JP2012021943A - Method for inspecting uterine cancer, test drug for uterine cancer and antibody for uterine cancer antigen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inspecting uterine cancer with which uterine cancer tissues can be detected with high sensitivity and specificity.SOLUTION: It has been found that the uterine cancer can be inspected with high sensitivity and specificity by detecting the expression of at least one gene selected from the group consisting of CRELD1 gene, GRK5 gene and SLC25A27 gene in a biological specimen separated from a human body.

Description

  The present invention relates to a test method for endometrial cancer, a test agent for endometrial cancer, and an antibody against endometrial cancer antigen.

  Endometrial cancer is one of the malignant tumors commonly found in the female genital tract worldwide. In 2004, 199000 new cases and 55000 deaths were reported (Non-patent Document 1). . In Japan, the incidence and mortality of endometrial cancer are increasing year by year. Despite the need to develop a test method for endometrial cancer in order to reduce the incidence and mortality, an effective test method for endometrial cancer has not yet been established (Non-Patent Literature). 2).

  A common symptom of endometrial cancer is irregular uterine bleeding. Uterine content removal (cervical dilatation and intrauterine curettage) has been used for many years as a standard method for examining endometrial cancer in women with malformed uterine bleeding. Many inspection methods for women who have caused abnormal uterine bleeding such as vaginal ultrasonography, uterine body cytodiagnosis, uterine curettage by aspiration, and uterine body sampling have been reported (Non-Patent Documents 3 to 5). However, only 10% of women are diagnosed with endometrial cancer based on irregular uterine bleeding (Non-Patent Documents 6 and 7), and organic abnormalities are observed in 60 to 70% of women. (Non-Patent Document 8). As described above, these inspection methods have problems in sensitivity and specificity.

  On the other hand, in endometrial cancer, it has been reported that many types of biomolecules such as STC2 (stanniocalcin 2), MSH2 (mutSomolog 2), and MUC1 (mucin 1) are highly expressed (patents). Literature 1-5, non-patent literature 9-20), and using these as biomarkers, it is attempted to examine endometrial cancer. However, even in this method, it is difficult to clearly distinguish the endometrial cancer tissue from the normal uterine body.

JP-T-2007-515957 Special table 2003-515535 gazette Japanese National Patent Publication No. 9-506509 Japanese National Patent Publication No. 11-506940 Japanese National Patent Publication No. 9-509822

The global burden of disease: 2004 update, World Health Organization, 2008 Cancer Statistics in Japan 2009, Foundation for Promotion of Cancer Research, 2009 Smith, R.A. A. , Cookinides, V .; & Brawley, O .; W. "CA Cancer J Clin", 2009, 59, 27-41. Holst, J. et al. Koskela, O .; & VonSchoultz, B.M. , "Ann Chir Gyneacol", 1983, 72, 274-277 Gredmark, T .; , Kvint, S .; Havel, G .; & Mattsson, L .; A. , “Br J Obstet Gyneecol”, 1995, 102, 133-136. Choo, Y .; C. Mak, K .; C. , Hsu, C.I. , Wong, T .; S. & Ma, H .; K. , “Obset Gynecol”, 1985, 66, 225-228. Gull, B.M. Karlsson, B .; Milsom, I .; & Granberg, S.M. , “Am J Obstet Gynecol”, 2003, 188, 401-408. Kondo, E .; , Et al. , “Cytopathology”, 2008, 19, 28-33. Kipp, B.M. R. , Et al. "Cancer", 2008, 114, 228-235 Cheung, A.M. N. Chiu, P .; M.M. & Khoo, U.S.A. S. "Hum Pathol", 1997, 28, 91-94. Torenbeek, R.A. , Et al. , “Histopathology”, 1998, 32, 20-27. Shih, H .; C. , Et al. "Hum Pathol", 2003, 34, 471-478. Chu, P.A. G. Arber, D .; A. & Weiss, L .; M.M. "Am J Clin Pathol", 2003, 120, 64-70. Lau, S .; K. , Weiss, L .; M.M. & Chu, P.A. G. "Am J Clin Pathol", 2004, 122, 61-69. Hong, S .; C. , Et al. , "J Obstet Ginaecol Res", 2006, 32, 379-386. Kobel, M .; , Et al. , “Mod Pathol”, 2006, 19, 581-587. Galgano, M .; T.A. , Hampton, G .; M.M. & Frierson, H .; F. , Jr. , "Mod Pathol", 2006, 19, 847-853. Modica, I. et al. , Et al. "Am J Surg Pathol", 2007, 31, 744-751. Tanabbe, K .; , Et al. , "Cancer Sci", 2008, 99, 1125-1130. Zheng, W.H. , Et al. , “Am J Surg Pathol”, 2008, 32, 304-315.

  The present invention has been made in view of the above-described problems of the prior art, and an object of the present invention is to provide a method for detecting endometrial cancer, which can detect endometrial cancer tissue with high sensitivity and specificity, and the uterus. The purpose is to provide a test for body cancer. A further object of the present invention is to provide an antibody against endometrial cancer antigen useful for such a test.

  In order to solve the above problems, the present inventors first selected a cDNA encoding a protein expressed on the cell surface or secreted from the cell by the signal sequence strap method (SST-REX method). Next, monoclonal antibodies against the protein encoded by the selected cDNA were prepared, and their specificity for endometrial cancer, including known antibodies, was examined. As a result, CRELD1 protein, GRK5 protein, SLC25A27 protein, MSH2 protein, and STC2 protein are highly expressed in endometrial cancer, but not expressed in normal uterine body, and antibodies against these proteins are used. Thus, it was found that endometrial cancer tissue can be detected with high sensitivity and specificity. Furthermore, the present inventors have succeeded in determining the amino acid sequences of the variable regions of the light and heavy chains for each monoclonal antibody against CRELD1 protein, GRK5 protein, SLC25A27 protein, and STC2 protein, and completed the present invention. It came to do.

That is, the present invention relates to a test method for endometrial cancer targeting a molecule specifically expressed in endometrial cancer tissue, a test agent for endometrial cancer, and antibodies useful for these tests. The following invention is provided.
(1) A method for examining endometrial cancer, comprising detecting the expression of at least one gene selected from the group consisting of CRELD1 gene, GRK5 gene, and SLC25A27 gene in a biological sample isolated from humans,
(2) At least one gene selected from the group consisting of CRELD1 gene, GRK5 gene, and SLC25A27 gene and at least one gene selected from the group consisting of MSH2 gene and STC2 gene in a biological sample isolated from human A method for examining endometrial cancer, characterized by detecting the expression of
(3) A method for examining endometrial cancer, comprising detecting expression of a CRELD1 gene, GRK5 gene, SLC25A27 gene, MSH2 gene, and STC2 gene in a biological sample isolated from a human,
(4) The test method according to any one of (1) to (3), wherein expression of the gene is detected at a protein level,
(5) The test method according to (4), wherein detection is performed using an antibody,
(6) A diagnostic agent for endometrial cancer characterized by comprising any one of the following (a) to (c) as an active ingredient:
(A) at least one antibody selected from the group consisting of an antibody that binds to CRELD1 protein, an antibody that binds to GRK5 protein, and an antibody that binds to SLC25A27 protein (b) an antibody that binds to CRELD1 protein and binds to GRK5 protein A combination of at least one antibody selected from the group consisting of an antibody and an antibody that binds to SLC25A27 protein, and at least one antibody selected from the group consisting of an antibody that binds to MSH2 protein and an antibody that binds to STC2 protein ( c) A combination of an antibody that binds to CRELD1 protein, an antibody that binds to GRK5 protein, an antibody that binds to SLC25A27 protein, an antibody that binds to MSH2 protein, and an antibody that binds to STC2 protein.
(7) The antibody according to any one of the following (a) to (d): (a) the amino acid sequence according to SEQ ID NO: 1 to 3 or at least one of the amino acid sequences, wherein one or more amino acids are A light chain variable region comprising a substituted, deleted, added and / or inserted amino acid sequence and at least one of the amino acid sequences set forth in SEQ ID NOs: 4 to 6 or the amino acid sequence are substituted. An antibody that retains a heavy chain variable region containing a deleted, added and / or inserted amino acid sequence and binds to CRELD1 protein (b) the amino acid sequence of SEQ ID NOs: 7 to 9 or at least of the amino acid sequence A light chain variable region comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted, and SEQ ID NO: A heavy chain variable region comprising at least one of the amino acid sequences according to 10 to 12 and / or at least one of the amino acid sequences, wherein one or more amino acids are substituted, deleted, added and / or inserted, and GRK5 Antibody binding to protein (c) An amino acid sequence represented by SEQ ID NOs: 13 to 15 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A light chain variable region comprising the amino acid sequence of SEQ ID NOs: 16 to 18 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences An antibody that retains the heavy chain variable region and binds to the SLC25A27 protein (d) SEQ ID NOs: 19-2 A light chain variable region comprising the amino acid sequence according to 1 or at least one of the amino acid sequences, wherein the light chain variable region comprises an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted; And a heavy chain variable region comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences described in 1. and bound to STC2 protein Antibody to
And (8) the antibody according to any one of the following (a) to (d): (a) the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or these A light chain variable region comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences; and the amino acid sequence set forth in SEQ ID NO: 28 or the amino acid sequence An amino acid sequence from which a signal sequence has been removed, or at least one of these amino acid sequences, and a heavy chain variable region containing an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted. An antibody that binds to CRELD1 protein (b) the amino acid sequence set forth in SEQ ID NO: 30 or the amino acid sequence A light chain variable region comprising an amino acid sequence from which the signal sequence has been removed from the sequence, or at least one of these amino acid sequences, wherein one or more amino acids are substituted, deleted, added and / or inserted; In the amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and / or inserted. An antibody that retains the heavy chain variable region containing the determined amino acid sequence and binds to the GRK5 protein (c) the amino acid sequence set forth in SEQ ID NO: 34, an amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or these One or more amino acids in at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence in which no acid is substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NO: 36 or an amino acid sequence from which the signal sequence has been removed, or these An antibody that retains a heavy chain variable region comprising an amino acid sequence in which one or a plurality of amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences of and binds to the SLC25A27 protein (d) In the amino acid sequence of No. 38 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and / or inserted. A light chain variable region comprising the amino acid sequence and the amino acid set forth in SEQ ID NO: 40 A heavy chain comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequence or the amino acid sequence from which the signal sequence is removed, or at least one of these amino acid sequences An antibody that retains a variable region and binds to STC2 protein.

  According to the test method of the present invention, endometrial cancer tissue can be detected with high sensitivity and specificity. Antibodies against the proteins encoded by the CRELD1 gene, GRK5 gene, SLC25A27 gene, MSH2 gene, and STC2 gene are excellent uterine endometrial cancer screening agents. By using these antibodies in combination, the sensitivity of endometrial cancer testing can be dramatically increased.

It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of anti-ACT038-617 antibody (2D1E12I antibody, anti-CRELD1 monoclonal antibody) and endometrial cancer by immunohistochemical staining. It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of anti-ACT038-617 antibody (2D1E12I antibody, anti-CRELD1 monoclonal antibody) and a normal uterine body by the immunohistochemical staining method. It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of anti-ACT043-0113 antibody (2F11C3 antibody, anti-GRK5 monoclonal antibody) and endometrial cancer by the immunohistochemical staining method. It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of anti-ACT043-0113 antibody (2F11C3 antibody, anti-GRK5 monoclonal antibody) and a normal uterine body by the immunohistochemical staining method. It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of an anti- MSH2 monoclonal antibody and endometrial cancer by the immunohistochemical staining method. It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of an anti- MSH2 monoclonal antibody and a normal uterine body by the immunohistochemical dyeing | staining method. It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of an anti-ACT067-0116 antibody (3A8B14 antibody, anti-SLC25A27 monoclonal antibody) and endometrial cancer by the immunohistochemical staining method. It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of anti-ACT067-0116 antibody (3A8B14 antibody, anti-SLC25A27 monoclonal antibody) and a normal uterine body by the immunohistochemical staining method. It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of anti-ACT004-262 antibody (2D4C4 antibody, anti-STC2 monoclonal antibody) and endometrial cancer by the immunohistochemical staining method. It is an optical microscope observation photograph which shows the result of having analyzed the reactivity of anti-ACT004-262 antibody (2D4C4 antibody, anti-STC2 monoclonal antibody) and a normal uterine body by the immunohistochemical staining method. It is a figure which shows the result of having analyzed the reactivity of anti-ACT038-617 antibody (2D1E12I antibody) and Ba / F3 cell which expresses CRELD1 gene with a flow cytometer. It is a figure which shows the result of having analyzed the reactivity of anti-ACT043-0113 antibody (2F11C3 antibody) and Ba / F3 cell which expresses GRK5 gene with the flow cytometer. It is a figure which shows the result of having analyzed the reactivity of anti-ACT067-0116 antibody (3A8B14 antibody) and Ba / F3 cell which expresses SLC25A27 gene with the flow cytometer. It is a figure which shows the result of having analyzed the reactivity of anti-ACT004-262 antibody (2D4C4 antibody) and Ba / F3 cell which expresses STC2 gene with the flow cytometer. It is a figure which shows the amino acid sequence of the variable region of an anti-ACT038-617 antibody (2D1E12I antibody) and the result of CDR prediction. It is a figure which shows the amino acid sequence of the variable region of an anti-ACT043-0113 antibody (2F11C3 antibody), and the result of CDR prediction. It is a figure which shows the amino acid sequence of the variable region of an anti-ACT067-0116 antibody (3A8B14 antibody) and the result of CDR prediction. It is a figure which shows the amino acid sequence of the variable region of an anti-ACT004-262 antibody (2D4C4 antibody), and the result of CDR prediction.

The present invention relates to a biological sample isolated from a human,
(1) Expression of at least one gene selected from the group consisting of CRELD1 gene, GRK5 gene, and SLC25A27 gene,
(2) Expression of at least one gene selected from the group consisting of CRELD1 gene, GRK5 gene, and SLC25A27 gene, and at least one gene selected from the group consisting of MSH2 gene and STC2 gene,
Or (3) expression of CRELD1 gene, GRK5 gene, SLC25A27 gene, MSH2 gene, and STC2 gene,
A method for examining endometrial cancer is provided.

  In the present invention, “biological sample” refers to cells, tissues, body fluids, and the like derived from living bodies that are targets for detecting the expression of the CRELD1 gene and the like, and “isolated from humans” It means a state in which the cells, tissues, body fluids, etc. are completely isolated from the living body from which the cells, tissues, body fluids, etc. are extracted. The method for extracting the biological sample is not particularly limited, and a known method can be used. Examples include uterine content removal (cervical dilation and intrauterine curettage), uterine curettage by aspiration, and tampon insertion into the vagina. Among these methods, tampon insertion into the vagina is preferable from the viewpoint that cells detached from endometrial cancer or the like can be removed without invading the living body. In addition, the “living body” for extracting cells, tissues, body fluids and the like is not limited to patients with endometrial cancer, but can also be used for healthy individuals (including those who may have endometrial cancer).

  In the present invention, the “CRELD1 gene” for detecting expression is typically ACCESSION No. The “GRK5 gene” is typically a gene consisting of a DNA sequence specified by NM — 015513. NM_005308.2 is a gene consisting of a DNA sequence, and “SLC25A27 gene” is typically an ACCESSION No. NM_004277 is a gene consisting of a DNA sequence specified by NM_004277, and “MSH2 gene” is typically a gene consisting of a DNA sequence specified by ACCESSION NM_000021.1, and “STC2 gene” is typically , ACCESSION No. NM_003714.2 is a gene consisting of a DNA sequence specified by NM_003714.2. However, the DNA sequence of a gene can be mutated in nature (ie, non-artificially) due to such mutations. Therefore, in the present invention, such a natural mutant can also be a detection target.

  Further, as described above, the method for examining endometrial cancer in the present invention detects the expression of at least one gene selected from the group consisting of CRELD1 gene, GRK5 gene, and SLC25A27 gene. The expression of one or two genes may be detected, and all three genes may be detected, but from the viewpoint of high sensitivity to endometrial cancer, all three genes may be detected. preferable. Moreover, when detecting the expression of one gene and examining endometrial cancer, it is preferable to target the CRELD1 gene from the viewpoint of high sensitivity to endometrial cancer.

  In the method for examining endometrial cancer in the present invention, the detection of the expression of at least one gene selected from the group consisting of CRELD1 gene, GRK5 gene, and SLC25A27 gene and the association with endometrial cancer are already known. Detection of the expression of a gene (at least one gene selected from the group consisting of MSH2 gene and STC2 gene) can be combined. In the present invention, endometrial cancer can be examined with the highest sensitivity by detecting the expression of all of the CRELD1 gene, GRK5 gene, SLC25A27 gene, MSH2 gene, and STC2 gene ( (See Table 4).

  Moreover, in the test method for endometrial cancer in the present invention, even when the expression of any one gene selected from the group consisting of CRELD1 gene, GRK5 gene, SLC25A27 gene, MSH2 gene, and STC2 gene is detected, Endometrial cancer can be tested with extremely high specificity (see Table 4).

  In the present invention, “detecting gene expression” means both detection of the presence or absence of gene expression and detection of the degree of expression. The expression level of a gene can be grasped as an absolute amount or a relative amount. In the case of grasping the relative amount, for example, it can be determined by comparing with the gene expression level of the prepared standard sample. The “standard sample” is a sample in which whether or not the target gene is expressed is specified in advance. For example, a pathological tissue in which a site where endometrial cancer already exists can be used as the standard sample of the present invention. Moreover, the tissue (normal tissue) which does not suffer from endometrial cancer can also be used as the standard sample of the present invention.

  Further, in the present invention, “gene expression” means both transcription and translation of a gene. Therefore, “detection of gene expression” in the present invention includes both detection at the mRNA level and protein level.

  A known technique can be used for detection of gene expression in the present invention. Examples of methods for detecting at the mRNA level include RT-PCR, DNA microarray analysis, Northern blotting, in situ hybridization, dot blot, and RNase protection assay. As a method for detecting at the protein level, for example, antibodies such as immunohistochemical staining, imaging cytometry, flow cytometry, ELISA, radioimmunoassay, immunoprecipitation, immunoblotting, antibody array, etc. are used. The method of detection (immunological technique) is mentioned. In the present invention, from the viewpoint of simplicity, a method of detecting at the protein level is preferable, and a method of detecting using an antibody (immunological technique) is particularly preferable.

  In the detection method (immunological technique) using the antibody in the present invention, an antibody that binds to CRELD1 protein (anti-CRELD1 protein antibody), an antibody that binds to GRK5 protein (anti-GRK5 protein antibody), and an antibody that binds to SLC25A27 protein (Anti-SLC25A27 protein antibody), an antibody that binds to MSH2 protein (anti-MSH2 protein antibody), and an antibody that binds to STC2 protein (anti-STC2 protein antibody) are used, and the antibody is brought into contact with the protein to which each antibody binds. The CRELD1 protein and the like are detected using the binding (binding amount) of each antibody to each protein as an index. Here, “contact” means placing the antibody and the protein under physiological conditions that allow the antibody to recognize the target protein. For example, when using the antibody to stain the protein on the cell surface, immerse cells, tissues, body fluids, etc. separated from a human living body (subject) in a solution containing the antibody, or The solution containing the antibody is sufficiently dropped or sprayed on the cells, tissues, body fluids, etc., and the cells are placed under physiological conditions where the antibodies can recognize the proteins present in the cells, tissues, body fluids, etc. means.

  The “CRELD1 protein” that is the target of the antibody of the present invention is typically ACCESSION No. NP_056328 is a protein consisting of an amino acid sequence, and “GRK5 protein” is typically ACCESSION No. NP_005299 is a protein consisting of an amino sequence, and “SLC25A27 protein” is typically ACCESSION No. NP_004268.1 is a protein consisting of the amino acid sequence specified, and “MSH2 protein” is typically ACCESSION No. The “STC2 protein” is typically a protein having the amino acid sequence described in NP — 000022.1. It is a protein consisting of the amino acid sequence specified by NP_003705. In addition to a protein having such a typical amino acid sequence, a protein may naturally have a mutated amino acid. Therefore, the “protein” targeted by the antibody of the present invention includes such natural mutants. A natural variant usually consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the above typical amino acid sequence. The substitution, deletion, insertion or addition of the amino acid sequence is generally within 10 amino acids (for example, within 5 amino acids, within 3 amino acids, 1 amino acid).

  The “antibody” used in the test method of the present invention may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody. “Antibodies” also includes all classes and subclasses of immunoglobulins. “Polyclonal antibodies” are antibody preparations comprising different antibodies directed against different epitopes. The “monoclonal antibody” means an antibody (including an antibody fragment) obtained from a substantially homogeneous antibody population. In contrast to polyclonal antibodies, monoclonal antibodies are those that recognize a single determinant on an antigen. The antibody of the present invention is preferably a monoclonal antibody. The antibodies of the invention are antibodies that have been separated and / or recovered (ie, isolated) from components of the natural environment. In the present invention, the “functional fragment” of an antibody means a part (partial fragment) of an antibody that specifically recognizes a target protein. Specifically, Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers thereof.

  If the antibody of the present invention is a polyclonal antibody, an immunized animal is immunized with an antigen (such as a target protein, a partial peptide thereof, or a cell expressing these), and conventional means (eg, salting out, (Centrifuge separation, dialysis, column chromatography, etc.). Monoclonal antibodies can be prepared by a hybridoma method or a recombinant DNA method.

  The hybridoma method typically includes the method of Kohler and Milstein (Kohler & Milstein, Nature, 256: 495 (1975)). The antibody-producing cells used in the cell fusion step in this method are animals (eg, mice, rats, hamsters, rabbits, monkeys) immunized with an antigen (such as a target protein, a partial peptide thereof, or a cell expressing these). Goat) spleen cells, lymph node cells, peripheral blood leukocytes and the like. It is also possible to use antibody-producing cells obtained by allowing an antigen to act on the above-mentioned cells or lymphocytes previously isolated from an unimmunized animal in a medium. As the myeloma cells, various known cell lines can be used. The antibody-producing cells and myeloma cells may be of different animal species as long as they can be fused, but are preferably of the same animal species. The hybridoma is produced, for example, by cell fusion between a spleen cell obtained from a mouse immunized with an antigen and a mouse myeloma cell, and a hybridoma that produces a monoclonal antibody specific for the target protein is obtained by subsequent screening. Obtainable. A monoclonal antibody against the target protein can be obtained by culturing the hybridoma or from the ascites of the mammal to which the hybridoma has been administered.

  In the recombinant DNA method, a DNA encoding the antibody or peptide of the present invention is cloned from a hybridoma, a B cell or the like and incorporated into an appropriate vector, which is then introduced into a host cell (for example, a mammalian cell line, E. coli, yeast cell, insect). Cells, plant cells, etc.) to produce the antibody of the present invention as a recombinant antibody (for example, PJ Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoc). Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandam AM et al., Eur. J. Biochem. 192: 767-775 (1990). ). In the expression of the DNA encoding the antibody of the present invention, DNA encoding the heavy chain or light chain may be separately incorporated into an expression vector to transform the host cell. Host cells may be transformed into a single expression vector (see WO94 / 11523). The antibody of the present invention can be obtained in a substantially pure and uniform form by culturing the above host cell, separating and purifying it from the host cell or culture medium. For the separation and purification of the antibody, the methods used in the usual purification of polypeptides can be used. If transgenic animals (such as cows, goats, sheep or pigs) in which an antibody gene is incorporated are produced using transgenic animal production technology, a large amount of monoclonal antibody derived from the antibody gene is produced from the milk of the transgenic animal. It is also possible to obtain.

The antibody used in the test method of the present invention is preferably an anti-ACT038-617 antibody (2D1E12I antibody), an anti-ACT043-0113 antibody (2F11C3 antibody), an anti-ACT067-0116 antibody (3A8B14 antibody) described in this Example, A light chain variable region comprising light chains CDR1 to CDR3 of an anti-ACT004-262 antibody (2D4C4 antibody) (hereinafter sometimes simply referred to as “the antibody described in this Example”) and a heavy chain comprising heavy chains CDR1 to CDR3. An antibody or an amino acid sequence variant thereof that retains a chain variable region. Specifically, it is the following antibody.
<Anti-CRELD1 protein antibody>
(A) a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 1 to 3 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NOs: 4 to 6 or at least one of the amino acid sequences, wherein one or more amino acids are substituted, deleted, added and / or inserted. That binds to CRELD1 protein <anti-GRK5 protein antibody>
(B) a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 7 to 9 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10 to 12 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences; That binds to GRK5 protein <anti-SLC25A27 protein antibody>
(C) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 13 to 15 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 16 to 18 or at least one of the amino acid sequences, wherein one or more amino acids are substituted, deleted, added and / or inserted. That binds to SLC25A27 protein <anti-STC2 protein antibody>
(D) the light chain variable region comprising the amino acid sequence of SEQ ID NOs: 19 to 21 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 22 to 24 or at least one of the amino acid sequences, wherein one or more amino acids are substituted, deleted, added and / or inserted. And an antibody that binds to STC2 protein.

The antibody used in the test method of the present invention is particularly preferably an antibody or an amino acid sequence variant thereof that retains the light chain variable region and heavy chain variable region of the antibody described in this example. Specifically, it is the following antibody.
<Anti-CRELD1 protein antibody>
(A) In the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence in which the signal sequence is removed from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 28, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Alternatively, an antibody that retains a heavy chain variable region containing an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted and binds to CRELD1 protein <anti-GRK5 protein antibody>
(B) the amino acid sequence set forth in SEQ ID NO: 30 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 32, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Alternatively, an antibody that retains a heavy chain variable region containing an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and binds to GRK5 protein <anti-SLC25A27 protein antibody>
(C) the amino acid sequence set forth in SEQ ID NO: 34 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 36, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Alternatively, an antibody that retains a heavy chain variable region containing an amino acid sequence in which a plurality of amino acids are substituted, deleted, added, and / or inserted and binds to SLC25A27 protein <anti-STC2 protein antibody>
(D) In the amino acid sequence set forth in SEQ ID NO: 38 or an amino acid sequence in which the signal sequence is removed from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 40, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Alternatively, an antibody that retains a heavy chain variable region containing an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted and binds to STC2 protein. Anti-MSH2 protein antibody was used in, for example, this Example Commercially available antibodies or amino acid sequence variants thereof can also be used.

  Amino acid sequence variants of the antibodies of the invention can be made by introducing mutations into DNA encoding the antibody chain or by peptide synthesis. The site where the amino acid sequence of the antibody is modified may be the constant region of the heavy chain or light chain of the antibody as long as it has an activity equivalent to that of the antibody before modification, and the variable region (framework region and CDR). Modification of amino acids other than CDR is considered to have a relatively small effect on the binding affinity with the antigen, but at present, the amino acid of the CDR is modified to screen for an antibody having an increased affinity for the antigen. Methods are known (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-493 (2008), WO 2002/051870, J. Biol. Chem., 280: 24880-24887 (2005), Protein Engineering, Design & Selection, 21: 345-351 (2008)).

  The number of amino acids to be modified is preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids (eg, within 2 amino acids, 1 amino acid). The amino acid modification is preferably a conservative substitution. In the present invention, “conservative substitution” means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention belongs. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), neutral amino acids, amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy groups Amino acids with amino acids (serine / threonine), amino acids with sulfur (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / (Tryptophan). The amino acid sequence variant preferably has an antigen-binding activity equivalent to that of the target antibody (typically, the antibody described in this example). The binding activity to the antigen can be evaluated, for example, by preparing Ba / F3 cells expressing the antigen and analyzing the reactivity with the antibody sample with a flow cytometer (see Example 3 described later). In addition, as described above, the binding activity to the antigen can be evaluated by immunostaining of endometrial cancer tissues described in Example 4 described later.

  Once the antibody described in this example is obtained, those skilled in the art will identify peptide regions (epitope) on the protein that the antibody recognizes and prepare various antibodies that bind to that region. be able to. The epitope of an antibody can be determined by a well-known method such as examining binding to an overlapping synthetic oligopeptide obtained from the amino acid sequence of a target protein (for example, Ed Harlow and D. Lane, Using Antibodies). , A Laboratory Manual, Cold Spring Harbor Laboratory Press, US Pat. No. 4,708,871). A peptide library by phage display can also be used for epitope mapping. Whether two antibodies bind to the same or sterically overlapping epitopes can be determined by competition assays.

  In addition, as an antibody used in the test method of the present invention, an antibody to which a labeling substance is bound can be used. By detecting the label, the amount of antibody bound to the target protein can be directly measured. The labeling substance is not particularly limited as long as it can bind to an antibody and can be detected by a chemical or optical method. For example, peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin, and radioactive substances.

  In addition to directly measuring the amount of antibody bound to the target protein using an antibody bound to the labeling substance, a method using a secondary antibody bound to the labeling substance or a method of binding the secondary antibody to the labeling substance. An indirect detection method such as a method using a polymer can also be used. Here, the “secondary antibody” is an antibody exhibiting specific binding to the antibody of the present invention. For example, when the antibody of the present invention is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as the secondary antibody. Labeled secondary antibodies that can be used are commercially available for antibodies derived from various biological species such as rabbits, goats, and mice, and appropriate secondary antibodies are available depending on the biological species from which the antibody of the present invention is derived. Can be selected and used in the present invention. Instead of the secondary antibody, protein G or protein A to which a labeling substance is bound can be used.

  According to the immunohistochemical staining method of the present invention, the presence or absence of endometrial cancer, the location and situation of endometrial cancer (metastasis site, status of metastasis, invading new tissue) It is useful for the detection of endometrial cancer that is difficult to distinguish visually from the normal, especially early detection of endometrial cancer. is there. Furthermore, imaging cytometry and flow cytometry are useful in that quantitative evaluation of endometrial cancer-derived cells stained with the antibody of the present invention can be performed automatically.

  In the immunological technique of the present invention, a biological sample separated from a human can be stained and analyzed by using a plurality of types of antibodies labeled with different fluorescent dyes in combination.

  Information obtained by implementing the above method for persons other than endometrial cancer patients, that is, those who have not been certified endometrial cancer, is used to evaluate the presence or absence of endometrial cancer, etc. Available to: Based on the method of the present invention, endometrial cancer can be diagnosed based on an index that is excellent in objectivity such as staining.

  On the other hand, information obtained by carrying out the above method for patients with endometrial cancer can be used for evaluation or grasping of the patient's disease state, evaluation of therapeutic effects, and the like. For example, if the method of the present invention is performed in parallel with the treatment of endometrial cancer, the therapeutic effect can be evaluated based on the resulting information. Specifically, by carrying out the method of the present invention after drug administration, changes in the staining properties in the pathological tissue can be examined, and the therapeutic effect can be determined from the change in increase or decrease in the stained site. Thus, you may utilize the method of this invention for the monitoring of a therapeutic effect.

  The diagnosis of endometrial cancer in a subject is usually performed by a doctor (including those who have received instructions from the doctor; the same applies hereinafter), but the expression of the CRELD1 gene or the like in a pathological tissue obtained by the method of the present invention. The data on the amount is useful for a doctor's diagnosis. Therefore, the method of the present invention can also be expressed as a method of collecting and presenting data useful for diagnosis by a doctor.

  The present invention also provides a test for endometrial cancer characterized by comprising the antibody of the present invention or a combination thereof as an active ingredient. The antibody used in the test agent of the present invention may be labeled as described above. The test agent of the present invention can contain other components acceptable as a composition in addition to the antibody component. Examples of such other components include carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, labeled compounds, and secondary antibodies. It is done. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As a preservative, sodium azide, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  In addition to the above-described test agent of the present invention, a substrate necessary for detection of a label, a positive control or negative control, or a buffer used for dilution or washing of a sample can be combined, and a kit for detecting endometrial cancer It can also be. Further, when an unlabeled antibody is used as an antibody preparation, those labeled with a substance that binds to the antibody (for example, secondary antibody, protein G, protein A, etc.) can be combined. Further, the kit for detecting endometrial cancer can include instructions for using the kit.

Furthermore, the present invention relates to a light chain variable region comprising the light chains CDR1 to CDR3 of the antibody described in this Example, an antibody having a heavy chain variable region comprising the heavy chains CDR1 to CDR3, or an amino acid sequence variant thereof. Also provide. Specifically, it is the following antibody.
<Anti-CRELD1 protein antibody>
(A) a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 1 to 3 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NOs: 4 to 6 or at least one of the amino acid sequences, wherein one or more amino acids are substituted, deleted, added and / or inserted. That binds to CRELD1 protein <anti-GRK5 protein antibody>
(B) a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 7 to 9 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10 to 12 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences; That binds to GRK5 protein <anti-SLC25A27 protein antibody>
(C) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 13 to 15 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 16 to 18 or at least one of the amino acid sequences, wherein one or more amino acids are substituted, deleted, added and / or inserted. That binds to SLC25A27 protein <anti-STC2 protein antibody>
(D) the light chain variable region comprising the amino acid sequence of SEQ ID NOs: 19 to 21 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 22 to 24 or at least one of the amino acid sequences, wherein one or more amino acids are substituted, deleted, added and / or inserted. And an antibody that binds to STC2 protein.

Furthermore, the present invention also provides an antibody or an amino acid sequence variant thereof that retains the light chain variable region and heavy chain variable region of the antibody described in this Example. Specifically, it is the following antibody.
<Anti-CRELD1 protein antibody>
(A) In the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence in which the signal sequence is removed from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 28, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Alternatively, an antibody that retains a heavy chain variable region containing an amino acid sequence in which a plurality of amino acids are substituted, deleted, added, and / or inserted, and binds to CRELD1 protein. It encodes the amino acid sequence set forth in SEQ ID NO: 26 The DNA sequence of the gene is shown in SEQ ID NO: 25 and described in SEQ ID NO: 28 The DNA sequence of the gene encoding the amino acid sequence is shown in SEQ ID NO: 27.
<Anti-GRK5 protein antibody>
(B) the amino acid sequence set forth in SEQ ID NO: 30 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 32, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Alternatively, an antibody that retains a heavy chain variable region containing an amino acid sequence in which a plurality of amino acids are substituted, deleted, added, and / or inserted and binds to GRK5 protein. Note that the amino acid sequence of SEQ ID NO: 30 is encoded. The DNA sequence of the gene is shown in SEQ ID NO: 29, and the amino acid described in SEQ ID NO: 32 The DNA sequence of the gene encoding the no acid sequence is shown in SEQ ID NO: 31.
<Anti-SLC25A27 protein antibody>
(C) the amino acid sequence set forth in SEQ ID NO: 34 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 36, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Alternatively, an antibody that retains a heavy chain variable region containing an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted and binds to the SLC25A27 protein. Note that the amino acid sequence of SEQ ID NO: 34 is encoded. The DNA sequence of the gene is shown in SEQ ID NO: 33 and described in SEQ ID NO: 36. The DNA sequence of the gene encoding the listed amino acid sequence is shown in SEQ ID NO: 35.
<Anti-STC2 protein antibody>
(D) In the amino acid sequence set forth in SEQ ID NO: 38 or an amino acid sequence in which the signal sequence is removed from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 40, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Alternatively, an antibody that retains a heavy chain variable region containing an amino acid sequence in which a plurality of amino acids are substituted, deleted, added, and / or inserted, and binds to the STC2 protein. The amino acid sequence of SEQ ID NO: 38 is encoded. The DNA sequence of the gene is shown in SEQ ID NO: 37, and the amino acid described in SEQ ID NO: 40 The DNA sequence of the gene encoding the no acid sequence is shown in SEQ ID NO: 39.

  These antibodies can be used in the above-described method for examining endometrial cancer of the present invention, but may also be used for research purposes for detecting a target protein or for treating endometrial cancer. . When used for human therapeutic purposes, chimeric antibodies, humanized antibodies, or human antibodies are preferred.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example.

[Example 1] Implementation of SST-REX SST-REX was performed from various human cells in order to comprehensively obtain information on membranes or secretory genes expressed on the cell surface.

(1) Preparation of cDNA 2 × 10 ⁷ human-derived cancer cells were suspended in 1 ml of Trizol ^(R) (manufactured by Invitrogen, catalog number: 15596-026) and left for 5 minutes, and 200 μl of chloroform was added. After suspending for 15 seconds, the mixture was centrifuged at 12,000 × g for 15 minutes. The supernatant after centrifugation and 500 μl of isopropanol were mixed, and then centrifuged at 12,000 × g for 10 minutes. The obtained pellet was washed with 80% ethanol to obtain 200 μg or more of total RNA, and used for the subsequent experiments.

  All of the obtained total RNA was dissolved in 100 μl of water, and 3 μg of mRNA was obtained using FastTrack 2.0 mRNA Isolation kit (manufactured by Invitrogen, catalog number: K1593-02). Double-stranded cDNA was prepared from the obtained mRNA using SuperScript ™ Choice System (Invitrogen, catalog number: 18090-019).

(2) Integration of cDNA sequence into pMX-SST vector (chimerization)
In order to incorporate the obtained cDNA into the retroviral vector pMX-SST, 5 μg of pMX-SST vector (see Nature Biotechnology 17: 487-490 (1999)) was used at 45 ° C. in a 100 μl reaction system at 45 ° C. Time cut processing. All the reaction solutions were electrophoresed on a 1% agarose gel, and a DNA fragment having a length of about 5000 bases corresponding to the vector site was excised. Further, a DNA fragment having a length of about 5000 bases was purified using Wizard ^(R) SV Gel and PCR Clean-Up System (promega, catalog number: A9282). The DNA fragment thus obtained was treated with restriction enzyme treatment of pMX-SST vector with BstXI, and prepared to be an aqueous solution containing 50 nanograms per μl.

The previously prepared double-stranded cDNA has blunt ends and cannot be directly bound to pMX-SST treated with BstXI. Therefore, an operation was performed to give the DNA sequence after cleavage of the BstXI restriction enzyme to both ends of the double-stranded cDNA. Double-stranded cDNA was dissolved in an aqueous BstXI Adapter solution in which 9 μg of BstXI Adapter (manufactured by Invitrogen, catalog number: N408-18) was dissolved in 10 μl of water. Ligation High (TOYOBO, code number: LGK-201) 5 μl was added thereto, suspended, and reacted at 16 ° C. for 16 hours to bind BstXI Adapter and double-stranded cDNA. Thereafter, a DNA fragment having a chain length of about 400 bases or less was removed using a size fractionation column attached to SuperScript ™ Choice System (manufactured by Invitrogen, catalog number: 18090-019). Thereafter, 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added and mixed by inversion, followed by centrifugation at 20,400 × g for 30 minutes. The precipitate obtained by removing the supernatant after centrifugation was dissolved in 15 μl of water and electrophoresed on a 1.5% agarose gel. Thereafter, a gel containing a conjugate of a double-stranded cDNA fragment having a length of about 500 to about 4000 bases and BstXI Adapter was cut out, Wizard ^(R) SV Gel and PCR Clean-Up System (manufactured by promega). , Catalog number: A9292), and the conjugate of the double-stranded cDNA and BstXI Adapter was purified.

  50 ng of pMX-SST vector treated with BstXI restriction enzyme, the total amount of the obtained double-stranded cDNA and BstXI Adapter conjugate, and T4 DNA ligase were treated with 20 μl reaction system at room temperature for 3 hours, and pMX-SST The vector was subjected to restriction enzyme treatment with BstXI and the above conjugate was ligated. The composition of the reaction solution was adjusted according to the booklet.

(3) Amplification of cDNA Library A cDNA library constructed using the pMX-SST vector was introduced into E. coli and amplified. 5 μg of tRNA, 12.5 μl of 7.5 M sodium acetate, and 70 μl of ethanol were added to the cDNA library, mixed by inversion, centrifuged at 20,400 × g for 30 minutes, and the supernatant was discarded to obtain a precipitate. To the resulting precipitate, 500 μl of 70% ethanol was added, and centrifuged at 20,400 × g for 5 minutes. The supernatant was discarded, and the resulting precipitate was dissolved in 10 μl of water. In order to amplify cDNA in E. coli, 2 μl of this was mixed with 23 μl of competent cells (manufactured by Invitrogen, catalog number: 18920-015), electroporated under conditions of 1.8 kV, and 1 ml of SOC medium The whole amount was suspended. This operation was performed twice, and the SOC medium in which Escherichia coli was suspended was cultured with shaking at 37 ° C. for 90 minutes. Thereafter, the entire amount of the culture solution was put into 500 ml of LB medium containing 100 μg of ampicillin per ml of the medium, and cultured with shaking at 37 ° C. for 16 hours.

  In order to confirm the number of cDNA libraries introduced into Escherichia coli and the chain length of the cDNA bound to the pMX-SST vector, 5 μl of the culture solution was taken out and plated on an LB agar medium containing 50 μg / ml ampicillin.

As a result, growth of 150 colonies was observed on the LB agar medium plated with 5 μl. As a result, it was considered that there were 1.5 × 10 ⁷ independent cDNA libraries in 500 ml of the culture solution. In addition, a plasmid was extracted from any 16 of the colonies, treated with restriction enzyme BstXI, and the treated product was electrophoresed on a 1% agarose gel to measure the length of cDNA on the pMX-SST vector. did. As a result, the average value was about 1000 bases.

Harvested from the remaining ^{culture, 10 NucleoBond (R) AX 500} columns ( Nippon Genetics Co., catalog number: 740574) was used to purify the plasmid, and established the amplified cDNA library system.

(4) Packaging of cDNA library and implementation of SST-REX method In order to produce a retrovirus containing a pMX-SST retroviral vector RNA in which a gene derived from the cDNA library has been incorporated, the virus packaging cell Plat-E (Gene Ther. 7 (12): 1063-6 (2000) Jun) 2 × 10 ⁶ cells were suspended in a 6 cm dish containing 4 ml of DMEM medium (Wako, code number: 044-29765), and 5% CO at 37 ° C. _The culture was performed under the conditions of ₂ for 24 hours. On the other hand, 100 μl of opti-MEM (manufactured by GIBCO, product number: 31985070) and 9 μl of Fugene (manufactured by Roche, product code: 18144443) were mixed and left at room temperature for 5 minutes, then 3 μg of cDNA library was added, and 15 Left at room temperature for minutes. A solution containing the cDNA library was dropped onto the cultured Plat-E cells, and after 24 hours, the supernatant was replaced and the culture was continued under the same conditions. Further, the supernatant after 24 hours was filtered through a 0.45 μm filter.

0.5 ml of the obtained filtration supernatant was added to a 10 cm dish containing 9.5 ml of RPMI-1640 (manufactured by Kojin Bio Inc.) medium containing 4 × 10 ⁶ Ba / F3 cells.

  Furthermore, 10 μl of polybrene (manufactured by CHEMICON, catalog number: TR-1003-G) and 10 ng of IL-3 were added and cultured for 24 hours. Thereafter, the cells were washed three times with RPMI-1640 medium, suspended in 200 ml of fresh RPMI-1640 medium, and spread on 20 96-well plates to an equal volume, and selection based on the autonomous proliferation ability of Ba / F3 cells and Attempted cloning. Cells that grew after 10 to 20 days were selected based on SST-REX, and the culture was further continued until the cells grew to full wells.

(5) Analysis of gene product obtained by SST-REX Half the amount of cells obtained from each well was expanded and used as stock cells. Further, by culturing stock cells, transfectant Ba / F3 cells (hereinafter also referred to as “SST clonal cells”) expressing the incorporated cDNA-derived peptide molecules outside the cells are immunized for antibody production. The cells were used as original cells and cells to be screened. From the remaining half of the cells obtained from each well, the genome was extracted and sequenced, and the gene derived from the introduced cDNA was analyzed. In the sequence, PCR was performed on the obtained genome using LA taq DNA polymerase (TaKaRa, product number: # RR002) or PrimeSTAR MAX DNA polymerase (TaKaRa, product number: # R045A). It was. The following sequences were used as PCR primers.

SST3 'side -T7 5'-TAATACGACTCACTATAGGGGCGCGCAGCTGTAAACGGGTAG-3' (SEQ ID NO: 41)
SST5 ′ side-T3 5′-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3 ′ (SEQ ID NO: 42)

The PCR product ^{Wizard (R) SV Gel and PCR} Clean-Up System (promega Corporation, product number: A9282) was purified by using a.

Thereafter, the purified PCR product was sequenced using BigDye Terminator v3.1 Cycle sequencing (manufactured by ABI, catalog number: 4337456) and DNA sequencer ABI3100XL. The following primers were used as sequence primers.

SST5 ′ side-T3 5′-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3 ′ (SEQ ID NO: 42)

The sequence data obtained were BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) and SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/ ) Was used for analysis.

[Example 2] Preparation of monoclonal antibody As an immunized animal, mouse BALB / c was used. First, as an immunostimulant, an equal amount of TiterMax Gold (manufactured by Alexis Biochemicals, catalog number: ALX-510-002-L010) was used. 50 μl of an emulsified mixture with PBS was administered. On the next day, 5 × 10 ⁶ SST clonal cells were administered as immunogen cells, and immunogen cells were injected four times every two days. About 2 weeks after the first immunization, the excised secondary lymphoid tissue was ground to obtain a cell population containing antibody-producing cells. These cells and fusion partner cells were mixed, and a hybridoma] was prepared by cell fusion using polyethylene glycol (MERCK, catalog number: 1.09727.0100). Mouse fusion myeloma cells P3U1 (P3-X63-Ag8.U1) were used as fusion partner cells.

  The hybridoma is HAT (manufactured by Sigma-Aldrich, product number: H0262), 5% BM-condimed (manufactured by Roche, catalog number: 663573), 15% FBS, 1% penicillin / streptimimycin solution (manufactured by GIBCO). , Catalog number: 15140-122, product name: Pencillin-streptomycin liquid (hereinafter abbreviated as “P / S”)) and cultured in RPMI 1640 (Wako) selective medium for 10-14 days. Next, hybridomas that react with the immunogen cells by flow cytometry shown in Example 3 and do not react with Ba / F3 cells (negative control cells) that do not contain the antigen gene in the immunogen cells were selected. Monocloning was performed by limiting dilution to obtain a hybridoma clone that produced an antibody against a gene derived from SST-REX expressed by immunogenic cells.

  The obtained hybridoma is the required amount HT (product name: HT media supplement (50X) Hybrid-Max, manufactured by Sigma-Aldrich, product number: H0137), RPMI-1640 containing 15% FBS, 1% P / S solution. Medium was used to maintain.

  Acquisition of the purified antibody from the monocloned hybridoma was performed as follows. The hybridoma was acclimated to a serum-free medium (product name: Hybridoma-SFM, manufactured by GIBCO, catalog number: 12045-076), expanded, and cultured for a certain period to obtain a culture supernatant. Subsequently, the IgG fraction contained in this culture supernatant was purified using Protein A Sepharose (GE Healthcare, code number: 17-1279-03), MAPS-II binding buffer (BIO-RAD, catalog number: 153-). 6161), MAPS-II elution buffer (manufactured by BIO-RAD, catalog number: 153-6162). The eluted IgG was dialyzed against PBS to obtain a purified antibody fraction.

[Example 3] Antibody screening using flow cytometry The reactivity of the obtained antibody with various cells (Ba / F3 cells expressing the target gene, Ba / F3 cells not expressing the target gene, etc.) Analysis was performed using flow cytometry.

In this example, PBS containing 0.5% BSA and 2 mM EDTA was used as the cell suspension buffer and the subsequent washing buffer. Various cells (target cells) to be reacted with the antibody are prepared in a 96-well plate (BD Falcon, catalog number: 353911) so that the cell suspension contains 5 × 10 ⁴ cells per well to 100 μl. And dispensed.

  50 μl of hybridoma culture supernatant or 2 μg / ml purified antibody (hereinafter referred to as “antibody solution”) was added to each sample of the cell suspension to react the antibody with the cells. As the isotype control of the antibody solution, mouse IgG1 (manufactured by BioLegend, catalog number: 400412), mouse IgG2a (manufactured by BioLegend, catalog number: 400224), mouse IgG2b (manufactured by BioLegend, catalog number: 400324) each 2 μg / A wash buffer containing 1 ml was used. After the hybridoma culture supernatant and the target cells were reacted at room temperature for 30 minutes, the culture supernatant was removed by centrifugation at 700 × g for 2 minutes, 100 μl of washing buffer was further added, and centrifugation at 700 × g for 2 minutes was performed again. To remove the supernatant and wash the cells.

  Next, 50 μl of Goat anti-mouse IgG, F (ab ′) 2-PE (Beckman Coulter, IM0855) diluted 200-fold with a washing buffer is added to the washed cell pellet as a secondary antibody for detection. And allowed to react for 30 minutes at room temperature in the dark. After the reaction, the supernatant was removed by centrifugation at 700 × g for 2 minutes, 100 μl of washing buffer was further added, and the supernatant was removed again by centrifugation at 700 × g for 2 minutes to wash the cells. Thereafter, the cells were suspended with an appropriate amount of washing buffer, and the reactivity between the antibody and the cells was analyzed by flow cytometry (manufactured by Beckman Coulter, product number: FC500MPL).

  In the measurement of reactivity, a gate was set to select live cells from the measured values of forward scatter and side scatter. Measure the fluorescence intensity of PE based on the reactivity with the antibody against the selected live cells, and the reactivity of the isotype control is the standard, but the reactivity is recognized to the immunogen cells, but the negative control cells Hybridoma cells that produce culture supernatants that are not reactive with the above were selected as candidate clones.

Example 4 Establishment of Monoclonal Antibody Set for Detecting Endometrial Cancer Hybridoma culture supernatant (monoclonal antibody) obtained by the screening and a commercially available antibody described below (assuming high expression in endometrial cancer) In order to select antibodies having high specificity for endometrial cancer from among the reported antibodies that recognize antigens, screening by immunohistochemical staining was performed.

Immunohistochemical staining was performed as follows.
(Immunohistochemical staining)
A frozen section having a thickness of 5 μm was obtained from a frozen tissue embedded with an OCT compound (Sakura Finetech Co., Ltd.). The tissues used were cancer tissues after surgery through the Ethics Committee of the National Cancer Center and normal tissues around them. The obtained frozen section was immersed in a 4% paraformaldehyde solution at 4 ° C. for 10 minutes to fix tissues and the like in the frozen section and rinsed with ultrapure water. It was immersed in a 3% hydrogen peroxide solution / methanol solution for 30 minutes to inhibit the activity of endogenous peroxidase and rinsed twice with PBS for 5 minutes. It was immersed in 5% skim milk / PBS for 30 minutes at room temperature to inhibit nonspecific protein adsorption. After removing skim milk / PBS, the following were added and incubated for 1 hour at room temperature.
-Culture supernatant of hybridoma obtained by the screening (1: 250)
-Commercial mouse-derived monoclonal antibody Anti-CA125 monoclonal antibody (manufactured by Covance, 1:40), anti-CA15-3 monoclonal antibody (manufactured by Cosmo Bio, 1: 625, anti-CD44v6 monoclonal [CD44 variant 6] antibody (Millipore) 1: 250), anti-CD64 monoclonal antibody, anti-Cdk2 [cyclin-dependent kinase 2] monoclonal antibody, anti-Ezrin monoclonal antibody, anti-MSH2 monoclonal antibody, anti-MUC1 monoclonal antibody, and anti-Syndecan1 monoclonal antibody (manufactured by Santa Cruz) , 1:50), anti-EpCAM1 monoclonal antibody (AbD Serotec, 1: 250), anti-FcγRI [Fc fragment of IgG r eceptor] monoclonal antibody (R & D systems, 1: 250), anti-panCEACAM [pan carcinoma immunogen cell adhesion molecule] monoclonal antibody (Alexis, 1: 250)
・ A commercially available goat-derived polyclonal antibody anti-HE4 [human epidimidis protein 4] polyclonal antibody, anti-HP1γ [heterochromatin protein 1γ] polyclonal antibody, anti-IMP3 [insulin-like growth protein polyfactor 3 mRNA factor -Null antibody and anti-XTRP3 [X transporter protein 3] polyclonal antibody (manufactured by Santa Cruz, 1:50)
Polyclonal antibodies derived from commercially available rabbits Anti-Midkine polyclonal antibodies (Abcam, 1: 250) and anti-Calpain 13 polyclonal antibodies (Santa Cruz, 1:50)
In the description of the hybridoma culture supernatant and antibody, the numerical values in parentheses indicate the dilution rate when each antibody or the like is used. The anti-EpCAM monoclonal antibody was used as a positive control because it can strongly stain the epithelium of endometrial cancer tissue and normal endometrium.

  After incubation with the above antibodies and the like, the cells were rinsed twice with PBS for 5 minutes, and the sections were incubated with HRP-labeled secondary antibody for 30 minutes at room temperature. Then, after further rinsing twice with PBS for 5 minutes, it was incubated with DAB + (3,3'-diaminobenzidine tetrahydrochloride) substrate kit (manufactured by Dako) for 5 minutes. Finally, the section was rinsed, counterstained with a hematoxylin solution, and magnified 10 times with an optical microscope and observed.

Evaluation of immunohistochemical staining was performed by gynecological specialists who were not informed about the patient's prognosis. Further, the staining intensity was evaluated based on the following criteria: (−) not stained or weakly stained (+) moderately stained (2+) strongly stained.

  Using the immunohistochemical staining method, first, as a first-stage screening, frozen sections (N = 1) of endometrial cancer tissues and frozen sections of normal endometrium using 104 hybridoma culture supernatants. (N = 3) immunohistochemical staining was performed. The results obtained are shown in Table 1. In Tables 1 to 3, “EC” indicates “uterine body cancer tissue”, “N” indicates “normal uterine body”, and the shaded items are culture supernatants selected in each screening. Or it shows that it is an antibody. As a result of screening in the first stage, as a candidate for a monoclonal antibody capable of detecting endometrial cancer, it is possible to recognize an antigen that is not expressed in normal uterine but only in endometrial cancer. 36 monoclonal antibodies contained in 32 hybridoma supernatants were selected. The number of hybridoma supernatants and the number of monoclonal antibodies do not match because some hybridoma supernatants contain two or more monoclonal antibodies.

  Next, as a second stage screening, 18 types of antibodies recognizing antigens that are reported to be highly expressed in endometrial cancer were added to the 36 types of monoclonal antibodies, and a total of 54 types of antibodies were used. Immunohistochemical staining of frozen sections (N = 13) of endometrial cancer tissues and frozen sections (N = 10) of normal uterine bodies was performed. The results obtained are shown in Table 2. As a result of the second stage screening, a total of 21 types of antibodies that did not react with normal uterine bodies and reacted with several endometrial cancers were selected.

  Finally, in the third stage screening, a frozen section of endometrial cancer tissue (N = 15) and a frozen section of normal uterine body (N = 12) were added to the frozen section used in the second stage screening. Immunostaining of frozen sections of endometrial cancer tissue (N = 28) and frozen sections of normal uterine body (N = 22) was performed using the 21 types of antibodies, and never expressed in normal uterine bodies. Thus, an antibody capable of recognizing an antigen expressed only in endometrial cancer and staining cancer cells, not in the stromal site of cancer, was selected. The results are shown in FIGS. In addition, the scale bar in FIGS. 1-10 shows 200 micrometers, and in Table 3, the antibody attached | subjected with the asterisk (*) shows the antibody which dye | stains the stromal region of cancer strongly, and double asterisk (**) shows The attached antibody indicates an antibody that weakly stains the stromal site of cancer. As a result, five types of monoclonal antibodies were selected as monoclonal antibodies that do not react with normal endometrium and can detect only endometrial cancer.

Of the four antibodies obtained by screening by immunohistochemical staining, the SST clone cells used as antigens of the obtained antibodies are as follows: (1) ACT038-617_2D1E12I: Homo sapiens cysteine-rich with EGF-like domains 1 (CRELD1), transcript variant 2, mRNA (NM — 015513)
(2) ACT043-113_2F11C3: Homo sapiens G protein-coupled receptor kinase 5 (GRK5), mRNA (NM_005308)
(3) ACT067-116_3A8B14: Homo sapiens solution carrier family 25, member 27 (SLC25A27), nuclear gene encoding mitochondral protein_N, mRNA (N)
(4) ACT004-262_2D4C4: Homo sapiens stannocalcin 2 (STC2), mRNA (NM_003714.2).

  Further, the reactivity of each of the four monoclonal antibodies with each SST clonal cell used as an antigen was verified with a flow cytometer. The obtained results are shown in FIGS. As is clear from these results, it was confirmed that each monoclonal antibody reacts with each antigen.

  Moreover, as a result of determining the isotype of the produced antibody using an isostrip kit (Roche, product number: 1493027), the anti-ACT067-116 monoclonal antibody is IgM / k, and the other three are all IgG2a / k. there were. Then, immunohistochemical staining of 28 endometrial cancer tissues was performed using the 5 types of monoclonal antibodies. The results obtained are shown in Table 4.

  CREDD1 (identified to cysteine-rich with EGF-like domains 1), GRK5 (G-protein-coupled receptor kinase 5), MSH2, SLC25A27 (solute carrier family 25) The positive rates in immunohistochemical staining of endometrial cancer tissues using the monoclonal antibody of the present invention were 60.7% (17/28), 32.1% (9/28), and 35.7%, respectively. (10/28), 21.4% (6/28), and 35.7% (10/28). When all of these monoclonal antibodies were used in combination, the positive rate in immunohistochemical staining of endometrial cancer tissue was 85.7% (24/28). On the other hand, as a result of immunostaining of 22 normal uterine bodies, the negative rate in immunostaining of normal uterine bodies was 100% (22/22) for all the five monoclonal antibodies. As is apparent from these results, the antibody of the present invention is an excellent antibody capable of clearly distinguishing between endometrial cancer and normal uterine body, and by using these antibodies in combination. It is possible to provide a test agent or test method for endometrial cancer having high specificity (sensitivity) to endometrial cancer.

[Example 5] To clarify the genetic sequence of the antibody variable region determination method variable region of the four monoclonal antibodies, resulting antibody-producing cell hybridoma cells 2 × 10 ⁶ pieces of Trizol (invitrogen, Inc., catalog number: 15596-026) suspended in 1 ml and allowed to stand for 5 minutes, 200 μl of chloroform was added, suspended for 15 seconds, and then centrifuged at 12,000 × g for 15 minutes to obtain a supernatant. This supernatant was mixed with 500 μl of isopropanol, and then centrifuged at 12,000 × g for 10 minutes. The obtained pellet was washed with 80% ethanol to obtain 40 μg of total RNA. The whole amount was dissolved in 20 μl of water. Among them, using a solution for 5 μg of total RNA, a double-stranded cDNA was prepared from the total RNA using SuperScript ™ Choice System (manufactured by Invitrogen, catalog number: 18090-019). The obtained double-stranded cDNA was ethanol precipitated, and then the 5 'end and 3' end of the double-stranded cDNA were bound using LigationHigh (manufactured by TOYOBO, code number: LGK-201), 1 μl of which was used as a template. Went. Primers designed for the heavy and light chain constant regions were used. Primer sequences are as follows.

That is, the heavy chain 5′-side gtccacgagggtgctgcacaat (SEQ ID NO: 43) is used for sequencing the heavy chain of the anti-ACT038-617 antibody, anti-ACT043-113 antibody, and anti-ACT004-262 antibody (IgG2a / k).
Heavy chain 3 ′ side aggtcaaggtcactggctcaggg (SEQ ID NO: 44)
In addition, the heavy chain 5′-side ctctcagcatggaaggagag (SEQ ID NO: 45) was used for sequencing the heavy chain of the anti-ACT067-116 antibody (IgM / k).
Heavy chain 3 ′ side gatatccctggagtactcag (SEQ ID NO: 46)
Furthermore, for sequencing of the light chains of the four monoclonal antibodies, the light chain 5 ′ aagatggatacagtttgggtc (SEQ ID NO: 47)
Light chain 3 ′ side tgtcaagagctttacaacagga (SEQ ID NO: 48)
It was used.

  The PCR product was electrophoresed on a 1.5% gel, and then cut out and purified. Sequencing was performed using the purified DNA. For the light chain, the purified DNA was cloned and then sequenced. The determined light chain variable region nucleotide sequence is SEQ ID NO: 25, 29, 33, 37, the amino acid sequence is SEQ ID NO: 26, 30, 34, 38, and the heavy chain variable region nucleotide sequence is SEQ ID NO: : 27, 31, 35 and 39, the amino acid sequences are shown in SEQ ID NOs: 28, 32, 36 and 40.

  Moreover, about the amino acid sequence of these variable regions, the sequence analysis in the site of "Andrew CR Martin's Bioinformatics Group" of UCL (http://www.bioinf.org.uk/abysis/tools/analyze.cgi) The CDR regions were identified according to the criteria described in “Table of CDR Definitions” (http://www.bioinf.org.uk/abs/#kabatnum). The results of CDR prediction and the light chain and heavy chain signal sequences are shown in FIGS. Further, the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are represented by SEQ ID NOs: 1-3, 7-9, 13-15, and 19-21, and the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain are represented by SEQ ID NO: 4. ~ 6, 10-12, 16-18, 22-24.

  As described above, according to the test method for endometrial cancer of the present invention, endometrial cancer tissue can be detected with high sensitivity and specificity. The antibody of the present invention is an excellent drug for such examination. In the method for examining endometrial cancer according to the present invention, by using cells detached from endometrial cancer as a specimen, a test can be easily performed without invading the living body. As described above, the test method and test agent for endometrial cancer are extremely useful medically.

Claims (8)

  A method for examining endometrial cancer, comprising detecting the expression of at least one gene selected from the group consisting of a CRELD1 gene, a GRK5 gene, and an SLC25A27 gene in a biological sample isolated from a human.   Expression of at least one gene selected from the group consisting of CRELD1 gene, GRK5 gene, and SLC25A27 gene and at least one gene selected from the group consisting of MSH2 gene and STC2 gene in a biological sample isolated from human A method for examining endometrial cancer characterized by detecting.   A method for examining endometrial cancer, comprising detecting expression of a CRELD1 gene, a GRK5 gene, an SLC25A27 gene, an MSH2 gene, and an STC2 gene in a biological sample isolated from a human.   The test method according to any one of claims 1 to 3, wherein gene expression is detected at a protein level.   The test method according to claim 4, wherein the detection is performed using an antibody. A test for endometrial cancer comprising any one of the following (a) to (c) as an active ingredient: (a) an antibody that binds to CRELD1 protein, an antibody that binds to GRK5 protein, and SLC25A27 protein At least one antibody selected from the group consisting of antibodies that bind to (b) at least one antibody selected from the group consisting of antibodies that bind to CRELD1 protein, antibodies that bind to GRK5 protein, and antibodies that bind to SLC25A27 protein And at least one antibody selected from the group consisting of an antibody that binds to MSH2 protein and an antibody that binds to STC2 protein (c) an antibody that binds to CRELD1 protein, an antibody that binds to GRK5 protein, and binds to SLC25A27 protein Antibody, antibody binding to MSH2 protein, and STC2 protein A combination of binding antibody. The antibody according to any one of the following (a) to (d): (a) one or more amino acids in the amino acid sequence represented by SEQ ID NOs: 1 to 3 or at least one of the amino acid sequences is substituted or deleted; A light chain variable region comprising a deleted, added and / or inserted amino acid sequence, and the amino acid sequence set forth in SEQ ID NOs: 4 to 6 or at least one of the amino acid sequences, one or more amino acids are substituted or deleted An antibody that retains a heavy chain variable region comprising an added and / or inserted amino acid sequence and binds to CRELD1 protein (b) in the amino acid sequence of SEQ ID NOs: 7 to 9 or at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence wherein one or more amino acids are substituted, deleted, added and / or inserted; and SEQ ID NO: 10 12 or a heavy chain variable region comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in at least one of the amino acid sequences described in No. 12 and the GRK5 protein Binding antibody (c) A light chain comprising an amino acid sequence represented by SEQ ID NOs: 13 to 15 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences A heavy chain comprising a chain variable region and an amino acid sequence of SEQ ID NOs: 16 to 18 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences An antibody that retains the variable region and binds to the SLC25A27 protein (d) SEQ ID NOS: 19-21 Or a light chain variable region comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in at least one of the amino acid sequences or the amino acid sequences, and SEQ ID NOS: 22-24. An antibody that retains a heavy chain variable region containing an amino acid sequence or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in at least one of the amino acid sequences and binds to an STC2 protein. The antibody according to any one of the following (a) to (d): (a) the amino acid sequence of SEQ ID NO: 26, an amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least of these amino acid sequences In any case, a light chain variable region comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NO: 28 or a signal sequence is removed from the amino acid sequence And a heavy chain variable region containing an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in at least one of these amino acid sequences, the CRELD1 protein Binding antibody (b) The amino acid sequence set forth in SEQ ID NO: 30 or a sequence derived from the amino acid sequence A light chain variable region comprising an amino acid sequence from which a null sequence has been removed, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of these amino acid sequences; and SEQ ID NO: : In the amino acid sequence described in 32, or an amino acid sequence in which the signal sequence is removed from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or inserted An antibody that retains a heavy chain variable region containing an amino acid sequence and binds to GRK5 protein (c) the amino acid sequence set forth in SEQ ID NO: 34, an amino acid sequence obtained by removing a signal sequence from the amino acid sequence, or an amino acid sequence thereof 1 or more amino acids are substituted in at least one of A light chain variable region comprising a deleted, added and / or inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 36, an amino acid sequence from which the signal sequence has been removed, or at least one of these amino acid sequences In any case, an antibody that retains a heavy chain variable region comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted, and binds to SLC25A27 protein (d) described in SEQ ID NO: 38 Or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence, or at least one of these amino acid sequences, an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted A light chain variable region; and the amino acid sequence set forth in SEQ ID NO: 40 or A heavy chain variable region comprising an amino acid sequence from which the signal sequence has been removed from the amino acid sequence, or a heavy chain variable region comprising at least one of these amino acid sequences, wherein one or more amino acids are substituted, deleted, added and / or inserted And an antibody that binds to STC2 protein.