HK1165445A - Diagnosis and treatment of cancer using anti-lgr7 antibody - Google Patents

Description

Diagnosis and treatment of cancer using anti-LGR 7 antibody

Technical Field

The present invention relates to an antibody that binds to LGR7 protein, a method for diagnosing cancer, a method for treating cancer, and an anti-cancer agent.

Background

The LGR7 molecule is a protein encoded by the ENSG00000171509 gene of Ensembl ID of human chromosome 4q32, and is a member of LGR family (leucine-rich GPCR family, hereinafter referred to as LGR family) of hormone receptor family of G protein-coupled seven-transmembrane binding protein, as is clear from the characteristics of its amino acid sequence (non-patent document 1). It is registered in RefSeq as NM _021634/NP _ 067647. A sequence in which the 70 th amino acid is changed from Leu to Met has also been reported (patent document 1). Also, 3 splice variants were reported, LGR7.1(AY899848.1) having exon 6a inserted between exon 6 and exon 7, and exon 15a inserted between exon 15 and exon 16. LGR7.2(AY899849.1) forms the gene structure for deletion of exons 12, 13. LGR7.10(AY899850.1) lacks exon 3. Members of the LGR family are divided into 3 groups, the first group being hormone receptors of FSHR (LGR1), LHCGR (LGR2) or TSHR (LGR3), the second group being ligand-undefined LGR4, LGR5 or LGR6, and the third group being LGR7 or LGR8 with relaxin, insulin-like peptide 3(INSL3) or the like as a ligand (non-patent document 2). Both hetero-peptides are known to act as ligands, primarily transmitting cAMP-mediated signals into the cell. The LGR family has a structure formed by seven transmembrane protein regions and an N-terminally long extracellular region, in which 9-17 repeats of a leucine-rich region of about 25 amino acid residues are present (LRR). LGR7 has 10 LRRs (non-patent document 1). Further, an LDL-a domain not present in other LGR family molecules is present at the N-terminus immediately before the LRR (non-patent document 3). It has been reported that: LDL-a is essential for signal transmission and also involved in intramembrane transport (trafficking) of LGR7 (non-patent document 4). According to analysis of TSHR and the like, the following results are obtained: in the LGR family molecule, a ligand binds with high affinity to its extracellular LRR and to the 2nd extracellular loop region, thereby performing signaling coupled to a G protein (non-patent document 5). As for the ligand binding to LGR7, relaxin is known, and in the case of human, relaxin 2 and relaxin 3 are known, but a ligand having a higher binding ability is relaxin 2, and it is considered that it functions as a ligand of LGR7 in vivo (non-patent documents 6 and 7).

As for relaxin as a ligand, a relationship between it and Thyroid cancer (Thyroid cancer) and prostate cancer (prostate cancer) has been reported (non-patent document 8). It has been reported that: in prostate cancer, androgen-independent proliferation is promoted in a prostate cancer cell line LNCaP into which p53 in which R of the amino acid at position 273 is mutated to H is introduced (non-patent document 9). H2 relaxin rose in this cell, suggesting that: it is involved in the expression of H2 relaxin and the progression of prostate cancer, and p53 of R273H binds directly to the promoter of H2 relaxin, inducing the expression of PSA via androgen receptor. However, no paper reporting the association of LGR7 with cancer exists.

On the other hand, patent documents report that LGR7 gene expression is higher in uterine cancer and ovarian cancer than in normal tissues, and report on the association of LGR7 with cancer, but in all documents, the anticancer effect using an antibody has not been confirmed, and it is not clear whether or not LGR 7-expressing cancer can be treated with an antibody (patent documents 2 to 5).

Among ovarian cancers, clear cell adenocarcinomas are known as a type of cancer in which chemotherapy is not easily effective (non-patent documents 10 and 11). Sequoyishan et al report: the response rate to chemotherapy containing cisplatin or taxane compounds as standard therapy was 72.5% in serous adenocarcinoma, while the response rate was 11.1% in clear cell adenocarcinoma (non-patent document 10). On the other hand, the number of patients with clear cell adenocarcinoma has recently increased, and it has been reported that clear cell adenocarcinoma accounts for 22% of the total ovarian carcinomas in Japan, as described in journal Kouzo 57 No. 11 1711 (2005). This is in contrast to the number reported as 6% in overseas Report FIGO Annual Report 1998. In addition, according to the Japanese woman reports that the ratio of clear cell adenocarcinoma to total epithelial malignant tumor in ovarian cancer was 4% in 1971-1977 and about 10% in 1978-1983, whereas it was more than 20% and increased in 2002, it is desired to develop a therapy against clear cell adenocarcinoma.

Documents of the prior art

Patent document

Patent document 1: WO 9948921

Patent document 2: US 2005107595

Patent document 3: WO 2003016487

Patent document 4: WO 2003093827

Patent document 5: WO 2005107396

Non-patent document

Non-patent document 1: hsu, S.Y. et al, molec. Endocr., 14, 1257-

Non-patent document 2: hsueh A.J.W. et al, Journal of Endocrinology, 187, 333-

Non-patent document 3: bathgate RA. et al, Pharmacol Rev, 58, 7-31(2006)

Non-patent document 4: kern A. et al, Endocrinology, 148, 1181-

Non-patent document 5: kristiansen K., Pharmacology & Therapeutics, 103, 21-80(2004)

Non-patent document 6: halls M.L. et al, British Journal of Pharmacology, 150, 677-

Non-patent document 7: van Der Westhuizen, E.T. et al, Current Drug Targets, 8, 91-104(2007)

Non-patent document 8: Hombach-Klonisch S. et al, American Journal of Pathology 169, 617-632(2006)

Non-patent document 9: vinall R.L. et al, Oncogene, 25, 2082-

Non-patent document 10: sugiyama T et al Cancer, 88, 2584(2000)

Non-patent document 11: proceedings of ASCO.2003; 1797, Chicago

Disclosure of Invention

Problems to be solved by the invention

The invention aims to: provided are novel antibodies that bind to LGR7 protein, novel methods for diagnosing cancer, novel methods for treating cancer, novel cell proliferation inhibitors, and anticancer drugs.

Means for solving the problems

The inventors of the present invention found that: in clear cell adenocarcinoma cells in ovarian cancer, not only the gene of LGR7 but also its protein is highly expressed. Even in ovarian cancer, LGR7 was limited to a cancer closely related to clear cell adenocarcinoma, which has not been reported so far.

In addition, the present inventors produced monoclonal antibodies against LGR7 protein.

The present inventors also found that the anti-LGR 7 antibody has ADCC activity on LGR 7-expressing cells when measuring the antibody-dependent cellular cytotoxicity (ADCC) activity of the anti-LGR 7 antibody. In addition, when the complement-dependent cell-mediated cytotoxicity (CDC) activity was measured, it was also found that the anti-LGR 7 antibody had CDC activity on LGR 7-expressing cells. Furthermore, when the anti-LGR 7 antibody was administered to a xenograft tumor model mouse, it was confirmed that the anti-LGR 7 antibody showed a tumor reduction effect. From the above knowledge, the present inventors found that: the anti-LGR 7 antibody is effective for the diagnosis, prevention and treatment of primary or metastatic ovarian clear cell adenocarcinoma, and thus the present invention has been completed. More specifically, the present inventors have found that: the anti-LGR 7 antibody is effective as a means for treating or diagnosing a cancer with increased LGR7 expression, represented by ovarian clear cell adenocarcinoma, and the present invention has been completed.

That is, the present invention provides antibodies that bind to LGR7 protein. The present invention also provides antibodies that bind to LGR7 protein and have cytotoxic activity against cells expressing LGR7 protein. Preferably, the cytotoxic activity is ADCC activity. The invention also provides anti-LGR 7 antibodies that bind a cytotoxic substance.

The present invention also provides a pharmaceutical composition comprising an antibody that binds to LGR7 protein as an active ingredient. The present invention also provides a cell proliferation inhibitor containing an antibody that binds to LGR7 protein as an active ingredient. The present invention also provides an anti-cancer agent comprising an antibody that binds to LGR7 protein as an active ingredient.

Alternatively, the present invention provides a pharmaceutical composition comprising an antibody that binds to LGR7 protein and a pharmaceutically acceptable carrier. More specifically, the present invention provides the following inventions [1] to [23 ].

[1] An antibody that binds to LGR7 protein and has cell proliferation inhibitory activity against cells expressing LGR7 protein.

[2] [1] the antibody, wherein the cell proliferation inhibitory activity is cytotoxic activity.

[3] [2] the antibody, wherein the cytotoxic activity is an antibody-dependent cytotoxic activity.

[4] [2] the antibody, wherein the cytotoxic activity is complement-dependent cytotoxic activity.

[5] The antibody according to any one of [1] to [4], wherein the antibody is an antibody to which a cytotoxic substance binds.

[6] [5] the antibody having an internalization activity.

[7] The antibody according to any one of [1] to [6], which inhibits cancer cell proliferation.

[8] [7] the antibody according to any one of the above methods, wherein the cancer cell is an ovarian cancer clear cell.

[9] The antibody according to any one of the following (1) to (29):

(1) an antibody (22DA6 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of CDR2 shown in SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 7;

(2) an antibody (22DA6 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 10, the amino acid sequence of CDR2 of SEQ ID NO: 11, and the amino acid sequence of CDR3 shown in SEQ ID NO: 12;

(3) an antibody (22DA6) comprising the H chain of (1) and the L chain of (2);

(4) an antibody (22DA7 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 15, the amino acid sequence of CDR2 of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 17;

(5) an antibody (22DA7 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 20, the amino acid sequence of CDR2 of SEQ ID NO: 21 and the amino acid sequence of SEQ ID NO: 22;

(6) an antibody (22DA7) comprising the H chain of (4) and the L chain of (5);

(7) an antibody (22DA17 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 27, an amino acid sequence of seq id no;

(8) an antibody (22DA17 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31 and the amino acid sequence of SEQ ID NO: 32;

(9) an antibody (22DA17) comprising the H chain of (7) and the L chain of (8);

(10) an antibody (22DA22 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 35, the amino acid sequence of CDR2 of SEQ ID NO: 36 and the amino acid sequence of SEQ ID NO: 37, or a pharmaceutically acceptable salt thereof;

(11) an antibody (22DA22 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 40, the amino acid sequence of CDR2 of SEQ ID NO: 41 and the amino acid sequence of SEQ ID NO: 42;

(12) an antibody (22DA22) comprising the H chain of (10) and the L chain of (11);

(13) an antibody (22DA23 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 45, and the amino acid sequence of CDR2 shown in SEQ ID NO: 46 and the amino acid sequence of SEQ ID NO: 47 in a sequence listing;

(14) an antibody (22DA23 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 50, the amino acid sequence of CDR2 of SEQ ID NO: 51, and the amino acid sequence of SEQ ID NO: 52;

(15) an antibody (22DA23) comprising the H chain of (13) and the L chain of (14);

(16) an antibody (22DA24 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 55, the amino acid sequence of CDR2 of SEQ ID NO: 56, and the amino acid sequence of SEQ ID NO: an amino acid sequence according to 57;

(17) an antibody (22DA24 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 60, the amino acid sequence of CDR2 of SEQ ID NO: 61, and the amino acid sequence of SEQ ID NO: 62, an amino acid sequence as described in 62;

(18) an antibody (22DA24) comprising the H chain of (16) and the L chain of (17);

(19) an antibody (22SD7 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 65, the amino acid sequence of CDR2 of SEQ ID NO: 66 and the amino acid sequence of SEQ ID NO: 67, or a nucleotide sequence thereof;

(20) an antibody (22SD7 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 70, the amino acid sequence of CDR2 of SEQ ID NO: 71 and the amino acid sequence of SEQ ID NO: 72, an amino acid sequence thereof;

(21) an antibody (22SD7) comprising the H chain of (19) and the L chain of (20);

(22) an antibody (22SD11 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 75, the amino acid sequence of CDR2 of SEQ ID NO: 76, and the amino acid sequence of SEQ ID NO: 77;

(23) an antibody (22SD11 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 80, the amino acid sequence of CDR2 of SEQ ID NO: 81 and the amino acid sequence of CDR3 as SEQ ID NO: 82;

(24) an antibody (22SD11) comprising the H chain of (22) and the L chain of (23);

(25) an antibody (22SD48 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 85, and SEQ ID NO: 86 and the amino acid sequence of CDR3 as shown in SEQ ID NO: 87;

(26) an antibody (22SD48 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 90, the amino acid sequence of CDR2 of SEQ ID NO: 91 and the amino acid sequence of CDR3 as set forth in SEQ ID NO: 92 in a sequence listing;

(27) an antibody (22SD48) comprising the H chain of (25) and the L chain of (26);

(28) an antibody having the same activity as the antibody according to any one of (1) to (27);

(29) an antibody that recognizes the same epitope as that recognized by the antibody according to any one of (1) to (27).

[10] The antibody according to any one of [1] to [9], which has a human constant region.

[11] [10] the antibody which is a chimeric antibody, a humanized antibody or a human antibody.

[12] The antibody according to any one of [1] to [11], which is a fucose-deficient antibody.

[13] A pharmaceutical composition comprising the antibody according to any one of [1] to [12] as an active ingredient.

[14] A cell growth inhibitor comprising the antibody according to any one of [1] to [12] as an active ingredient.

[15] An anticancer agent comprising the antibody according to any one of [1] to [12] as an active ingredient.

[16] [15] the anticancer agent according to any of the above aspects, wherein the cancer to be treated is ovarian cancer.

[17] [16] the anticancer agent according to any of the above aspects, wherein the ovarian cancer is clear cell adenocarcinoma.

[18] A method of diagnosing cancer, comprising: detecting the LGR7 protein or a gene encoding the LGR7 protein.

[19] A method of diagnosing cancer, comprising: LGR7 protein was detected.

[20] [19] the diagnostic method according to any one of the above methods, wherein the LGR7 protein is detected using an antibody that binds to LGR7 protein.

[21] A method of diagnosing cancer, the method comprising the steps of:

(a) a step of providing a sample collected from a subject;

(b) a step of detecting LGR7 protein contained in the sample of (a) using an antibody that binds to LGR7 protein.

[22] A method of diagnosing cancer, the method comprising the steps of:

(a) a step of administering an antibody having a binding activity to LGR7 protein and labeled with a radioisotope to a subject;

(b) and detecting the accumulation of the radioisotope.

[23] The diagnostic method according to any one of [18] to [22], wherein the cancer to be diagnosed is ovarian cancer.

[24] [23] the diagnostic method according to any one of the above methods, wherein the ovarian cancer is clear cell adenocarcinoma.

Drawings

FIG. 1-1 is a graph showing the expression profile of LGR7(1552715_ a _ at) in U133 Plus2.0 expression data of 10 histological details of ovarian cancer, namely 4 clear cell carcinomas, 2 serous adenocarcinomas, 3 intimal adenocarcinomas, 1 carcinosarcoma, 10 normal tissues, 4 fetal tissues, 4 ovarian cancer cell lines, and 87 ovarian cancers including 3 clear cell carcinomas as disclosed by International Genomics Consortium (IGC). The symbols described in the sample name of IGC represent the following meanings. C: clear cell adenocarcinoma; e: intima-like gonadal cancer; s: serous adenocarcinoma; m: mucinous adenocarcinoma; o: other cancers; b: benign serous cystadenoma (Benign serous cystadenoma of Borderline malignance). JHOC-5, MCAS, RMG-1 and RMUG-S, TKY-nu are ovarian cancer cell strains, wherein RMG-1 is a cell strain derived from clear cell adenocarcinoma.

Fig. 1-2 is a diagram showing an expression profile of LGR7(1552715_ a _ at).

Fig. 1 to 3 are diagrams showing an expression profile of LGR7(1552715_ a _ at).

FIG. 2 is a photograph showing the results of subjecting cell lysates to SDS-PAGE, and detecting the expression of LGR7 in HA-LGR7/BaF3#48 and HA-LGR7/DG44#24 by WB using an anti-HA antibody (HA-7). Lanes are as follows, lane 1: BaF 3; lane 2: HA-LGR7/BaF3# 48; lane 3: DG 44; lane 4: HA-LGR7/DG44# 24.

FIG. 3 is a graph showing the results of measuring ADCC activity by Cr release using HA-LGR7/DG44 as a target cell and NK92mFcR3 as an effector cell.

FIG. 4 is a graph showing the results of measurement of complement-dependent cytotoxic activity by simultaneously reacting an anti-LGR 7 antibody with baby rabbit complement using HA-LGR7/DG44 as a target cell. The ratio of cells having 7-AAD incorporated into cells on which each antibody acts was regarded as the intensity of CDC activity.

FIG. 5 is a graph showing the results of determining the number of living cells by WST8 analysis after allowing Mab-ZAP (Saprin-labeled anti-mouse antibody) to act on each monoclonal antibody using HA-LGR7/DG44 as a target cell. The higher the value of the vertical axis, the higher the number of living cells.

FIG. 6 is a graph showing that 22DA17, 22DA23 have crossability with mouse LGR7, based on FACS reactivity relative to HA-mLGR7/BaF 3. The vertical axis represents the value of GeoMean.

FIG. 7 is a graph showing the results of competitive FACS analysis using biotinylated antibodies Bio-22DA17, Bio-22DA 22. Whether a non-labeled antibody is an antibody recognizing a different epitope is analyzed according to whether it competes for the epitope. It was confirmed that 22DA12 and 22DA22 are antibodies recognizing epitopes different from those of other antibodies. The solid line shows the effect of biotinylated antibody, and the shaded peaks show the effect of reacting FITC-labeled anti-mouse antibody.

Fig. 8 shows the results of the efficacy test using the mouse xenograft model. Changes in tumor volume caused by antibody administration were plotted against days post tumor implantation. The solid line shows the results of the negative control group administered with PBS, the dotted square line shows the results of the group administered with 10mg/kg of antibody FTKODA23, and the dotted triangular line shows the results of the group administered with 2mg/kg of antibody FTKODA 23.

Detailed Description

LGR7

In the present invention, LGR7 is a protein of an LGR family member of the seven-transmembrane type. The amino acid sequence of human LGR7 and the gene sequence encoding the sequence are disclosed in NCBI accession Nos. NP-067647 (SEQ ID NO: 1) and NM-021634 (SEQ ID NO: 2), respectively. LGR7 used in the present invention may be a splice variant or mutant. In the present invention, the meaning of LGR7 protein includes both full-length protein and fragments thereof. A fragment refers to a polypeptide containing any region of LGR7 protein, which may not have the function of the native LGR7 protein. Examples of fragments are: a fragment comprising the extracellular region of LGR7 protein. The extracellular region of LGR7 protein is represented in SEQ ID NO: 1 corresponds to the 1-404 th, 462-485 th, 549-581 th and 648-661 th amino acid sequences. The transmembrane region is shown in SEQ ID NO: the amino acid sequence of 1 corresponds to the 405-427 th, 439-461 th, 486-508 th, 529-548 th, 582-604 th, 625-647 th and 662-681 th positions.

Production of anti-LGR 7 antibody

The anti-LGR 7 antibody used in the present invention is not limited in its origin, kind, shape, etc. as long as it binds to LGR7 protein. Specifically, known antibodies such as non-human animal antibodies (e.g., mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used. In the present invention, a monoclonal antibody or a polyclonal antibody can be used as the antibody, but a monoclonal antibody is preferable. The binding of the antibody to LGR7 protein is preferably specific. In addition, when the anti-LGR 7 antibody used in the present invention is an antibody recognizing human LGR7, it may be an antibody specifically recognizing human LGR7, or an antibody recognizing LGR7 (e.g., mouse LGR7) derived from another animal at the same time.

The anti-LGR 7 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody by a known method. The anti-LGR 7 antibody used in the present invention is particularly preferably a monoclonal antibody derived from a mammal. Monoclonal antibodies from mammals include: monoclonal antibodies produced by the hybridoma; and monoclonal antibodies produced by a host transformed with an expression vector containing an antibody gene by a genetic engineering method.

Basically, hybridomas producing monoclonal antibodies can be prepared as follows by using known techniques. First, LGR7 protein was used as a sensitizing antigen, which was immunized according to a conventional immunization method. The hybridoma is obtained by fusing an immune cell obtained from an immunized animal with a known parent cell according to a conventional cell fusion method. Then, a hybridoma producing an anti-LGR 7 antibody can be selected by screening cells producing the target antibody from the hybridoma by a usual screening method.

Specifically, the monoclonal antibody can be produced, for example, as follows. First, by expressing LGR7 gene, LGR7 protein serving as a sensitizing antigen for obtaining antibodies can be obtained. The nucleotide sequence of the LGR7 gene is disclosed in NCBI accession No. NM-021634 (SEQ ID NO: 2) and the like. That is, a gene sequence encoding LGR7 is inserted into a known expression vector, an appropriate host cell is transformed, and then the target human LGR7 protein can be purified from the host cell or culture supernatant according to a known method. Purified native LGR7 protein may also be used as such. In addition, as used in the present invention, a fusion protein in which a desired partial polypeptide of the LGR7 protein is fused with a different polypeptide can also be used as an immunogen. For producing a fusion protein as an immunogen, for example, an Fc fragment of an antibody, a peptide tag, or the like can be used. The vector for expressing the fusion protein can be prepared as follows: the fusion gene is prepared by inserting a desired gene encoding two or more polypeptide fragments into an expression vector by in-frame fusion. Methods for producing fusion proteins are described in Molecular Cloning 2nd ed. (Sambrook, J et al, Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1989).

The LGR7 protein thus purified can be used as a sensitizing antigen for immunization of mammals. Partial peptides of LGR7 may also be used as sensitizing antigens. For example, the following peptides can be used as the sensitizing antigen.

A peptide obtained by chemical synthesis based on the amino acid sequence of human LGR 7;

a peptide obtained by inserting a part of LGR7 gene into an expression vector and expressing the same;

a peptide obtained by decomposing LGR7 protein with a protease.

There is no limitation on the region and size of LGR7 used as a partial peptide. Preferred regions may be selected from the amino acid sequences constituting the extracellular domain of LGR7 (positions 1-404, 462-485, 549-581, and 648-661 in the amino acid sequence of SEQ ID NO: 1). The number of amino acids constituting the peptide as the sensitizing antigen is preferably at least 3 or more, for example, 5 or more or 6 or more. More specifically, a peptide having 8 to 50, preferably 10 to 30 residues can be used as the sensitizing antigen.

The mammal immunized with the sensitizing antigen is not particularly limited. In order to obtain a monoclonal antibody by the cell fusion method, it is preferable to select an immunized animal in consideration of suitability to a parent cell for cell fusion. Usually, rodents are preferred as the immunized animals. Specifically, a mouse, rat, hamster, or rabbit may be used as the immunized animal. Furthermore, monkeys and the like can be used as the immunized animals.

The animal can be immunized with the sensitizing antigen according to a known method. For example, the conventional methods are: immunization of mammals can be carried out by intraperitoneal or subcutaneous injection of a sensitizing antigen. Specifically, the sensitizing antigen is administered to the mammal a plurality of times every 4 to 21 days. The sensitizing antigen is diluted with PBS (phosphate buffered saline) or physiological saline at an appropriate dilution ratio and used for immunization. Also, the sensitizing antigen can be administered with an adjuvant. For example, the sensitizing antigen can be prepared by mixing and emulsifying the sensitizing antigen with Freund's complete adjuvant. In addition, an appropriate carrier can be used for immunization with a sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as the sensitizing antigen, it is preferable to immunize the antigen by binding to a carrier protein such as albumin or keyhole limpet hemocyanin (keyhole limpet hemocyanin).

On the other hand, monoclonal antibodies can also be obtained by DNA Immunization (DNA Immunization). DNA immunization refers to a method of imparting immunostimulation by administering a vector DNA constructed in such a manner that a gene encoding an antigen protein (for example, SEQ ID NO: 2) can be expressed in an immunized animal to the immunized animal, and allowing the immune antigen to be expressed in the immunized animal. The following advantages are expected to be obtained in DNA immunization as compared with the ordinary immunization method of administering a protein antigen.

Can maintain the structure of such a membrane protein as LGR7 and confer immunostimulation

Without purification of the immunizing antigen

On the other hand, it is difficult to combine DNA immunization with an immunostimulating means such as an adjuvant. Since LGR7 has such a structural feature as a seven-fold transmembrane type stereostructure, it is expected to be difficult to elicit an immune response while maintaining a naturally occurring structure in vivo. It was an unexpected result that a monoclonal antibody that binds to the protein LGR7 belonging to the LGR family, for which it is difficult to obtain an antibody due to such structural characteristics, was actually obtained by DNA immunization.

When the monoclonal antibody of the present invention is obtained by DNA immunization, first, a DNA expressing LGR7 protein is administered to the immunized animal. The DNA encoding LGR7 can be synthesized by a known method such as PCR. The resulting DNA is inserted into an appropriate expression vector, and then administered to an immunized animal. As the expression vector, for example, a commercially available expression vector such as pcDNA3.1 can be used. The method of administering the carrier to the organism may also be a method generally used. For example, DNA immunization can be performed by injecting gold particles having an expression vector adsorbed thereto into cells via a gene gun (gene gun).

After the mammal is immunized in this manner and the increase in the amount of the desired antibody in the serum is confirmed, immune cells are collected from the mammal for cell fusion. Particularly preferably, spleen cells are used as immune cells.

The cells fused with the above immune cells are mammalian myeloma cells. Preferably, the myeloma cells are provided with appropriate selection markers for selection. Selectable markers refer to the property of being viable (or not) under specific culture conditions. Among the selection markers, hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency), thymidine kinase deficiency (hereinafter abbreviated as TK deficiency), and the like are known. HGPRT-or TK-deficient cells have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells do not synthesize DNA in HAT selective medium and die, but when fused with normal cells, they can continue DNA synthesis by a salvage pathway of normal cells, and therefore can proliferate in HAT selective medium.

HGPRT-deficient or TK-deficient cells can be selected in a medium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG) or 5' -bromodeoxyuridine, respectively. The normal cells die by taking the pyrimidine analog into DNA, but the cells lacking the enzyme can survive in a selective medium because they do not take the pyrimidine analog. Furthermore, a selection marker called G418 resistance provides resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via a neomycin resistance gene. Various myeloma cells suitable for cell fusion are known. For example, the following myeloma cells can be used for producing the monoclonal antibody of the present invention.

P3(P3x63Ag8.653)(J.Immunol.(1979)123，1548-1550)

P3x63Ag8U.1(Current Topics in Microbiology and Immunology(1978)81，1-7)

NS-1(Kohler.G. and Milstein, C.Eur.J.Immunol. (1976)6, 511-519)

MPC-11(Margulies. D.H. et al, Cell (1976)8, 405-415)

SP2/0(Shulman, M. et al, Nature (1978)276, 269-270)

FO (de St.Groth, S.F. et al, J.Immunol.methods (1980)35, 1-21)

S194(Trowbridge，I.S.J.Exp.Med.(1978)148，313-323)

R210(Galfre, G. et al, Nature (1979)277, 131-

Cell fusion of immune cells with myeloma cells can be carried out basically according to known Methods, for example, the method of Kohler and Milstein et al (Kohler. G. and Milstein, C., Methods Enzymol. (1981)73, 3-46), etc.

More specifically, for example, cell fusion can be carried out in the presence of a cell fusion promoter in a normal nutrient medium. For example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like can be used as the fusion promoter. In order to further improve the fusion efficiency, an auxiliary agent such as dimethyl sulfoxide may be added as necessary.

The ratio of the immune cells to the myeloma cells can be arbitrarily set. For example, it is preferable that the immune cells are 1 to 10 times as many as myeloma cells. The culture solution for cell fusion can be, for example: RPMI1640 culture medium and MEM culture medium suitable for proliferation of myeloma cell line, and conventional culture medium for culturing the cells. Further, a serum replacement fluid such as Fetal Calf Serum (FCS) may be added to the culture fluid.

Cell fusion can be formed as follows: a predetermined amount of immune cells and myeloma cells are mixed well in a culture solution, and a PEG solution heated to about 37 ℃ in advance is further mixed to form fused cells (hybridomas) of interest. In the cell fusion method, PEG having an average molecular weight of about 1000 to 6000, for example, can be added at a concentration of 30 to 60% (w/v). Then, the appropriate culture medium as exemplified above is added in order, centrifuged to remove the supernatant, and this operation is repeated to remove the cell fusion agent and the like which are not favorable for the growth of the hybridoma.

The hybridoma obtained in this manner can be selected by using a selection medium corresponding to a selection marker possessed by the hybridoma to be used for cell fusion. For example, cells deficient in HGPRT or TK can be selected by culturing in HAT medium (medium containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have succeeded in cell fusion with normal cells can selectively proliferate in HAT culture medium. The culture is continued for a sufficient time using the HAT culture solution in order to cause the death of cells (non-fused cells) other than the target hybridoma. Specifically, the target hybridoma can be selected, usually by culturing for several days to several weeks. Then, screening of hybridomas producing the target antibody and single cloning can be performed by performing a conventional limiting dilution method. Alternatively, an antibody recognizing LGR7 may also be produced according to the method described in international publication WO 03/104453.

The screening of the target antibody and the single clone can be preferably carried out according to a screening method based on a known antigen-antibody reaction. For example, the antigen is bound to beads made of polystyrene or the like or a carrier such as a commercially available 96-well microtiter plate, and then reacted with a culture supernatant of hybridoma. The carrier is then washed and then reacted with a secondary antibody or the like labeled with an enzyme. When the culture supernatant contains the target antibody that reacts with the sensitizing antigen, the secondary antibody binds to the carrier via the antibody. Finally, by detecting the secondary antibody bound to the carrier, the presence or absence of the antibody of interest in the culture supernatant can be determined. Hybridomas producing desired antibodies capable of binding to antigens can be cloned by limiting dilution or the like. In this case, not only the antigen used for immunization but also LGR7 protein having substantially the same property can be suitably used as the antigen. For example, a cell strain expressing LGR7, an extracellular domain of LGR7, or an oligopeptide comprising a partial amino acid sequence constituting the region may be used as an antigen.

In addition to the method of obtaining the above-mentioned hybridoma by immunizing an animal other than a human with an antigen, the target antibody can be obtained by antigen-sensitizing human lymphocytes. Specifically, first, human lymphocytes were sensitized with LGR7 protein in vitro. The immunosensitized lymphocytes are then fused with the appropriate fusion partner. As the fusion partner, for example, a human-derived myeloma cell having a permanent division ability can be used (see Japanese patent publication No. Hei 1-59878). The anti-LGR 7 antibody obtained by this method is a human antibody having a binding activity to LGR7 protein.

Also, an anti-LGR 7 human antibody can be obtained by administering LGR7 protein as an antigen to a transgenic animal having all the components of a fully human antibody gene, or by immunizing with a DNA constructed in such a manner that LGR7 is expressed in the animal. Antibody-producing cells of an immunized animal can be immortalized by cell fusion with an appropriate fusion partner or by treatment such as EB virus infection. A human antibody against LGR7 protein can be isolated from the immortalized cells obtained by such procedures (see International publications WO 94/25585, WO93/12227, WO 92/03918 and WO 94/02602). Furthermore, by cloning immortalized cells, cells producing antibodies specific to the desired reaction can also be cloned. When a transgenic animal is used as an immunized animal, the animal's immune system recognizes human LGR7 as a foreign body. Therefore, a human antibody against human LGR7 can be easily obtained.

The monoclonal antibody-producing hybridoma thus prepared can be subcultured in a conventional culture medium. The hybridomas can also be stored in liquid nitrogen for long periods of time.

The hybridoma is cultured according to a conventional method, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma may be administered to a mammal having a suitability for the hybridoma to proliferate the hybridoma, and the monoclonal antibody may be obtained as ascites thereof. The former method is suitable for obtaining an antibody of high purity.

In the present invention, an antibody encoded by an antibody gene cloned from an antibody-producing cell can also be used. The cloned antibody gene is inserted into an appropriate vector and then introduced into a host, so that it can be expressed in the form of an antibody. Methods for the isolation of antibody genes, the introduction of vectors, and the transformation of host cells have been established (see, e.g., Vandamm, A.M. et al, Eur.J.biochem. (1990)192, 767-775).

For example, a cDNA encoding the variable region (V region) of an anti-LGR 7 antibody can be obtained from a hybridoma cell producing the anti-LGR 7 antibody. Thus, total RNA is typically first extracted from the hybridoma. As a method for extracting mRNA from cells, for example, the following method can be employed.

Guanidine ultracentrifugation (Chirgwin, J.M. et al, Biochemistry (1979)18, 5294-5299); the AGPC method (Chomczynski, P. et al., anal. biochem. (1987)162, 156-159).

The extracted mRNA can be purified by using an mRNA purification kit (manufactured by GE healthcare biosciences) or the like. Alternatively, a kit for directly extracting total mRNA from cells is also commercially available, such as a QuickPrep mRNA purification kit (manufactured by GE Healthcare Biosciences). Using this kit, total mRNA can also be obtained from hybridomas. A cDNA encoding the V region of the antibody can be synthesized from the resulting mRNA using reverse transcriptase. An arbitrary 15 to 30-base sequence selected from sequences common to mouse antibody genes can be used as a primer. Specifically, by using a peptide having SEQ ID NO: 97 to 100 to obtain cDNA encoding the V region of the antibody. The cDNA can be synthesized by using AMV reverse transcriptase First Strand cDNA Synthesis Kit (AMV reverse transcriptase First-Strand cDNA Synthesis Kit) (manufactured by Biochemical industries, Ltd.). For the synthesis and amplification of cDNA, 5 '-Ampli FINDER kit (manufactured by Clontech) and 5' -RACE method using PCR (Frohman, M.A. et al, Proc. Natl.acid.Sci.USA (1988)85, 8998-9002, Belyavsky, A. et al, Nucleic Acids Res (1989)17, 2919-2932) can be used. In the above-mentioned cDNA synthesis process, appropriate restriction enzyme sites described below may be introduced into both ends of the cDNA.

The target cDNA fragment was purified from the resulting PCR product, followed by ligation with vector DNA. After preparing a recombinant vector as described above, introducing it into E.coli or the like, and selecting a colony, a desired recombinant vector can be prepared from the E.coli which forms the colony. The nucleotide sequence of the cDNA can be confirmed by a known method, for example, the dideoxynucleotide chain termination method.

In addition, in order to obtain a gene encoding an antibody variable region, a cDNA library can be used. First, a cDNA library is obtained by synthesizing cDNA using mRNA extracted from antibody-producing cells as a template. In the synthesis of cDNA libraries, it is convenient to use commercially available kits. In fact, since only a small number of cells produce a very small amount of mRNA, direct purification of mRNA results in a low yield. Therefore, usually, vector RNA which is clearly free of antibody gene is added and then purified. Alternatively, when a certain amount of RNA can be extracted, even only RNA from antibody-producing cells can be extracted efficiently. For example, when RNA is extracted from antibody-producing cells of 10 or more, or 30 or more, preferably 50 or more, it may not be necessary to add carrier RNA.

The resulting cDNA library was used as a template, and the antibody gene was amplified by PCR. Primers for amplifying antibody genes by the PCR method are well known. For example, primers for amplifying human antibody genes can be designed according to the disclosure of the article (J.mol.biol. (1991)222, 581-597) and the like. These primers form different nucleotide sequences in each immunoglobulin subclass. Therefore, when a cDNA library of an unknown subclass is used as a template, the PCR method is performed in consideration of all the possibilities.

Specifically, for example, in order to obtain a gene encoding human IgG, primers capable of amplifying genes encoding γ 1 to γ 5 as heavy chains, and κ chain and λ chain as light chains can be used. For amplifying the variable region gene of IgG, a primer annealing to a portion corresponding to the hinge region is generally used as the 3' -side primer. In addition, primers corresponding to each subclass can be used as primers on the 5' side.

PCR products obtained from the primers for gene amplification of each subclass of heavy and light chains were made into independent libraries. Using the library so synthesized, immunoglobulins comprising a combination of heavy and light chains can be reconstituted. The antibody of interest can be screened using the binding activity of the reconstituted immunoglobulin to LGR7 as an index.

For example, in order to obtain an anti-LGR 7 antibody, it is further preferable that the binding of the antibody to LGR7 is specific. Antibodies that bind to LGR7 can be screened, for example, as follows.

(1) A step of contacting an antibody containing a V region encoded by the resulting cDNA with LGR 7;

(2) a step of detecting the binding of LGR7 to the antibody; and

(3) a step of selecting an antibody that binds to LGR7.

Methods for detecting binding of antibodies to LGR7 are well known. Specifically, the test antibody was reacted with LGR7 immobilized on a carrier, and then reacted with a labeled antibody that recognizes the antibody. When the labeled antibody on the carrier is detected after washing, it can be confirmed that the test antibody binds to LGR7. For labeling, enzymatically active proteins such as peroxidase and β -galactosidase, or fluorescent substances such as FITC can be used. To assess the binding activity of the antibodies, fixed samples of LGR7 expressing cells may also be used.

As a method for screening an antibody using a binding activity as an index, a panning method using a phage vector can be used. When antibody genes are obtained as a library of subclasses of heavy and light chains in the above manner, a screening method using a phage vector is advantageous. The genes encoding the variable regions of the heavy and light chains may form a single chain fv (scFv) by ligation with appropriate linker sequences. When a gene encoding scFv is inserted into a phage vector, a phage expressing scFv on the surface can be obtained. When the phage is contacted with a target antigen and the phage bound to the antigen is recovered, DNA encoding scFv having a target binding activity can be recovered. This procedure is repeated as necessary to concentrate scFv having a target binding activity.

In the present invention, the polynucleotide encoding the antibody may encode the entire length of the antibody, or may encode a part of the antibody. A portion of an antibody refers to any portion of an antibody molecule. Hereinafter, an antibody fragment may be used as a term indicating a part of an antibody. Preferred antibody fragments of the invention include the complementary Chain Determining Regions (CDRs) of an antibody. It is further preferred that the antibody fragment of the invention comprises all 3 CDRs which make up the variable region.

After obtaining a cDNA encoding the V region of the target anti-LGR 7 antibody, the cDNA was digested with restriction enzymes that recognize restriction enzyme sites inserted into both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that are less likely to occur in the nucleotide sequences constituting the antibody genes. To further insert a single copy of the digested fragment into the vector in the correct orientation, it is preferred to provide a restriction enzyme with a sticky end. An antibody expression vector can be obtained by inserting the above-described digested cDNA encoding the V region of the anti-LGR 7 antibody into an appropriate expression vector. In this case, a chimeric antibody can be obtained by in-frame fusion of a gene encoding the constant region (C region) of an antibody and a gene encoding the above V region. A chimeric antibody herein means that the constant region and the variable region are derived from different sources. Therefore, in addition to mouse-human and the like heterochimeric antibodies, human-human homochimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can also be constructed by previously inserting the above V region gene into an expression vector into which a DNA encoding a constant region is integrated.

Specifically, for example, a restriction enzyme recognition sequence of a restriction enzyme digesting a V region gene may be prepared in advance on the 5' side of an expression vector holding a DNA encoding a constant region (C region) of a desired antibody. The two were digested with the same combination of restriction enzymes and subjected to in-frame fusion to construct a chimeric antibody expression vector.

To prepare the anti-LGR 7 antibody for use in the present invention, the antibody gene may be inserted into an expression vector to be expressed under the control of an expression control region. Expression control regions for expressing antibodies include, for example, enhancers and promoters. Subsequently, an appropriate host cell is transformed with the expression vector, whereby a recombinant cell expressing a DNA encoding an anti-LGR 7 antibody can be obtained.

When expressing the antibody gene, DNAs encoding the heavy chain (H chain) and light chain (L chain) of the antibody may be inserted into another expression vector, respectively. The vector having the H chain and the L chain inserted therein is simultaneously transformed (co-transfected) into the same host cell, so that the antibody molecule having the H chain and the L chain can be expressed. Alternatively, the host cell may be transformed by inserting DNA encoding the H chain and the L chain into a single expression vector (see International publication WO 94/11523).

Various combinations of hosts and expression vectors for introducing isolated antibody genes into appropriate hosts to produce antibodies are known. These expression systems are all applicable to the present invention. When eukaryotic cells are used as the host, animal cells, plant cells or fungal cells can be used. Specifically, the animal cells usable in the present invention include the following:

(1) mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, HEK293, Ba/F3, HL-60, Jurkat, SK-HEP 1, etc.;

(2) an amphibian cell: xenopus laevis egg cells, and the like; and

(3) insect cells: sf9, sf21, Tn5, and the like.

Alternatively, as plant cells, expression systems of antibody genes produced by cells of Nicotiana (Nicotiana) such as Nicotiana tabacum are known. In transformation of plant cells, cells cultured from callus may be used.

Further, as the fungal cell, the following cells can be used.

Yeast: genus Saccharomyces such as Saccharomyces cerevisiae and genus Pichia such as methylotrophic yeast (Pichia pastoris);

filamentous fungi: aspergillus (Aspergillus) such as Aspergillus niger.

Alternatively, expression systems using antibody genes from prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E.coli) and Bacillus subtilis can be used in the present invention.

When mammalian cells are used, the polyA signal can be expressed by functionally binding to a commonly used promoter, an antibody gene to be expressed, and the 3' -side downstream thereof. For example, the promoter/enhancer may be the human cytomegalovirus early promoter/enhancer.

In addition, a promoter/enhancer derived from a mammalian cell such as a viral promoter/enhancer or human elongation factor 1 α (HEF1 α) can be used for antibody expression. Examples of viruses that can use promoters and enhancers include: retroviruses, polyoma viruses, adenoviruses, simian virus 40(SV40), and the like.

When the SV40 promoter/enhancer is used, the method of Mulligan et al (Nature (1979)277, 108) can be used. In addition, the HEF1 α promoter/enhancer can be readily used for expression of a gene of interest using the method of Mizushima et al (nucleic acids Res. (1990)18, 5322).

In the case of E.coli, the gene can be expressed by functionally combining a commonly used promoter, a signal sequence for antibody secretion, and the antibody gene to be expressed. Examples of the promoter include lacZ promoter and araB promoter. When the lacZ promoter is used, the method of Ward et al (Nature (1989)341, 544 to 546; FASEBJ. (1992)6, 2422 to 2427) can be employed. Alternatively, the araB promoter can be used for expression of a target gene by the method of Better et al (Science (1988)240, 1041 to 1043).

When the signal sequence for antibody secretion is produced in the periplasm of E.coli, the pel B signal sequence may be used (Lei, S.P. et al, J.Bacteriol. (1987)169, 4379). Next, the antibody produced in the periplasm is isolated, and then the structure of the antibody can be refolded to have a desired binding activity by using a protein modifier such as guanidine hydrochloride of urea.

When an antibody is produced using an animal cell, it is preferable to use a signal sequence of a heavy chain gene or a light chain gene of the antibody as a signal sequence necessary for secretion to the outside of the cell. Furthermore, a signal sequence possessed by a secretory protein such as IL-3 or IL-6 can be used.

As the origin of replication to be inserted into the expression vector, those derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV) and the like can be used. Furthermore, in order to amplify the gene copy number in the host cell system, a selection marker may be inserted into the expression vector. Specifically, the following selection markers can be used:

an Aminoglycoside Phosphotransferase (APH) gene;

a Thymidine Kinase (TK) gene;

e.coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene;

dihydrofolate reductase (dhfr) gene, and the like.

These expression vectors are introduced into host cells, and then the transformed host cells are cultured in vitro or in vivo to produce the target antibody. The host cell can be cultured according to a known method. For example, DMEM, MEM, RPMI1640, IMDM may be used as the culture medium, and a serum replacement solution such as Fetal Calf Serum (FCS) may be used in combination.

The antibody expressed and produced as described above can be purified by using known methods used in conventional protein purification, either alone or in an appropriate combination. For example, Antibodies can be isolated and purified by appropriately selecting and combining affinity columns such as protein A columns, chromatography columns, filters, ultrafiltration, salting out, dialysis, and the like (Antibodies A Laboratory Manual. Ed Harbor, David Lane, Cold Spring Harbor Laboratory, 1988).

In addition, in the production of recombinant antibodies, transgenic animals may be used in addition to the above-described host cells. That is, a gene encoding an antibody of interest is introduced into an animal, and the antibody can be obtained from the animal. For example, fusion genes can be constructed by inserting antibody genes in-frame into genes encoding proteins produced by traits in milk. As the protein secreted in milk, for example, goat β casein or the like can be used. A DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat. The goat which received the embryo gives rise to a transgenic goat from which the desired antibody can be obtained as a fusion protein with a milk protein in the milk produced by the transgenic goat (or its offspring). In addition, in order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones may be suitably used for the transgenic goat (Ebert, K.M. et al, Bio/Technology (1994)12, 699-702).

The C region of the recombinant antibody of the present invention may be the C region of an antibody derived from an animal other than human. For example, the H chain C region of a mouse antibody may be C.gamma.1, C.gamma.2 a, C.gamma.2 b, C.gamma.3, C.mu.C.delta.C.alpha.1, C.alpha.2, C.epsilon.C.and the L chain C region may be C.kappa.C.lambda.C. Further, as the antibody of an animal other than a mouse, an antibody of rat, rabbit, goat, sheep, camel, monkey, or the like can be used. Their sequences are well known. The C region may be modified in order to improve the stability of the antibody or its production.

In the present invention, when the antibody is administered to a human, a genetically modified recombinant antibody can be prepared in order to reduce the antigenicity of the human. Recombinant antibodies include, for example: chimeric antibodies, humanized antibodies, and the like. These modified antibodies can be prepared by a known method.

A chimeric antibody is an antibody in which variable regions and constant regions of different origins are linked to each other. For example, an antibody comprising the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody is a mouse-human-xenochimeric antibody. A recombinant vector for expressing a chimeric antibody can be prepared by ligating a DNA encoding a variable region of a mouse antibody with a DNA encoding a constant region of a human antibody and inserting the ligated DNA into an expression vector. The chimeric antibody produced in the culture can be obtained by culturing the recombinant cell transformed with the vector and expressing the inserted DNA. The C region of a human antibody is used for the C regions of the chimeric antibody and the humanized antibody.

For example, in the H chain, C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4, C.mu.C.delta.C.alpha.1, C.alpha.2 and C.epsilon.can be used as the C region. In the L chain, C.kappa.and C.lambda.can be used as the C region. The amino acid sequence of the above-mentioned C region and the nucleotide sequence encoding the amino acid sequence are known. To improve the stability of the antibody itself or of the antibody production, the C region of the human antibody may be modified.

Generally, a chimeric antibody is composed of a V region derived from an antibody derived from an animal other than a human and a C region derived from a human antibody. In contrast, a humanized antibody is composed of Complementarity Determining Regions (CDRs) of an antibody derived from an animal other than a human, Framework Regions (FRs) derived from a human antibody, and a C region derived from a human antibody. The humanized antibody has reduced antigenicity in humans and is therefore useful as an active ingredient of the therapeutic agent of the present invention.

Antibody variable regions are typically composed of 3 Complementarity Determining Regions (CDRs) sandwiched between 4 Framework Regions (FRs). CDRs are essentially the regions that determine the binding specificity of an antibody. The amino acid sequences of the CDRs are rich in diversity. On the one hand, the amino acid sequences constituting the FRs often show high homology even among antibodies having different binding specificities. Thus, the binding specificity of an antibody can be grafted to other antibodies, usually by grafting CDRs.

Humanized antibodies are also known as reshaped (reshaped) human antibodies. Specifically, humanized antibodies and the like in which CDRs of an animal other than a human, for example, a mouse antibody are grafted onto a human antibody are known. Conventional genetic recombination methods for obtaining humanized antibodies are also known.

Specifically, as a method for grafting CDRs of a mouse antibody into human FRs, for example, overlap Extension pcr (overlap Extension pcr) is known. In the overlap sequence extension PCR, the grafted nucleotide sequence encoding the CDR of the mouse antibody is added to primers for synthesizing the FR of the human antibody. Primers for 4 FRs were prepared. In general, when a mouse CDR is grafted to a human FR, it is advantageous to select a human FR having high homology with the mouse FR in order to maintain the function of the CDR. That is, it is generally preferable to use a human FR comprising an amino acid sequence having high amino acid sequence homology with the FR adjacent to the grafted mouse CDR.

In addition, the linked nucleotide sequences are designed to be linked in frame with each other. Human FRs were synthesized using the respective primers. As a result, a product in which DNA encoding the mouse CDR was added to each FR was obtained. The nucleotide sequences encoding the mouse CDRs of each product were designed to overlap each other. Subsequently, the overlapping CDR portions of the product synthesized using the human antibody gene as a template are annealed to each other, and a complementary strand synthesis reaction is performed. By this reaction, human FRs are linked via mouse CDR sequences.

Finally, the full-length V region gene to which 3 CDRs and 4 FRs are ligated is amplified using primers that anneal to their 5 'and 3' ends and are appended with appropriate restriction enzyme recognition sequences. The DNA obtained as described above and a DNA encoding a human antibody C region are inserted into an expression vector and fused in frame, thereby preparing a human antibody expression vector. The vector is introduced into a host to create recombinant cells, and the recombinant cells are cultured to express a DNA encoding a humanized antibody, thereby producing the humanized antibody in the culture of the cultured cells (see European patent publication EP 239400 and International publication WO 96/02576).

By qualitatively or quantitatively measuring and evaluating the binding activity of the humanized antibody prepared as above to an antigen, the FR of a human antibody in which the CDR forms a good antigen binding site when linked via the CDR can be appropriately selected. If necessary, the amino acid residues of the FR may be replaced so that the CDRs of the reshaped human antibody form an appropriate antigen binding site. For example, a mutation of an amino acid sequence can be introduced into an FR by using a PCR method for grafting a mouse CDR into a human FR. Specifically, a mutation of a part of the nucleotide sequence may be introduced into a primer annealing to FR. The FR synthesized by using such a primer has a mutation in the nucleotide sequence introduced therein. By measuring and evaluating the binding activity of the mutant antibody in which the amino acid has been substituted to the antigen by the above-described method, a mutant FR sequence having desired properties can be selected (Sato, K. et al, Cancer Res, 1993, 53, 851-856).

In addition, methods for obtaining human antibodies are also known. For example, human lymphocytes are sensitized in vitro with a desired antigen or cells expressing a desired antigen. Subsequently, the sensitized lymphocytes are fused with human myeloma cells to obtain a desired human antibody having an antigen-binding activity (see Japanese examined patent publication (Kokoku) No. 1-59878). For example, U266 or the like can be used for human myeloma cells as a fusion partner.

The desired human antibody can be obtained by immunizing a transgenic animal having all the components of a fully human antibody gene with the desired antigen (see International publications WO93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735). Also, a technique for obtaining a human antibody by panning using a human antibody library is known. For example, phage display can be used to express a human antibody V region as a single chain antibody (scFv) on the surface of a phage, thereby selecting a phage that binds to an antigen. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of a human antibody that binds to the antigen can be determined. After the DNA sequence of the scFv that binds to the antigen has been determined, the V region sequence is fused in frame with the desired human antibody C region sequence, and then inserted into an appropriate expression vector, thereby preparing an expression vector. The human antibody can be obtained by introducing the expression vector into the above-exemplified preferred expression cells and expressing the gene encoding the human antibody. These methods are already known (International publications WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388).

Therefore, as one of preferable embodiments of the antibody used in the present invention, an antibody having a human constant region can be mentioned.

The antibody of the present invention may be one that binds to LGR7 protein, and includes not only bivalent antibodies represented by IgG but also monovalent antibodies or multivalent antibodies represented by IgM. Multivalent antibodies of the invention include: multivalent antibodies with identical antigen binding sites, or multivalent antibodies with a portion or completely different antigen binding sites. The antibody of the present invention is not limited to the full-length molecule of the antibody as long as it binds to LGR7 protein, and may be a low-molecular antibody or a modified product thereof.

Low molecular antibodies include antibody fragments in which a portion of a full-length antibody (e.g., full-length IgG, etc.) is deleted. So long as it has a binding ability to LGR7 antigen, partial deletion of the antibody molecule is allowed. The antibody fragment of the present invention preferably contains a heavy chain variable region (VH) and/or a light chain variable region (VL). Furthermore, the antibody fragment of the present invention preferably contains CDRs. The amino acid sequence of VH or VL may include substitutions, deletions, additions and/or insertions. Furthermore, VH and/or a part of VL may be deleted as long as they have a binding ability to LGR7 antigen. In addition, the variable region may be chimeric or humanized. Specific examples of the antibody fragment include: fab, Fab ', F (ab') 2, Fv, etc. Specific examples of the low-molecular antibody include: fab, Fab ', F (ab') 2, Fv, scFv (single chain Fv), diabody, sc (Fv)2 (single chain (Fv)2), scFv-Fc, and the like. Multimers (e.g., dimers, trimers, tetramers, multimers) of these antibodies are also included in the low molecular antibodies of the invention.

The antibody fragment can be obtained by treating an antibody with an enzyme to produce an antibody fragment. Enzymes for producing antibody fragments, such as papain, pepsin, and plasmin, are known. Alternatively, genes encoding these antibody fragments are constructed, introduced into expression vectors, and then expressed in appropriate host cells (see, e.g., Co, M.S. et al, J.Immunol. (1994)152, 2968-2976, Better, M. and Horwitz, A.H.methods in Enzymology (1989)178, 476-496, Plcuethroughout, A. and Skerra, A.methods in Enzymology (1989)178, 476-496, Lamoyi, E. Methods in Enzymology (1989)121, 652-663, Rousseaux, J. et al, Methods in Enzymology (1989)121, 663-669, Bird, R.E. et al, TIBTECH (1991)9, 137).

The digestion enzyme cleaves a specific site of the antibody fragment, and the antibody fragment of the following specific structure is administered. When the antibody fragment obtained by the above enzymatic hydrolysis is subjected to a genetic engineering method, any part of the antibody can be deleted.

And (3) papain digestion: f (ab)2 or Fab

And (3) pepsin digestion: f (ab ') 2 or Fab'

Plasmin digestion: facb

Therefore, the low molecular weight antibody of the present invention may be an antibody fragment in which any region is deleted as long as it has binding affinity to LGR7. In particular, in the treatment of cell proliferative diseases such as cancer of the present invention, it is preferable that the antibody maintains its effector activity. That is, the preferred low molecular antibodies of the present invention have both binding affinity to LGR7 and effector function. Effector functions of an antibody include ADCC activity and CDC activity. The therapeutic antibody of the present invention particularly preferably has ADCC activity and/or CDC activity as effector functions.

Diabodies (diabodies) refer to bivalent antibody fragments constructed by gene fusion (Holliger P et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP 404,097; WO 93/11161 et al). Diabodies are dimers consisting of two polypeptide chains. Typically, the polypeptide chains that make up the dimer, each VL and VH in the same chain, are joined by a linker. The linker in diabodies is typically so short that VL and VH cannot bind to each other. Specifically, the amino acid residues constituting the linker are, for example, about 5 residues. Thus, VL and VH encoded on the same polypeptide chain cannot form a single chain variable fragment, but rather form a dimer with another single chain variable fragment. As a result, the diabody has two antigen binding sites.

scFv can be obtained by linking the H chain V region and L chain V region of an antibody. In scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, J.S. et al, Proc. Natl.Acid.Sci.U.S.A.1988, 85, 5879-5883.). The H chain V region and the L chain V region in the scFv may be derived from any of the antibodies described in the present specification. The peptide linker connecting the V regions is not particularly limited. For example, any single-chain peptide containing about 3 to 25 residues can be used as a linker. Specifically, for example, a peptide linker described later can be used.

The V regions of both strands can be ligated by, for example, the PCR method described above. In order to connect V regions by the PCR method, first, a DNA encoding all or a desired partial amino acid sequence is used as a template in the following DNA.

A DNA sequence encoding an antibody H chain or H chain V region; and

a DNA sequence encoding an antibody L chain or L chain V region.

The DNAs encoding the V regions of the H chain and the L chain are amplified by PCR using a pair of primers having sequences corresponding to the sequences at both ends of the DNA to be amplified. Next, a DNA encoding the peptide linker moiety is prepared. DNA encoding a peptide linker can also be synthesized using PCR. The primer used at this time is added with a nucleotide sequence that can be ligated to the amplification product of each V region that is additionally synthesized, on the 5' side. Then, PCR reaction was carried out using each of the DNAs [ H chain V region DNA ] - [ peptide linker DNA ] - [ L chain V region DNA ] and a primer for assembly PCR.

The primer for assembly PCR comprises a combination of a primer that anneals to the 5 '-side of [ H chain V region DNA ] and a primer that anneals to the 3' -side of [ L chain V region DNA ]. That is, the assembly of PCR primers is a primer set capable of amplifying DNA encoding the full-length sequence of scFv to be synthesized. On the other hand, [ peptide linker DNA ] is added with a nucleotide sequence that can be ligated to each V region DNA. As a result, the DNAs are ligated together, and PCR primers are assembled to finally produce scFv in full length as an amplified product. DNA encoding scFv is prepared once, and an expression vector containing the DNA and a recombinant cell transformed with the expression vector can be obtained according to a conventional method. As a result, the obtained recombinant cell is cultured to express DNA encoding the scFv, thereby obtaining the scFv.

scFv-Fc is a low Molecular antibody obtained by fusing an Fc region to an scFv comprising the H chain V region and L chain V region of an antibody (Cellular & Molecular Immunology 2006; 3: 439-443). The source of scFv used for scFv-Fc is not particularly limited, and, for example, scFv derived from IgM can be used. The source of Fc is also not particularly limited, and for example, mouse IgG (e.g., mouse IgG2 a) and human IgG (e.g., human IgG 1) can be used. Accordingly, examples of preferable modes of scFv-Fc include: the scFv fragment of the IgM antibody and CH2 (e.g., C γ 2) and CH3 (e.g., C γ 3) of mouse IgG2a are linked via the hinge region (H γ) of mouse IgG2a to form scFv-Fc, or the scFv fragment of the IgM antibody and CH2 and CH3 of human IgG1 are linked via the hinge region of human IgG1 to form scFv-Fc.

sc (fv)2 is a low-molecular antibody in which two VH and two VL are connected by a linker or the like to form a single chain (Hudson et al, J Lmmonl. methods 1999; 231: 177-189). sc (fv)2 can be prepared, for example, by linking an scFv with a linker.

Preferred are antibodies having the following characteristics: the 2 VH and 2 VL are arranged in the order of VH, VL, VH and VL ([ VH ] -linker- [ VL ] -linker- [ VH ] -linker- [ VL ]) with the N-terminal side of the single-chain polypeptide as the base.

The order of the 2 VH and 2 VL is not particularly limited to the above arrangement, and may be arranged in any order. For example, the following arrangements are included:

[ VL ] -linker- [ VH ] -linker- [ VL ]

[ VH ] -linker- [ VL ] -linker- [ VH ]

[ VH ] -linker- [ VL ]

[ VL ] -linker- [ VH ]

[ VL ] -linker- [ VH ] -linker- [ VL ] -linker- [ VH ]

As the linker for linking the antibody variable region, any peptide linker that can be introduced by genetic engineering, or a chemically synthesized linker (e.g., a linker disclosed in protein engineering, 9(3), 299 to 305, 1996) can be used. Peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited, and may be appropriately selected by those skilled in the art according to the purpose. The amino acid residue constituting the peptide linker is usually 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, and particularly preferably 12 to 18 amino acids (for example, 15 amino acids).

The amino acid sequence constituting the peptide linker may be any sequence as long as it does not inhibit the binding action of scFv. For example, in the case of a peptide linker, the following amino acid sequence can be used.

Ser

Gly·Ser

Gly·Gly·Ser

Ser·Gly·Gly

Gly·Gly·Gly·Ser(SEQ ID NO：101)

Ser·Gly·Gly·Gly(SEQ ID NO：102)

Gly·Gly·Gly·Gly·Ser(SEQ ID NO：103)

Ser·Gly·Gly·Gly·Gly(SEQ ID NO：104)

Gly·Gly·Gly·Gly·Gly·Ser(SEQ ID NO：105)

Ser·Gly·Gly·Gly·Gly·Gly(SEQ ID NO：106)

Gly·Gly·Gly·Gly·Gly·Gly·Ser(SEQ ID NO：107)

Ser·Gly·Gly·Gly·Gly·Gly·Gly(SEQ ID NO：108)

(Gly·Gly·Gly·Gly·Ser(SEQ ID NO：101))n

(Ser·Gly·Gly·Gly·Gly(SEQ ID NO：102))n

[ n is an integer of 1 or more ]

Regarding the amino acid sequence of the peptide linker, those skilled in the art can appropriately select it according to the purpose. For example, n, which determines the length of the peptide linker, is usually 1 to 5, preferably 1 to 3, and more preferably 1 or 2.

Therefore, a particularly preferred embodiment of sc (fv)2 in the present invention is, for example, sc (fv)2 below.

[ VH ] -peptide linker (15 amino acids) - [ VL ] -peptide linker (15 amino acids) - [ VH ] -peptide linker (15 amino acids) - [ VL ]

Alternatively, chemically synthesized linkers (chemical crosslinkers) can also be used to link the V regions. In the present invention, a crosslinking agent generally used for crosslinking a peptide compound or the like can be used. For example, the following chemical crosslinking agents are well known. These crosslinking agents are all commercially available.

N-hydroxysuccinimide (NHS);

disuccinimidyl suberate (DSS);

bis (sulfosuccinimidyl) suberate (BS 3);

dithiobis (succinimidyl propionate) (DSP);

dithiobis (sulfosuccinimidyl propionate) (DTSSP);

ethylene glycol bis (succinimidyl succinate) (EGS);

ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS);

disuccinimidyl tartrate (DST);

disulfosuccinimidyl tartrate (sulfo-DST);

bis [2- (succinimidyloxycarbonyloxy) ethyl ] sulfone (BSOCOES); and

bis [2- (sulfosuccinimidyloxycarbonyloxy) ethyl ] sulfone (sulfo-BSOCOES), and the like.

To link 4 antibody variable regions, typically 3 linkers are required. The same or different linkers may be used for the plurality of linkers. In the present invention, the preferred low molecular weight antibody is a diabody or sc (fv) 2. In order to obtain such a low molecular weight antibody, the antibody may be treated with an enzyme such as papain or pepsin to produce an antibody fragment; alternatively, DNAs encoding these antibody fragments can be constructed, introduced into expression vectors, and then expressed in appropriate host cells (see, for example, Co, M.S. et al, J.Immunol. (1994)152, 2968-2976; Better, M. and Horwitz, A.H., Methods Enzymol. (1989)178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989)178, 497-515; Lamori, E., Methods Enzymol. (1986)121, 652-663; Rousseaux, J. et al, Methods Enzymol. (1986)121, 663-669; 137, R.E. and Bilker, B.W., Waldnol. (1991).

The antibody of the present invention includes not only monovalent antibodies but also multivalent antibodies. The multivalent antibody of the present invention includes a multivalent antibody having an identical antigen binding site, or a multivalent antibody having a portion or an entirely different antigen binding site.

As the modified antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) can be used. Furthermore, the antibody may be bound to a cytotoxic substance such as a chemotherapeutic drug, a toxic peptide, or a radioactive chemical. The antibody modified product (hereinafter referred to as an antibody conjugate) can be obtained by chemically modifying the obtained antibody. It is noted that methods for modifying antibodies have been established in the art. As described later, it can also be obtained in the form of a molecule such as a bispecific antibody (bispecific antibody) designed by gene recombination technology to recognize not only LGR7 protein but also cytotoxic substances such as chemotherapeutic drugs, toxic peptides, radioactive chemical substances, and the like. The "antibody" in the present invention also includes these antibodies.

Examples of the chemotherapeutic agent that binds to the anti-LGR 7 antibody and exerts cytotoxic activity include the following chemotherapeutic agents: azalipine, anastrozole, azacytidine, bleomycin, bortezomib (bortezomib), bryostatin-1, busulfan, camptothecin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin, irinotecan, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, actinomycin D, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin glucuronide, epirubicin, ethisterol, estramustine, etoposide glucuronide, floxuridine, fludarabine, flutamide, fluorouracil, flumethasterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, folinic acid, lomustine, mecgestone acetate, medroxyprogesterone acetate, Melphalan, mercaptopurine, methotrexate, mitoxantrone, plicamycin, mitomycin, mitotane, phenylbutyrate, prednisone, procarbazine, paclitaxel, pentostatin, semustine, streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uramustine, vinblastine, vinorelbine, vincristine.

In the present invention, the preferred chemotherapeutic agent is a low molecular chemotherapeutic agent. After the low-molecular chemotherapeutic drug is combined with the antibody, the possibility of interfering the function of the antibody is small. In the present invention, the low-molecular chemotherapeutic agent generally has a molecular weight of 100 to 2000, preferably 200 to 1000. The chemotherapeutic agents exemplified herein are all low molecular chemotherapeutic agents. These chemotherapeutic agents in the present invention include prodrugs that are converted in vivo to the active chemotherapeutic agent. Prodrug activation may be either enzymatic or non-enzymatic.

In addition, antibodies can be modified with toxic peptides. Examples of toxic peptides are the following: diphtheria toxin A chain (Langon J.J., et al, Methods in Enzymology, 93, 307, 308, 1983), Pseudomonas aeruginosa exotoxin (Nature Medicine, 2, 350, 353, 1996), ricin A chain (Fulton R.J., et al, J.biol.Chem., 261, 5314. sub.5319, 1986; SivamG., et al, Cancer Res., 47, 3169, 3173, 1987; Cumber A.J., et al, J.Immunol.methods, 135, 15-24, 1990; Wawrynczak E.J., et al, Cancer Res., 50, 7519, 7562, 1990; Ghee V., et al, J.munol.223, 142, Met 230, 1991), ricin A chain (Warne J.52, 19819, 7519, 1990), ricin A.31, 1987; Warne J.E.31, 1987; Warne. sub.31, W.23, 1987, W.E.J.J.23, 1983; Warwyzynczak. sub.E.J., K., 1983; Warne. sub.23, W.E.E.J.J.J.J.J.E.J.J.J.E.J.J., 1983, W.E.J.E.J., K, 1983, K, et al, W.E.S. K, 1987; thorpe P.E., et al, Cancer Res., 47, 5924-; cumber a.j. et al, j.immunol.methods, 135, 15-24, 1990; wawrzynczak e.j., et al, cancer res, 50, 7519-; blognesi a., et al, clin.exp.immunol., 89, 341- & 346, 1992), Pokeweed Antiviral Proteins (PAPs) (blognesi a., et al, clin.exp.immunol., 89, 341- & 346, 1992), brillidin (blognesi a., et al, clin.exp.immunol., 89, 341- & 346, 1992), saporin (blognesi a., et al, clin.exp.immunol., 89, 341- & 346, 1992), momordica charantia (momordin) (Cumber a.j., j.immunol.immunol.methods, 135, 15-24, 1990; wawrzynczak e.j., et al, Cancer res, 50, 7519-; borognesi A., et al, Clin. exp. Immunol., 89, 341-346, 1992), Momordica poison protein (Borognesi A., et al, Clin. exp. Immunol., 89, 341-346, 1992), dianilin 32 (Borognesi A., et al, Clin. exp. Immunol., 89, 341-346, 1992), dianilin 30(Stirpe F., Barbieri L., FEBSleeter 195, 1-8, 1986), Polynella poison protein II (Stirpe F., Barbieri L., BS FEBS letters 195, 1-8, 1986), mistletoe poison protein (Stirbieri F., FEBS leiter L., FEBS leiter 195, 1-8, 1986), Polynelle poison protein (Stirspe F., FES 1-195, FES 2, FES 1-8, FES.195), wheat mistletoe poison protein (FEI, FES.195, FES.E.F., FES.195, FES.E.E., FES.1-8, FES.E.K.195, FES.E.E.E.E.E., FES.K.K.K, FES.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K., FEBS letter195, 1-8, 1986), trichosanthen (Casellas P., et al, Eur. J. biochem.176, 581-588, 1988; bolognesi a, et al, clin. exp. immunol., 89, 341- & 346, 1992).

In the present invention, radioactive chemical means chemical containing radioactive isotope. The radioisotope is not particularly limited, and any radioisotope can be used, for example, radioisotope can be used³²P、¹⁴C、¹²⁵I、³H、¹³¹I、¹⁸⁶Re、¹⁸⁸Re, and the like.

In another embodiment, one or more low molecular weight chemotherapeutic agents may be used to modify the antibody in combination with the toxic peptide. The anti-LGR 7 antibody may be conjugated to the low molecular weight chemotherapeutic agent described above using covalent or non-covalent bonds. Methods for producing antibodies to which such chemotherapeutic drugs are bound are known.

Also, the protein drug or toxin can be bound to the antibody by genetic engineering methods. Specifically, for example, a recombinant vector can be constructed by fusing a DNA encoding the toxic peptide described above in frame with a DNA encoding an anti-LGR 7 antibody and inserting the fused DNA into an expression vector. The anti-LGR 7 antibody that binds to a toxic peptide can be obtained as a fusion protein by introducing the vector into an appropriate host cell, and then culturing the resulting transformed cell to express the recombinant DNA. In order to obtain a fusion protein with an antibody, a protein drug or toxin is usually placed on the C-terminal side of the antibody. Peptide linkers may also be interposed between the antibody and the proteinaceous drug or toxin.

Also, the antibody of the present invention may be a bispecific antibody (bispecific antibody). Bispecific antibodies are antibodies that have variable regions that recognize different epitopes within the same antibody molecule. In the present invention, bispecific antibodies may have antigen binding sites that recognize different epitopes on LGR7 molecules. Such bispecific antibodies can bind to 2 molecules of the antibody molecule relative to 1 molecule of LGR7. As a result, a more potent cytotoxic effect is expected.

Alternatively, bispecific antibodies can also be formed in which one antigen binding site recognizes LGR7 and the other antigen binding site recognizes a cytotoxic agent. The cytotoxic substance specifically includes a chemotherapeutic drug, a toxic peptide, a radioactive chemical substance, or the like. Such bispecific antibodies bind to LGR 7-expressing cells while capturing cytotoxic substances. As a result, the cytotoxic substance can be allowed to act directly on the LGR 7-expressing cells. That is, the bispecific antibody recognizing a cytotoxic substance can specifically kill tumor cells and inhibit the proliferation of tumor cells.

In the present invention, bispecific antibodies recognizing antigens other than LGR7 may also be combined. For example, bispecific antibodies recognizing a non-LGR 7 antigen, which is specifically expressed on the cell surface of cancer cells as a target as LGR7, can be combined.

Methods for making bispecific antibodies are well known. For example, a bispecific antibody can be prepared by binding two antibodies recognizing different antigens. The antibody to be bound may be 1/2 molecules each having an H chain and an L chain, or 1/4 molecules containing only an H chain. Alternatively, a fusion cell producing a bispecific antibody can be prepared by fusing hybridomas producing different monoclonal antibodies. Also, bispecific antibodies can be prepared by genetic engineering methods.

For measuring the antigen binding activity of the antibody (Antibodies A Laboratory Manual. EdHarlow, David Lane, Cold Spring Harbor Laboratory, 1988), a known method can be used. For example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), fluoroimmunoassay, or the like can be used.

The antibody of the present invention may be an antibody modified with a sugar chain. It is known that the cytotoxic activity of an antibody can be enhanced by modifying the sugar chain of the antibody. As an antibody having a modified sugar chain, for example, the following antibodies are known.

Antibodies modified by glycosylation (WO 99/54342, etc.);

fucose-deficient antibodies attached to sugar chains (WO 00/61739, WO 02/31140, WO2006/067913, etc.);

antibodies having sugar chains with bisecting GlcNAc (WO 02/79255, etc.), and the like.

When the antibody of the present invention is used for therapeutic purposes, the antibody is preferably an antibody having cytotoxic activity.

Examples of the cytotoxic activity in the present invention include: antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and the like. In the present invention, CDC activity refers to cytotoxic activity produced by the complement system. The ADCC activity is an activity in which, when a specific antibody is attached to a cell surface antigen of a target cell, a cell (e.g., an immune cell) having an Fc γ receptor binds to its Fc site via the Fc γ receptor, thereby injuring the target cell.

Whether an anti-LGR 7 antibody has ADCC activity or CDC activity can be determined using well-known methods (e.g., Current protocols in Immunology, chapter7.immunologic studios in humans, Editor, John E, Coligan et al, John Wiley & Sons, inc., (1993), etc.).

Specifically, first, effector cells, a complement solution, and target cells are prepared.

(1) Preparation of Effector cells

Spleens were excised from CBA/N mice and the like, and splenocytes were isolated in RPMI1640 medium (manufactured by Invitrogen). After washing with the same medium containing 10% fetal bovine serum (FBS, Hyclone Co., Ltd.), the cell concentration was adjusted to 5X 10⁶The effector cells can be prepared by one cell per mL.

(2) Preparation of complement solution

A Complement solution was prepared by diluting young Rabbit Complement (Baby Rabbit Complement) (manufactured by CEDARLANE) 10-fold with a medium containing 10% FBS (manufactured by Invitrogen).

(3) Preparation of target cells

Contacting a cell expressing LGR7 protein with 0.2mCi⁵¹Cr-sodium chromate (manufactured by GE healthcare biosciences) was cultured together in a DMEM medium containing 10% FBS at 37 ℃ for 1 hour, so that the target cells could be radiolabeled. Cells expressing LGR7 protein may be used as cells transformed with a gene encoding LGR7 protein, ovarian cancer, etc. After radiolabeling, cells were washed 3 times with RPMI1640 medium containing 10% FBS to adjust the cell concentration to 2 × 10⁵Individual cells/mL, so that the target cell can be prepared.

ADCC activity and CDC activity can be measured by the following methods. For the ADCC activity, 50. mu.L of target cells and anti-LGR 7 antibody were added to a 96-well U-plate (Becton Dickinson) and reacted for 15 minutes on ice. Thereafter, 100. mu.L of effector cells were added and cultured in a carbon dioxide incubator for 4 hours. The final concentration of antibody was 0 or 10. mu.g/mL. After the culture, 100. mu.L of the supernatant was collected, and the radioactivity was measured by a GAMMA-ray counter (COBRAIIAUTO-GAMMA, model D5005, manufactured by Packard Instrument Company). The cytotoxic activity (%) can be calculated using the obtained value according to the formula (A-C)/(B-C). times.100. A represents the radioactivity (cpm) of each sample, B represents the radioactivity (cpm) of a sample to which 1% NP-40 (manufactured by nacalai tesque) was added, and C represents the radioactivity (cpm) of a sample containing only target cells.

On the other hand, when the CDC activity was measured, 50 μ L of each of the target cells and the anti-LGR 7 antibody was added to a 96-well flat-bottom plate (manufactured by Becton Dickinson), and reacted for 15 minutes on ice. Thereafter, 100. mu.L of the complement solution was added thereto, and the mixture was cultured in a carbon dioxide incubator for 4 hours. The final concentration of antibody was 0 or 3. mu.g/mL. After the culture, 100. mu.L of the supernatant was collected and measured for radioactivity by a gamma-ray counter. The cytotoxic activity can be calculated in the same manner as the measurement of ADCC activity.

When the cytotoxic activity of the antibody conjugate was measured, 50. mu.L of each of the target cells and the anti-LGR 7 antibody conjugate was added to a 96-well flat-bottom plate (manufactured by Becton Dickinson corporation) and reacted on ice for 15 minutes. Culturing in a carbon dioxide incubator for 1-4 hours. The final concentration of antibody was brought to 0 or 3. mu.g/mL. After the culture, 100. mu.L of the supernatant was collected and measured for radioactivity by a gamma-ray counter. The cytotoxic activity can be calculated in the same manner as the measurement of ADCC activity. Another embodiment of the antibody used in the present invention includes an antibody having an internalization activity. In the present invention, the "antibody having an internalization activity" refers to an antibody that is delivered into a cell (into cytoplasm, into vesicles, into other small organs, etc.) upon binding to LGR7 on the cell surface.

Whether or not an antibody has an internalizing activity can be confirmed by a method known to those skilled in the art, and for example, the following method can be used: a method in which an anti-LGR 7 antibody bound with a labeling substance is contacted with a cell expressing LGR7, followed by confirming whether or not the labeling substance is taken into the cell; a method in which an anti-LGR 7 antibody to which a cytotoxic substance is bound is contacted with a cell expressing LGR7, and then it is confirmed whether or not cell death of the LGR 7-expressing cell is induced. More specifically, whether or not the antibody has an internalizing activity can be confirmed by the method described in the following examples.

The antibody having an internalization activity can be used, for example, in the form of a pharmaceutical composition such as an anticancer drug by binding to the above cytotoxic substance.

Any antibody that recognizes LGR7 may be used as the antibody of the present invention. For example, preferable examples of the antibody include the antibodies described in the following (1) to (29). These antibodies may be, for example, full-length antibodies, low molecular antibodies, animal antibodies, chimeric antibodies, humanized antibodies, or human antibodies.

(1) An antibody (22DA6 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of CDR2 shown in SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 7;

(2) an antibody (22DA6 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 10, the amino acid sequence of CDR2 of SEQ ID NO: 11, and the amino acid sequence of CDR3 shown in SEQ ID NO: 12;

(3) an antibody (22DA6) comprising the H chain of (1) and the L chain of (2);

(4) an antibody (22DA7 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 15, the amino acid sequence of CDR2 of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 17;

(5) an antibody (22DA7 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 20, the amino acid sequence of CDR2 of SEQ ID NO: 21 and the amino acid sequence of SEQ ID NO: 22;

(6) an antibody (22DA7) comprising the H chain of (4) and the L chain of (5);

(7) an antibody (22DA17 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 27, an amino acid sequence of seq id no;

(8) an antibody (22DA17 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31 and the amino acid sequence of SEQ ID NO: 32;

(9) an antibody (22DA17) comprising the H chain of (7) and the L chain of (8);

(10) an antibody (22DA22 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 35, the amino acid sequence of CDR2 of SEQ ID NO: 36 and the amino acid sequence of SEQ ID NO: 37, or a pharmaceutically acceptable salt thereof;

(11) an antibody (22DA22 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 40, the amino acid sequence of CDR2 of SEQ ID NO: 41 and the amino acid sequence of SEQ ID NO: 42;

(12) an antibody (22DA22) comprising the H chain of (10) and the L chain of (11);

(13) an antibody (22DA23 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 45, and the amino acid sequence of CDR2 shown in SEQ ID NO: 46 and the amino acid sequence of SEQ ID NO: 47 in a sequence listing;

(14) an antibody (22DA23 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 50, the amino acid sequence of CDR2 of SEQ ID NO: 51, and the amino acid sequence of SEQ ID NO: 52;

(15) an antibody (22DA23) comprising the H chain of (13) and the L chain of (14);

(16) an antibody (22DA24 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 55, the amino acid sequence of CDR2 of SEQ ID NO: 56, and the amino acid sequence of SEQ ID NO: an amino acid sequence according to 57;

(17) an antibody (22DA24 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 60, the amino acid sequence of CDR2 of SEQ ID NO: 61, and the amino acid sequence of SEQ ID NO: 62, an amino acid sequence as described in 62;

(18) an antibody (22DA24) comprising the H chain of (16) and the L chain of (17);

(19) an antibody (22SD7 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 65, the amino acid sequence of CDR2 of SEQ ID NO: 66 and the amino acid sequence of SEQ ID NO: 67, or a nucleotide sequence thereof;

(20) an antibody (22SD7 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 70, the amino acid sequence of CDR2 of SEQ ID NO: 71 and the amino acid sequence of SEQ ID NO: 72, an amino acid sequence thereof;

(21) an antibody (22SD7) comprising the H chain of (19) and the L chain of (20);

(22) an antibody (22SD11 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 75, the amino acid sequence of CDR2 of SEQ ID NO: 76, and the amino acid sequence of SEQ ID NO: 77;

(23) an antibody (22SD11 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 80, the amino acid sequence of CDR2 of SEQ ID NO: 81 and the amino acid sequence of CDR3 as SEQ ID NO: 82;

(24) an antibody (22SD11) comprising the H chain of (22) and the L chain of (23);

(25) an antibody (22SD48 heavy chain) comprising an H chain having the amino acid sequence of SEQ ID NO: 85, and SEQ ID NO: 86 and the amino acid sequence of CDR3 as shown in SEQ ID NO: 87;

(26) an antibody (22SD48 light chain) comprising an L chain having the amino acid sequence of SEQ ID NO: 90, the amino acid sequence of CDR2 of SEQ ID NO: 91 and the amino acid sequence of CDR3 as set forth in SEQ ID NO: 92 in a sequence listing;

(27) an antibody (22SD48) comprising the H chain of (25) and the L chain of (26);

(28) an antibody having the same activity as the antibody according to any one of (1) to (27);

(29) an antibody that recognizes the same epitope as that recognized by the antibody according to any one of (1) to (27).

In the present invention, the term "having an equivalent activity to the antibody of the present invention" means that the binding activity to LGR7 and/or the cytotoxic activity against LGR 7-expressing cells are equivalent.

The method of introducing a mutation into a polypeptide is one of methods known to those skilled in the art for producing a polypeptide functionally equivalent to a certain polypeptide. For example, one skilled in the art can use site-specific mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods enzymol.100, 468-500, Kramer, W. et al. (1984) Nucleic Acids Res.12, 9441-9456, Kramer W, and Fritz HJ (1987) Methods Enzymol.154, 350-367, Kunkel, TA (1985) Proc Natl Acad Sci. USA.82, 488-492, Kunkel (1988) Methods enzymol.85, 2763-2766) to introduce appropriate mutations into the antibody of the present invention, and can thereby prepare an antibody functionally equivalent to the antibody. In addition, mutations of amino acids can also occur in nature. As described above, an antibody having an amino acid sequence in which 1 or more amino acids are mutated and functionally equivalent to the antibody is also included in the amino acid sequence of the antibody of the present invention.

In the above mutant, the number of amino acids to be mutated is usually within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (for example, within 5 amino acids).

Among the mutated amino acid residues, other amino acids that preserve the properties of the amino acid side chain are preferred. For example, based on the nature of the amino acid side chains, the following classifications are established:

a hydrophobic amino acid (A, I, L, M, F, P, W, Y, V);

a hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T);

an amino acid having an aliphatic side chain (G, A, V, L, I, P);

an amino acid having a hydroxyl-containing side chain (S, T, Y);

an amino acid having a sulfur atom-containing side chain (C, M);

an amino acid having a carboxylic acid and amide containing side chain (D, N, E, Q);

an amino acid (R, K, H) having a side chain containing a basic group;

having an amino acid with an aromatic side chain (H, F, Y, W)

(the amino acids are indicated in parentheses by the single letter symbols).

Polypeptides having an amino acid sequence modified by deletion, addition and/or substitution of 1 or more amino acid residues in an amino acid sequence are known to maintain their biological activity (Mark, D.F. et al, Proc. Natl. Acad. Sci. USA (1984)81, 5662-. That is, in general, in an amino acid sequence constituting a certain polypeptide, when amino acids classified into groups are substituted with each other, there is a high possibility that the activity of the polypeptide is maintained. In the present invention, the substitution between amino acids in the above-mentioned amino acid group is referred to as conservative substitution.

The invention also provides antibodies that bind to the same epitope as the anti-LGR 7 antibodies disclosed herein. That is, the present invention relates to an antibody that recognizes the same epitope as that recognized by 22DA6, 22DA7, 22DA17, 22DA22, 22DA23, 22DA24, 22SD7, 22SD11, and 22SD48, and use thereof. The antibody can be obtained, for example, by the following method.

The test antibody shares an epitope with an antibody, and this can be confirmed by competition of the two antibodies for the same epitope. Competition between antibodies is detected by cross-blocking assay (cross-blocking assay) or the like. For example, a competitive ELISA assay is a preferred cross-blocking assay.

Specifically, in the cross-blocking assay, LGR7 protein coated on the wells of the microplate is pre-incubated in the presence or absence of a candidate competing antibody, followed by addition of the anti-LGR 7 antibody of the invention. The amount of anti-LGR 7 antibody of the invention that binds to LGR7 protein in the well is indirectly related to the binding ability of the candidate competitor antibody (test antibody) that competitively binds to the same epitope. That is, the greater the affinity of the test antibody for the same epitope, the lower the amount of binding of the anti-LGR 7 antibody of the present invention to the LGR7 protein-coated pore, and the greater the amount of binding of the test antibody to the LGR7 protein-coated pore.

The amount of antibody bound to the well can be easily determined by labeling the antibody in advance. For example, biotin-labeled antibodies can be determined by using avidin peroxidase conjugates and appropriate substrates. Cross-blocking assays that utilize enzymatic labels such as peroxidase are particularly referred to as competitive ELISA assays. The antibody may be labeled with other labels that are detectable or determinable. Specifically, radioactive labels, fluorescent labels, and the like are known.

Alternatively, competitive FACS assays are also preferred cross-blocking assays.

Specifically, in a competition ELISA assay, LGR7 protein expressing cells were used instead of LGR7 protein coated on the wells of microtiter plates. After pre-incubation in the presence or absence of candidate competitor antibodies, the anti-LGR 7 antibodies of the invention labeled with biotin were added, followed by streptavidin/fluorescein conjugate, so that competition between the antibodies could be determined. Cross-blocking assays using flow cytometry are particularly referred to as competitive FACS assays. The antibody may be labeled with other fluorescent labeling substances that can be detected or measured.

Also, when the test antibody has a constant region from a species different from that of the anti-LGR 7 antibody of the present invention, any antibody bound to the well can also be measured by a labeled antibody recognizing any constant region. Alternatively, even if the antibodies are from the same species, when the classes (classes) are different, the antibodies bound to the wells can be measured using antibodies that recognize each class.

A candidate antibody that blocks binding of at least 20%, preferably at least 30%, and more preferably at least 50% of the anti-LGR 7 antibody binds to substantially the same epitope as, or competes for binding to the same epitope as, an anti-LGR 7 antibody of the invention, as compared to the binding activity obtained in a control assay performed in the absence of the candidate competing antibody. In the measurement of the epitope, the constant region of the antibody of the present invention may be replaced with the same constant region as that of the test antibody.

Pharmaceutical composition

Viewed from another aspect, the present invention provides a pharmaceutical composition comprising an antibody that binds to LGR7 protein as an active ingredient. Furthermore, the present invention relates to a cell proliferation inhibitor, particularly an anticancer agent, containing an antibody that binds to LGR7 protein as an active ingredient. The cell proliferation inhibitor and the anticancer agent of the present invention are preferably administered to a subject suffering from cancer or a subject likely to suffer from cancer. Since the expression level of LGR7 is very low in normal cells other than the brain but is increased in cancer cells, it is considered that: cancer cell-specific cytotoxicity can be obtained by administering an anti-LGR 7 antibody.

The anti-LGR 7 antibody used in the pharmaceutical composition (e.g., anticancer drug) of the present invention is not particularly limited, and may be any anti-LGR 7 antibody, for example, the above-described anti-LGR 7 antibody may be used.

In the present invention, "contains an antibody that binds to LGR7 as an active ingredient" means that it contains an anti-LGR 7 antibody as a main active ingredient, and the content of the anti-LGR 7 antibody is not limited.

When the disease to be treated by the pharmaceutical composition of the present invention is cancer, the cancer to be treated is not particularly limited, but ovarian cancer is preferred, and clear cell adenocarcinoma of ovarian cancer is particularly preferred. Cancer may be a primary lesion or a metastatic lesion.

The pharmaceutical composition of the present invention can be administered to a patient by any of oral and parenteral administration. Parenteral administration is preferred. The administration method specifically comprises the following steps: injection, nasal administration, pulmonary administration, transdermal administration, etc. Examples of administration by injection are: for example, the pharmaceutical composition of the present invention can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. The appropriate administration method can also be selected according to the age and symptoms of the patient. The dose can be selected, for example, from 0.0001mg to 1000mg per kg body weight per time. Alternatively, the dose may be selected, for example, from 0.001mg to 100000mg per patient. However, the pharmaceutical composition of the present invention is not limited to the above-mentioned dosage.

The Pharmaceutical composition of the present invention can be prepared into preparations according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark publishing company, Easton, U.S. A), and may contain pharmaceutically acceptable carriers or additives. Examples of the surfactant include a surfactant, an excipient, a colorant, a perfume, a preservative, a stabilizer, a buffer, a suspending agent, an isotonic agent, a binder, a disintegrant, a lubricant, a fluidity enhancer, and a flavoring agent. The carrier is not limited to these, and other commonly used carriers may be further used as appropriate. The carrier specifically comprises: light silicic anhydride, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, and the like.

The present invention also provides: a method of eliciting toxicity or a method of inhibiting cell proliferation in an LGR 7-expressing cell by contacting the LGR 7-expressing cell with an antibody that binds to an LGR7 protein.

The antibody used in the method of the present invention is not particularly limited, and for example, the above-mentioned antibody can be used. The cell to which the anti-LGR 7 antibody binds is not particularly limited as long as it is a cell expressing LGR7. Preferred LGR 7-expressing cells in the present invention are cancer cells. More preferably ovarian cancer cells. The method of the present invention is applicable to both primary and metastatic lesions of the above-mentioned cancers. Further preferred cancer cells are primary ovarian cancer cells and metastatic ovarian cancer cells.

In the present invention, "contact" can be performed, for example, by adding an antibody to a culture solution of an LGR 7-expressing cell cultured in an in vitro. In the present invention, "contacting" may also be performed by administering to a non-human animal in which LGR 7-expressing cells are transplanted into the body, or an animal having cancer cells endogenously expressing LGR7.

As a method for evaluating or measuring cytotoxicity induced in LGR 7-expressing cells by the contact with an anti-LGR 7 antibody, the following method is preferably employed. Methods for evaluating or measuring the cytotoxic activity in vitro include: the above-mentioned antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and the like. Whether an anti-LGR 7 antibody has ADCC activity or CDC activity can be determined using known methods (e.g., Current protocols in Immunology, Chapter7.Immunologic studios in humans, Editor, John E, Coligan et al, John Wiley & Sons, inc., (1993)) or not. When the activity was measured, a binding antibody having the same isotype as the anti-LGR 7 antibody but not having the cytotoxic activity was used as a control antibody in the same manner as the anti-LGR 7 antibody, and the activity was determined based on the fact that the anti-LGR 7 antibody showed a stronger cytotoxic activity than the control antibody.

The isotype of an antibody is defined by the sequence of the H chain constant region of the amino acid sequence of the antibody. In vivo, the isotype of an antibody is ultimately determined by class switching due to gene recombination on chromosomes that occurs when antibody-producing B cells mature. The differences in isotype are reflected in the differences in physiological, pathological function of the antibodies. Specifically, for example, it is known that the intensity of cytotoxic activity and the expression amount of antigen are simultaneously affected by the isotype of an antibody. Therefore, when the cytotoxic activity is measured, it is preferable to use an antibody used as a control in the same isotype as the test antibody.

In addition, for evaluation or measurement of cytotoxic activity in vivo, for example, LGR 7-expressing cancer cells are transplanted into the skin or subcutaneous of a non-human test animal, after which the test antibody is administered intravenously or intraperitoneally every day or several days from the day or the next day. Cytotoxic activity can be determined by measuring the size of the tumor daily. When a control antibody having the same isotype was administered in the same manner as in the in-vitro evaluation, the cytotoxic activity was determined based on the fact that the tumor size of the group to which the anti-LGR 7 antibody was administered was significantly smaller than that of the group to which the control antibody was administered. When a mouse is used as a non-human test animal, a nude mouse (nu/nu) whose T lymphocyte function is deleted by genetic deletion of thymus can be suitably used. By using this mouse, intervention of T lymphocytes in an animal to be tested can be excluded when evaluating and measuring cytotoxic activity of the administered antibody.

The present invention also provides a method for diagnosing cancer, which comprises: detecting the LGR7 protein or a gene encoding the LGR7 protein. LGR7 was found to be significantly overexpressed in various cancer tissues or cancer cell lines, whereas LGR7 was expressed in normal cells very lowly. Therefore, LGR7 is effective as a marker for specifically detecting cancer.

In one embodiment of the method of the present invention, the diagnosis of cancer is performed by detecting LGR7 protein in a test sample. The extracellular region of the LGR7 protein is preferably detected. Detection of LGR7 protein is preferably performed using an antibody that recognizes LGR7 protein.

One specific example of the diagnostic method of the present invention is a method for diagnosing cancer, which comprises the following steps.

(a) A step of providing a sample collected from a subject;

(b) a step of detecting LGR7 protein contained in the collected sample using an antibody that binds to LGR7 protein.

In the present invention, the detection includes quantitative or qualitative detection. For example, the qualitative detection may be the following assay:

determining only the presence or absence of LGR7 protein;

determining the presence or absence of an amount of LGR7 protein or greater;

an assay for comparing the amount of LGR7 protein with other samples (e.g., control samples, etc.).

And the quantitative determination may be: the concentration of LGR7 protein, the amount of LGR7 protein and the like.

The test sample in the present invention is not particularly limited as long as it is a sample that may contain LGR7 protein. Specifically, a sample collected from the body of a living body such as a mammal is preferable. More preferably, the sample is a sample collected from the inside of a human body. Specific examples of the test sample include: blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymphatic fluid, saliva, urine, tissue, ascites fluid, peritoneal lavage fluid, and the like. A preferred sample is a sample obtained from a test sample which is: a specimen or a cell culture solution obtained by fixing a tissue or a cell collected from a living body, and the like.

The cancer diagnosed according to the present invention is not particularly limited, and may be any cancer. Specifically ovarian cancer. In the present invention, both primary and metastatic lesions of the above-mentioned cancer can be diagnosed. Particularly preferred cancers in the present invention are primary ovarian cancer and metastatic ovarian cancer.

In the present invention, when LGR7 protein is detected in a test sample, cancer is diagnosed using the level as an indicator. Specifically, when the amount of LGR7 protein detected in the test sample is larger than that of a negative control or a healthy subject, it indicates that the subject has cancer or is likely to have cancer in the future. Namely, the present invention relates to a method for diagnosing cancer, which comprises the steps of:

(1) a step of detecting the expression level of LGR7 in a biological sample collected from the subject; and

(2) a step of indicating that the subject has cancer when the expression level of LGR7 detected in (1) is high as compared with a control.

In the present invention, the control refers to a sample as a reference for comparison, and includes a negative control and a biological sample of a healthy subject. Negative controls can be obtained by taking biological samples of healthy subjects and mixing them as needed. The level of LGR7 expression of the control can be measured in parallel with the level of LGR7 expression in a biological sample from the subject. Alternatively, the expression level of LGR7 in biological samples of a plurality of healthy subjects may be detected in advance, and then the standard expression level in healthy subjects may be statistically determined. Specifically, for example, the mean ± 2 × standard deviation (s.d.) or the mean ± 3 × standard deviation (s.d.) may also be used as the standard value. Statistically, the mean ± 2 × standard deviation (s.d.) includes values of 80% of healthy persons, and the mean ± 3 × standard deviation (s.d.) includes values of 90% of healthy persons.

Alternatively, the expression level of LGR7 in the control can be set using the ROC curve. The ROC curve (receiver operating characteristic curve) is a graph in which the vertical axis represents the detection sensitivity and the horizontal axis represents the false positive rate (i.e., "1-specificity"). In the present invention, when the reference value used for determining the expression level of LGR7 in a biological sample is continuously changed, by plotting the changes in sensitivity and false positive rate, an ROC curve can be obtained.

The "reference value" used for obtaining the ROC curve is a value that is temporarily used for statistical analysis. The "reference value" used to derive the ROC curve typically varies continuously over a range that can cover all reference values that are selectable. For example, the reference value may be varied between the minimum and maximum values of LGR7 measurement values of the analyzed population.

From the obtained ROC curve, a standard value expected to have desired detection sensitivity and accuracy can be selected. The standard value statistically set from the ROC curve or the like is also referred to as a cut-off value (cut-off value). In the method for detecting cancer based on the cutoff value, the expression level of LGR7 detected in (1) is compared with the cutoff value in step (2) above. Also, when the expression level of LGR7 detected in (1) is higher than the cutoff value, cancer of the subject is detected.

In the present invention, the expression level of LGR7 may be determined by any method. Specifically, by evaluating the amount of mRNA of LGR7, the amount of LGR7 protein, and the biological activity of LGR7 protein, the expression level of LGR7 can be known. The amount of mRNA or protein of LGR7 can be determined by the methods described herein.

In the present invention, any animal species expressing LGR7 protein may be used as the subject. For example, LGR7 protein is expressed in various mammals other than humans, such as chimpanzees (Pan trogloytes) (ENSPTRG00000016551), rhesus monkeys (Macaca mulatta) (ENSMMUG00000004647), mice (Mus musculus) (ENSMUSG00000034009), rats (Rattus norvegicus) (ENSRNOG00000024120), guinea pigs (Cavia porcellus) (ENSCPOG00000015517), dogs (Canis falaris) (ENSCAFG00000008672), and chickens (Gallus Gallus) (ENSGALG 00000009429). Thus, the subject of the present invention includes these animals. Particularly suitable subjects are humans. When an animal other than human is used as the subject, it is needless to say that the LGR7 protein of the animal species can be detected.

The method for detecting LGR7 protein contained in a test sample is not particularly limited, but detection is preferably carried out by the following immunological methods using an anti-LGR 7 antibody:

radioimmunoassay (RIA);

enzyme linked immunosorbent assay (EIA);

fluorescence Immunoassay (FIA);

a Luminescent Immunoassay (LIA);

immunoprecipitation (IP);

immunoturbidimetry (TIA);

western Blotting (WB);

immunohistochemistry (IHC); and

immunodiffusion method (SRID).

Among the above methods, Immunohistochemistry (IHC) is one of the preferred immunological assay methods as a method for diagnosing cancer, and the method comprises the steps of: tissues or cells taken from a patient suffering from cancer were fixed on the sections, and then LGR7 protein on the sections was detected. The above immunological methods such as Immunohistochemical (IHC) method are well known to those skilled in the art.

That is, LGR7 is a membrane protein whose specific expression is enhanced in cancer cells, and thus cancer cells or cancer tissues can be detected using an anti-LGR 7 antibody. The immunohistological analysis described above allows detection of cancer cells contained in cells or tissues collected from the body.

In another preferred mode, the anti-LGR 7 antibody can also be used to detect cancerous tissue in the body. Namely, the present invention relates to a method for detecting cancer, which comprises: (1) administering to the subject an antibody that binds to LGR7 protein, which is labeled with a label such as a radioisotope; and (2) detecting the accumulation of the label. To track the antibody administered into the body, the antibody may be labeled to enable detection. For example, the behavior of an antibody labeled with a fluorescent substance, a luminescent substance, or a radioisotope in the body can be tracked. The antibody labeled with a fluorescent substance or a luminescent substance can be observed with an endoscope or a laparoscope. As for the radioactive isotope, the localization of the antibody can be imaged by tracing the radioactivity thereof. In the present invention, localization of anti-LGR 7 antibodies in the body indicates the presence of cancer cells.

For the detection of cancer in the body, a positron emitting nuclide may be used as the radioisotope labeled with the antibody. For example, can use¹⁸F、⁵⁵Co、⁶⁴Cu、⁶⁶Ga、⁶⁸Ga、⁷⁶Br、⁸⁹Zr and¹²⁴i such positron emitting nuclides labeled antibodies. When an anti-LGR 7 antibody is labeled with these positron emitting nuclides, a known method (Acta Oncol.32, 825 to 830, 1993) can be used.

After an anti-LGR 7 antibody labeled with a positron emitting nuclide is administered to a human or an animal, the radioactive rays emitted from the radionuclide are measured from the outside of the body using a PET (positron emission tomography) device and converted into an image by a computer tomography method. PET is a device for noninvasively obtaining data relating to in vivo behavior of a drug and the like. The radiation intensity can be quantitatively imaged in the form of signal intensity using PET. By using PET in the manner described above, there is no need to collect a sample from the patient's bodyDetecting antigenic molecules that are highly expressed in a particular cancer. The anti-LGR 7 antibody may be labeled with a nuclide as described above, and may be used¹¹C、¹³N、¹⁵O、¹⁸F、⁴⁵Short-lived nuclides such as positron emitting nuclides like Ti are radiolabeled.

The production of short-lived nuclides using the above nuclides obtained by a medical cyclotron, and the development of techniques for producing short-lived radiolabeled compounds have been advanced. Using these techniques, anti-LGR 7 antibodies can be labeled with various radioisotopes. The anti-LGR 7 antibody administered to the patient accumulated in primary and metastatic foci according to the specificity of the anti-LGR 7 antibody for pathological tissues of various sites. When labeled with a positron-emitting nuclide, anti-LGR 7 antibody detects its radioactivity, thereby detecting the presence of the primary and metastatic foci based on the localization of the radioactivity. For the diagnostic use, gamma particles of 25 to 4000keV or an activity value of positron emission can be suitably used. In addition, when a suitable nuclide is selected and administered in a large amount, a therapeutic effect is expected. In order to obtain an anticancer effect by radiation, a nuclide to which gamma particles of 70 to 700keV or positron emission values are given can be used.

In another mode of the method of the present invention, the expression of the gene of LGR7 is detected. In the present invention, the gene to be detected is not particularly limited, but mRNA is preferred. In the present invention, the detection includes quantitative or qualitative detection. Examples of qualitative assays are, for example: the following measurement procedures were carried out.

Determining only the presence or absence of mRNA for LGR 7;

determining the presence or absence of an amount of LGR7 or more mRNA;

and a measurement for comparing the amount of mRNA of LGR7 with other samples (for example, a control sample).

And the quantitative determination may be: the measurement of the mRNA concentration of LGR7, the measurement of the mRNA amount of LGR7, and the like.

As the test sample in the present invention, any sample that may contain mRNA of LGR7 can be used. The sample is preferably collected from the body of a living body such as a mammal, and more preferably from the body of a human. Specific examples of the test sample include: blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymphatic fluid, saliva, urine, tissue, ascites fluid, peritoneal lavage fluid, and the like. The test sample of the present invention also includes a preferable sample, that is, a sample obtained from a test sample such as a specimen or a cell culture solution obtained by fixing a tissue or a cell collected from a living body.

When a sample obtained from a test sample, such as a specimen obtained by fixing tissue or cells collected from a living body or a cell culture solution, is used, the in situ hybridization method is preferably used. The in situ hybridization method has been developed as a method for confirming the presence or absence, distribution, and expression level of a specific DNA or RNA in a cell or tissue. The principle is to utilize the following properties: probe nucleic acids having nucleotide sequences complementary to specific nucleic acid sequences in cells specifically form complexes. Since the site of hybridization can be recognized by labeling the probe with a Radioisotope (RI), an antigenic substance (hapten), or the like in advance and detecting the label, the in situ hybridization method is used for detection of DNA, RNA, or the like in a cell. As the label of the probe, a label using RI is preferably used. As a more preferable example, a fluorescent label using a hapten such as biotin or digoxigenin which is a non-radioactive substance can be used. As a particularly preferred example, an assay using Fluorescence In Situ Hybridization (FISH) is used.

Examples of the cancer to be diagnosed include clear cell adenocarcinoma among ovarian cancers. In the present invention, both primary and metastatic lesions of the above-mentioned cancer can be diagnosed.

In the present invention, any animal species expressing LGR7 protein may be used as the subject. For example, various mammals other than mice, rats, rhesus monkeys, chimpanzees, and the like are known to express LGR7. Particularly suitable subjects are humans. In addition, when an animal species other than human is used as the subject, the mRNA of LGR7 of the animal species is detected.

The specific embodiment of the detection method is described below. First, a specimen is prepared from a subject. Then, the mRNA of LGR7 contained in the sample was detected. In the present invention, cDNA synthesized from mRNA can also be detected. In the present invention, when mRNA of LGR7 or cDNA encoding LGR7 is detected in a test sample, it is judged that cancer may be present. For example, when the amount of mRNA of LGR7 or cDNA encoding LGR7 detected in a test sample is larger than that of a negative control or a healthy person, it indicates that the subject has cancer or has a high possibility of having cancer in the future.

Methods for detecting mRNA are well known. In the present invention, for example, northern blotting, RT-PCR, DNA array method and the like can be specifically used.

The detection method of the present invention can be automatically performed by using various automatic inspection apparatuses. By automation, a plurality of samples can be inspected in a short time.

The present invention also provides a diagnostic reagent or kit for diagnosing cancer, which contains a reagent for detecting LGR7 protein in a test sample. The diagnostic reagent of the present invention contains at least an anti-LGR 7 antibody.

By combining the cancer diagnosis reagent of the present invention with other elements for detecting LGR7, a kit for diagnosing cancer can be prepared. That is, the present invention relates to a kit for diagnosing cancer, comprising: the antibody that binds to LGR7, and the reagent that detects the binding of the antibody to LGR7, may further contain a control sample comprising a biological sample comprising LGR7. The kit of the present invention may further comprise an instruction manual for instructing a measurement procedure.

All prior art documents cited in this specification are incorporated herein by reference.

Examples

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples.

[ example 1]Human LGR7 mRNA using Affymetrix U133 Plus2.0 array Expression analysis

After written approval was obtained at the subsidiary hospital of the department of medicine of tokyo university (japan), total RNA was extracted from 10 ovarian cancer surgical specimens collected and cryopreserved. At this time, the surgical specimen was embedded in an OCT compound (OCT compound), and the thinned surgical specimen was dissolved in TRIZOL (Invitrogen corporation), followed by total RNA extraction according to the method of the product appendix. At the same time, HE stained specimens were prepared and the presence of cancer was confirmed. The histologic details of the ovarian cancer cases were: 4 cases of clear cell adenocarcinoma, 2 cases of serous adenocarcinoma, 3 cases of intimal adenocarcinoma, and 1 case of cancerous edema. Using the total RNA of these samples, expression analysis was performed using Affymetrix U133 Plus2.0 array, and genes that were confirmed to be specifically highly expressed in clear ovarian cell adenocarcinoma were selected. Total RNA from normal tissue (Clontech) and ovarian cancer cell lines (purchased from ATCC, JCRB, Japan research) was used as the target.

As a selection criterion of a target molecule suitable for treating clear cell adenocarcinoma, 11761 probe set narrowed down to an expression value of 200 or more in at least 1 of 4 cases of ovarian clear cell adenocarcinomas. Next, in 4 cases of ovarian bright cell adenocarcinomas, the 197 probe group, in which the expression value at the 3 rd highest was compared with the expression value at the highest value in normal ovaries, peripheral blood, bone marrow, and main tissues (liver, kidney, lung, stomach, intestine, and pancreas), was further narrowed down to a ratio of 1.8 or more. Among them, LGR7 was selected as a molecule whose relationship with ovarian clear cell adenocarcinoma has not been reported yet and the ratio thereof showed the highest value. For selection, reference is made to the expression data of 87 ovarian cancers including 3 clear cell adenocarcinomas as disclosed by the International Genome Consortium (IGC). The signal values were plotted in ovarian cancer, ovarian cancer cell lines, and normal tissues of probe 1552715_ a _ at of LGR7, and the results are shown in fig. 1. Details of the sample names are shown in table 1.

LGR7 is specifically expressed in clear cell adenocarcinoma even in ovarian cancer, and is expected to be an antitumor agent targeting human LGR7 against this cancer species.

TABLE 1

brain	Normal tissue	Brain
			muscle	Normal tissue	Skeletal muscle
heart	Normal tissue	Heart and heart
			skin	Normal tissue	Skin(s)
lung	Normal tissue	Lung (lung)
			liver	Normal tissue	Liver disease
stomach	Normal tissue	Stomach (stomach)
			colon	Normal tissue	Large intestine
pancreas	Normal tissue	Pancreas (pancreas)
			kidney	Normal tissue	Kidney (A)
bone marrow	Normal tissue	Bone marrow
			perpheral blood	Normal tissue	Peripheral blood
ovary	Normal tissue	Ovary (LU) of human
			testis	Normal tissue	Testis
fetal brain	Normal tissue	Fetal brain
			fetal lung	Normal tissue	Fetal lung
fetal liver	Normal tissue	Fetal liver
			fetal colon	Normal tissue	Fetal large intestine
Ovary3Ca	Ovarian cancer enucleated tissue	Clear cell adenocarcinoma of ovary
			Ovary5Ca	Ovarian cancer enucleated tissue	Clear cell adenocarcinoma of ovary
Ovary7Ca	Ovarian cancer enucleated tissue	Clear cell adenocarcinoma of ovary
			Ovary19Ca	Ovarian cancer enucleated tissue	Clear cell adenocarcinoma of ovary
Ovary9Ca	Ovarian cancer enucleated tissue	Ovarian intimal adenocarcinoma
			Ovary13Ca	Ovarian cancer enucleated tissue	Ovarian intimal adenocarcinoma
Ovary17Ca	Ovarian cancer enucleated tissue	Ovarian intimal adenocarcinoma
			Ovary1Ca	Ovarian cancer enucleated tissue	Ovarian serous adenocarcinoma
Ovary15Ca	Ovarian cancer enucleated tissue	Ovarian serous adenocarcinoma
			Ovary11Ca	Ovarian cancer enucleated tissue	Sarcoma of ovarian cancer
CP_JHOC_5	Ovarian cancer cell line	Clear cell adenocarcinoma of ovary
			CP_MCAS	Ovarian cancer cell line	Ovarian mucinous adenocarcinoma
CP_RMG_1	Ovarian cancer cell line	Clear cell adenocarcinoma of ovary
			CP_RMUG_S	Ovarian cancer cell line	Ovarian mucinous adenocarcinoma
CP_TKY_nu	Ovarian cancer cell line	Undifferentiated adenocarcinoma of ovary

[ example 2]Establishment of full-Length human LGR 7-expressing cells

Based on NCBI accession numbers NP-067647 (SEQ ID NO: 1 (amino acid sequence)) and NM-021634 (SEQ ID NO: 2 (nucleotide sequence)), respectively, a full-length human LGR7cDNA was isolated by PCR method using human uterus QUICK-CLONE cDNA (Clontech) and cloned into pGEM-T Easy (Promega corporation), followed by addition of HA tag sequence at the N-terminus and cloning into a pMCN2i vector for mammalian cell expression.

A BioRad GenePulser was used to introduce a gene into cells derived from the ovary of Chinese hamster, namely, the DG44 cell line, to obtain the HA-LGR 7-expressing cell line HA-LGR7/DG # 24. The gene was introduced into mouse pro-B cells, Ba/F3, to give HA-LGR 7-expressing cell line HA-LGR/BaF3# 48. LGR7 expression was confirmed by western blotting using anti-HA-tagged antibody HA-7(Sigma company) (fig. 2).

A vector into which the LGR7 gene was introduced was constructed and used for DNA immunization. The expression vector pMCN is a vector which can express a gene inserted under the mouse CMV promoter (ACCESSION No. u68299) and which has a neomycin resistance gene integrated as a drug resistance marker. The LGR7 gene was cloned into pMCN according to a conventional method, thereby producing pMCN-LGR7 as an expression vector of LGR7.

[ example 3]Production of anti-LGR 7 monoclonal antibody by DNA immunization

DNA immunization was carried out by introducing a gene into a mouse using a Particle gun (GeneGun Particle) method. The method was performed according to the BioRad operating guidelines. A pellet for DNA immunization was prepared by mixing 1mm gold particles (BioRad) with pMCN-LGR7DNA and coating the mixture inside a tube. Gene transfer was performed by injecting pellets coated with pMCN-LGR7DNA into the abdominal skin of 6-week-old female MRL/lpr mice using a Helios Gene gun (BioRad) at a pressure of 200-. It is considered that the LGR7 protein is expressed by a gene introduced into keratinocytes, Langerhans cells, and dermal dendritic cells (dermaldendritic cells) present in the skin, and these cells become Antigen Presenting Cells (APC), resulting in immunity (Methods 31, 232-242 (2003); Immunization with DNA through the skin). DNA immunizations were performed 6 times at 1 week intervals. Final immunization refers to 100 ten thousand cells of LGR 7-expressing BaF3 cell line HA-LGR/BaF3#48 diluted in PBS for tail vein administration. The antibody titer was determined by FACS analysis using HA-LGR7/DG #24 cells. The reaction of the sera of the immunized mice was compared with the LGR7 protein expressed on the cell membrane surface of HA-LGR7/DG #24 cells. The most reactive mice were finally immunized and used for cell fusion. 3 days after the final immunization, the spleen cells were removed and mixed with the mouse myeloma cell P3-X63Ag8U1(P3U1, purchased from ATCC) to a ratio of 2: 1. Cell fusion was performed by adding PEG1500(Roche Diagnostics) slowly to prepare hybridoma cells. The concentration of PEG1500 was diluted by careful addition of RPMI1640 medium (GIBCO BRL Co.), followed by removal of PEG1500 by centrifugation. Next, the hybridoma cells were suspended in RPMI1640 medium (hereinafter referred to as HAT medium) containing 10% FBS, 1 xhat medium additive (SIGMA), 0.5 × BM-conditioned H1 hybridoma cloning additive (Roche Diagnostics), and then seeded on a 96-well plate to 200 μ L/well. Cell concentration at the time of inoculationThe number of P3U1 cells used was diluted, and hybridoma cells were plated in 96-well plates at 37 ℃ with 5% CO₂Medium, and cultured in HAT medium for about 1 week. Screening of hybridomas secreting antibodies into the culture supernatant was performed by flow cytometry.

[ example 4]]Preparation of sLGR7Fc

A fragment containing amino acids 1 to 555 of LGR7 protein was amplified by PCR, and then a vector was constructed so that the amplified fragment was expressed as a fusion protein with human Fc protein (nucleotide sequence, SEQ ID NO: 95, amino acid sequence, SEQ ID NO: 96). The constructed vector was introduced into DG44 cells, and cells capable of expressing the fusion protein of sLGR7Fc were selected as neomycin-resistant strains. The resulting cell strain was cultured in a large amount, and then the culture supernatant was recovered to purify the sLGR7Fc protein. sLGR7Fc protein, affinity-purified as an Fc fusion protein through a rProtein A column, is supplied to a protein-immunized antigen or a screening antigen of hybridoma.

[ example 5]anti-LGR 7 antibodies made using sLGR7Fc protein immunization

Mice were immunized subcutaneously by mixing 50 μ g of affinity purified sLGR7Fc protein with freund's complete adjuvant. Then, 50. mu.g of the double affinity-purified sLGR7Fc protein was mixed with Freund's incomplete adjuvant, and the resulting mixture was used to induce an antibody by subcutaneous immunization of a mouse, 25. mu.g of sLGR7Fc protein was injected into a mouse having the highest reactivity with LGR7 protein from the tail vein, and 3 days later, the spleen was removed from the mouse body and supplied to a mouse myeloma cell line P3X63Ag8U.1 and cell fusion, and a hybridoma was prepared in the same manner as in [ example 3 ].

[ example 6]Evaluation of binding Activity Using FACS (flow cytometry)

Using the resulting hybridomas, binding to human LGR7/DG44 cells was assessed by flow cytometry. The suspension was suspended in FACS buffer (2% FBS/PBS/0.05% NaN)₃) The human LGR 7-expressing cell line in (1) was diluted to 1X 10 with FACS buffer⁶Each cell/mL, then, 50. mu.L/well of Falcon353 was injected910 in a round bottom 96 well plate. The culture supernatant of the hybridoma diluted to an appropriate concentration was added to the well to which the cells were added, and allowed to react for 60 minutes on ice. Next, the cells were washed 1 time with FACS buffer. Goat F (ab') as a secondary antibody was added to the wells to which the cells were added₂The fragment anti-mouse IgG Fc γ -FITC (Beckman Coulter) was then allowed to react on ice for 30 minutes. After the reaction, the supernatant was removed by centrifugation, and then the cells suspended in 100. mu.L of FACS buffer were subjected to flow cytometry. FACS Calibur (Becton Dickinson) was used in flow cytometry. The binding activity of the cells contained in a population of living cells was evaluated by selecting a histogram of FL1, with threshold values set in the population based on the dot blots of forward angle scatter (forward scatter) and side angle scatter (side scatter).

When the supernatant of the hybridoma was reacted with each of DG44 cells inducing LGR7 expression and DG44 as a parent strain, a hybridoma specifically reacting with LGR 7-expressing cells was obtained. Hybridomas from these wells were monoclonalized using limiting dilution. Isotypes of each antibody were analyzed using an isosbrop (registered trademark) mouse monoclonal antibody isotype kit (Roche Diagnostics). As a result, 22DA6, 22DA7, 22DA11, 22DA12, 22DA23, and 22DA24 were IgG 1. 22DA4, 22DA10, 22SD7, 22SD25, 22SD31 and 22SD48 are IgG2a, and 22DA17 and 22DA20 are IgG2 b. The monoclonal hybridomas were expanded and then purified from the culture supernatants using protein G columns according to the protocol. The purified antibody is subjected to protein quantification by DC protein assay or the like.

[ example 7]Determination of ADCC Activity of anti-LGR 7 monoclonal antibody

ADCC activity of cells that forcibly express DG44 against LGR7 in the human LGR7 monoclonal antibody was investigated using a chromium release method. The target cells were cultured for several hours in a culture medium (CHO-SSFM II (Invitrogen)) supplemented with chromium-51, after which the culture medium was removed, the cells were washed with the culture medium, and the cells suspended in a fresh culture medium were added to a 96-well round bottom plate to a volume of 1X 10⁴Individual cells/well. Then, antibody is added to make the final concentrationTo effector cells (NK-92(ATCC, CRL-2407)) approximately 5 times as much as target cells in each well, recombinant cells (WO 2008/093688) forcibly expressing a chimeric protein containing the extracellular region of mouse Fc-gamma receptor 3 (NM-010188) and the transmembrane and intracellular regions of human gamma chain (NM-004106) were added, to the extent of 1. mu.g/mL or 0.1. mu.g/mL. The plates were kept at 5% CO₂The mixture was allowed to stand at 37 ℃ for 4 hours in an incubator. After standing, the plate was centrifuged, a predetermined amount of supernatant was recovered from each well, radioactivity was measured by a gamma-ray counter Wallac 1480, and specific chromium release rate (%) was determined by the following equation.

Specific chromium release rate (%) - (A-C). times.100/(B-C)

Wherein A represents radioactivity in each well, B represents an average value of radioactivity released into the medium after cell lysis at a final concentration of 1% Nonidet P-40, and C represents an average value of radioactivity when only the medium was added.

As shown in fig. 3, among the anti-human LGR7 monoclonal antibodies used in the test, in particular, 22DA6, 22DA7, 22DA17, 22DA22, 22DA23, 22DA24, 22SD7, 22SD25, and 22SD38 induced very strong ADCC activity against human LGR 7-expressing cells. This result shows that: antibody treatment was very effective against tumors targeting human LGR7.

[ example 8]Determination of CDC Activity of anti-LGR 7 monoclonal antibody

CDC activity was determined by using the degree of uptake of 7-AAD in the cells producing cytotoxicity as an index.

LGR7 expressing DG44 cells were reacted with monoclonal antibodies at a concentration of 10. mu.g/mL for 30 minutes at 4 ℃. Next, young Rabbit Complement (CEDARLANE LABORATORIES) was added to the reaction mixture to a final concentration of 10%, and the reaction was continued at 37 ℃ for 90 minutes. 7-AAD (Beckman Coulter) was added to a final concentration of 1. mu.g/mL, followed by standing at room temperature for 10 minutes. Thereafter, the cells were washed with FACS buffer, and then the proportion of cells producing cytotoxicity was analyzed with FACS Calibur. The value of% FL3 indicates the proportion of injured cells stained with 7-AAD, and in DG44 cells expressing HA-LGR7, as shown in fig. 4, multiple anti-LGR 7 antibodies showed complement-dependent cytotoxic (CDC) activity.

[ example 9]Cloning of antigen genes

The sequences of antibody variable region genes were determined for 9 hybridomas 22DA6, 22DA7, 22DA17, 22DA22, 22DA23, 22DA24, 22SD7, 22SD11, and 22SD48 that exhibited ADCC activity and CDC activity. For the gene of the antibody, total RNA extracted from each hybridoma producing the anti-LGR 7 antibody was used for amplification by the RT-PCR method. Using RNeasy Plant Mini kit (QIAGEN), 1X 10⁷Total RNA was extracted from individual cell hybridomas. The RACE library was constructed using 1. mu.g of total RNA by using SMART RACE cDNA amplification kit (CLONTECH). The antibody gene was amplified using synthetic oligonucleotides MHC-IgG1(SEQ ID NOS: 97, GGGCCAGTGGATAGACAGATG), MHC-IgG2a (SEQ ID NOS: 98, CAGGGGCCAGTGGATAGACCGATG), MHC-IgG2b (SEQ ID NOS: 99, CAGGGGCCAGTGGATAGACTGATG) complementary to the constant region sequence of mouse IgG1 or synthetic oligonucleotides kappa (SEQ ID NOS: 100, GCTCACTGGATGGTGGGAAGATG) complementary to the nucleotide sequence of mouse kappa chain constant region, to thereby amplify a gene fragment on the 5' -terminal side of the gene encoding the antibody produced by the hybridoma. The reverse transcription reaction was carried out at 42 ℃ for 1 hour and 30 minutes. The 50. mu.L of PCR solution contained 5. mu.L of 10 XDavantage 2PCR buffer, 5. mu.L of 10 XDUART Mix, 0.2mM dNTPs (dATP, dGTP, dCTP, dTTP), 1. mu.L of Advantage 2 polymerase Mix (prepared above by CLONTECH), 2.5. mu.L of reverse transcription reaction product, 10pmol of synthetic oligonucleotide MHC-IgG1, MHC-IgG2a, MHC-IgG2b, or κ. The PCR reaction was performed as follows: after 30 seconds of reaction at an initial temperature of 94 ℃, 5 cycles of reaction at 94 ℃ for 5 seconds and at 72 ℃ for 3 minutes were repeated 5 times, 5 cycles of reaction at 94 ℃ for 5 seconds, at 70 ℃ for 10 seconds and at 72 ℃ for 3 minutes were repeated 5 times, and 25 cycles of reaction at 94 ℃ for 5 seconds, at 68 ℃ for 10 seconds and at 72 ℃ for 3 minutes were repeated 25 times. Finally, the reaction product was heated at 72 ℃ for 7 minutes. Each PCR product was purified from agarose gel using QIAquick gel extraction kit (prepared by QIAGEN). After that time, the user can use the device,the PCR product was cloned into pGEM-T Easy vector (manufactured by Promega) and the nucleotide sequence thereof was determined.

The nucleotide sequence of the H chain variable region of 22DA6 is set forth in SEQ ID NO: 3. the amino acid sequence is shown in SEQ ID NO: 4, the nucleotide sequence of the variable region of L is shown in SEQ ID NO: 8. the amino acid sequence is shown in SEQ ID NO: 9. the amino acid sequence of the heavy chain CDR1 of 22DA6 is shown in SEQ ID NO: 5. the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 6. the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 7. the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 10. the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 11. the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 12.

the nucleotide sequence of the H chain variable region of 22DA7 is set forth in SEQ ID NO: 13. the amino acid sequence is shown in SEQ ID NO: 14. the nucleotide sequence of the L chain variable region is shown in SEQ ID NO: 18. the amino acid sequence is shown in SEQ ID NO: 19. the amino acid sequence of the heavy chain CDR1 of 22DA7 is shown in SEQ ID NO: 15. the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 16. the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 17. the amino acid sequence of light chain CDR1 is set forth in SEQ id no: 20. the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 21. the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 22.

the nucleotide sequence of the H chain variable region of 22DA17 is set forth in SEQ ID NO: 23. the amino acid sequence is shown in SEQ ID NO: 24, the nucleotide sequence of the L chain variable region is set forth in SEQ ID NO: 28. the amino acid sequence is shown in SEQ ID NO: 29. the amino acid sequence of the heavy chain CDR1 of 22DA17 is shown in SEQ ID NO: 25. the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 26. the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 27. the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 30. the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 31. the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 32.

the nucleotide sequence of the H chain variable region of 22DA22 is set forth in SEQ ID NO: 33. the amino acid sequence is shown in SEQ ID NO: 34. the nucleotide sequence of the L chain variable region is shown in SEQ ID NO: 38. the amino acid sequence is shown in SEQ ID NO: 39. the amino acid sequence of the heavy chain CDR1 of 22DA22 is shown in SEQ ID NO: 35. the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 36. the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 37. the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 40. the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 41. the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 42.

the nucleotide sequence of the H chain variable region of 22DA23 is set forth in SEQ ID NO: 43. the amino acid sequence is shown in SEQ ID NO: 44. the nucleotide sequence of the L chain variable region is shown in SEQ ID NO: 48. the amino acid sequence is shown in SEQ ID NO: 49. the amino acid sequence of the heavy chain CDR1 of 22DA23 is shown in SEQ ID NO: 45. the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 46. the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 47. the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 50. the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 51. the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 52.

the nucleotide sequence of the H chain variable region of 22DA24 is set forth in SEQ ID NO: 53. the amino acid sequence is shown in SEQ ID NO: 54. the nucleotide sequence of the L chain variable region is shown in SEQ ID NO: 58. the amino acid sequence is shown in SEQ ID NO: 59. the amino acid sequence of the heavy chain CDR1 of 22DA24 is shown in SEQ ID NO: 55. the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 56. the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 57. the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 60. the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 61. the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 62.

the nucleotide sequence of the H chain variable region of 22SD7 is set forth in SEQ ID NO: 63. the amino acid sequence is shown in SEQ ID NO: 64. the nucleotide sequence of the L chain variable region is shown in SEQ ID NO: 68. the amino acid sequence is shown in SEQ ID NO: 69. the amino acid sequence of the heavy chain CDR1 of 22SD7 is shown in SEQ ID NO: 65. the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 66. the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 67. the amino acid sequence of light chain CDR1 is set forth in SEQ id no: 70. the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 71. the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 72.

the nucleotide sequence of the H chain variable region of 22SD11 is set forth in SEQ ID NO: 73. the amino acid sequence is shown in SEQ ID NO: 74. the nucleotide sequence of the L chain variable region is shown in SEQ ID NO: 78. the amino acid sequence is shown in SEQ ID NO: 79. the amino acid sequence of the heavy chain CDR1 of 22SD11 is shown in SEQ ID NO: 75. the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 76. the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 77. the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 80. the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 81. the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 82.

the nucleotide sequence of the H chain variable region of 22SD48 is set forth in SEQ ID NO: 83. the amino acid sequence is shown in SEQ ID NO: 84. the nucleotide sequence of the L chain variable region is shown in SEQ ID NO: 88. the amino acid sequence is shown in SEQ ID NO: 89. the amino acid sequence of the heavy chain CDR1 of 22SD48 is shown in SEQ ID NO: 85. the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 86. the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 87. the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 90. the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 91. the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 92.

[ example 10]Cell killing effect by internalization using Mab-Zap

The killing ability against LGR 7-expressing cells was evaluated using a secondary antibody, Mab-Zap (manufactured by Advanced Targeting Systems), which binds to a toxin named saporin, as a model for the development of antibody drugs having the following mechanism of action: the antibody is bound with a toxin or the like, incorporated into (endocyto) cells after the intracellular antibody binds to the target cells, and then the target cells are killed by the action of the conjugated toxin. Cells BaF3 cells expressing HA-LGR7 were used. 100ng of antibody and 100ng of mab-Zap were added to each well at 37 ℃ in 5% CO₂The incubation was carried out in an incubator for 3 days, after which the number of living cells was analyzed by WST8 assay using living cell assay reagent SF (manufactured by Nakalai Tesque Co., Ltd.). The results are shown in FIG. 5. The value measured by WST8 was significantly lower in cells that acted simultaneously with Mab-ZAP, compared to a control antibody of the same isotype, and a cell killing effect was confirmed in all antibodies analyzed. This shows that: antibodies labelled with toxins or radioisotopesIncorporated into cells, can kill the cells into which they are incorporated.

[ example 11]Reactivity of anti-LGR 7 monoclonal antibody with mouse LGR7

Mouse LGR7 (nucleotide sequence, SEQ ID NO: 93, amino acid sequence, SEQ ID NO: 94) was inserted into an expression vector, a gene was introduced into BaF3 cells, and the resulting forced expression cell line HA-mLGR7/BaF3 was used to study the crossability with mouse LGR7 by flow cytometry. As shown in fig. 6, it was confirmed that 22DA17, 22DA23 showed crosswalk to LGR7 of mice.

[ example 12]Classification of epitopes by competitive FACS analysis

Epitopes were classified by competition FACS analysis. The antibody was biotinylated using a biotin protein labeling kit (Roche) according to the instructions. The following methods were utilized in the competitive FACS analysis: an excess amount of the non-labeled antibody was reacted in advance, then the biotinylated antibody was reacted, and the biotinylated antibody was detected by using FITC-labeled streptavidin. When the antibody recognizes the same epitope, the unlabeled antibody masks the epitope, the biotinylated antibody cannot approach the antigen, and the peak in FACS analysis shifts to the left. When the same antibody was used and the reaction was carried out in the order of unlabeled and biotinylated antibody, the position was shifted to the left side compared to the case where FITC-labeled anti-mouse antibody was used. On the other hand, in the case of an antibody recognizing a different epitope, since binding can be performed without competing with a non-labeled antibody, the shift to the left side is small. Several antibodies were biotinylated and then subjected to competition assays, some of which are shown in FIG. 7. The results of competitive FACS analysis using biotinylated antibodies Bio-22DA17, Bio-22DA22 showed: 22DA12 and 22DA22 are antibodies that recognize different epitopes from other antibodies.

[ example 13]Preparation of mouse IgG2a chimeric antibody with mouse IgG2aCH and CL

As a method for enhancing ADCC activity of an antibody, a method of modifying a sugar chain of an antibody is known. WO2006/067913 et al describe: antibodies having sugar chains without alpha-1, 6 core fucose were produced using CHO cells (CHO _ FTKO) from which fucose transporter genes were knocked out.

The procedure of example 9 was followed to amplify the H chain and L chain variable regions of the cloned anti-human LGR7 monoclonal antibody 22DA23 antibody gene by PCR, respectively, and then to link them with the H chain C region Cgamma 2a and L chain C region Ckappa of the mouse antibody, and insert them into an expression vector for mammalian cells so that they can be expressed as a chimeric molecule of mouse IgG2 a. The vector obtained was used to introduce a gene into fucose transporter deficient CHO cells CHO _ FTKO to establish a neomycin-resistant strain.

The chimeric antibody mouse IgG2a was purified from the culture supernatant using a protein A column in the presence of RPMI-1640/10% ultra-low IgG FBS (Invitrogen) in 500. mu.g/mL geneticin/penicillin-streptomycin according to the protocol. Purified antibody 22DA23-mIgG2a/FTPKO was supplied to a pharmacodynamic test in a mouse xenograft model.

[ example 14]Pharmacodynamic test using mouse xenograft model

Purified 22DA23-mIgG2a/FTPKO (FTKODA23) antibody was supplied to mice for pharmacodynamic testing. 1 piece of approximately 3mm square RMG-1 in vivo passaged tumor was subcutaneously transplanted into 7-week-old Scid female mice (Clea, Japan), and the tumors were divided into groups according to tumor volume and body weight 10 days after transplantation and subjected to the experiment. RMG-1 in vivo passaging tumors excised 42 days after subcutaneous implantation of RMG-1 cultured cells at 1e7 cells/subject were used. The FTKODA23 antibody was administered at 2mg/kg and 10mg/kg once per week to 1 group of 6 mice. The control group was given PBS (-). The first administration was performed 10 days after transplantation, the 2nd administration was performed 17 days after transplantation, the 3 rd administration was performed 24 days after transplantation, and the 4 th administration was performed 31 days after transplantation. Tumor volumes were measured 2 times per week and the final measurements were made 1 week after the 4 th dose, and the changes in tumor volume were plotted, and the results are shown in FIG. 8. The size of the tumor volume was compared between the control group and the antibody administration group. In the group administered with 10mg/kg, the TGI (tumor growth inhibition) was 37% on the average, and in the group administered with 2mg/kg, the TGI was 33% on the average, and the effect of tumor reduction was confirmed.

Industrial applicability

The anti-LGR 7 antibody of the present invention can exert an anti-cancer effect on LGR 7-expressing cells by utilizing antibody-dependent cytotoxic activity, complement-dependent cytotoxic activity, and the like, and can also bring cytotoxicity to LGR 7-expressing cells by binding toxins (cytotoxic substances), and thus is effective for diagnosis, prevention, and treatment of various primary or metastatic cancers.

The LGR7 protein specific antibody is specifically expressed in ovarian cancer, particularly in clear cell adenocarcinoma, and can be used as a diagnostic agent for the clear cell adenocarcinoma. The diagnostic agent of the present invention is effective for the diagnosis of primary or metastatic cancer. Specifically, by detecting LGR7 protein contained in a biological sample collected from a patient, the possibility of cancer can be determined. Alternatively, the presence of ovarian clear cell adenocarcinoma can be detected in an organism by detecting the distribution of LGR7 expressing cells in the organism.

The anti-LGR 7 antibody having cytotoxic activity according to the present invention is effective for the treatment or prevention of cancer expressing LGR7 protein. Specifically, according to the present invention, a cytotoxic agent or a cell proliferation inhibitor for cancer cells of an ovarian clear cell adenocarcinoma is provided. The cytotoxic agent or cell proliferation inhibitor of cancer cells of the present invention is applicable to both primary and metastatic cancers.

Also, the anti-LGR 7 antibody having cytotoxic activity according to the present invention is useful as a therapeutic agent for ovarian clear cell adenocarcinoma. In the present invention, the anti-LGR 7 antibody is also effective as a therapeutic agent for any one of primary and metastatic cancers.

Furthermore, the gene encoding an antibody and the recombinant cell transformed with the gene according to the present invention can be used for producing a recombinant antibody that exhibits the above-described effect or more suitably.

Claims (24)

1. An antibody that binds to LGR7 protein and has cell proliferation inhibitory activity against cells expressing LGR7 protein.

2. The antibody of claim 1, wherein the cell proliferation inhibitory activity is a cytotoxic activity.

3. The antibody of claim 2, wherein said cytotoxic activity is antibody-dependent cytotoxic activity.

4. The antibody of claim 2, wherein said cytotoxic activity is complement dependent cytotoxic activity.

5. The antibody of any one of claims 1 to 4, which is an antibody to which a cytotoxic substance binds.

6. The antibody of claim 5, which is an antibody having internalization activity.

7. The antibody according to any one of claims 1 to 6, which is an antibody that inhibits the proliferation of cancer cells.

8. The antibody according to claim 7, wherein the cancer cell is an ovarian cancer clear cell.

9. The antibody according to any one of the following (1) to (29):

(1) the antibody, 22DA6 heavy chain, contains an H chain having the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of CDR2 shown in SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 7;

(2) an antibody, 22DA6 light chain, comprising an L chain having the amino acid sequence of SEQ ID NO: 10, the amino acid sequence of CDR2 of SEQ ID NO: 11, and the amino acid sequence of CDR3 shown in SEQ ID NO: 12;

(3) 22DA6 which is an antibody comprising the H chain of (1) and the L chain of (2);

(4) the antibody, 22DA7 heavy chain, contains an H chain having the amino acid sequence of SEQ ID NO: 15, the amino acid sequence of CDR2 of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 17;

(5) an antibody, 22DA7 light chain, comprising an L chain having the amino acid sequence of SEQ ID NO: 20, the amino acid sequence of CDR2 of SEQ ID NO: 21 and the amino acid sequence of SEQ ID NO: 22;

(6) 22DA7 which is an antibody comprising the H chain of (4) and the L chain of (5);

(7) the antibody, 22DA17 heavy chain, contains an H chain having the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 27, an amino acid sequence of seq id no;

(8) an antibody, 22DA17 light chain, comprising an L chain having the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31 and the amino acid sequence of SEQ ID NO: 32;

(9) 22DA17 which is an antibody comprising the H chain of (7) and the L chain of (8);

(10) the antibody, 22DA22 heavy chain, contains an H chain having the amino acid sequence of SEQ ID NO: 35, the amino acid sequence of CDR2 of SEQ id no: 36 and the amino acid sequence of SEQ ID NO: 37, or a pharmaceutically acceptable salt thereof;

(11) an antibody, 22DA22 light chain, comprising an L chain having the amino acid sequence of SEQ ID NO: 40, the amino acid sequence of CDR2 of SEQ ID NO: 41 and the amino acid sequence of SEQ ID NO: 42;

(12) 22DA22 which is an antibody comprising the H chain of (10) and the L chain of (11);

(13) the antibody, 22DA23 heavy chain, contains an H chain having the amino acid sequence of SEQ ID NO: 45, and the amino acid sequence of CDR2 shown in SEQ id no: 46 and the amino acid sequence of SEQ ID NO: 47 in a sequence listing;

(14) an antibody, 22DA23 light chain, comprising an L chain having the amino acid sequence of SEQ ID NO: 50, the amino acid sequence of CDR2 of SEQ ID NO: 51, and the amino acid sequence of SEQ ID NO: 52;

(15) 22DA23 which is an antibody comprising the H chain of (13) and the L chain of (14);

(16) an antibody 22DA24 heavy chain comprising an H chain having the amino acid sequence of SEQ ID NO: 55, the amino acid sequence of CDR2 of SEQ ID NO: 56, and the amino acid sequence of SEQ ID NO: an amino acid sequence according to 57;

(17) an antibody, 22DA24 light chain, comprising an L chain having the amino acid sequence of SEQ ID NO: 60, the amino acid sequence of CDR2 of SEQ ID NO: 61, and the amino acid sequence of SEQ ID NO: 62, an amino acid sequence as described in 62;

(18) 22DA24 which is an antibody comprising the H chain of (16) and the L chain of (17);

(19) the antibody, 22SD7 heavy chain, contains an H chain having the amino acid sequence of SEQ ID NO: 65, the amino acid sequence of CDR2 of SEQ ID NO: 66 and the amino acid sequence of SEQ ID NO: 67, or a nucleotide sequence thereof;

(20) an antibody, 22SD7 light chain, comprising an L chain having the amino acid sequence of SEQ ID NO: 70, the amino acid sequence of CDR2 of SEQ ID NO: 71 and the amino acid sequence of SEQ ID NO: 72, an amino acid sequence thereof;

(21) 22SD7, which is an antibody comprising the H chain of (19) and the L chain of (20);

(22) the antibody, 22SD11 heavy chain, contains an H chain having the amino acid sequence of SEQ ID NO: 75, the amino acid sequence of CDR2 of SEQ ID NO: 76, and the amino acid sequence of SEQ ID NO: 77;

(23) an antibody, 22SD11 light chain, comprising an L chain having the amino acid sequence of SEQ ID NO: 80, the amino acid sequence of CDR2 of SEQ ID NO: 81 and the amino acid sequence of CDR3 as SEQ ID NO: 82;

(24) 22SD11 as an antibody comprising the H chain of (22) and the L chain of (23);

(25) the antibody, 22SD48 heavy chain, contains an H chain having the amino acid sequence of SEQ ID NO: 85, and SEQ ID NO: 86 and the amino acid sequence of CDR3 as shown in SEQ ID NO: 87;

(26) an antibody, 22SD48 light chain, comprising an L chain having the amino acid sequence of SEQ ID NO: 90, the amino acid sequence of CDR2 of SEQ ID NO: 91 and the amino acid sequence of CDR3 as set forth in SEQ ID NO: 92 in a sequence listing;

(27) 22SD48 as an antibody comprising the H chain of (25) and the L chain of (26);

(28) an antibody having the same activity as the antibody according to any one of (1) to (27);

(29) an antibody that recognizes the same epitope as that recognized by the antibody according to any one of (1) to (27).

10. The antibody of any one of claims 1 to 9 which has human constant regions.

11. The antibody of claim 10, which is a chimeric, humanized or human antibody.

12. The antibody of any one of claims 1 to 11 which is a fucose-deficient antibody.

13. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 12 as an active ingredient.

14. An inhibitor of cell proliferation comprising the antibody according to any one of claims 1 to 12 as an active ingredient.

15. An anticancer agent comprising the antibody according to any one of claims 1 to 12 as an active ingredient.

16. The anticancer agent according to claim 15, wherein the cancer to be treated is ovarian cancer.

17. The anticancer agent according to claim 16, wherein the ovarian cancer is clear cell adenocarcinoma.

18. A method of diagnosing cancer, comprising: detecting the LGR7 protein or a gene encoding the LGR7 protein.

19. A method of diagnosing cancer, comprising: LGR7 protein was detected.

20. The diagnostic method of claim 19, wherein detection of LGR7 protein is performed using an antibody that binds to LGR7 protein.

21. A method of diagnosing cancer, the method comprising the steps of:

(a) a step of providing a sample collected from a subject;

(b) a step of detecting LGR7 protein contained in the sample of (a) using an antibody that binds to LGR7 protein.

22. A method of diagnosing cancer, the method comprising the steps of:

(a) a step of administering to the subject an antibody having LGR7 protein binding activity and labeled with a radioisotope;

(b) and detecting the accumulation of the radioisotope.

23. The diagnostic method according to any one of claims 18 to 22, wherein the cancer to be diagnosed is ovarian cancer.

24. The diagnostic method according to claim 23, wherein the ovarian cancer is clear cell adenocarcinoma.