sequence represented by SEQ ID NO: 5, its amide, or a salt thereof;
(36) a method for preventing or treating cancer or anorexia nervosa, which comprises administering an effective amount of the antibody according to (1) or (9) to a mammal;
(37) Use of the antibody according to (1) or (9) above for producing a prophylactic / therapeutic agent for cancer or anorexia.

The antibody of the present invention has an extremely high binding ability to TGR23-2 ligand, can neutralize the intracellular [Ca ²⁺ ] elevating activity of TGR23-2 ligand, and also has a tumor growth inhibitory effect. Therefore, the antibody of the present invention suppresses the action of the TGR23-2 ligand to produce, for example, cancer (eg, colon cancer, colon cancer, rectal cancer, breast cancer, lung cancer, non-small cell lung cancer, prostate cancer, esophageal cancer, gastric cancer). , Liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, cervical cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, etc.) or prevention and treatment of anorexia Can be used as a safe medicine such as a drug and an appetite (appetite) promoter. Preferably, it is a prophylactic / therapeutic agent for cancer. Assays using the antibody of the present invention, for example, the C-terminal part of the TGR23-2 ligand (the C-terminal part of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or the salt thereof) The TGR23-2 ligand can be quantified with high sensitivity and specificity by a sandwich immunoassay using a monoclonal antibody that recognizes TGR23 and a monoclonal antibody that specifically recognizes the N-terminal end of TGR23-2 ligand. Therefore, for example, diagnosis of the above-mentioned diseases and the like is also possible. The antibody of the present invention can also be used for immunohistochemical staining of TGR23-2 ligand.

In the protein (polypeptide) in this specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling. The protein used in the present invention, including the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, may have a carboxyl group at the C-terminus, a carboxylate, an amide or an ester.
Examples of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 include (1) SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (2) an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; (3) The number (1-5) amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 are substituted with other amino acids And the like having a modified amino acid sequence.
Examples of the salt of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 include physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, , Alkali metals, etc.), and especially preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.

Examples of the partial peptide at the N-terminal side of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a salt thereof include:
(A) a polypeptide having (i) the first to third amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3,
(Ii) a polypeptide having the first to fourth amino acid sequences,
(Iii) a polypeptide having the first to fifth amino acid sequences,
(Iv) a polypeptide having the first to sixth amino acid sequences,
(V) a polypeptide having the first to seventh amino acid sequences,
(Vi) a polypeptide having the second to fourth amino acid sequences,
(Vii) a polypeptide having the second to fifth amino acid sequences,
(Viii) a polypeptide having the second to sixth amino acid sequences,
(Ix) a polypeptide having the second through seventh amino acid sequences,
(X) a polypeptide having the third to fifth amino acid sequences,
(Xi) a polypeptide having the third to sixth amino acid sequences,
(Xii) a polypeptide having the third to seventh amino acid sequences,
(Xiii) a polypeptide having the fourth to sixth amino acid sequences,
(Xiv) a polypeptide having the 4th to 7th amino acid sequence, (xv) a polypeptide having the 5th to 7th amino acid sequence, and (b) a 1st amino acid sequence represented by SEQ ID NO: 2. And a polypeptide having the 8th to 8th amino acid sequences.
As the partial peptide at the C-terminal side of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof, for example, the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 of,
(I) a polypeptide having the twelfth to fourteenth amino acid sequence,
(Ii) a polypeptide having the twelfth to fifteenth amino acid sequence,
(Iii) a polypeptide having the twelfth to sixteenth amino acid sequence,
(Iv) a polypeptide having the twelfth to seventeenth amino acid sequence,
(V) a polypeptide having the twelfth to eighteenth amino acid sequence,
(Vi) a polypeptide having the thirteenth to fifteenth amino acid sequence,
(Vii) a polypeptide having the thirteenth to sixteenth amino acid sequence,
(Viii) a polypeptide having the thirteenth to seventeenth amino acid sequence,
(Ix) a polypeptide having the amino acid sequence of positions 13-18,
(X) a polypeptide having the amino acid sequence of positions 14-16,
(Xi) a polypeptide having the amino acid sequence of positions 14-17,
(Xii) a polypeptide having the amino acid sequence of positions 14-18,
(Xiii) a polypeptide having the amino acid sequence of positions 15-17,
(Xiv) a polypeptide having the 15th to 18th amino acid sequence, and (xv) a polypeptide having the 16th to 18th amino acid sequence.

Examples of an antibody that specifically reacts with a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a partial peptide on the N-terminal side of a salt thereof include, for example, the polypeptide or Any substance that specifically reacts with the partial peptide on the N-terminal side of the salt may be used, and a monoclonal antibody is preferable. Specifically, a peptide containing the first to seventh amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1, for example, the first to eighth amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2 And an antibody (preferably a monoclonal antibody) that specifically reacts with a polypeptide having an amino acid sequence in which the ninth amino acid sequence of the amino acid sequence is substituted with Cys-NH ₂ is used. Of these, those that do not recognize the partial peptide at the C-terminal side of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a salt thereof are preferred.
More preferably, it is an antibody that neutralizes the activity of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a salt thereof.
Preferred specific examples include a monoclonal antibody represented by 23L-1Na.
Furthermore, the antibody specifically reacting with the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or the partial peptide on the N-terminal side of the salt thereof includes (a) (I) the first to third amino acid sequences, (ii) the first to fourth amino acid sequences, and (iii) the first amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. (Iv) 1st to 6th amino acid sequence, (v) 1st to 7th amino acid sequence, (vi) 2nd to 4th amino acid sequence, (vii) 2nd to 5th amino acid sequence (Viii) 2nd to 6th amino acid sequence, (ix) 2nd to 7th amino acid sequence, (x) 3rd to 5th amino acid sequence, (xi) 3rd to 6th amino acid sequence Amino acid sequence, (xii) third to seventh amino acid sequence, (xiii) fourth to sixth amino acid sequence Column, (xiv) the fourth to seventh amino acid sequences, (xv) the fifth to seventh amino acid sequences, and (b) the first to eighth amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2. An antibody specifically recognizing is used.

An antibody that specifically reacts with the polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a partial peptide on the C-terminal side of a salt thereof is, for example, SEQ ID NO: 2 or SEQ ID NO: 3 May be any as long as it specifically reacts with the partial peptide at the C-terminal side of the polypeptide having the amino acid sequence represented by or a salt thereof, preferably a monoclonal antibody. Specifically, for example, an antibody (preferably having an amino acid sequence represented by SEQ ID NO: 2 and specifically reacting with a polypeptide having an amino acid sequence having Cys added to position 19 of the amino acid sequence (preferably, , Monoclonal antibodies) and the like. Of these, those that do not recognize the N-terminal partial peptide of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof are preferred.
More preferred is an antibody that neutralizes the activity of a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof.
Preferred specific examples include a monoclonal antibody represented by 23L-2Ca.
Further, as an antibody that specifically reacts with a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a partial peptide on the C-terminal side of a salt thereof, SEQ ID NO: 2 or SEQ ID NO: (I) the 12th to 14th amino acid sequence of the amino acid sequence represented by 3, (ii) the 12th to 15th amino acid sequence, (iii) the 12th to 16th amino acid sequence, and (iv) the 12th to 16th amino acid sequences. 17th amino acid sequence, (v) 12th to 18th amino acid sequence, (vi) 13th to 15th amino acid sequence, (vii) 13th to 16th amino acid sequence, (viii) 13th to 17th amino acid sequence (Ix) amino acid sequence at positions 13-18, (x) amino acid sequence at positions 14-16, (xi) amino acid sequence at positions 14-17, (xii) amino acids at positions 14-18 Sequence, (xiii) fifteenth to 7 amino acid sequence, (xiv) a 15 to 18 amino acid sequence, and (xv) such as the 16-18 amino acid sequence specifically recognizing antibody is used.

Hereinafter, an antibody that specifically reacts with the N-terminal partial peptide of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a salt thereof, and SEQ ID NO: 2 or an antibody that specifically reacts with the partial peptide at the C-terminal side of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 3 or a salt thereof (hereinafter, both may be collectively referred to as the antibody of the present invention). The method for preparing the antigen and the method for producing the antibody are described below.
(1) Preparation of Antigen As an antigen used for preparing the antibody of the present invention, for example, a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or Any salt, such as a synthetic peptide having one or more antigen determinants identical to its salt (hereinafter sometimes referred to as a TGR23-2 ligand) can be used (hereinafter, these are simply referred to as TGR23-2 ligands). Ligand antigen).
TGR23-2 ligand is prepared from (a), for example, a known method or a method equivalent thereto from tissues or cells of mammals such as humans, monkeys, rats, and mice, and (b) a known method using a peptide synthesizer or the like. It is also produced by chemically synthesizing a peptide by a peptide synthesis method, and (c) culturing a transformant containing DNA encoding TGR23-2 ligand.
(A) When preparing a TGR23-2 ligand antigen from the mammalian tissue or cells, the tissue or cells are homogenized and then extracted with an acid or alcohol, and the extract is subjected to salting out, dialysis, Purification and isolation can be performed by combining chromatography such as gel filtration, reverse phase chromatography, ion exchange chromatography, and affinity chromatography.
(B) When chemically preparing a TGR23-2 ligand antigen, examples of the synthetic peptide include those having the same structure as the above-described naturally-purified TGR23-2 ligand antigen, represented by SEQ ID NO: 1. Peptides containing one or more of the same amino acid sequences as the amino acid sequence at an arbitrary position consisting of three or more, preferably eight or more amino acids in the amino acid sequence are used.
(C) When producing a TGR23-2 ligand using a transformant containing DNA, the DNA can be prepared by a known cloning method [eg, Molecular Cloning (2nd ed .; J. Sambrook et al., Cold Spring Harbor). Lab. Press, 1989)]. The cloning method includes (1) a transformant containing DNA encoding TGR23-2 ligand by hybridization from a cDNA library using a DNA probe or DNA primer designed based on the amino acid sequence of TGR23-2 ligand. Or (2) a method of obtaining a transformant containing DNA encoding TGR23-2 ligand by PCR using a DNA primer designed based on the amino acid sequence of TGR23-2 ligand.

Peptides as TGR23-2 ligand antigens are (1) polypeptides containing an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 according to a known peptide synthesis method, or (2) Can be produced by cleaving with a suitable peptidase.
As a method for synthesizing the peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can constitute the peptide with the remaining portion, and removing the protective group when the product has a protective group. Examples of the known condensation method and elimination of the protecting group include the methods described in the following (i) to (ii).
(I) Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) The Peptide, Academic Press, New York (1965)
After the reaction, the peptide can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and when it is obtained by a salt, it can be converted to a free form by a known method. .

  As the amide form of the peptide, a commercially available resin for peptide synthesis suitable for amide formation can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethylphenyl. Acetamide methyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like can be given. . Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target peptide according to various known condensation methods. At the end of the reaction, the peptide is cleaved from the resin, and at the same time, various protecting groups are removed to obtain the desired peptide. Alternatively, a partially protected peptide can be taken out using a chlorotrityl resin, an oxime resin, a 4-hydroxybenzoic acid-based resin, or the like, and the protecting group can be removed by a conventional means to obtain the desired peptide.

  For the condensation of the protected amino acids described above, various activating reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. Carbodiimides include DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like. Activation by these involves adding the protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt, etc.), or pre-protecting the amino acid as a symmetrical anhydride or HOBt ester or HOOBt ester. Can be added to the resin after activation. The solvent used for activation of the protected amino acid or condensation with the resin can be appropriately selected from solvents known to be usable for the peptide condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and sulfoxides such as dimethyl sulfoxide And tertiary amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. The reaction temperature is appropriately selected from a range known to be usable for the peptide bond formation reaction, and is usually appropriately selected from a range of about -20 to about 50 ° C. The activated amino acid derivative is usually used in about 1.5 to about 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When a sufficient condensation cannot be obtained even by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.

Examples of the protecting group for the amino group of the starting amino acid include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoro Acetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like can be mentioned. Examples of the carboxyl-protecting group include C _1-6 alkyl, C _3-8 cycloalkyl, C _7-14 aralkyl, 2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, Phenacyl and benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide and the like.
The hydroxyl groups of serine and threonine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, and groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group. . Examples of groups suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a tertiary butyl group.

Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl-Bzl, 2-nitrobenzyl, Br-Z, tert-butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, Bom, Bum, Boc, Trt, Fmoc and the like.
Examples of the activated carboxyl group of the raw material include the corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanophenol) Methyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, esters with HOBt)]. The activated amino group of the raw material includes, for example, the corresponding phosphoric amide.

As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane Examples include acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, and the like, and reduction with sodium in liquid ammonia. The elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of -20 to 40 ° C, but in the acid treatment, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-butanedithiol, Addition of a cation scavenger such as 2-ethanedithiol is effective. In addition, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is 1,2-ethanedithiol, 1,4-butane described above. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide, dilute ammonia or the like.
The protection and protection of the functional group which should not be involved in the reaction of the raw materials, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a peptide, first, after amidating the α-carboxyl group of the carboxyl-terminal amino acid, extending the peptide chain to a desired chain length on the amino group side, and then N-terminal of the peptide chain To produce a peptide (or amino acid) from which only the α-amino group protecting group has been removed and a peptide (or amino acid) from which only the C-terminal carboxyl group protecting group has been removed, and condense these peptides in a mixed solvent as described above. . Details of the condensation reaction are the same as described above. After purifying the protected peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude peptide. This crude peptide is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired peptide.
In order to obtain an ester of the peptide, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester of the desired peptide can be obtained in the same manner as the amide of the peptide.

The TGR23-2 ligand antigen can be used to directly immunize the insolubilized one. In addition, a complex obtained by binding or adsorbing a TGR23-2 ligand antigen to a suitable carrier may be immunized. The mixing ratio of the carrier (carrier) and the TGR23-2 ligand antigen (hapten) depends on the ratio of what kind and what ratio as long as the antibody can be efficiently produced with respect to the TGR23-2 ligand antigen bound or adsorbed to the carrier. May be bound or adsorbed, and a natural or synthetic polymer carrier commonly used in the production of an antibody against a hapten antigen is bound or adsorbed at a ratio of 0.1 to 100 to hapten 1 by weight. Can be used. Examples of the natural polymer carrier include serum albumin of mammals such as bovine, rabbit and human, and thyroglobulin of mammal such as bovine and rabbit, for example, hemoglobin of mammalian such as bovine, rabbit, human and sheep, and key. Whole limpet hemocyanin or the like is used. As the synthetic polymer carrier, for example, various latexes such as a polymer such as polyamino acids, polystyrenes, polyacryls, polyvinyls, and polypropylenes or a copolymer can be used.
Various coupling agents can be used for coupling the hapten and the carrier. For example, diazonium compounds such as bisdiazotized benzidine that crosslink tyrosine, histidine, and tryptophan, dialdehyde compounds such as glutaraldetite that crosslink amino groups, diisocyanate compounds such as toluene-2,4-diisocyanate, and crosslinks between thiol groups A dimaleimide compound such as N, N'-o-phenylenedimaleimide, a maleimide active ester compound that bridges an amino group and a thiol group, and a carbodiimide compound that bridges an amino group and a carboxyl group are conveniently used. Also, when cross-linking amino groups, a thiol group is introduced by reacting an active ester reagent having a dithiopyridyl group with one amino group (for example, SPDP), and then introducing a thiol group into the other amino group. After the introduction of a maleimide group with a maleimide active ester reagent, both can be reacted.

(2) Preparation of monoclonal antibody
The TGR23-2 ligand antigen is administered to a warm-blooded animal by itself, for example, by intraperitoneal injection, intravenous injection, subcutaneous injection, or the like, alone or together with a carrier or diluent at a site where antibody production is possible. . Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of the warm-blooded animal include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, chickens and the like, and mice are preferably used for preparing monoclonal antibodies.
When preparing a monoclonal antibody, a warm-blooded animal immunized with the TGR23-2 ligand antigen, for example, an individual having an antibody titer was selected from a mouse, and the spleen or lymph node was collected 2 to 5 days after the final immunization. By fusing the antibody-producing cells contained in the above with myeloma cells, an anti-TGR23-2 ligand monoclonal antibody-producing hybridoma can be prepared. The anti-TGR23-2 ligand antibody titer in the serum is measured, for example, by reacting the labeled TGR23-2 ligand described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, and the like, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of bone marrow cells to be used is usually about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. The cell fusion can be carried out efficiently by incubating at usually 20 to 40 ° C, preferably 30 to 37 ° C, usually for 1 to 10 minutes.

Various methods can be used for screening the anti-TGR23-2 ligand antibody-producing hybridoma. For example, the hybridoma culture supernatant is applied to a solid phase (eg, microplate) on which the TGR23-2 ligand or a partial peptide thereof is adsorbed directly or together with a carrier. Was added, and then an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (if the cells used for cell fusion are mice, an anti-mouse immunoglobulin antibody is used) or protein A was added, and the mixture was bound to the solid phase. A method for detecting an anti-TGR23-2 ligand monoclonal antibody, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding a TGR23-2 ligand labeled with a radioactive substance, an enzyme, or the like, A method for detecting the TGR23-2 ligand monoclonal antibody bound to the phase is exemplified. Screening and breeding of the anti-TGR23-2 ligand monoclonal antibody are usually performed by adding HAT (hypoxanthine, aminopterin, thymidine) and using a medium for animal cells (eg, RPMI1640) containing 10 to 20% fetal bovine serum. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-described measurement of the anti-TGR23-2 ligand antibody titer in the antiserum.
Separation and purification of the anti-TGR23-2 ligand monoclonal antibody can be performed by the same method as in the separation and purification of normal polyclonal antibodies (e.g., salting out, alcohol precipitation, isoelectric focusing, electrophoresis, iontophoresis). Adsorption / desorption method using an exchanger (eg DEAE), ultracentrifugation method, gel filtration method, antigen-bound solid phase or only antibody is collected using an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody Specific purification method].
As described above, the antibody of the present invention can be produced by culturing the hybridoma cells in vivo or in vitro of a warm-blooded animal and collecting the antibody from the body fluid or culture.

  In addition, a hybridoma producing an anti-TGR23-2 ligand antibody that reacts with a partial region of TGR23-2 ligand, and an anti-TGR23-2 ligand monoclonal antibody that reacts with TGR23-2 ligand but does not react with the partial region Screening of the produced hybridoma can be performed, for example, by measuring the binding between a peptide corresponding to a partial region thereof and an antibody produced by the hybridoma.

[1] TGR23-2 ligand quantification method using the antibody of the present invention, cancer diagnosis method, etc. The TGR23-2 ligand quantification method (immunoassay) will be described in more detail below.
By using the antibody of the present invention, measurement of TGR23-2 ligand or detection by tissue staining or the like can be performed. For these purposes, the antibody molecule itself may be used, or F (ab ') 2, Fab' or Fab fraction of the antibody molecule may be used.
The measuring method using the antibody of the present invention is not particularly limited, and the amount of the antibody, the antigen, or the antibody-antigen complex corresponding to the amount of the antigen (eg, the amount of TGR23-2 ligand) in the liquid to be measured is determined. Any measurement method may be used as long as it is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, a sandwich method, a competitive method, an immunometric method, a nephelometry and the like are used, but a sandwich method and a competitive method described later are particularly preferable in terms of sensitivity and specificity, and a sandwich method is particularly preferable.

(1) Sandwich method After reacting the antibody of the present invention, the labeled antibody of the present invention, and a test solution which are insolubilized on a carrier, the activity of the labeling agent is measured to determine the TGR23-2 in the test solution. Quantify the ligand. Preferably,
(I) an antibody that specifically reacts with the partial peptide on the N-terminal side of the TGR23-2 ligand insolubilized on the carrier, the C-terminal side of the labeled TGR23-2 ligand (SEQ ID NO: 2 or SEQ ID NO: 3 After reacting an antibody specifically reacting with the polypeptide containing the amino acid sequence represented or the partial peptide at the C-terminal side of a salt thereof and a test solution, the activity of the labeling agent is measured. Method for quantifying TGR23-2 ligand in a test solution,
(Ii) Specific to the partial peptide at the C-terminal side of the TGR23-2 ligand insolubilized on the carrier (C-terminal side of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or the salt thereof) Reacting an antibody that specifically reacts, an antibody that specifically reacts with the partial peptide on the N-terminal side of the labeled TGR23-2 ligand, and a test solution, and then measuring the activity of the labeling agent. Examples include a method for quantifying TGR23-2 ligand in a test solution.
More preferably, the antibody that specifically reacts with the partial peptide at the N-terminal side of the TGR23-2 ligand is a monoclonal antibody represented by 23L-1Na, and specifically binds to the partial peptide at the C-terminal side of the TGR23-2 ligand. A quantitative method in which the reacting antibody is a monoclonal antibody represented by 23L-2Ca.

In the sandwich method, a test solution is reacted with an insolubilized antibody of the present invention (primary reaction), and further a labeled antibody of the present invention is reacted (secondary reaction). By measuring the activity of TGR23-2, the amount of TGR23-2 ligand in the test solution can be determined. The primary reaction and the secondary reaction may be performed at the same time or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be. In the method for measuring TGR23-2 ligand by the sandwich method, for example, the antibody used in the primary reaction contains the amino acid sequence represented by C-terminal side of TGR23-2 ligand (SEQ ID NO: 2 or SEQ ID NO: 3). When the partial peptide at the C-terminal side of the polypeptide or its salt is recognized, the antibody used in the secondary reaction is preferably an antibody that recognizes other than the partial peptide at the C-terminal side (that is, the N-terminal side), When the antibody used in the reaction recognizes the partial peptide at the N-terminal side of the TGR23-2 ligand, the antibody used in the secondary reaction is an antibody that recognizes other than the partial peptide at the N-terminal side (that is, the C-terminal side). Is preferably used.
As a specific example, a monoclonal antibody represented by 23L-1Na and a monoclonal antibody represented by 23L-2Ca are used. These antibodies are preferably used after being labeled with, for example, horseradish peroxidase (HRP).

(2) Competition method An antibody of the present invention, a test solution, and a labeled TGR23-2 ligand are allowed to react competitively, and the ratio of the labeled TGR23-2 ligand bound to the antibody is measured. Then, the TGR23-2 ligand in the test solution is quantified.
This reaction method is performed using, for example, a solid-phase method.
As a specific example, an anti-mouse IgG antibody (manufactured by ICN / CAPPEL) is used as an immobilized antibody, and i) an antibody of the present invention (eg, 23L-1Na, 23L- 2Ca), ii) TGR23-2 ligand labeled with horseradish peroxidase (HRP), and iii) a test solution. After the reaction, the HRP activity adsorbed on the solid phase is measured, and the TGR23-2 ligand is added. Quantify.
(3) Immunometric method In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of the labeled antibody of the present invention, and then the solid phase and the liquid phase are separated. Alternatively, the antigen in the test solution is allowed to react with an excessive amount of the labeled antibody of the present invention, and then the immobilized antigen is added, and the unreacted labeled antibody of the present invention is immobilized on the solid phase. After binding, the solid and liquid phases are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
(4) Nephrometry In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In the above (1) to (4), examples of the labeling agent used in the measurement method using a labeling substance include radioisotopes (eg, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] ], [ ³² P], [ ³³ P], [ ³⁵ S], etc.), fluorescent substance [eg, cyanine fluorescent dye (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience)) , Fluorescamine, fluorescein isothiocyanate, etc.), enzymes (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, Lucigenin), biotin, lanthanide elements, and the like. Further, a biotin-avidin system can be used for binding the antibody to the labeling agent.
For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, and synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass.

In applying these individual immunological measurement methods to the method of the present invention, no special conditions, operations, and the like need to be set. The TGR23-2 ligand measurement system may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews and compendiums [for example, “Radio immunoassay” edited by Hiro Irie (Kodansha, published in 1974) and “Radio immunoassay” edited by Hiro Irie. (Kodansha, published in 1979), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Publishing, published in 1978), Eiji Ishikawa et al., "Enzyme Immunoassay" (second edition) (Medical Publishing, Showa 57) Ed. Ishikawa et al., “Enzyme Immunoassay” (3rd edition) (Medical Publishing, published in 1987), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), 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: Monooclonal) Antibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemic al Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (see Academic Press). Therefore, when a measurement system for TGR23-2 ligand is constructed by the sandwich immunoassay of the present invention, the method is not limited to the examples described later.
As described above, since the antibody of the present invention can quantify TGR23-2 ligand with high sensitivity, it is useful for elucidating further physiological functions of TGR23-2 ligand and diagnosing diseases involving TGR23-2 ligand. . Specifically, by measuring the amount of TGR23-2 ligand contained in body fluids (blood, plasma, serum, urine, follicular fluid, spinal fluid, semen, etc.) using the antibody of the present invention, TGR23- Diseases involving 2 ligands, for example, cancer (eg, colon cancer, colon cancer, rectal cancer, breast cancer, lung cancer, non-small cell lung cancer, prostate cancer, esophagus cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, Bladder cancer, uterine cancer, ovarian cancer, cervical cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, etc.), anorexia nervosa, hyperphagia, etc. Can be diagnosed.
For example, in the diagnosis of colon cancer, TGR23-2 ligand in body fluids is quantified, and when the amount of TGR23-2 ligand is higher than normal, for example, the blood concentration is about 100 fmol / ml or more, preferably about 150 fmol / ml or more. If fmol / ml or more, colon cancer is diagnosed.

[2] Pharmaceuticals Containing Antibody of the Present Invention The antibody of the present invention has a medium activity of TGR23-2 ligand (eg, TGR23 binding activity, TGR23 cell stimulating activity, tumor growth activity, eating behavior suppressing activity, etc.). For example, a disease associated with a TGR23-2 ligand, such as cancer (eg, colon cancer, colon cancer, rectal cancer, breast cancer, lung cancer, non-small cell lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, Spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, cervical cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, etc.) or prophylactic / therapeutic agents such as anorexia nervosa, appetite (appetite) It can be used as a safe medicine such as an accelerator. Preferably, it is a prophylactic / therapeutic agent for cancer.
The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.

For example, as a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. Dosage forms. Such injections are prepared according to a known method, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used. Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, or the like is used, and benzyl benzoate, benzyl alcohol, or the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually 5 to 500 mg, especially 5 to 100 mg, for each dosage unit. Other dosage forms preferably contain 10 to 250 mg of the above antibody.
Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the antibody.
The therapeutic / prophylactic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity, and can be used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form in humans or mammals (eg, rat, rabbit, sheep, pig, Cattle, cats, dogs, monkeys, etc.) orally or parenterally. The dose varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used for the treatment of adult colon cancer, the antibody of the present invention is used in a single dose, usually 0.01 to 0.01%. Parenteral administration of about 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day It is convenient. In the case of oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.

In the specification of the present invention, when an amino acid or the like is indicated by an abbreviation, it is based on an abbreviation by IUPAC-IUB Commision on Biochemical Nomenclature or an abbreviation commonly used in the art, and examples thereof are described below. When amino acids may have optical isomers, L-form is indicated unless otherwise specified.
Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: serine Thr: threonine Cys: cysteine Met: methionine Glu: glutamic Asp: aspartic acid Lys: lysine Arg: arginine-histine arginine thyrosine arginine thyrosine arginine : Tryptophan Pro: proline Asn: asparagine Gln: glutamine TFA: trifluoroacetic acid DMF: N, N-dimethylformamide SPDP: 3- (2-pyridyldithio) propionic acid N-succinimidyl GMBS: N- (4-maleimidobutyryloxy ) Succinimide BSA: Bovine serum albumin BTG: Bovine thyroglobulin EIA: Enzyme immunoassay HPLC: Reversed phase high performance liquid chromatography -HRP: Horseradish peroxidase FBS: fetal bovine serum d-FBS: dialyzed fetal bovine serum TMB: 3,3 ', 5,5'-tetramethylbenzidine NMP: N-methylpyrrolidone Boc: tertiary butoxycarbonyl Fmoc : 9-fluorenylmethoxycarbonyl DCC: N, N'-dicyclohexylcarbodiimide Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl tBu: tertiary butyl Trt: trityl Tos: paratoluenesulfonyl DIEA: N, N'-diisopropylethylamine HOBt: 1-hydroxybenzotriazole HOAt: 1-hydroxy-7-azabenzotriazole PyAop: 7-azabenzotriazol-1-yloxytrispirolidinophos
Phonium hexafluorophosphate Clt: 2-chlorotrityl BHA: ^{Benzhydrylamine} Thr (Ψ ^{Me. Me} Pro): 2,2-dimethyl, 5-methyl-1,3-oxazolidine
-4-carboxylic acid

The SEQ ID NOs used herein represent the amino acid sequences of the following peptides.
[SEQ ID NO: 1]
2 shows the amino acid sequence of human TGR23-2 ligand.
[SEQ ID NO: 2]
2 shows the amino acid sequence of rat TGR23-2 ligand.
[SEQ ID NO: 3]
2 shows the amino acid sequence of mouse TGR23-2 ligand.
[SEQ ID NO: 4]
[Cys ¹⁹ ] shows the amino acid sequence of rat TGR23-2 ligand (1-19).
[SEQ ID NO: 5]
[Cys ⁹ ] shows the amino acid sequence of rat TGR23-2 ligand (1-9).

The hybridoma cell 23L-1N obtained in Example 1 described below has been used since February 26, 2003 at 1-1, Higashi 1-1, Tsukuba, Ibaraki Prefecture, Chuo No. 6 (Postal Code 305-8566) Location Deposited at the Patent Organism Depositary under the accession number FERM BP-8302.
The hybridoma cell 23L-2C obtained in Example 1 described below has been used since February 26, 2003 at 1-1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki Prefecture, Chuo No. 6 (Zip code 305-8566) Location Deposited at the Patent Organism Depositary under the accession number FERM BP-8303.
In addition, the antibody obtained from each hybridoma cell is represented by adding "a" to the cell name.

Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but these do not limit the scope of the present invention.
1% BSA, 0.4M NaCl, and 0.05% 2mM EDTA · Na [ethylenediamine-N, N, N ', N'-tetraacetic acid disodium salt dihydrate (Ethylenediamine-N, N, N , N'-tetraacetic acid, disodium salt, dihydrate), DOJINDO] is referred to as buffer C.

Example 1
_{^{(1) [Cys 19 -NH 2}} ] obtained in Preparation Example 4 of an immunogen comprising a rat TGR23-2 ligand _{^{(1-19) [Cys 19 -NH 2}} ] rat TGR23-2 ligand (1-19) And a complex with Keyhole Limpet Hemocianin (KLH) was prepared and used as an immunogen.
That is, 20 mg of KLH is dissolved in 1.4 ml of a 0.1 M phosphate buffer (pH 6.5), mixed with 100 μl of a DMF solution containing 2.2 mg (8 μmol) of N- (γ-maleimidobutyryloxy) succinimide (GMBS), The reaction was performed at room temperature for 40 minutes. After the reaction, the mixture was fractionated on a Sephadex G-25 column, and then 15 mg of KLH having a maleimide group introduced therein and 3.75 mg of [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) were mixed at 4 ° C. For 1 day. After the reaction, the solution was dialyzed against physiological saline at 4 ° C. for 3 days.
(2) Immunity
To a BALB / C female mouse 6 to 8 weeks old, the [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) -KLH complex obtained in the above (1) was added in an amount of about 70 μg / animal. Immunization subcutaneously with complete Freund's adjuvant. Thereafter, the same amount of immunogen was boosted twice or three times with incomplete Freund's adjuvant every three weeks.
(3) Preparation of HRP-labeled [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) and HRP (for enzyme immunoassay) And Boehringer Mannheim) to form a label for enzyme immunoassay (EIA).
That is, 9.2 mg (180 nmol) of HRP was dissolved in 0.95 ml of a 0.1 M phosphate buffer (pH 6.5), mixed with 50 μl of a DMF solution containing 0.504 mg (1.8 μmol) of GMBS, and reacted at room temperature for 30 minutes. Thereafter, fractionation was performed using a Sephadex G-25 column. The thus prepared maleimide group-introduced HRP (6.4 mg, 126 nmol) and the [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) 0.35 mg (378 nmol) obtained in Reference Example 4 were used. And reacted at 4 ° C. for 1 day. After the reaction, fractionation was performed using an Ultrogel AcA44 (manufactured by LKB-Pharmacia) column to obtain HRP-labeled [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19).

(4) Measurement of antibody titer in antiserum of mice immunized with [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) -KLH complex [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand ( 1-19) -KLH complex was immunized twice at three-week intervals, and one week later, blood was collected from the fundus of the eye to collect blood. The blood was further centrifuged at 12,000 rpm for 15 minutes at 4 ° C., and the supernatant was recovered to obtain an antiserum. The antibody titer in the antiserum was measured by the following method. First, a 0.1 M carbonate buffer (pH 9.6) solution containing 100 μg / ml of an anti-mouse immunoglobulin antibody (IgG fraction, manufactured by Kappel) was added to a 96-well microplate to prepare an anti-mouse immunoglobulin antibody-bound microplate. , And left at 4 ° C for 24 hours. Next, after washing the plate with phosphate buffered saline (PBS, pH 7.4), 300 μl of PBS containing 25% Block Ace (Snow Brand Milk Products) was added to block excess binding sites in each well. Noted and treated at 4 ° C for at least 24 hours.
To each well of the obtained anti-mouse immunoglobulin antibody-bound microplate, 50 μl of buffer C and 100 μl of antiserum against the complex diluted with buffer C were added, and the mixture was reacted at 4 ° C. for 16 hours. Next, after washing the plate with PBS, 100 μl of HRP-labeled [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) (diluted 400-fold with buffer C) prepared in (3) above was added. The reaction was carried out at room temperature for 1 day. Next, after the plate is washed with PBS, the enzymatic activity on the solid phase is measured using a TMB (3,3 ', 5,5'-tetramethylbenzidine) microwell peroxidase substrate system (KIRKEGAARD & PERRY LAB, INC, Funakoshi Pharmaceutical Co., Ltd.). (Handling) 100 μl was added and the reaction was carried out at room temperature for 10 minutes. After stopping the reaction by adding 100 μl of 1M phosphoric acid, the absorbance at 450 nm was measured with a plate reader (BICHROMATIC, manufactured by Dainippon Pharmaceutical Co., Ltd.).
The results are shown in FIG. Increased antibody titers against [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) were observed in the antisera of six of eight immunized mice.

次に、上記で得られたハイブリドーマを限界希釈法によるクローニングに付した。クローニングに際しては、フィーダー細胞としてBALB/Cマウスの胸腺細胞を5×10⁵個／ウェルになるように加えた。クローニング後、ハイブリドーマを、あらかじめミネラルオイル0.5mlを腹腔内投与されたマウス（BALB/C）に1〜3×10⁶セル/匹を腹腔内投与したのち、6〜20日後に抗体含有腹水を採取した。
モノクローナル抗体は、得られた腹水よりプロテイン-Aカラムにより精製した。腹水6〜20mlを等量の結合緩衝液〔3.5M NaCl、0.05％ NaN₃を含む1.5Mグリシン（pH9.0）〕で希釈したのち、あらかじめ結合緩衝液で平衡化したリコンビナントプロテイン-A-アガロース（Repligen社製）カラムに供し、特異抗体を溶離緩衝液〔0.05％NaN₃を含む0.1Mクエン酸緩衝液（pH3.0）〕で溶出した。溶出液をPBSに対して4℃、2日間透析したのち、0.22μmのフィルター（ミリポア社製）により除菌濾過し、4℃あるいは-80℃で保存した。
モノクローナル抗体のクラス・サブクラスの決定に際しては、精製モノクローナル抗体結合固相を用いるエンザイム−リンクトイムノソーベントアッセイ（ELISA）法を行った。すなわち、抗体2μg/mlを含む0.1M炭酸緩衝液（pH9.6）溶液を96ウェルマイクロプレートに100μlずつ分注し、4℃で24時間放置した。上記（１）に記載の方法に従って、ウェルの余剰の結合部位をブロックエースでふさいだのち、アイソタイプタイピングキット(Mouse-Typer^TMSub-Isotyping Kit バイオラッド社製)を用いるELISAによって固相化抗体のクラス、サブクラスを調べた。得られた3種のモノクローナル抗体はいずれもH鎖がIgG1、L鎖がκであった。 (5) Preparation of anti-TGR23-2 ligand monoclonal antibody The final immunization was performed by intravenously inoculating 50 μg of immunogen dissolved in 0.2 ml of physiological saline intravenously into a mouse showing a relatively high antibody titer. Done. The spleen was removed from the mouse 4 days after the final immunization, pressed with a stainless mesh, filtered, and suspended in Eagle's Minimum Essential Medium (MEM) to obtain a spleen cell suspension. As a cell used for cell fusion, BALB / C mouse-derived myeloma cell P3-X63.Ag8.U1 (P3U1) was used (Current Topics in Microbiology and Imnology, Vol. 81, page 1, 1978).
Cell fusion was carried out according to the original method (Nature, 256, 495, 1975).
The spleen cells and P3U1 were each washed three times with serum-free MEM, mixed with spleen cells and P3U1 at a ratio of 5: 1, and centrifuged at 800 rpm for 15 minutes to precipitate the cells. After sufficiently removing the supernatant, the precipitate was lightly loosened, 0.3 ml of 45% polyethylene glycol (PEG) 6000 (manufactured by Kochlite) was added, and the mixture was allowed to stand still in a 37 ° C warm water bath for 7 minutes to perform fusion. After the fusion, MEM was added to the cells at a rate of 2 ml / min, and a total of 15 ml of MEM was added, followed by centrifugation at 600 rpm for 15 minutes to remove the supernatant. The cell precipitate was suspended in GIT medium (Wako Pure Chemical Industries) (GIT-10% FCS) containing 10% fetal bovine serum so that P3U1 was 2 × 10 ⁵ per ml, and a 24-well multi-dishes ( (Limbro) was seeded in 192 wells at 1 ml / well. After seeding, cells were cultured at 37 ° C. in a 5% CO 2 incubator. After 24 hours, 1 ml of GIT-10% FCS medium (HAT medium) containing HAT (hypoxanthine 1 × 10 ⁻⁴ M, aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 × 10 ⁻³ M) per well The HAT selection culture was started by adding each of the HATs. The HAT selective culture was continued by discarding 1 ml of the old solution 3 days, 6 and 9 days after the start of the culture, and then adding 1 ml of HAT medium. Hybridoma growth was observed 9 to 14 days after cell fusion. When the culture broth turned yellow (approximately 1 × 10 ⁶ cells / ml), the supernatant was collected and the antibody was purified according to the method described in (4) above. The titer was measured.
As an example of selecting a hybridoma antibody-producing cell line derived from a mouse immunized with [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) -KLH complex, mouse Nos. 2 and 6 (FIG. 1)), the antibody production state of the obtained hybridoma is shown in FIGS. 2 to 4. From the obtained antibody-producing hybridomas, a total of three types of hybridomas shown in Table 1 were selected.

Next, the hybridoma obtained above was subjected to cloning by the limiting dilution method. At the time of cloning, thymocytes of BALB / C mice were added as feeder cells at 5 × 10 ⁵ cells / well. After cloning, the hybridoma was intraperitoneally administered to mice (BALB / C) previously administered with 0.5 ml of mineral oil intraperitoneally at 1 to 3 × 10 ⁶ cells / animal, and antibody-containing ascites was collected 6 to 20 days later did.
The monoclonal antibody was purified from the obtained ascites by a protein-A column. An equal volume of binding buffer ascites 6~20ml [3.5 M NaCl, 0.05% 1.5M glycine (pH 9.0) containing NaN _3] After dilution with, a recombinant protein -A- agarose equilibrated in advance with binding buffer (Repligen) column, and the specific antibody was eluted with an elution buffer [0.1 M citrate buffer containing 0.05% NaN ₃ (pH 3.0)]. The eluate was dialyzed against PBS at 4 ° C. for 2 days, sterilized and filtered through a 0.22 μm filter (Millipore), and stored at 4 ° C. or −80 ° C.
In determining the class and subclass of the monoclonal antibody, an enzyme-linked immunosorbent assay (ELISA) using a solid phase bound with the purified monoclonal antibody was performed. That is, a 0.1 M carbonate buffer (pH 9.6) solution containing 2 μg / ml of the antibody was dispensed in 100 μl portions into a 96-well microplate and left at 4 ° C. for 24 hours. According to the method described in the above (1), the excess binding site of the well is covered with a block ace, and then the immobilized antibody is immobilized by ELISA using an isotype typing kit (Mouse-Typer ^™ Sub-Isotyping Kit BioRad). Checked class and subclass. In all three types of the obtained monoclonal antibodies, the H chain was IgG1 and the L chain was κ.

Example 2
Examination of antibody recognition site using competitive enzyme immunoassay [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) -KLH complex It investigated by the method of.
First, the antibody titers of the three types of the obtained monoclonal antibody solutions were examined by the method described in Example 1- (4) above, and the binding amount of the labeled product was determined to be the saturation binding amount as the antibody concentration used in the competition method-EIA. The antibody concentration that resulted in about 50% was determined (about 10-50 ng / ml). Next, (i) 23L-1Na was 50 ng / ml, 23L-1Ca was 10 ng / ml, or 23L-2Ca was 30 ng / ml in the anti-mouse immunoglobulin antibody-bound microplate described in Example 1- (4) above. ng / ml 50 μl of anti-rat TGR23-2 ligand (1-19) antibody solution diluted with buffer C, (ii) 50 μl of human TGR23-2 ligand solution diluted with buffer C, rat TGR23-2 ligand 50 μl solution, 50 μl mouse TGR23-2 ligand solution, or 50 μl [Cys ⁹ -NH ₂ ] rat TGR23-2 ligand (1-9) solution (Example 8 described later), and (iii) Example 1- (3) 50 μl of the HRP-labeled [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) (diluted 250-fold with buffer C) obtained in the above step was added to the anti-mouse immunoglobulin antibody-bound microplate, Allowed to react for hours. After the reaction, the plate was washed with PBS and the enzyme activity on the anti-mouse immunoglobulin antibody-bound microplate was measured by the method described in Example 1- (4) above.
Table 1 shows the results. From these results, the recognition sites of the three types of antibodies were considered as follows.
23L-1Na has reactivity with human TGR23-2 ligand, rat TGR23-2 ligand and mouse TGR23-2 ligand, and also reacts with [Cys ⁹ -NH ₂ ] rat TGR23-2 ligand (1-9) . Therefore, the recognition site of 23L-1Na is a common site of these peptides, human TGR23-2 ligand (1-7) (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3). (1st to 7th amino acid sequence).
23L-1Ca is reactive with rat TGR23-2 ligand, but reacts with human TGR23-2 ligand, mouse TGR23-2 ligand, and [Cys ⁹ -NH ₂ ] rat TGR23-2 ligand (1-9) Has no reactivity. Therefore, the recognition site of 23L-1Ca is a rat TGR23-2 ligand (9-12) centered on valine at position 10 (the ninth to twelfth amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2). It is believed that there is.
23L-2Ca is reactive with rat TGR23-2 ligand and mouse TGR23-2 ligand, but not with human TGR23-2 ligand and [Cys ⁹ -NH ₂ ] rat TGR23-2 ligand (1-9) Has no reactivity. Therefore, the recognition site of 23L-2Ca is determined by rat TGR23-2 ligand (15-18) and mouse TGR23-2 ligand (15-18) centering on arginine at position 16 (SEQ ID NO: 2 and SEQ ID NO: 3). 15th to 18th amino acid sequences of the amino acid sequence shown).

Next, as an example, the results of the competition method-EIA of each monoclonal antibody are shown in FIGS. 5, 6, and 7.
23L-1Na is human TGR23-2 ligand, from a standard curve of rat TGR23-2 ligand and mouse TGR23-2 ligand concentration giving a ratio (B / B ₀₎ = 0.5 for the maximum response, human and mouse TGR23-2 The ligand was found to be 60 nM, and the rat TGR23-2 ligand was found to be 100 nM (FIG. 5). From these results, it is considered that 23L-1Na shows high reactivity with any of human TGR23-2 ligand, rat TGR23-2 ligand and mouse TGR23-2 ligand.
23L-1Ca is human TGR23-2 ligand, from a standard curve of rat TGR23-2 ligand and mouse TGR23-2 ligand concentration giving a ratio (B / B ₀₎ = 0.5 for the maximum response, rats TGR23-2 ligand It is 20 nM and shows no reactivity with human TGR23-2 ligand and mouse TGR23-2 ligand (FIG. 6). From these results, it is considered that 23L-1Ca shows high reactivity only with rat TGR23-2 ligand.
23L-2Ca is human TGR23-2 ligand, from a standard curve of rat TGR23-2 ligand and mouse TGR23-2 ligand concentration giving a ratio (B / B ₀₎ = 0.5 for the maximum response, rats TGR23-2 ligand 20 nM, mouse TGR23-2 ligand is 40 nM, and shows no reactivity with human TGR23-2 ligand (FIG. 7). From these results, it is considered that 23L-2Ca shows high reactivity only to rat TGR23-2 ligand and mouse TGR23-2 ligand.

Example 3
Preparation of HRP-labeled anti-TGR23-2 ligand monoclonal antibody (23L-2Ca-HRP)
50 μl of DMF containing 0.59 μmol of GMBS was added to 0.1 M phosphate buffer (pH 6.8) containing 8.91 mg (59.4 nmol) of the 23L-2Ca purified fraction, and reacted at room temperature for 40 minutes. The reaction solution was separated on a Sephadex G-25 column (eluent, 0.1 M phosphate buffer, pH 6.7) to obtain 6.23 mg of an antibody fraction into which a maleimide group was introduced. Next, 60 μl of DMF containing 6.42 μmol of SPDP was added to 1.14 ml of 0.02 M phosphate buffer (including 0.15 M NaCl) (pH 6.8) containing 17.1 mg (428 nmol) of HRP, and reacted at room temperature for 40 minutes. Was. Next, 0.4 ml of 0.1 M acetate buffer (pH 4.5) containing 64.2 μmol of dithiothreitol was added, and reacted at room temperature for 20 minutes. Then, a Sephadex G-25 column (eluent, 0.1 mM containing 2 mM EDTA) was added. (M phosphate buffer, pH 6.0) to obtain 9.2 mg of HRP into which SH groups were introduced. Next, 8 mg of the HRP into which the SH group was introduced and 3 mg of the antibody fraction into which the maleimide group was introduced were mixed, concentrated to about 0.5 ml with a Collodion bag (manufactured by Sartorius), and then at 4 ° C. for 16 hours. I left it. The reaction solution was applied to a SephacrylS-300HR column (Pharmacia) equilibrated with 0.1 M phosphate buffer (pH 6.5) to purify the 23L-2Ca-HRP complex fraction.

Example 4
Sandwich method-EIA
A 0.1 M carbonate buffer (pH 9.6 solution) containing 15 μg / ml of the purified monoclonal antibody 23L-1Na obtained in Example 1 was dispensed in 100 μl portions into a 96-well microplate and left at 4 ° C. for 24 hours. Excess binding sites in the wells were inactivated by adding 400 μl of Block Ace diluted 4-fold with PBS.
To the prepared plate, 100 μl of a standard solution of human TGR23-2 ligand, rat TGR23-2 ligand and mouse TGR23-2 ligand diluted with buffer C was added and reacted at 4 ° C. for 24 hours. After washing with PBS, 100 μl of 23L-2Ca-HRP (diluted 3,000-fold with buffer C) prepared in Example 3 above was added, and reacted at 4 ° C. for 24 hours. After washing with PBS, the enzyme activity on the solid phase was measured using the TMB microwell peroxidase substrate system according to the method described in Example 1- (4) (enzyme reaction for 30 minutes).
FIG. 8 shows the results.
This sandwich method-EIA can detect rat TGR23-2 ligand at 3 fmol / ml and mouse TGR23-2 ligand at 10 fmol / ml, and reacts with human TGR23-2 ligand up to 1000 fmol / mL. Did not. Therefore, the sandwich method-EIA using 23L-1Na as a solid-phase antibody and 23L-2Ca-HRP as a label detects rat TGR23-2 and mouse TGR23-2 ligand with extremely high sensitivity and very selectively. It turns out that it is possible.

Example 5
23L-1Na and 23L-2Ca neutralize the biological activity of mouse TGR23-2 ligand
The neutralizing activity of 23L-1Na and 23L-2Ca against mouse TGR23-2 ligand was determined by increasing the intracellular [Ca ²⁺ ] concentration using TGR23-2 expressing CHO cells described in Reference Example 1 of WO 02/31145. Using FLIPR (Molecular Devices) as an index, measurement was performed.
Dulbecco's modified Eagle medium (DMEM) medium containing dialysis-treated fetal bovine serum (hereinafter referred to as dFBS) (JRH BIOSCIENCES) containing TGR23-2 expressing CHO cells at 3 × 10 ⁴ cells / ml (Nissui Pharmaceutical Co., Ltd.) ) (10% dFBS-DMEM) and inoculate 200 μl per well into a 96-well plate for FLIPR (Black plate clear bottom, Costar) using a pipettor (4 × 10 ⁴ cells / 200 μl / well) ) And cultured overnight at 37 ° C. in a 5% CO ₂ incubator before use (hereinafter referred to as a cell plate). FLIPR assay buffer [Nissui Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9.8 g, sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, pH 7.4 with 6M sodium hydroxide solution, fill up to 1 L, filter sterilize 20 ml), 200 μl of 250 mM probesecid (SIGMA), and 210 μl of fetal bovine serum (FBS). Also, two vials (50 μg) of Fluo 3-AM (Dojindo Chemical Laboratories) were dissolved in 40 μl of dimethyl sulfoxide and 40 μl of 20% Pluronic acid (Molecular Probes), and this was dissolved in H / HBSS [Hepes buffered solution]. Hanks Balance Solution (Nissui Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9.8 g, sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, adjusted to pH 7.4 with sodium hydroxide solution, then filter sterilized) 20 ml, 250 mM After adding to a H / HBSS-probeside-FBS solution containing 200 μl of probesecid and 200 μl of FBS, mixing and dispensing 100 μl of each well into a cell plate from which the culture solution was removed using an 8-pipette, 5% CO ₂ Incubated at 37 ° C. for 1 hour in an incubator (dye loading). 23L-1Na and 23L-2Ca were diluted in 120 μl of Hanks' / HBSS containing 2.5 mM probesecid and 0.2% BSA, and incubated with mouse TGR23-2 ligand (5.0 × 10 ⁻¹⁰ M) at 37 ° C. for 1 hour, 5 μl of each fraction was transferred to a 96-well plate for FLIPR (V-Bottom plate, Coster) (hereinafter referred to as a sample plate). After dye loading of the cell plate was completed, the cell plate was washed four times with a washing buffer obtained by adding 2.5 mM probesecid to Hanks' / HBSS using a plate washer (Molecular Devices), and 100 μl of the washing buffer was left after washing. . The cell plate and the sample plate were set on the FLIPR, and the measurement was performed (the FLIPR transfers 50 μl of the sample from the sample plate to the cell plate).
FIG. 9 shows the results.
Thus, the activity of mouse TGR23-2 ligand (5.0 × 10 ⁻¹⁰ M) was increased to about 74% at 23 × 1 ⁻⁹ M at 5 × 10 ⁻⁹ M, which was 10 times higher, and 5L at 5 × 10 ⁻⁹ M, which was 100 times higher. It can be seen that the suppression was about 15% at x 10 ^-8 M. 23L-2Ca increases the activity of mouse TGR23-2 ligand (5.0 × 10 ⁻¹⁰ M) to about 65% at 5 × 10 ⁻⁹ M at 10-fold molar concentration and 5 × 10 ^{− at} 100-fold molar concentration. It turns out that it suppressed to about 12% at ⁸ M.
From the above results, it was revealed that 23L-1Na and 23L-2Ca neutralize the intracellular [Ca ²⁺ ] concentration increasing activity of mouse TGR23-2 ligand, and these antibodies neutralize mouse TGR23-2 ligand. It turns out that it can be used as an antibody.

マウスの血液血漿中に27.6±3.76 fmol/ml（mean±SEM，n=22）の高濃度のマウスTGR23-2リガンドが検出された。 Example 6
Quantification of mouse TGR23-2 ligand in plasma Male Balb / C mouse (6-week-old) plasma was diluted 2-fold with the same amount of buffer C, and the mouse TGR23-2 was subjected to the sandwich method-EIA described in Example 4 above. The ligand was quantified.
Table 2 shows the results.

A high concentration of mouse TGR23-2 ligand at 27.6 ± 3.76 fmol / ml (mean ± SEM, n = 22) was detected in mouse blood plasma.

Example 7
Effect of TGR23-2 ligand neutralizing antibody administration on tumor growth in nude mice
The effect of administration of 23L-2Ca showing neutralizing activity on TGR23-2 ligand on tumor growth in human colon cancer cell line LS 174T tumor-bearing nude mice was examined.
The colon cancer cell line LS 174T dissolved in PBS was subcutaneously administered to the left abdomen of female nude mice (BALB / cAnN-nu, 6 weeks old) at a rate of 2 × 10 ⁶ cells / 200 μl / mouse. On the 10th day after administration, the tumor scale was measured, and 24 out of 42 mice were selected so that the tumor size became uniform, and 12 mice were divided into two groups. From the day of grouping, 23L-2Ca and an anti-human metastin monoclonal antibody (KIS-1Na) having the same IgG subclass structure (IgG1, κ) as 23L-2Ca as a control antibody (Journal of Clinical Endocrinology & Metabolism, vol. 88, 914-919, 2003) was intraperitoneally administered to mice at a dose of 3 mg / kg / day for 14 consecutive days. The scale of the tumors were measured every other day, the tumor volume was calculated by (major axis) × (minor diameter) ^2/2.
The results are shown in FIG.
From this, a significant decrease in the tumor volume of LS 174T was observed in the 23L-2Ca-administered group from the second day to the 17th day of administration.

Example 8
[Cys ⁹ ] Production of rat TGR23-2 ligand (1-9) amide (SEQ ID NO: 5) A commercially available p-methyl BHA resin (0.80 mmol / g resin) was placed in a reaction soda of a peptide synthesizer ABI 430A. Boc-Strategy (NMP-HOBt) Boc amino acid derivatives of Boc-Cys (MeBzl), Boc-Ser (Bzl), Boc-Gly, Boc-Val, Boc-Asn, Boc-Arg (Tos) Were introduced in the order of the sequence to obtain the desired protective peptide resin. After stirring 0.21 g of this resin in 1.5 ml of p-cresol and 10 ml of anhydrous hydrogen fluoride at 0 ° C. for 60 minutes, the hydrogen fluoride was distilled off under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered. The precipitate was extracted with 50% aqueous acetic acid, the insoluble portion was removed, the extract was concentrated sufficiently, and then applied to a Sephadex (trademark) G-25 column (2.0 × 80 cm) filled with 50% aqueous acetic acid. The mixture was developed with a solvent, and the main fractions were collected and freeze-dried to obtain 40 mg of a white powder. A half amount was applied to a chromatography column (2.6 × 60 cm) packed with LiChroprep (trade name) RP-18, washed with 200 ml of 0.1% TFA water, and 300 ml of 0.1% TFA water and 300 ml of 33% acetonitrile water containing 0.1% TFA were used. A linear gradient elution was performed, and the main fraction was collected and freeze-dried to obtain 10 mg of the desired peptide.
M ⁺ 925.5 by mass spectrometry (calculated value: 925.0)
HPLC elution time: 11.5 minutes Elution conditions Column: Wakosi15C18T (4.6 x 100mm)
Eluent: Solution A: 0.1% TFA water, Solution B: Acetonitrile containing 0.1% TFA, A / B: 95/5 to 45/55, linear concentration gradient elution (25 minutes)
Flow rate: 1.0 ml / min

Reference Example 1
Production of human TGR23-2 ligand Commercially available Boc-Ser (Bzl) -OCH ₂ -PAM resin was placed in a reaction vessel of a peptide synthesizer ACT90, swollen with DCM, Boc was removed with TFA, and neutralized with DIEA. This resin was suspended in NMP, and Boc-Lys (Cl-Z) was condensed with HOBt-DIPCI. After the reaction, the presence or absence of a free amino group was examined by a ninhydrin test. When the ninhydrin test was positive, the same amino acid was condensed again. Even after the recondensation, when the ninhydrin test was positive, it was acetylated with acetic anhydride. Repeat this cycle, Boc-Ala, Boc-Arg (Tos), Boc-Gln, Boc-Phe, Boc-Ser (Bzl), Boc-Thr (Bzl), Boc-Lys (Cl-Z), Boc-Lys (CL-Z), Boc-Met, Boc-Gly, Boc-Thr (Bzl), Boc-Gly, Boc-Val, Boc-Gly, Boc-Asn, Boc-Arg (Tos), Boc-Phe, Boc- Ser (Bzl) was condensed in the sequence order to obtain 0.24 g of the desired protected peptide resin. The resin was stirred with 1.5 ml of p-cresol in about 15 ml of hydrogen fluoride at 0 ° C. for 60 minutes, then the hydrogen fluoride was distilled off under reduced pressure, and diethyl ether was added to the residue, followed by filtration. Water and acetic acid were added to the filtrate to extract the peptide, which was separated from the resin. The extract was concentrated, applied to a Sephadex (trademark) G-25 column (2.0 × 80 cm) filled with 50% acetic acid, developed with the same solvent, and the main fraction was collected and lyophilized. An aliquot (45 mg) was applied to a reversed-phase chromatography column (2.6 x 60 cm) packed with LiChroprep ™ RP-18, washed with 200 ml of 0.1% TFA water, 300 ml of 0.1% TFA water and containing 0.1% TFA 25 A linear gradient elution was performed using 300 ml of acetonitrile water, and the main fractions were collected and lyophilized to obtain 12.7 mg of the desired peptide.
ESI-MS: molecular weight MW 2188.0 (theoretical value 2187.5)
HPLC elution time 10.6 min Column conditions:
Column: Wakosil 5C18T 4.6 x 100mm
Eluent: Linear concentration gradient elution to A / B: 95/5 to 45/55 using solution A-0.1% TFA water and solution B-acetonitrile containing 0.1% TFA (25 minutes)
Flow rate: 1.0 ml / min

Reference Example 2
Production of rat TGR23-2 ligand Commercially available 2-chlorotrityl resin (Clt resin, 1.33 mmol / g) with Fmoc-Ala-OH introduced into Fmoc-Ala-O-Clt resin (0.638 mmol / g) 0.25 mmol for peptide The mixture was placed in a reaction vessel of a synthesizer ABI 433A, and solid-phase synthesis was performed using the Fmoc / DCC / HOBt method. As the side chain protecting group of the Fmoc amino acid, a Pbf group was used for Arg, a tBu group for Ser, a Boc group for Lys, and a Trt group for Asn. The other amino acids used were unprotected in the side chain, and peptide chains were introduced from Arg at position 17 in the above sequence to Ser at position 14 in the N-terminal direction. The obtained Fmoc-rat TGR23-2 ligand (14-18) -O-Clt resin (0.25 mmol) was converted to Fmoc-Lys (Boc) -Thr (Psi (Me, Me) pro) -OH (manufactured by NOVA, product). No. 05-20-1116) 381.1 mg (0.625 mmol), PyAOP 326.1 mg (0.625 mmol), HOAt 85.1 mg (0.625 mmol), DIEA 435.5 ml (2.5 mmol), Lys 12th, Thr 13th Was introduced. Then, using the obtained Fmoc- [Thr (Psi (Me, Me) pro) ¹³ ] -rat TGR23-2 ligand (12-18) -O-Clt resin, solid-phase synthesis by a peptide synthesizer was resumed. A peptide chain was introduced from position Lys to position 1 Ser in the N-terminal direction in order to obtain 573.5 mg of the desired protected peptide resin.
After stirring all of this resin (0.25 mmol) in a mixture of 9 ml of TFA, thioanisole, m-cresol, water, triisopropylsilane, ethandithiol (80: 5: 5: 5: 2.5: 2.5) for 90 minutes, Ether was added to the reaction solution to precipitate a white powder, and after centrifugation, the operation of removing the supernatant was repeated three times. The residue was extracted with water and freeze-dried to obtain 219.4 mg of a white powder. The obtained crude peptide was subjected to preparative HPLC using a YMC Pack R & D-ODS-5-B S-5, 120A column (30 x 250 mm) to obtain a solution A: 0.1% TFA-water and a solution B: 0.1% TFA. A / B: 90/10 to 70/30 with acetonitrile was used to perform linear concentration gradient elution (60 minutes), and fractions containing the target compound were collected and freeze-dried to obtain a white powder.
After dissolving all the obtained powders in water and mixing, the solution was added with 3 ml of a resin obtained by chlorinating the ion exchange resin AG1 x 8 100-200 mesh chloride form manufactured by BIO-RAD for 3 ml, and the mixture was stirred for 20 minutes and filtered. After removing the resin and impurities, it was freeze-dried to obtain 107.0 mg of white powder as acetate.
ESI-MS: M ⁺ 1954.2 (theoretical 1954.2)
HPLC elution time: 15.2 minutes Elution conditions Column: YMC AM 301 (4.6 x 100mm)
Eluent: Solution A: 0.1% TFA-water, Solution B: Acetonitrile containing 0.1% TFA, linear concentration gradient elution from A / B: 100/0 to 50/50 (25 minutes)
Flow rate: 1.0 ml / min

Reference Example 3
Production of mouse TGR23-2 ligand Commercially available 2-chlorotrityl resin (Clt resin, 1.12 mmol / g) with Fmoc-Gln (Trt) -OH introduced Fmoc-Gln (Trt) -O-Clt resin (0.408 mmol / g) Using 0.25 mmol, solid phase synthesis was carried out in the same manner as in the production of rat TGR23-2 ligand described in Reference Example 1 to obtain 324.7 mg of the desired protected peptide resin.
100 mg of this resin was treated in the same manner as in Reference Example 1 to obtain 50.0 mg of a white powder. The obtained crude peptide was subjected to preparative HPLC using a YMC SH-343-5 S-5, 120A column (20 x 250 mm), and the solution A: 0.1% TFA-water, and the solution B: acetonitrile containing 0.1% TFA. A / B: A linear gradient elution was performed (92 min) from 92/8 to 72/28, and fractions containing the target compound were collected and freeze-dried to obtain 22.6 mg of a white powder.
ESI-MS: M ⁺ 2182.8 (theoretical 2182.5)
HPLC elution time: 14.1 minutes Elution conditions Column: YMC AM 301 (4.6 x 100 mm)
Eluent: Solution A: 0.1% TFA-water, Solution B: Acetonitrile containing 0.1% TFA, linear concentration gradient elution from A / B: 100/0 to 50/50 (25 minutes)
Flow rate: 1.0 ml / min

Reference example 4
[Cys ¹⁹ ] Production of Rat TGR23-2 Ligand (1-19) (SEQ ID NO: 4) Commercially available 0.25 mmol of H-Cys (Trt) -O-Clt resin (0.63 mmol / g) was prepared using a peptide synthesizer ABI 433A. The resultant was placed in a reaction soda and subjected to solid-phase synthesis using the Fmoc / DCC / HOBt method. As the side chain protecting group of the Fmoc amino acid, a Pbf group was used for Arg, a tBu group for Ser, a Boc group for Lys, and a Trt group for Asn. The other amino acids used were those without side chain protection, and peptide chains were introduced from Ala at position 18 in the above sequence to Ser at position 14 in the N-terminal direction. After removing the Fmoc group from half (0.125 mmol) of the obtained Fmoc-rTGR23L (14-18) -O-Clt resin (0.25 mmol) with 20% piperidine / DMF, Fmoc-Lys (Boc) -Thr ( ^{処理 Me.Me} Pro) -OH (manufactured by NOVA) 304.9 mg (0.5 mmol), PyAOP 260.9 mg (0.5 mmol), 0.5 M HOAt / DMF solution 1 ml (0.5 mmol), DIEA 326.6 μl (1.88 mmol) As a result, Lys at position 12 and Thr at position 13 were introduced. Then, using the obtained Fmoc- [Thr (Ψ ^Me.Me Pro) ¹³ , Cys ¹⁹ ] rat TGR23-2 ligand (12-19) -O-Clt resin, solid-phase synthesis by a peptide synthesizer was resumed. A peptide chain was sequentially introduced from position Lys to position 1 Ser in the N-terminal direction to obtain 234.0 mg of the desired protected peptide resin.
After stirring 100 mg of this resin in 1.5 ml of a mixed solution of TFA, thioanisole, m-cresol, H ₂ O, triisopropylsilane, ethandithiol (80: 5: 5: 5: 2.5: 2.5) for 90 minutes at room temperature, the reaction solution Ether was added to the mixture to precipitate a white powder. After centrifugation, the operation of removing the supernatant was repeated three times. The residue was extracted with an aqueous acetic acid solution, and lyophilized. The obtained white powder was subjected to preparative HPLC using a YMC SH-343-5 S-5, 120A column (20 × 250 mm) to obtain a liquid A: 0.1% TFA-water. A / B: A / B: 86/14 to 76/24 with acetonitrile containing 0.1% TFA was subjected to linear concentration gradient elution (60 minutes), and fractions containing the target compound were collected and lyophilized to give 26.1 mg of a white powder. Got.
By mass spectrometry (M + H) ⁺ 2056.6 (calculated 2057.1)
HPLC elution time 15.3 minutes Elution conditions Column: YMC-AM301 (4.6 x 100mm)
Eluent: Solution A: 0.1% TFA-water, Solution B: Acetonitrile containing 0.1% TFA, A / B: 100/0 to 50/50, linear concentration gradient elution (25 minutes)
Flow rate: 1.0ml / min

Reference example 5
Effects of Human TGR23-2 Ligand on Food Intake by Intraventricular Administration of Rats Rats were bred at a lighting time of 8 to 20 hours at room temperature of 25 ° C. Eight-week-old male Wistar rats (body weight at the time of surgery: 260 to 280 g) were anesthetized by intraperitoneal administration of pentobarbital 50 mg / kg, and fixed to a rat stereotaxic apparatus. The incisor bar was 3.3 mm below the interoral line. The skull was exposed, and a hole was drilled in the bone using a dental drill to implant a guide cannula AG-8 (0.4 mm id, 0.5 mm outside diameter, Acom) into the lateral ventricle. In addition, anchor screws were buried in three places around it. A stainless steel guide cannula AG-8 was inserted so that its tip was located in the upper part of the lateral ventricle. According to the atlas of Paxinos and Watson (1998), the localization coordinates were AP: -0.8 mm, L: 1.5 mm, H: -4.5 mm from Bregma. The guide cannula was fixed to the skull with dental cement and anchor screws. A stainless steel dummy cannula AD-8 (outer diameter 0.35 mm, Acom) was inserted into the guide cannula and fixed with a cap nut (Acom). After surgery, rats were housed in individual cages and acclimated to powdered diet for one week.
After the guide cannula has been implanted, it is bred for approximately one week, and is allowed to recover after surgery and adaptation to powdered feed. The cap nut and dummy cannula attached to the skull of the rat are removed, and a PTFE (polytetrafluoroethylene) tube is used instead. A stainless steel microinjection cannula AMI-9 (0.17 mm inner diameter, 0.35 mm outer diameter, Acom) connected to (length 50 cm, inner diameter 0.1 mm, outer diameter 0.35 mm, Acom) was inserted. The length of the microinjection cannula was adjusted so that its tip 1 mm was exposed from the guide cannula. One of the PTFE tubes was connected to a micro syringe pump, and Otsuka distilled water and distilled water in which human TGR23-2 ligand was dissolved were injected into the lateral ventricle at a flow rate of 5 μl / min for a total of 10 μl (10 nmol / rat). Two minutes after the end of the injection, the microinjection cannula was removed, and the dummy cannula was fixed again with the cap nut. Infusions were performed between 19:00 and 20:00, after which time food intake was measured over time using a food intake measure Feed-Scale (Columbus) every 30 minutes up to 4 hours after administration.
The results are shown in FIG.
From this, a significant (p <0.05) decrease in food consumption was observed in the human TGR23-2 ligand administration group at 1.5 and 2 hours after administration, as compared to the control group.

FIG. 4 shows the results of measuring antibody titers in antisera of mice immunized with [Cys ¹⁹ -NH ₂ ] rat TGR23-2 ligand (1-19) -BTG complex. In the figure,-◇-is mouse No. 1,-□-is mouse No. 2,-△-is mouse No. 3,-○-is mouse No. 4,-◆-is mouse No. 5,-■. -Indicates mouse No. 6,-▲-indicates mouse No. 7, and-●-indicates mouse No. 8. [Cys ¹⁹ -NH ₂ ] shows a state in which a hybridoma derived from a mouse immunized with the rat TGR23-2 ligand (1-19) -BTG complex is producing an antibody (result of absorption analysis). [Cys ¹⁹ -NH ₂ ] shows a state in which a hybridoma derived from a mouse immunized with the rat TGR23-2 ligand (1-19) -BTG complex is producing an antibody (result of absorption analysis). [Cys ¹⁹ -NH ₂ ] shows a state in which a hybridoma derived from a mouse immunized with the rat TGR23-2 ligand (1-19) -BTG complex is producing an antibody (result of absorption analysis). The results of the competition method-EIA of 23L-1Na are shown. In the figure,-●-indicates the reactivity with human TGR23-2 ligand,-○-indicates the reactivity with rat TGR23-2 ligand, and-■-indicates the reactivity with mouse TGR23-2 ligand. 23 shows the results of 23L-1Ca competition method-EIA. In the figure,-●-indicates the reactivity with human TGR23-2 ligand,-○-indicates the reactivity with rat TGR23-2 ligand, and-■-indicates the reactivity with mouse TGR23-2 ligand. 23 shows the results of 23L-2Ca competition method-EIA. In the figure,-●-indicates the reactivity with human TGR23-2 ligand,-○-indicates the reactivity with rat TGR23-2 ligand, and-■-indicates the reactivity with mouse TGR23-2 ligand. FIG. 9 shows the results of a sandwich method EIA using 23L-1Na and 23L-2Ca-HRP. In the figure,-●-indicates the reactivity with human TGR23-2 ligand,-○-indicates the reactivity with rat TGR23-2 ligand, and-■-indicates the reactivity with mouse TGR23-2 ligand. Fig. 4 shows the neutralizing effect of mouse TGR23-2 ligand on TGR23-2 expressing CHO cells in the presence of 23L-1Na or 23L-2Ca in increasing the intracellular [Ca2 ⁺ ] concentration. Mouse TGR23-2 ligand (5 × ^{10 −10} M) is reacted with 23L-1Na or 23L-2Ca at a molar ratio of 1: 1, 1:10, 1: 100 or 1: 1000 at room temperature for 1 hour. ^{Shows the} activity of increasing intracellular [Ca ²⁺ ] concentration. In the figure, the black bars show the control of intracellular [Ca ²⁺ ] concentration increasing activity in TGR23-2 expressing CHO cells when 23L-1Na and 23L-2Ca coexist with mouse TGR23-2 ligand, respectively. (When no antibody is added). FIG. 4 shows tumor growth curves in human colon cancer cell line LS 174T tumor-bearing nude mice by administration of 23L-2Ca exhibiting neutralizing activity against TGR23-2 ligand and a control antibody. In the figure,-●-shows the growth curve of the group administered with 23L-2Ca, and-○-shows the growth curve of the group administered with the control antibody (KIS-1Na). The values indicate mean ± standard deviation (mean ± SE) (n = 12). * Indicates that the P value is 0.05 or less as compared with the control antibody (KIS-1Na) administration group. ** indicates that the P value is 0.01 or less compared to the control antibody (KIS-1Na). [Fig. 4] Fig. 4 shows temporal changes in food consumption every 30 minutes up to 4 hours after administration of human TGR23-2 ligand or distilled water into a rat lateral ventricle. In the figure,-●-indicates a group administered with human TGR23-2 ligand, and-○-indicates a group administered with distilled water. Values are shown as mean ± standard error (n = 8). * Indicates a significant (p <0.05) difference from the distilled water administration group.

Claims (37)

An antibody that specifically reacts with a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a partial peptide on the N-terminal side of a salt thereof. The antibody according to claim 1, which specifically reacts with a peptide having the first to seventh amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. 1st to 3rd, 1st to 4th, 1st to 5th, 1st to 6th, 1st to 7th of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 , 2nd-4th, 2nd-5th, 2nd-6th, 2nd-7th, 3rd-5th, 3rd-6th, 3rd-7th, 4th-6th, The antibody according to claim 1, which specifically reacts with a peptide having the fourth to seventh or fifth to seventh amino acid sequence. The antibody according to claim 1, wherein the antibody does not recognize a partial peptide at the C-terminal side of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a salt thereof. The antibody according to claim 1, which has a neutralizing activity against a peptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The antibody according to claim 1, which is labeled. The antibody according to claim 1, which is a monoclonal antibody. The monoclonal antibody according to claim 7, which is designated as 23L-1Na which can be produced from a hybridoma cell designated as 23L-1N (FERM BP-8302). An antibody that specifically reacts with a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a partial peptide on the C-terminal side of a salt thereof. The antibody according to claim 9, which specifically reacts with a peptide having the 15th to 18th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3. The 12th to 14th, 12th to 15th, 12th to 16th, 12th to 17th, 12th to 18th, and 13th to 15th amino acids of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 Th, 13th to 16th, 13th to 17th, 13th to 18th, 14th to 16th, 14th to 17th, 14th to 18th, 15th to 17th, 15th to 18th or The antibody according to claim 9, which specifically reacts with a peptide having the 16th to 18th amino acid sequences. The antibody according to claim 9, which does not recognize a partial peptide at the N-terminal side of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or a salt thereof. The antibody according to claim 9, which has a neutralizing activity against a peptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3. The antibody according to claim 9, which is labeled. The antibody according to claim 9, which is a monoclonal antibody. The monoclonal antibody according to claim 15, which is designated as 23L-2Ca which can be produced from a hybridoma cell designated as 23L-2C (FERM BP-8303). A medicament comprising the antibody according to claim 1 or 9. The medicament according to claim 17, which is an agent for preventing or treating cancer or anorexia nervosa. A diagnostic agent comprising the antibody according to claim 1 or / and / or 9. A method for quantifying a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a salt thereof, wherein the antibody according to claim 1 is used. The method according to claim 20, further comprising using the antibody according to claim 9. A method for diagnosing a disease involving the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof, comprising using the antibody according to claim 1. A method for diagnosing cancer, comprising using the antibody according to claim 1. The diagnostic method according to claim 22 or 23, further comprising using the antibody according to claim 9. A method for quantifying a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof, wherein the antibody according to claim 9 is used. 2. The antibody according to claim 1 is competitively reacted with a test solution and a labeled polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a salt thereof. And measuring the ratio of the labeled polypeptide or a salt thereof bound to the antibody, wherein the ratio is represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the test solution. For determining a polypeptide containing an amino acid sequence or a salt thereof. 10. The antibody according to claim 9, which is allowed to competitively react with the test solution and a labeled polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof. A polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 in a test solution or a polypeptide thereof, wherein the ratio of the bound labeled polypeptide or a salt thereof is measured. Determination of salt. (1) measuring the activity of the labeling agent after reacting the antibody of claim 1 insoluble on a carrier, the labeled antibody of claim 9 and a test solution, or
(2) A test solution comprising reacting the antibody of claim 9 insoluble on a carrier, the labeled antibody of claim 1 and a test solution, and then measuring the activity of the labeling agent. Of the polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof. A hybridoma cell that produces the monoclonal antibody according to claim 7. 30. The hybridoma cell of claim 29, designated 23L-1N (FERM BP-8302). The method for producing a monoclonal antibody according to claim 7, wherein the hybridoma cell according to claim 29 is cultured in vivo or in vitro, and the monoclonal antibody according to claim 7 is collected from the body fluid or culture. A hybridoma cell that produces the monoclonal antibody according to claim 15. 33. The hybridoma cell of claim 32, designated 23L-2C (FERM BP-8303). The method for producing a monoclonal antibody according to claim 15, wherein the hybridoma cell according to claim 32 is cultured in vivo or in vitro, and the monoclonal antibody according to claim 15 is collected from the body fluid or culture. A polypeptide having the amino acid sequence represented by SEQ ID NO: 5, an amide thereof, or a salt thereof. A method for preventing or treating cancer or anorexia nervosa, comprising administering an effective amount of the antibody according to claim 1 or 9 to a mammal. Use of the antibody according to claim 1 or 9 for producing a prophylactic / therapeutic agent for a prophylactic / therapeutic agent for cancer or anorexia nervosa.

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