JP2021042960A - Method for differentiating malignant mesothelioma and anti-CLDN15 antibody for differentiating malignant mesothelioma - Google Patents

Abstract

To provide a biomarker for differentiating malignant mesothelioma and a method for differentiating malignant mesothelioma.SOLUTION: A biomarker consisting of a claudin 15 (CLDN15) gene or a CLDN15 protein for differentiating malignant mesothelioma is provided.SELECTED DRAWING: None

Description

The present invention relates to a biomarker for differentiating malignant mesothelioma and a method for differentiating malignant mesothelioma.

Malignant mesothelioma is a highly lethal malignant tumor derived from mesothelial cells and has been increasing worldwide in recent years (Non-Patent Document 1). The mesothelium is a membranous tissue composed of two layers, a monolayer of mesothelium cells and a connective tissue, and is a pleura, a peritoneum, a pericardium, and a testis sheath (Pleura). It forms a serosa such as the Testicular sheath membrane.

Malignant mesothelioma is histologically classified into three types: epithelioid mesothelioma, sarcomatoid mesothelioma, and biphasic mesothelioma.

There are still many unclear points about the pathogenesis of malignant mesothelioma, but it has been clarified that malignant pleural mesothelioma is particularly closely related to asbestos exposure (non-patent). Document 2; Non-Patent Document 3). It is known that malignant pleural mesothelioma usually develops after an incubation period of 30 to 40 years after the initial exposure to asbestos (Non-Patent Document 4; Non-Patent Document 5). The incidence of malignant mesothelioma is expected to peak in Japan in the latter half of the 2020s, and the development of diagnostic and therapeutic methods is expected to become even more important in the future.

Since malignant mesothelioma shows various tissue patterns similar to various malignant tumors, its differential diagnosis is difficult. In general, in order to distinguish malignant mesothelioma from other tumors, it is necessary to confirm by immunohistochemical staining that at least two kinds of positive markers for mesothelium are positive and at least two kinds of negative markers are negative. Yes (Non-Patent Document 6). Specifically, calretinin, cytokeratin 5/6 (cytokeratin 5/6; CK5 / 6), Wilms tumor 1 (WT1), and podoplanin are used as positive markers. Although these positive markers are relatively effective in differentiating epithelial malignant mesothelioma, they are less sensitive in differentiating sarcoma-type malignant mesothelioma and stain only a small number of tumor cells. It has become a problem (Non-Patent Document 7). Therefore, the development of new biomarkers that are effective in differentiating malignant mesothelioma is required.

Robinson, B. W. and Lake, R. A., N Engl J Med, 2005, 353 (15), 1591-603. Yap, T.A. et al., Nat Rev Cancer, 2017, 17 (8), 475-488. Bianchi, C. and Bianchi, T., Ind Health, 2007, 45 (3), 379-87. Wagner, J.C. et al., Br J Ind Med, 1960, 17, 260-71. Robinson, B. W. et al., Lancet, 2005, 366 (9483), 397-408. Travis W. D. et al., J Thorac Oncol, 2015, 10 (9), 1243-1260. Husain A.N. et al., Arch Pathol Lab Med, 2018, 142 (1), 89-108.

An object of the present invention is to provide a biomarker for differentiating malignant mesothelioma and a method for differentiating malignant mesothelioma.

As a result of diligent research for a new biomarker for differentiating malignant mesothelioma, the present inventors have found that the claudin 15 (CLDN15) gene or CLDN15 protein can be an extremely good biomarker. , The present invention has been completed. The present invention is based on this finding and provides the following.

(1) A biomarker consisting of a claudin 15 (CLDN15) gene or a CLDN15 protein for differentiating malignant mesothelioma.
(2) Anti-CLDN15 antibody or fragment thereof for differentiating malignant mesothelioma.
(3) The anti-CLDN15 antibody or fragment thereof according to (2), wherein the anti-CLDN15 antibody or fragment thereof recognizes an epitope contained in the intracellular domain on the C-terminal side of the CLDN15 protein.
(4) The anti-CLDN15 antibody or fragment thereof according to (3), wherein the epitope comprises the amino acid sequence shown in SEQ ID NO: 1.
(5) The anti-CLDN15 antibody or a fragment thereof is a heavy chain variable containing CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 2, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 3, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 4. (2) to (2) to (2) to include a region and a light chain variable region containing CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 5, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 7. The anti-CLDN15 antibody or fragment thereof according to any one of 4).
(6) The anti-CLDN15 according to (5), wherein the anti-CLDN15 antibody or fragment thereof contains a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 9. Antibodies or fragments thereof.
(7) A malignant mesothelioma differential kit containing the anti-CLDN15 antibody according to any one of (2) to (6) or a fragment thereof for detecting the biomarker according to (1).
(8) A method for differentiating malignant mesothelioma, which is a detection step for detecting a transcript of CLDN15 gene or CLDN15 protein in a sample derived from a region suspected to be a malignant tumor in and around the infiltration of a subject, cells. A determination step for determining whether or not the sample contains a malignant tumor based on any one or more of atypical, structural atypia, infiltration, and metastasis, and a determination step in which the sample contains a malignant tumor and the malignant tumor thereof. The above-mentioned method including a differentiation step of differentiating a subject suffering from malignant mesothelioma when a transcript of the CLDN15 gene or a CLDN15 protein is detected therein.
(9) The detection step is a step of detecting the CLDN15 protein, wherein the CLDN15 protein is detected using the anti-CLDN15 antibody or a fragment thereof according to any one of (2) to (6), (8). ).
(10) The detection step is a step of further detecting another positive marker protein for differentiating malignant mesothelioma and / or another negative marker protein for differentiating malignant mesothelioma in the sample, and the discrimination step is a step of detecting the other negative marker protein for differentiating malignant mesothelioma. When CLDN15 protein is detected in the malignant tumor and the other positive marker protein for differentiating malignant mesothelioma is detected and / or when other negative marker protein for differentiating malignant mesothelioma is not detected. The method according to (9), which is a step of differentiating the subject from having malignant mesothelioma.

According to the biomarker for differentiating malignant mesothelioma of the present invention and the discrimination method using the marker, it is possible to discriminate the presence or absence of malignant mesothelioma in the subject.

It is a figure which immunohistochemically stained the tissue specimen of epithelial type malignant pleural mesothelioma collected from the patient of malignant pleural mesothelioma with anti-CLDN15 antibody and anti-carretinin antibody. In the figure, H & E indicates hematoxylin / eosin staining, αCLDN15 indicates anti-CLDN15 antibody, and αCal indicates anti-calretinin antibody. (+) Or (-) in the figure indicates that each of the indicated markers was positive or negative. The scale bar indicates 50 μm. It is a figure which immunohistochemically stained the tissue sample of the biphasic type malignant pleural mesothelioma collected from the malignant pleural mesothelioma patient with anti-CLDN15 antibody and anti-carretinin antibody. In the figure, H & E indicates hematoxylin / eosin staining, αCLDN15 indicates anti-CLDN15 antibody, and αCal indicates anti-calretinin antibody. "Biphase (E)" and "biphasic (S)" mean epithelial and sarcomatous components in biphasic malignant pleural mesothelioma, respectively. (+) Or (-) in the figure indicates that each of the indicated markers was positive or negative. The scale bar indicates 50 μm. It is a figure which immunohistochemically stained the histological specimen of sarcoma type malignant pleural mesothelioma collected from the patient of malignant pleural mesothelioma with anti-CLDN15 antibody and anti-carretinin antibody. In the figure, H & E indicates hematoxylin / eosin staining, αCLDN15 indicates anti-CLDN15 antibody, and αCal indicates anti-calretinin antibody. (+) Or (-) in the figure indicates that each of the indicated markers was positive or negative. The scale bar indicates 50 μm. It is a figure of the immunohistochemical staining which shows the antigen specificity and the antigen reactivity of the anti-CLDN15 antibody used in this Example. CLDN1, CLDN4, CLDN5, CLDN6, CLDN9, and CLDN15 shown in the upper left of each panel indicate HEK293 cells expressing their respective CLDN proteins. Vector control shows HEK293 cells into which a plasmid vector containing no CLDN gene has been introduced. ΑCLDN15 shown in the lower right of each panel indicates that the anti-CLDN15 antibody was used as the primary antibody, and αCLDN15 (-) indicates a negative control in which the primary antibody was not used. The scale bar indicates 50 μm. It is a figure which shows the epitope mapping of the anti-CLDN15 antibody. Twenty types of amino acid (alanine or threonine) substitutions (mut1 to mut20) at the site corresponding to the antigen peptide (amino acid region in the range indicated by the box) in the C-terminal amino acid sequence of the human CLDN15 protein were prepared, and the amino acid substitutions were made. Western blots were performed using extracts of HEK293T cells expressing each body. Venus was a loading control and was detected using an anti-GFP antibody.

1. 1. Biomarker for differentiating malignant mesothelioma 1-1. Overview The first aspect of the present invention is a biomarker (biomarker for differentiating malignant mesothelioma) used for differentiating malignant mesothelioma. The biomarker for differentiating malignant mesothelioma of the present invention comprises a claudin 15 (CLDN15) gene, a transcript of the CLDN15 gene (CLDN15 transcript) or a CLDN15 protein that can be expressed in malignant mesothelioma. By using the CLDN15 transcript or CLDN15 protein as a biomarker for malignant mesothelioma, the prevalence of malignant mesothelioma can be differentiated with high sensitivity.

1-2. Definitions The terms frequently used herein are defined below.
As mentioned above, "mesothelium" is a membranous tissue composed of two layers of monolayer mesothelium cells and connective tissue, and serosa such as pleura, peritoneum, pericardium, and testicular sheath. Is forming. The parietal pleura and visceral pleura are on the pleura, and the parietal peritoneum and visceral peritoneum and pericardium are on the peritoneum. ), As well as the testicular sheath membrane.

"Malignant mesothelioma" is a malignant tumor derived from the mesothelial cells. The breakdown by the site of malignant mesothelioma is 80-85% for the pleura, 10-15% for the malignant peritoneum, 1% for the pericardium or testicular serosa, and Malignant pleural mesothelioma is the most common. .. Of the malignant pleural mesothelioma, 70-80% are associated with asbestos exposure, but the detailed pathogenic mechanism is unknown. Malignant pleural mesothelioma has a very poor prognosis. The 5-year survival rates for stage I, stage II, stage III, and stage IV are 14.6%, 4.5%, 8.0%, and 0.0%, respectively.

Malignant mesothelioma is classified into three histological types: "epithelial type (Epithelioid mesothelioma)", "sarcomatoid mesothelioma", and "biphasic mesothelioma". In epithelial malignant mesothelioma, cubic tumor cells show duct-like, pseudopapillary, or sheet-like structures and proliferate and infiltrate. Sarcoma-type malignant mesothelioma shows proliferation of spiny tumor cells associated with collagen fiber proliferation and proliferates and infiltrates. Biphasic malignant mesothelioma is a mixture of epithelial type and sarcoma type, and is characterized in that both epithelial type and sarcoma type tumor components account for 10% or more of the entire tumor. The histological breakdown of malignant mesothelioma is epithelial type (about 70%), biphasic type (about 20%), and sarcoma type (about 10%). Malignant mesothelioma has various histological types and is difficult to distinguish from other malignant tumors and Leydig cell tumors. Here, other malignant tumors include various primary malignant tumors of the lung and metastatic malignant tumors. Examples include, but are not limited to, lung adenocarcinoma, squamous cell carcinoma of the lung, sarcomatoid carcinoma of the lung, and metastatic malignancies such as ovarian serous adenocarcinoma and synovial sarcoma.

As used herein, the term "differentiation" is used to determine whether or not a subject has malignant mesothelioma, or whether or not the subject has malignant mesothelioma or other malignant tumors. To do.

As used herein, the term "subject" refers to a human individual who provides a sample and is subjected to a test. In principle, it is an individual, but the present specification may sometimes include tissues and cells derived from humans. Further, the individual may be not only a healthy body but also a patient having some kind of disease (for example, malignant tumor) or an individual who may be affected by the disease (for example, malignant tumor).

As used herein, the term "healthy body" refers to a human individual who does not suffer from at least malignant mesothelioma, preferably a human individual who does not suffer from any disease. However, in the present specification, healthy human cells are also included in a healthy body in a broad sense. Therefore, if it is in a healthy state not only at the individual level but also at the cellular level, such as a normal part of a tissue collected from a patient with malignant mesothelioma, it is referred to as a healthy body.

In the present specification, the "biomarker used for differentiating malignant mesothelioma (biomarker for differentiating malignant mesothelioma)" refers to differentiating the presence or absence of malignant mesothelioma, or malignant mesothelioma and others. A biomarker that can be differentiated from malignant tumors. Specifically, the biomarkers for differentiating malignant mesothelioma include the CLDN15 gene, CLDN15 transcript or CLDN15 protein, and known biomarkers for differentiating malignant mesothelioma. In addition, the biomarkers for differentiating malignant mesothelioma are a positive marker that distinguishes the patient from malignant mesothelioma when the marker is positive, and a malignant mesothelioma when the marker is negative. Includes both negative markers that distinguish it from suffering from. In addition, the biomarker for differentiating malignant mesothelioma shall include a gene and any product (for example, a transcript and a translation product) that can be produced by gene expression. In the present specification, in particular, when the biomarker for differentiating malignant mesothelioma is a gene, it is referred to as "marker gene for differentiating malignant mesothelioma", and when the biomarker for differentiating malignant mesothelioma is a protein. It is called "marker protein for differentiating malignant mesothelioma".

In the conventional differentiation of malignant mesothelioma, as positive markers for epithelial mesothelioma, calretinin, cytokeratin 5/6 (cytokeratin 5/6; CK5 / 6), Wilms tumor 1 (WT1), podoplanin ( podoplanin) etc. are used. Here, "cytokeratin 5/6" means cytokeratin 5 (cytokeratin 5; CK5) and / or cytokeratin 6 (cytokeratin 6; CK6). Since the antibody usually used for differentiation is a mixed antibody capable of detecting both cytokeratin 5 and cytokeratin 6, such a notation is generally adopted, and this notation also follows this notation in principle. AE1 / 3, carretinin, WT1, podoplanin and the like are used as positive markers for sarcoma-type mesothelioma. Of these, carretinin is a good marker for epithelial mesothelioma, but the positive rate for sarcoma-type mesothelioma is about 30%, and the sensitivity is low. In addition, since these positive markers can be expressed in other malignant tumors, their use alone is not sufficient for differentiating malignant mesothelioma. For example, it is known that carretinin is expressed in lung adenocarcinoma, ovarian serous adenocarcinoma, Leydig cell tumor, etc., WT1 is ovarian serous adenocarcinoma, and podoplanin is also expressed in lung adenocarcinoma. Therefore, negative markers are also used in combination with positive markers in the differentiation of malignant mesothelioma.

For example, in the differentiation between malignant mesothelioma and lung adenocarcinoma, CEA (Carcinoembryonic antigen), TTF-1 (Thyroid transcription factor-1), claudin 4 (CLDN4), etc. are used as negative markers for malignant mesothelioma. .. In addition, in the differentiation between malignant mesothelioma and lung squamous epithelial cancer, p63, p40, desmocollin-3, etc. are used as negative markers for malignant mesothelioma.

In general, the diagnosis of malignant mesothelioma requires that at least two positive markers are positive and two negative markers are negative. On the other hand, if only one of the positive markers is positive or if the negative marker is positive, the possibility of false positives or false negatives cannot be ruled out, so careful diagnosis is required.

"Claudin (CLDN) protein" is a general term for four transmembrane proteins essential for the formation of tight junctions. Claudin has a function of sealing adjacent cells such as between epithelial cells and vascular endothelial cells and preventing various molecules from passing between the cells. Claudins form a family of more than 24 species in humans and mice and exhibit tissue-specific or cell-specific expression patterns. Claudins are also known to show abnormal expression in various oncogenes (Oliveira, SS and Morgado-Diaz, JA, Cell Mol Life Sci, 2007, 64 (1), 17-28; Osanai, M. et al., Pflugers Arch, 2017, 469 (1), 55-67; Tabaries, S. and Siegel PM, Oncogene, 2017, 36 (9), 1176-1190). Only two reports have been reported on claudin expression in normal lining tissue (Inai T. et al., Arch Histol Cytol., 2005, 68 (5), 349-360; Markov et al., Respir Physiol. Neurobiol., 2011, 175 (3), 331-5), there is insufficient knowledge about its expression profile.

The "CLDN15 protein" is a member of the claudin family and is a highly conserved protein among species. In humans, it is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 18 and consisting of 228 amino acid residues. In the present specification, the CLDN15 protein basically refers to the CLDN15 protein derived from humans, but 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% with respect to the amino acid sequence shown in SEQ ID NO: 18. Includes a mutant CLDN15 protein having 98% or more, 99% or more identity, or having one or more amino acids added, deleted, or substituted to the amino acid sequence shown in SEQ ID NO: 18. It shall be a protein.

In the present specification, the term "plurality" means, for example, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 10. 8 pieces, 2-7 pieces, 2-6 pieces, 2-5 pieces, 2-4 pieces or 2-3 pieces. Further, in the present specification, "amino acid identity" means the ratio (%) of the number of matching amino acid residues to the total number of amino acid residues of the two amino acid sequences to be compared. Specifically, the two amino acid sequences are aligned, and if necessary, gaps are appropriately inserted in one or both of them. At this time, 1 gap is counted as 1 amino acid residue in the total number of amino acid residues. Amino acid sequence alignment can be performed using, for example, known programs such as Blast, FASTA, Clustal W (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-. 5877; Altschul, S.F. et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, WR et al., 1988, Proc. Natl. Acad. Sci. USA, 85 : 2444-2448). If the total number of amino acid residues differs between the two amino acid sequences to be compared, the longer one is taken as the total number of amino acid residues. It is calculated by dividing the number of the same amino acid residues when the degree of amino acid matching is highest in the two amino acid sequences to be compared by the total number of amino acid residues.

As used herein, the term "substitution (of amino acids)" refers to a group of conservative amino acids having similar properties such as charge, side chain, polarity, and aromaticity among the 20 amino acids that make up a natural protein. Refers to replacement. For example, uncharged polar amino acids (Gly, Asn, Gln, Ser, Thr, Cys, Tyr) with low side chains, branched amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile). , Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Asn, Gln, Thr, Ser, Tyr, Cys), basic amino acids (Asn, Gln, Thr, Ser, Tyr, Cys) Arg, Lys, His), substitutions within the aromatic amino acid group (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferable because it is known that the properties of peptides are unlikely to change.

1-3. Composition The biomarker for differentiating malignant mesothelioma of this embodiment consists of CLDN15 gene, CLDN15 transcript or CLDN15 protein. The biomarker for differentiating malignant mesoderma consisting of the CLDN15 gene, CLDN15 transcript or CLDN15 protein of the present invention is shown by SEQ ID NO: 19, an mRNA consisting of an RNA sequence corresponding to the CLDN15 cDNA sequence (base sequence shown by SEQ ID NO: 19). Variant CLDN15 having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with respect to the RNA sequence corresponding to the base sequence. A mutant CLDN15 mRNA in which one or more nucleotides are added, deleted, or substituted to the mRNA or the RNA sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 19, or CLDN15 consisting of the amino acid sequence shown in SEQ ID NO: 18. Mutations having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with respect to the protein and the amino acid sequence shown by SEQ ID NO: 18. A type CLDN15 protein, or a mutant CLDN15 protein in which one or more amino acids are added, deleted, or substituted with respect to the amino acid sequence shown in SEQ ID NO: 18.

The malignant mesothelioma differentiated using the biomarker for differentiating malignant mesothelioma consisting of the CLDN15 gene or CLDN15 protein of the present invention is epithelial malignant mesothelioma, sarcoma-type malignant mesothelioma, and biphasic malignant medium. It may be any mesothelioma.

In addition, the malignant mesothelioma differentiated using the biomarker for differentiating malignant mesothelioma consisting of the CLDN15 gene, CLDN15 transcript or CLDN15 protein of the present invention is the pleura (for example, parietal pleura and / or visceral peritoneum). , Peritoneum (eg, parietal pleura and / or visceral peritoneum), pericardium (epicardium), and / or malignant mesothelioma of the testis sheath. For example, the malignant mesothelioma to be differentiated by the biomarker for differentiating malignant mesothelioma of the present invention may be a malignant mesothelioma other than the pleura.

By using the biomarker for differentiating malignant mesothelioma of this embodiment, it is possible to discriminate the presence or absence of malignant mesothelioma in the subject with high sensitivity. In particular, by using the biomarker for differentiating malignant mesothelioma of this embodiment in combination with other biomarkers for differentiating malignant mesothelioma, the presence or absence of malignant mesothelioma in the subject can be differentiated with high sensitivity. Can be done.

2. Anti-CLDN15 antibody for differentiating malignant mesothelioma or a fragment thereof 2-1. Overview A second aspect of the present invention is an anti-CLDN15 antibody for differentiating malignant mesothelioma (anti-CLDN15 antibody for differentiating malignant mesothelioma) or a fragment thereof. The anti-CLDN15 antibody of the present invention or a fragment thereof is a malignant mesothelioma in a subject by detecting the CLDN15 protein, which is a biomarker for differentiating malignant mesothelioma according to the first aspect, which can be expressed in malignant mesothelioma. Can be differentiated.

2-2. Structure The anti-CLDN15 antibody of the present invention or a fragment thereof will be specifically described below.

(1) Anti-CLDN15 antibody “Anti-CLDN15 antibody” refers to an antibody that exhibits immunoreactivity to a fragment containing a CLDN15 protein or an epitope.
The species from which the anti-CLDN15 antibody of the present invention is derived is not particularly limited. Antibodies derived from birds and mammals are preferred. For example, chickens, ostriches, mice, rats, guinea pigs, rabbits, goats, donkeys, ovis aries, camels, horses, humans and the like can be mentioned.

The anti-CLDN15 antibody of the present invention may be either a monoclonal antibody or a polyclonal antibody as long as it is an antibody that recognizes the CLDN15 protein and exhibits immunoresponsiveness. A monoclonal antibody having a stable antibody titer is preferable. As used herein, the term "polyclonal antibody" refers to a group of different immunoglobulins that can specifically bind to and recognize an antigen. Further, in the present specification, the term "monoclonal antibody" refers to a framework region (hereinafter referred to as "FR") and a complementarity determining region (hereinafter referred to as "CDR"). A single type of immunoglobulin that contains, specifically binds to and recognizes an antigen, or at least one set of light chain variable regions ( _VL regions) and heavy chain variable regions (V) contained in an immunoglobulin. Refers to a recombinant antibody or synthetic antibody that includes the _{H region).}

When the anti-CLDN15 antibody is composed of immunoglobulin molecules, the immunoglobulin is in any class (eg IgG, IgE, IgM, IgA, IgD and IgY) or any subclass (eg IgG1, IgG2, IgG3, IgG4, It can be IgA1, IgA2).

The position of the epitope of the CLDN15 protein recognized by the anti-CLDN15 antibody of the present invention is not particularly limited. For example, it may be an epitope contained in the intracellular domain on the C-terminal side. The C-terminal intracellular domain of the CLDN15 protein corresponds to positions 186 to 228 in the human CLDN15 protein consisting of the amino acid sequence shown in SEQ ID NO: 18. Specific examples of the epitope include the epitopes (SEQ ID NO: 1) shown in Table 1 below. This epitope corresponds to positions 219 to 226 in the human CLDN15 protein consisting of the amino acid sequence shown in SEQ ID NO: 18. Specific examples of such a monoclonal antibody include the rat anti-human CLDN15 monoclonal antibody clone 2C11 described in Example 1 described later. The 2C11 antibody has a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 9. According to Kabat's rules (Kabat EA, et al., 1991, Sequences of proteins of immunological interest, Vol.1, eds. 5, NIH publication), in the heavy chain variable region of the 2C11 antibody, CDR1 was assigned to SEQ ID NO: 2. It consists of the amino acid sequences shown, CDR2 consists of the amino acid sequence shown in SEQ ID NO: 3, and CDR3 consists of the amino acid sequence shown in SEQ ID NO: 4. Further, in the light chain variable region of the 2C11 antibody, CDR1 consists of the amino acid sequence shown in SEQ ID NO: 5, CDR2 consists of the amino acid sequence shown in SEQ ID NO: 6, and CDR3 consists of the amino acid sequence shown in SEQ ID NO: 7.

Examples of the nucleic acid (nucleotide) encoding the amino acid sequence shown in SEQ ID NO: 8 corresponding to the heavy chain variable region of the 2C11 antibody include a nucleic acid consisting of the base sequence shown in SEQ ID NO: 16. Further, as a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 9 corresponding to the light chain variable region of the 2C11 antibody, for example, a nucleic acid consisting of the base sequence shown in SEQ ID NO: 17 can be mentioned. Further, examples of the base sequence encoding CDR1, CDR2, and CDR3 of the heavy chain variable region in the 2C11 antibody include nucleic acids consisting of the base sequences shown in SEQ ID NOs: 10, 11, and 12, respectively. In addition, examples of the base sequences encoding CDR1, CDR2, and CDR3 of the light chain variable region of the 2C11 antibody include nucleic acids consisting of the base sequences shown in SEQ ID NOs: 13, 14, and 15, respectively.

"Recombinant antibody" refers to a chimeric antibody or a humanized antibody. A "chimeric antibody" is an antibody produced by combining amino acid sequences of antibodies derived from different animals, in which the constant region (C region) of a certain antibody is replaced with the C region of another antibody. For example, an antibody in which the C region of a rat monoclonal antibody is replaced with the C region of a human antibody is applicable. To give a specific example, the heavy chain variable region of a human antibody against an arbitrary antigen is replaced with the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 in the above-mentioned 2C11 antibody, and the light chain variable region of a human antibody is also replaced. Can be mentioned as an antibody obtained by substituting a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 9. This can reduce the immune response to the antibody in the human body. A "humanized antibody" is a mosaic antibody in which the CDR in a human antibody is replaced with the CDR in an antibody derived from a non-human mammal. The variable region (V region) of the immunoglobulin molecule consists of four FRs (FR1, FR2, FR3 and FR4) and three CDRs (CDR1, CDR2 and CDR3) from the N-terminal side. It is configured by concatenating in the order of CDR3-FR4. Of these, FR is a relatively conserved region that constitutes the skeleton of the variable region, and CDR directly contributes to the antigen-binding specificity of the antibody. The humanized antibody is, for example, a set of CDR1, CDR2 and CDR3 in the light chain or heavy chain of a rat-derived anti-CLDN15 antibody and a set of CDR1, CDR2, and CDR3 in the light chain or heavy chain of a human antibody against any antigen. By substituting each with CDR3, it can be constructed as a human antibody that inherits the antigen-binding specificity of the rat anti-CLDN15 antibody. Specific examples include CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 2 derived from the heavy chain in the above-mentioned 2C11 antibody, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 3, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 4. Is replaced with the heavy chain CDR1, CDR2, and CDR3 of the human antibody, respectively, and CDR1 consisting of the amino acid sequence shown by SEQ ID NO: 5 derived from the light chain in the above-mentioned 2C11 antibody, CDR2 consisting of the amino acid sequence shown by SEQ ID NO: 6, and Examples thereof include antibodies obtained by substituting CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 7 with the light chains CDR1, CDR2, and CDR3 of human antibodies, respectively. Since such a humanized antibody is derived from a human antibody other than CDR, the immune reaction against the antibody in the human body can be reduced more than that of a chimeric antibody.

"Synthetic antibody" refers to an antibody synthesized chemically or by using a recombinant DNA method. For example, an antibody newly synthesized using the recombinant DNA method can be mentioned. Specific examples thereof include scFv (single chain Fragment of variable region), diabody, triabody, tetrabody and the like. In an immunoglobulin molecule, a set of variable regions (light chain variable region V _L and heavy chain variable region V _H ) that form a functional antigen binding site are located on separate polypeptide chains, the light chain and the heavy chain. To do. _{scFv is a synthetic antibody having a structure in which V L} and V _H are linked by a flexible linker of sufficient length and contained in a single polypeptide chain in an immunoglobulin molecule and having a molecular weight of about 35 kDa or less. Within scFv, a set of variable regions can self-assemble with each other to form one functional antigen-binding site. The scFv can be obtained by incorporating the recombinant DNA encoding it into a vector using a known technique and expressing it. A diabody is a molecule with a structure based on the dimeric structure of scFv (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448). For example, if the length of the linker is shorter than about 12 amino acid residues, the two variable regions within the scFv cannot self-assemble, but by interacting the two scFvs to form a diabody, one scFv. V _{L can be assembled with V H of the} other sc Fv to form two functional antigen binding sites. Furthermore, by adding a cysteine residue to the C-terminal of scFv, a disulfide bond between two scFvs becomes possible, and a stable diabody can be formed. Thus, the diabody is a divalent antibody fragment. Triabodies and tetrabodies are trivalent and tetravalent antibodies having a trimer and tetramer structure based on the scFv structure similar to diabody, respectively. Diabodies, triabodies, and tetrabodies may be multispecific antibodies. The “multispecific antibody” refers to a multivalent antibody, that is, an antibody having a plurality of antigen binding sites in one molecule, in which each antigen binding site binds to a different epitope. For example, in the diabody, a bispecific antibody (Bispecific antibody) in which each antigen-binding site binds to a different epitope can be mentioned. Specifically, for example, in the case of the anti-CLDN15 antibody of the present invention, one antigen-binding site binds to an epitope consisting of the amino acid sequence shown in SEQ ID NO: 1, and the other antigen-binding site is the amino acid sequence shown in SEQ ID NO: 1. A diabody that binds to an epitope on a CLDN15 protein other than the epitope consisting of is applicable.

The anti-CLDN15 antibody of the present invention can also be modified. The term "modification" as used herein includes functional modification required for antigen-specific binding activity such as glycosylation and labeling modification required for antibody detection.

Glycosylation modifications on the anti-CLDN15 antibody are performed to regulate the affinity of the anti-CLDN15 antibody for the target CLDN15 protein. Specifically, for example, in the FR of the anti-CLDN15 antibody, a modification that causes loss of glycosylation at the site by introducing a substitution into an amino acid residue constituting glycosylation to remove the glycosylation site can be mentioned. ..

Labeling of anti-CLDN15 antibody includes, for example, fluorescent dyes (FITC, Rhodamine, Texas Red, Cy3, Cy5), fluorescent proteins (eg PE, APC, GFP), enzymes (eg horseradish peroxidase, alkaline phosphatase, glucose oxidase). ), Radioisotope (eg, ³ H, ¹⁴ C, ³⁵ S) or labeling with biotin or (streptavidin) avidin.

The anti-CLDN15 antibody of the present invention ^{preferably has a dissociation constant of 10-7} M or less with the CLDN15 protein. It preferably has a high affinity of 10 ^{-8 M or less, more preferably 10 -9} M or less, and particularly preferably 10 ^-10 M or less. The dissociation constant can be measured using a technique known in the art. For example, it may be measured by the Biacore system (GE Healthcare) using the speed evaluation kit software.

(2) Fragment As used herein, the term "fragment thereof" refers to an antibody fragment consisting of a part of an anti-CLDN15 antibody and exhibiting an immune response to a CLDN15 protein in the same manner as the anti-CLDN15 antibody. For example, Fab, F (ab') ₂ , Fab', etc. are applicable.

Fab is an antibody fragment produced IgG molecules are cleaved at the N-terminus side from the disulfide bonds in the hinge portion Papain, H chain constant region: constituting (heavy chain constant region is denoted by the following C _H) 3 _{It is composed of C H} 1 and V _H adjacent to _{V H} in one domain (C _H 1, C _H 2, C _H 3), and a full-length L chain.

F (ab') ₂ is a dimer of Fab'generated by pepsin cleaving the IgG molecule on the C-terminal side of the disulfide bond at the hinge. Fab'has a slightly longer H chain than Fab because it includes a hinge portion, but has a structure substantially equivalent to that of Fab. Fab'can be obtained by reducing F (ab') ₂ under mild conditions and breaking the disulfide link in the hinge region. Since all of these antibody fragments include an antigen-binding site, they have the ability to specifically bind to an antigen epitope.

2-3. Preparation of anti-CLDN15 antibody The anti-CLDN15 antibody of the present invention can be obtained by a conventional method in the art. Further, if the amino acid sequence of the monoclonal antibody is clear, it can be prepared by using a chemical synthesis method or a recombinant DNA technique based on the amino acid sequence. In addition, monoclonal antibodies can also be obtained from hybridomas that produce the antibody.

The antigenic peptide that can be used as an immunogen for the anti-CLDN15 antibody of the present invention is any part of the CLDN15 protein consisting of the amino acid sequence shown in SEQ ID NO: 18 (hereinafter referred to as "CLDN15 antigen peptide"). For example, the antigen peptide that can be used as an immunogen for the anti-CLDN15 antibody of the present invention is a part located near the carboxy terminal of the human CLDN15 protein consisting of the amino acid sequence shown in SEQ ID NO: 18, and is the amino acid shown in SEQ ID NO: 1. Examples thereof include peptides comprising an epitope consisting of a sequence, for example, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 20. This CLDN15 antigen peptide corresponds to positions 205 to 228 in the human CLDN15 protein consisting of the amino acid sequence shown in SEQ ID NO: 18. The CLDN15 antigen peptide can be prepared, for example, using a chemical synthesis method or a DNA recombination technique.

3. 3. Malignant mesothelioma differentiation kit 3-1. Overview A third aspect of the present invention is a malignant mesothelioma differentiation kit for detecting a biomarker for differentiating malignant mesothelioma. The malignant mesothelioma differentiation kit of the present invention contains the anti-CLDN15 antibody of the second aspect or a fragment thereof having immune responsiveness as an essential component, and is a marker protein for differentiating malignant mesothelioma other than the CLDN15 protein (hereinafter, "" An antibody against an antibody (hereinafter referred to as "another antibody for differentiating malignant mesothelioma") against "another marker protein for differentiating malignant mesothelioma" or a fragment thereof having immunoresponsiveness is included as a selective component. According to the malignant mesothelioma differentiation kit of the present invention, a biomarker for differentiating malignant mesothelioma that may be present in a sample derived from a subject can be detected with high sensitivity.

3-2. Configuration 3-2-1. Essential Components The malignant mesothelioma differentiation kit of this embodiment contains the anti-CLDN15 antibody of the second aspect or a fragment thereof as an essential component.
The anti-CLDN15 antibody contained in the malignant mesothelioma differentiation kit of the present invention may be a single type or a plurality of types. When a plurality of anti-CLDN15 antibodies are contained, for example, at least one is an antibody exhibiting immunoresponsiveness to an epitope consisting of the amino acid sequence shown in SEQ ID NO: 1, and the other anti-CLDN15 antibody is shown in SEQ ID NO: 1. Antibodies that recognize epitopes on CLDN15 proteins other than epitopes consisting of amino acid sequences are preferred.

3-2-2. Selected components The malignant mesothelioma differentiation kit of this embodiment has one or more types of selected components, for example, 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, and 9 types. It may further contain a type, 10 types, 11 types, 12 types, or 12 or more types of other malignant mesothelioma differential antibodies or fragments thereof having immunoresponsiveness. Here, the other antibody for differentiating malignant mesothelioma may be any antibody as long as it can improve the sensitivity for differentiating malignant mesothelioma when used in combination with the above-mentioned anti-CLDN15 antibody. It may be either an antibody against a positive marker of the tumor or an antibody against a negative marker. For example, the malignant mesothelioma differentiation kit of this embodiment has antibodies against positive markers of 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, or 6 or more types of malignant mesothelioma as selective constituents. Alternatively, it has an immunoresponsive fragment and / or an antibody or immune responsiveness to negative markers of 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, or 6 or more types of malignant mesothelioma. The fragment may be further included. Specific examples of antibodies against positive markers include antibodies against AE1 / 3, carretinin, cytokeratin 5, cytokeratin 6, cytokeratin 5/6, WT1, or podoplanin. Specific examples of antibodies against negative markers include antibodies against CEA, TTF-1, CLDN4, p63, p40, or desmocollin 3.

The other antibody for differentiating malignant mesoderma may be either a monoclonal antibody or a polyclonal antibody as described in the second aspect, and the immunoglobulin constituting the antibody is in any class or any subclass. It may be derived from any animal including mammals and birds, and may be an artificially produced antibody, for example, a recombinant antibody, a synthetic antibody, or an antibody fragment. The morphology of these antibodies has been described in the second aspect, and detailed description thereof will be omitted here.

When the selected component of the malignant mesothelioma differentiation kit of this embodiment contains an anti-carretinine antibody, the anti-carletinine antibody recognizes a human carretinin protein having the amino acid sequence shown in SEQ ID NO: 21 and exhibits immunoresponsiveness. As long as it is, there is no particular limitation. For example, a rabbit anti-calretinin antibody (Life Technologies; product number 081211) can be used.

In addition, when the selected component of the malignant mesothelioma differentiation kit of this embodiment contains an anti-cytokeratin 5 antibody, the anti-cytokeratin 5 antibody recognizes a human cytokeratin 5 protein having the amino acid sequence shown in SEQ ID NO: 22. , As long as it is an antibody exhibiting immune responsiveness, it is not particularly limited.

When the selected component of the malignant mesothelioma differentiation kit of this embodiment contains an anti-cytokeratin 6 antibody, the anti-cytokeratin 6 antibody recognizes and immunizes the human cytokeratin 6 protein having the amino acid sequence shown in SEQ ID NO: 23. The antibody is not particularly limited as long as it is a responsive antibody.

When an anti-cytokeratin 5/6 antibody is included in the selected component of the malignant mesodenal tumor differential kit of this embodiment, the anti-cytokeratin 5/6 antibody recognizes both cytokeratin 5 and cytokeratin 6 and provides an immune response. As long as it is an antibody showing sex, it is not particularly limited. For example, an anti-CK5 / 6 antibody (DAKO; product number M7237; trade name Monoclonal Mouse Anti-Human Cytokeratin 5/6 (Clone. D5 / 16 B4)), which is a mixed antibody, can be used.

When the selected component of the malignant mesothelioma differentiation kit of this embodiment contains an anti-WT1 antibody, the anti-WT1 antibody recognizes the human WT1 protein having the amino acid sequence shown in SEQ ID NO: 24 and exhibits immunoresponsiveness. As long as it is, there is no particular limitation. For example, anti-WT1 antibody (Leica; product number PA0562)
; Product name BOND Ready-to-Use Primary Antibody Wilms'Tumor (WT49)), Anti-WT1 antibody (Novocastra; Product number NCL-L-WT1-562; Product name Liquid Mouse Monoclonal Antibody Wilms' Tumor), or Anti-WT1 antibody (Dako; product number M3561; product name Monoclonal Mouse Anti-Human Wilms' Tumor 1 (WT1) (Clone 6F-H2)) can be used.

When the selected component of the malignant mesothelioma differentiation kit of this embodiment contains an anti-podopranin antibody, the anti-podopranine antibody recognizes a human podoplanin protein having the amino acid sequence shown in SEQ ID NO: 25 and exhibits immunoresponsiveness. As long as it is an antibody, it is not particularly limited. For example, a D2-40 antibody (DAKO; product number M3619; trade name monoclonal Mouse anti-Human D2-40 (Clone D2-40)) can be used.

When the selected component of the malignant mesothelioma differentiation kit of this embodiment contains an anti-CEA antibody, the anti-CEA antibody is not particularly limited. For example, an anti-CEA antibody (DAKO; product number M7072; trade name Monoclonal Mouse Anti-Human Carcinoembryonic Antigen (Clone Il-7)) can be used.

When the anti-TTF-1 antibody is included in the selected component of the malignant mesothelioma differentiation kit of this embodiment, the anti-TTF-1 antibody is not particularly limited. For example, an anti-TTF-1 antibody (DAKO; product number M3575; trade name Monoclonal Mouse Anti-Thyroid Transcription Factor (Clone 8G7G3 / 1)) can be used.

When the anti-CLDN4 antibody is included in the selected component of the malignant mesothelioma differentiation kit of this embodiment, the anti-CLDN4 antibody is not particularly limited. For example, an anti-CLDN4 antibody (Thermo Fisher Scientific; product number 32-9400; trade name Claudin 4 Monoclonal Antibody (3E2C1)) can be used.

When the anti-p63 antibody is included in the selected component of the malignant mesothelioma differentiation kit of this embodiment, the anti-p63 antibody is not particularly limited. For example, an anti-p63 antibody (Abcam; product number ab735; trade name Anti-p63 antibody [4A4]) or an anti-p63 antibody (Santa Cruz; product number sc-8431; trade name p63 (4A4)) can be used.

When the selected component of the malignant mesothelioma differentiation kit of this embodiment contains an anti-p40 antibody, the anti-p40 antibody is not particularly limited. For example, an anti-p40 antibody (Abcam; product number ab172731; trade name Anti-p40-DeltaNp63 antibody [BC28]) can be used.

When the selected component of the malignant mesothelioma differentiation kit of this embodiment contains an anti-desmocollin 3 antibody, the anti-desmocollin 3 antibody is not particularly limited.

The above-mentioned anti-CLDN15 antibody and / or other antibody for differentiating malignant mesothelioma may be labeled with a fluorescent dye, a luminescent substance, or the like, if necessary.

The malignant mesothelioma differentiation kit of the present invention includes an antibody for differentiating malignant mesothelioma and other reagents necessary for differentiating malignant mesothelioma, such as a buffer and a secondary antibody, and a manual used for detection and result identification. May include.

4. Method for distinguishing malignant mesothelioma 4-1. Outline A fourth aspect of the present invention is a method for differentiating malignant mesothelioma. The method for differentiating malignant mesothelioma of the present invention is to discriminate whether or not a subject has malignant mesothelioma by detecting the expression of CLDN15 protein or CLDN15 gene in a sample derived from a subject. Can be done. The method for differentiating malignant mesothelioma of this embodiment can also be used as a method for assisting in differentiating the disease of malignant mesothelioma of a subject.

4-2. Method The method for differentiating malignant mesothelioma of the present invention is a detection step for detecting a transcript of CLDN15 gene or CLDN15 protein in a sample derived from a region suspected to be a malignant tumor in and around the infiltration of a subject, a cell atypia, A determination step of determining whether or not the sample contains a malignant tumor based on any one or more of structural atypia, infiltration, and metastasis, and a determination step in which the sample contains a malignant tumor and is contained in the malignant tumor. When a transcript of the CLDN15 gene or CLDN15 protein is detected, it includes a differentiation step of differentiating the subject from malignant mesothelioma.

In one embodiment, the detection step is a step of detecting the CLDN15 protein, where the CLDN15 protein can be detected using the anti-CLDN15 antibody or fragment thereof according to the second aspect.

Further, the detection step is a step of detecting another positive marker protein for differentiating malignant mesothelioma and / or another negative marker protein for differentiating malignant mesothelioma in the sample, and the differentiating step is the step of detecting the malignant. Subject when CLDN15 protein is detected in the tumor and the other positive marker protein for differentiating malignant mesothelioma is detected and / or when other negative marker protein for differentiating malignant mesothelioma is not detected. May be a step of differentiating from suffering from malignant mesothelioma.

For example, the detection step further comprises one or more types, for example, one type, two types, three types, four types, five types, six types, or six or more types of positive markers for differentiating malignant mesothelioma in the sample. In the step of detecting a protein and / or one or more types, for example, one type, two types, three types, four types, five types, six types, or six or more types of other negative marker proteins for differentiating malignant mesothelioma. Yes, in the differentiation step, the CLDN15 protein is detected in the malignant tumor, and at least one or more of the other positive marker proteins for differentiating malignant mesothelioma, for example, one type, two types, three types, and 4 types. When 5 types, 6 types, or 6 or more types are detected, and / or at least one of other negative marker proteins for differentiating malignant mesothelioma, for example, 1 type, 2 types, 3 types, If 4 types, 5 types, 6 types, or 6 or more types are not detected, the subject may be differentiated as having malignant mesothelioma.

Here, in the above-described embodiment, the other positive marker protein for differentiating malignant mesothelioma refers to a positive marker protein for malignant mesothelioma other than the CLDN15 protein. Specific examples of other positive marker proteins for differentiating malignant mesothelioma include AE1 / 3, carretinin, cytokeratin 5/6, WT1, or podoplanin. Further, the other negative marker protein for differentiating malignant mesothelioma refers to a negative marker protein for malignant mesothelioma. Specific examples of other negative marker proteins for differentiating malignant mesothelioma include CEA, TTF-1, CLDN4, p63, p40, or desmocollin 3.

Hereinafter, the detection step, the determination step, and the discrimination step will be specifically described. The order of the detection step and the determination step may come first. For example, the detection step may come first, or the determination step may come first. Alternatively, the detection step and the determination step may be performed at the same time.

4-2-1. Detection step The "detection step" is to measure the expression level of a biomarker for differentiating malignant mesothelioma in a sample derived from a region suspected to be a malignant tumor in and around the inner skin of the subject, and use the measured value as the measured value. Based on this, it is a step of determining whether the biomarker is positive or negative (hereinafter, referred to as “positive / negative determination”).

As used herein, the term "sample" refers to a sample, a healthy body, or a group of healthy bodies, which is used in the method for distinguishing malignant mesothelioma of the present embodiment, and corresponds to, for example, a tissue or a cell. The "tissue" and "cell" referred to here correspond to tissues and cells derived from the inner skin of the subject and its surroundings. For example, the tissues or cells derived from the mesothelium and its surroundings include the pleura (for example, parietal pleura or visceral peritoneum), peritoneum (for example, parietal pleura or visceral peritoneum), and pericardium (epicardium). Alternatively, it may be any tissue or cell derived from the testicular pleura and its surroundings.

In the present specification, "a sample derived from a region suspected to be a malignant tumor in and around the mesothelium" is not only the tissue or cells of the mesothelium but also located in the vicinity of the mesothelium and is suspected to be malignant mesothelioma. It shall include tissues or cells. For example, the sample used for the method for differentiating malignant mesothelioma of this embodiment may be a sample derived from a region suspected to be a malignant tumor in and around the mesothelium other than the pleura. In addition, the sample used for the method for differentiating malignant mesothelioma of this embodiment is preferably a sample collected by biopsy or excised by surgery. Particularly preferably, it is a tissue or cell derived from a region suspected to be a malignant tumor in and around the lining collected by biopsy. In addition, these tissues or cells may be those embedded in paraffin after formalin fixation (FFPE: Formalin-Fixed Paraffin Embedded).

Samples may be obtained by biopsy or surgical removal by surgery to obtain tissue or cells. The amount of sample required for the method for differentiating malignant mesothelioma of this embodiment is not particularly limited. A biopsy material may be used, although at least 10 μg, preferably at least 0.1 mg, is desirable for tissues or cells. The sample can be prepared and processed as needed so that the expression level of the biomarker for differentiating malignant mesothelioma can be measured. For example, if the sample is a tissue or a cell, homogenization treatment, cell lysis treatment, removal of impurities by centrifugation or filtration, addition of a protease inhibitor, or the like can be mentioned. Details of these processes can be found in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press for reference.

As used herein, the "expression level of a biomarker for differentiating malignant mesothelioma" refers to the expression level of a transcript or translation product of a marker gene for differentiating malignant mesothelioma. Specifically, the expression level of the biomarker for differentiating malignant mesothelioma is the amount of transcript of the marker gene for differentiating malignant mesothelioma (for example, the amount of mRNA) or the amount of its translation product (for example, the amount of protein). It can be obtained as a measured value by the measurement of.

As used herein, the "measured value" is a value obtained by measuring the expression level. The measured value may be an absolute value in which the amount of mRNA or protein in the sample is expressed in units such as ng (nanogram) or μg (microgram), or the absorbance with respect to the control value, the fluorescence intensity of the labeled molecule, etc. It may be a relative value represented by, or it may be a score (score) calculated by a certain calculation formula from an expression pattern (for example, a staining pattern) in a sample.

Hereinafter, (1) measurement of the expression level of the biomarker for differentiating malignant mesothelioma, and (2) determination of positive / negative of the biomarker, which constitute this detection step, will be described.

(1) Measurement of Expression Level of Biomarker for Differentiation of Malignant Mesothelioma Hereinafter, a method for measuring the expression level of a transcript or translation product of a marker gene for differentiation of malignant mesothelioma will be specifically described.

(1-1) Measurement of Transcription Product The measurement of the transcript of the marker gene for differentiating malignant mesoderma is the measurement of the amount of mRNA or the amount of cDNA obtained by reverse transcription from mRNA. May be good. Generally, for the measurement of a transcript of a gene, a method of measuring the expression level of the gene as an absolute value or a relative value by using a nucleotide containing all or a part of the base sequence of the above gene as a primer or a probe is adopted. ..

The primer or probe of this embodiment is usually composed of a natural nucleic acid such as DNA or RNA. DNA that is highly stable, easy to synthesize, and inexpensive is particularly preferred. Further, if necessary, a natural nucleic acid can be combined with a chemically modified nucleic acid or a pseudo-nucleic acid. Examples of chemically modified nucleic acids and pseudo-nucleic acids include PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid; registered trademark), methylphosphonate-type DNA, phosphorothioate-type DNA, 2'-O-methyl-type RNA and the like. In addition, primers and probes are fluorescent substances and / or quenching substances, ^{labeling substances such as radioisotopes (for example, 32} P, ³³ P, ³⁵ S), biotin or (streptavidin) avidin, magnetic beads, etc. It may be labeled or modified with a modifying substance. The labeling substance is not limited, and commercially available substances can be used. For example, as a fluorescent substance, FITC, Texas, Cy3, Cy5, Cy7, Cyanine3, Cyanine5, Cyanine7, FAM, HEX, VIC, fluorescamine and its derivatives, rhodamine and its derivatives and the like can be used. As a quencher substance, AMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3 and the like can be used. The labeling position of the labeling substance in the primer and the probe may be appropriately determined according to the characteristics of the modifying substance and the purpose of use. In general, it is often modified to the 5'or 3'end. Further, one primer and probe molecule may be labeled with one or more labeling substances. Labeling of these substances on nucleotides can be performed by known methods.

The nucleotide used as a primer or probe may be either a sense strand of a marker gene for differentiating malignant mesothelioma or a nucleotide consisting of an antisense strand.

The base length of the primer or probe is not particularly limited. In the case of a probe, if it is used in the hybridization method described later, the full length of the marker gene for differentiating malignant mesothelioma (for example, in the case of the CLDN15 gene, the full length of the sequence of SEQ ID NO: 19), preferably 15 From base length or more to malignant mesothelioma differentiation marker gene total length, more preferably from base length or more to malignant mesothelioma differentiation marker gene total length, and more preferably from base length or more to malignant mesothelioma differentiation marker gene total length If used in a microarray, it has a length of 10 to 200 bases, preferably 20 to 150 bases, and more preferably 30 to 100 bases. Generally, the longer the probe, the higher the hybridization efficiency and the higher the sensitivity. On the other hand, the shorter the probe, the lower the sensitivity, but conversely, the higher the specificity. On the other hand, in the case of a primer, each of the forward primer and the reverse primer may be 10 to 50 bp, preferably 15 to 30 bp.

The preparation of the above-mentioned primers or probes is known to those skilled in the art and can be prepared, for example, according to the method described in Green & Sambrook, Molecular Cloning (2012) described above. It is also possible to provide sequence information to a nucleic acid synthesis contract manufacturer and manufacture it on a contract basis.

The measurement of the transcript of the marker gene for differentiating malignant mesothelioma may be any known nucleic acid quantification method and is not particularly limited. For example, a hybridization method or a nucleic acid amplification method can be mentioned.

The "hybridization method" uses a nucleic acid fragment having a base sequence complementary to all or part of the base sequence of the target nucleic acid to be detected as a probe, and utilizes the base pairing between the nucleic acid and the probe. , A method for detecting and quantifying a target nucleic acid or a fragment thereof. In this embodiment, the target nucleic acid corresponds to mRNA or cDNA of a marker gene for differentiating malignant mesothelioma, or a fragment thereof. In general, the hybridization method is preferably performed under stringent conditions in order to eliminate unintended nucleic acids that hybridize non-specifically. The above-mentioned low salt concentration and high stringent conditions at high temperature are more preferable. Several methods with different detection means are known for the hybridization method, and for example, Northern blotting (Northern hybridization method), microarray method, surface plasmon resonance method, or quartz crystal microbalance method is preferable. ..

The "Northern blot method" is the most common method for analyzing gene expression. Total RNA or mRNA prepared from a sample is separated by electrophoresis on an agarose gel or polyacrylamide gel under denaturing conditions, and transferred to a filter ( This is a method of detecting a target nucleic acid using a probe having a base sequence specific to the target RNA after blotting). By labeling the probe with a suitable marker such as a fluorescent dye or radioisotope, for example, a chemiluminescent analyzer (eg, Light Capture; Atto), a scintillation counter, an imaging analyzer (eg, FUJIFILM). : BAS series) and other measuring devices can also be used to quantify the target nucleic acid. The Northern blot method is a well-known and well-known technique in the art, and for example, the above-mentioned Green, M.R. and Sambrook, J. (2012) may be referred to.

In the "microarray method", a nucleic acid fragment complementary to all or part of the base sequence of a target nucleic acid is arranged on a substrate as a probe at a high density in a small spot shape, and the target nucleic acid is contained in a immobilized microarray or microchip. This is a method of reacting a sample to detect and quantify a nucleic acid hybridized to a base spot by fluorescence or the like. The target nucleic acid may be either RNA such as mRNA or DNA such as cDNA. Detection and quantification can be achieved by detecting and measuring fluorescence based on hybridization of a target nucleic acid or the like with a microplate reader or a scanner. From the measured fluorescence intensity, the amount of mRNA or cDNA or their abundance ratio to reference mRNA (reference mRNA) can be determined. The microarray method is also a well-known technique in the art. For example, the DNA microarray method (DNA microarray and latest PCR method (2000), supervised by Masaaki Muramatsu, Hiroyuki Nawa, Shujunsha) and the like may be referred to.

The "Surface Plasmon Resonance (SPR) method" is well known in the art. See, for example, Kazuhiro Nagata and Hiroshi Handa, Real-time Analytical Experimental Method for Biological Interactions, Springer Fairlark Tokyo, Tokyo, 2000.

The "quartz crystal microbalance (QCM) method" is well known in the art, for example, Christopher J. et al., 2005, Self-Assembled Monolayers of a Form of Nanotechnology, Chemical Review, 105: See 1103-1169, Toyoe Moriizumi, Takamichi Nakamoto, (1997) Sensor Engineering, Shokodo.

The "nucleic acid amplification method" refers to a method in which a specific region of a target nucleic acid is amplified by a nucleic acid polymerase using a forward / reverse primer. For example, the PCR method (including the RT-PCR method), the NASBA method, the ICAN method, and the LAMP® method (including the RT-LAMP method) can be mentioned. The PCR method is preferable. A quantitative nucleic acid amplification method such as a real-time RT-PCR method is used as a method for measuring a gene transcript using a nucleic acid amplification method. Further known real-time RT-PCR methods include an intercalator method using SYBR (registered trademark) Green and the like, a Taqman (registered trademark) probe method, a digital PCR method, and a cycling probe method. There may be. All of these are known methods and are also described in appropriate protocols in the art, so you can refer to them.

A method for quantifying gene transcripts by real-time RT-PCR will be briefly described below with an example. The real-time RT-PCR method is a reaction system in which the amplification product of PCR is specifically fluorescently labeled using the cDNA prepared by the reverse transcription reaction from the mRNA in the sample, and the fluorescence intensity derived from the amplification product is detected. This is a nucleic acid quantification method in which PCR is performed using a temperature cycler device equipped with a function. The amount of amplification product of the target nucleic acid in the reaction is monitored in real time, and the result is regression-analyzed by computer. Methods for labeling the amplification product include a method using a fluorescently labeled probe (for example, the TaqMan (registered trademark) PCR method) and an intercalator method using a reagent that specifically binds to double-stranded DNA. The TaqMan (registered trademark) PCR method uses a probe in which the 5'end is a quencher substance and the 3'end is a fluorescent dye. Normally, the 5'end quencher substance suppresses the fluorescent dye at the 3'end, but when PCR is performed, the probe is degraded by the 5'→ 3'exonuclease activity of Taq polymerase. As a result, the suppression of the quencher substance is released, and the fluorescence is emitted. The amount of fluorescence reflects the amount of amplification product. Since the number of cycles (CT) when the amplification product reaches the detection limit and the initial template amount are inversely related, the real-time measurement method quantifies the initial template amount by measuring CT. The absolute value of the initial template amount of an unknown sample can be calculated by measuring CT using several stages of known amounts of templates and preparing a calibration curve. As the reverse transcriptase used in RT-PCR, for example, M-MLV RTase, ExScript RTase (TaKaRa), Super Script II RT (Thermo Fisher Scientific) and the like can be used.

The reaction conditions for real-time PCR are generally based on known PCR methods, the base length of the nucleic acid fragment to be amplified, the amount of nucleic acid for the template, the base length and Tm value of the primer to be used, and the optimum reaction of the nucleic acid polymerase to be used. Since it varies depending on the temperature, optimum pH, etc., it may be appropriately determined according to these conditions. As an example, the denaturation reaction is usually carried out at 94 to 95 ° C. for 5 seconds to 5 minutes, the annealing reaction is carried out at 50 to 70 ° C. for 10 seconds to 1 minute, and the extension reaction is carried out at 68 to 72 ° C. for 30 seconds to 3 minutes. The elongation reaction can be repeated for about 15 to 40 cycles as one cycle. When using a kit commercially available from the manufacturer, in principle, the protocol attached to the kit may be followed.

Nucleic acid polymerases used in real-time PCR are DNA polymerases, especially heat resistant DNA polymerases. Various types of such nucleic acid polymerases are commercially available, and they can also be used. For example, Taq DNA polymerase attached to the Applied Biosystems TaqMan MicroRNA Assays Kit (Thermo Fisher Scientific) can be mentioned. In particular, such a commercially available kit is useful because a buffer or the like optimized for the activity of the attached DNA polymerase is attached.

(1-2) Measurement of translation product The method for measuring the protein amount of the translation product (marker protein for differentiation of malignant mesothelioma) of the marker gene for differentiation of malignant mesothelioma may be a peptide quantification method, and is not particularly limited. do not do. For example, known quantification methods such as immunological detection method, aptamer analysis method, and mass spectrometry can be used. Hereinafter, each quantitative method will be described.

(A) Immunological detection method An "immunological detection method" is a method for quantifying a target molecule using an antibody or a binding fragment thereof that specifically binds to the target molecule. Immunological detection methods include, for example, enzyme immunoassay (including ELISA and EIA methods), radioimmunoassay, radioimmunoassay (RIA), luminescent immunoassay, and surface plasmon resonance (SPR). , Crystal transducer microbalance (QCM) method, immunoturbidimetric method, latex agglutination immunoassay, latex turbidimetric method, hemagglutination reaction, particle agglutination reaction method, gold colloid method, capillary electrophoresis method, western blot method or immunity A histochemical method (immunostaining method) or the like is known, but any detection method may be used in this method. Immunohistochemistry is preferred, but not limited.

When immunohistochemistry is used, the method for quantifying the expression level of the marker protein for differentiating malignant mesothelioma is not limited. For example, it may be quantified as a score calculated using a certain formula based on the observation of the staining pattern of the tissue section. Such a scoring method is well known in the art, and for example, the IRS method (Immunoreactive scoring method) described in Remmele et al., 1986 may be modified and used. For example, after masking the patient background, the staining intensity (Signal intensity; I) of the marker protein for differentiating malignant mesothelioma was determined to be I = 0 (negative), I = 1 (weak), I = 2 ( It is classified into 4 stages of moderate) and I = 3 (strong), and the percentage of positive staining regions (P) in the tumor region is determined based on the percentage of staining range (r), P = 0. (R <1%), P = 1 (1% ≤ r <10%), P = 2 (10% ≤ r <30%), P = 3 (30% ≤ r <50%), P = 4 ( It is classified into 5 stages of r ≧ 50%). Next, the IRS can be calculated as IRS (Immunoreactive score) = S × P and used as the measured value. In addition, based on the obtained IRS, score 0 (IRS = 0), score 1+ (IRS = 1, 2), score 2+ (IRS = 3, 4, 6), score 3+ (IRS = 8,) It may be classified into 9, 12).

As described in the second aspect, the antibody used in this step may be either a monoclonal antibody or a polyclonal antibody, and the immunoglobulin constituting the antibody may be in any class or any subclass. It may be derived from any animal, including mammals and birds, and may be an artificially produced antibody, such as a recombinant antibody, a synthetic antibody, or an antibody fragment. The morphology of these antibodies has been described in the second aspect, and detailed description thereof will be omitted here.

More specifically, when the marker protein for differentiating malignant mesothelioma to be measured in this step is CLDN15 protein, the anti-CLDN15 antibody that can be used in this step is as described in the second aspect. Detailed description will be omitted.

When the marker protein for differentiating malignant mesothelioma to be measured in this step is another marker protein for differentiating malignant mesothelioma, the other antibody for differentiating malignant mesothelioma that can be used in this step is the third aspect. Follow the description in. Therefore, detailed description here will be omitted.

(B) Aptamer analysis method The "aptamer analysis method" is a method for quantifying a marker protein for differentiating malignant mesothelioma, which is a target molecule, using an aptamer that strongly and specifically binds to a target substance by its three-dimensional structure. .. Aptamers can be roughly classified into nucleic acid aptamers and peptide aptamers according to the type of the molecule, but any aptamer may be used.

"Nucleic acid aptamer" refers to an aptamer composed of nucleic acids. The nucleic acid constituting the nucleic acid aptamer may be DNA, RNA, or a combination thereof. If desired, chemically modified nucleic acids such as PNA, LNA / BNA, methylphosphonate DNA, phosphorothioate DNA, and 2'-O-methyl RNA can also be included. In the present invention, anti-CLDN15 RNA aptamer, anti-CLDN15 DNA aptamer and the like can be mentioned.

Nucleic acid aptamers can be prepared by using a marker protein for differentiating malignant mesothelioma or a part thereof as a target molecule by a method known in the art, for example, a SELEX (systematic evolution of ligands by exponential enrichment) method. The SELEX method is a known method, and a specific method may be performed according to, for example, Pan et al. (Proc. Natl. Acad. Sci. U.S.A., 1995, 92: 11509-11513).

A peptide aptamer is an aptamer composed of amino acids, and is a 1 to 6 kDa peptide molecule that recognizes the surface structure of a specific target molecule and specifically binds to it, like an antibody. In the present invention, anti-CLDN15 peptide aptamers and the like can be mentioned. The peptide aptamer may be prepared based on a production method known in the art. For example, Whaley, S.R., et al., Nature, 2000, 405, 665-668 can be referred to. Usually, it can be prepared by using a phage display method or a cell surface display method.

The antibody or aptamer may be labeled as required. As the label, a labeling substance known in the art may be used. For antibody and peptide aptamers, for example, fluorescent dyes (fluorescein, FITC, rhodamine, Texas red, Cy3, Cy5), fluorescent proteins (eg PE, APC, GFP), enzymes (eg horseradish peroxidase, alkaline phosphatase, glucose). It can be labeled with oxidase), radioactive isotopes (eg, ³ H, ¹⁴ C, ³⁵ S) or biotin or (strept) avidin. In the case of nucleic acid aptamers, for example, radioisotopes (eg ³² P, ³ H, ¹⁴ C), DIG, biotin, fluorescent dyes (eg FITC, Texas, cy3, cy5, cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, TAMRA), or luminescent material (eg, acridinium ester). Antibodies and aptamers labeled with a labeling substance can be useful tools in detecting aptamers bound to a target protein.

(C) Mass Spectrometry The "mass spectrometry" includes high-speed liquid chromatograph mass spectrometry (LC-MS), high-speed liquid chromatograph tandem mass spectrometry (LC-MS / MS), and gas chromatograph mass spectrometry (GC). -MS), gas chromatograph tandem mass spectrometry (GC-MS / MS), capillary electrophoresis mass spectrometry (CE-MS) and ICP mass spectrometry (ICP-MS).

The immunological detection method, the aptamer analysis method, and the mass spectrometry method are all known techniques in the art, and may be performed according to these methods. For example, Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Christopher J., et al., 2005, Chemical Review, 105 : 1103-1169; Iijima Y. et al., 2008 ,. The Plant Journal, 54,949-962; Hirai M. et al., 2004, Proc Natl Acad Sci USA, 101 (27) 10205-10210; Sato S, et Al., 2004 ,, The Plant Journal, 40 (1) 151-163; Shimizu M. et al., 2005, Proteomics, 5,3919-3931. In addition, peptide quantification kits are commercially available from each manufacturer, and they can also be used.

(2) Determining positive / negative of biomarker for differentiating malignant mesothelioma Next, positive / negative of biomarker for differentiating malignant mesothelioma in the sample is determined based on the measured value obtained in (1). To do.

The method for determining positive / negative based on the measured value is not limited. For example, a method of determining a cutoff value for the expression level of a biomarker for differentiating malignant mesothelioma and determining positive (high expression) / negative (low expression) based on the cutoff value can be mentioned. That is, a predetermined value is set as a cutoff value, and if the measured value is equal to or more than that value, the expression of the biomarker for differentiating malignant mesothelioma is positive, and conversely, if it is less than the cutoff value, it is determined to be negative. be able to.

The cutoff value is a boundary value for classifying a measured value into positive or negative. The cutoff value can usually be calculated based on the prevalence of the disease and the sensitivity and specificity calculated from the receiver operating characteristic curve. The method of setting the cutoff value is not particularly limited.

For example, the cutoff value is the measured value of a sample derived from a healthy body not suffering from malignant mesothelioma, or the average value of the measured values of a sample derived from a healthy body group as a cutoff value. When it is higher than, it can be determined to be positive.

Alternatively, 1.5 times or more, 2 times or more, 3 times or more, 4 times the average value of the measured value of the sample derived from a healthy body not suffering from malignant mesothelioma or the measured value of the sample derived from a healthy body group. As described above, 5 times or more, or 6 times or more is set as the cutoff value, and when the measured value of the subject is higher than the cutoff value, it can be determined as positive.

It is also possible to classify the measured values obtained from the control group by percentile and use the percentile value used for the classification as the cutoff value. For example, if the 95th percentile of the measured value obtained from the control is set as the cutoff value, the value above that value is positive, and the value below that value is negative, if the measured value of the subject is 95th percentile or more, it is determined to be positive. be able to.

The measured value of the healthy body as a control is different from the measured value of the subject, and it is not always necessary to measure the measured value each time. For example, if the amount of the sample used for the measurement, the measurement method of the biomarker for differentiating malignant mesothelioma, and the measurement conditions are kept constant, the previously measured measurement value of the control healthy body can be reused.

4-2-2. Judgment step The "judgment step" is a sample derived from a region suspected to be a malignant tumor in and around the inner skin of a subject based on any one or more of cell atypia, structural atypia, infiltration, and metastasis. This is a step of determining whether or not a malignant tumor is contained in the tumor.

As used herein, the term "cell atypia" refers to a distance from a normal cell structure, specifically, an increase in the ratio of nuclear vesicles, an irregular size of cells or nuclei, an irregular karyotype, an increase in nuclear chromatin, and a small nucleolus. Refers to the growth or increase of the body, the increase of fission images, and / or the appearance of abnormal fission images.

As used herein, a "structural atypia" is a distance from a normal tissue structure, that is, an irregularity of the tissue structure. In this embodiment, the method for detecting cell atypia or structural atypia is not limited. For example, it can be visualized using hematoxylin and eosin staining.

"Invasion" is the continuous progression of a malignant tumor while destroying the surrounding normal tissues and organs, and can be determined by making the boundary with the surrounding tissues unclear.
"Metastasis" is the discontinuous progression of a malignant tumor to an organ distant from the primary lesion.

4-2-3. Differentiation step The "differentiation step" is based on the detection result of the biomarker for differentiating malignant mesothelioma obtained in the detection step and the judgment result of whether or not the malignant tumor obtained in the judgment step is included. , A step of differentiating the prevalence of malignant mesothelioma in a subject.

In the differentiation step of the method for differentiating malignant mesothelioma of the present invention, a malignant tumor is contained in a sample derived from a region suspected to be a malignant tumor in and around the mesothelioma of a subject, and the CLDN15 gene is contained in the malignant tumor. When a transcript or CLDN15 protein is detected, the subject can be differentiated from having malignant mesothelioma.

Further, in the differentiation step of the method for differentiating malignant mesothelioma of the present invention, a malignant tumor is contained in a sample derived from a region suspected to be a malignant tumor in and around the inner skin of a subject, and the malignant tumor contains If the CLDN15 protein is detected and the other positive marker protein for differentiating malignant mesothelioma is detected and / or if no other negative marker protein for differentiating malignant mesothelioma is detected, the subject is malignant. It may be differentiated from having mesothelioma.

For example, in the differentiation step of the method for differentiating malignant mesothelioma of the present invention, a malignant tumor is contained in a sample derived from a region suspected to be a malignant tumor in and around the inner skin of a subject, and CLDN15 is included in the malignant tumor. A protein is detected, and at least one or more of the other positive marker proteins for differentiating malignant mesothelioma, for example, one type, two types, three types, four types, five types, six types, or six or more types are detected. And / or at least one or more of other negative marker proteins for differentiating malignant mesothelioma, such as one, two, three, four, five, six, or six or more. If not detected, the subject may be differentiated as suffering from malignant mesothelioma.

Further, in the differentiation step of the method for differentiating malignant mesothelioma of the present invention, when the CLDN15 protein is detected in the sample and no other positive marker protein for differentiating malignant mesothelioma is detected, the subject is malignant. It may be differentiated from having mesothelioma. For example, in the differentiation step of the method for differentiating malignant mesothelioma of the present invention, when CLDN15 protein is detected in the sample and carretinin protein is not detected, the subject is said to have malignant mesothelioma. It may be differentiated.

Alternatively, in the differentiation step of the method for differentiating malignant mesothelioma of the present invention, when the CLDN15 protein is detected in the sample and another negative marker protein for differentiating malignant mesothelioma is detected, the subject is the malignant mesothelium. It may be differentiated from having a tumor.

4-3. Effect According to the method for differentiating malignant mesothelioma of this embodiment, by examining a biological sample removed by biopsy or surgery, it is possible to accurately determine whether the subject who provided the sample is malignant mesothelioma or other malignant tumor. Can be differentiated into. By the method for differentiating malignant mesothelioma of this aspect, which has a high accuracy rate, it is possible to make a definitive diagnosis of malignant mesothelioma, recognize that it is a pathological condition different from other malignant tumors, and risk recurrence and treatment. There is an advantage that it can be dealt with in consideration of the judgment of the law.

In addition, as described in Example 2 of the present application, there are cases of malignant mesothelioma in which only one of the CLDN15 protein and the carretinine protein is positive. Therefore, by using the CLDN15 protein as a biomarker for differentiating malignant mesothelioma, it is extremely possible. Malignant mesothelioma can be differentiated with high sensitivity.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is merely an example, and the present invention is not limited to the scope described in the Examples.

<Example 1: Preparation of rat anti-CLDN15 monoclonal antibody>
(Purpose)
Raise a rat anti-CLDN15 monoclonal antibody.

(Method and result)
Monoclonal antibodies against the human CLDN15 protein were prepared by the following method according to the rat iliac lymph node method (Kishiro Y et al., Cell Struct Funct, 1995, 20 (2), 151-6).

(1) Preparation of antigen peptide A peptide (SEQ ID NO: 20) in which one cysteine is added to the amino terminus of a peptide consisting of 16 amino acid residues near the carboxy terminus of the human CLDN15 protein was commissioned and synthesized (Eurofin) and dissolved in PBS. The solution was 5 mg / mL. Imject Maleimide-activated mcKLH (Thermo Fisher Scientific; 77606) 2 mg was dissolved in 200 μL of ultrapure water to prepare a 10 mg / mL KLH solution mixed with a peptide solution and reacted at room temperature for 2 hours. To remove EDTA from the KLH solution, 150 mL of PBS was used as an external solution and dialyzed three times. The obtained solution was used as an antigen solution. Using two 2 mL luer-lock glass syringes and a connecting tube, 250 μL of antigen solution, 250 μL of PBS and 1 mL of Freund's complete adjuvant (Sigma-Aldrich; F5881) were mixed to prepare an antigen emulsion.

(2) Immunity An 8-week-old female Wistar rat was anesthetized and 100 μL of antigen emulsion was intradermally injected into the footpads of both hind limbs for immunization.

(3) Preparation of 50% polyethylene glycol (PEG) solution
5 g of PEG4000 (Merck; 109727) is dissolved by heating in an autoclave and mixed with 8 mL of Dalbecko's Modified Eagle's Medium (DMEM) (Sigma-Aldrich; D5796) and 0.4 mL of dimethyl sulfoxide (Sigma-Aldrich; D2650). To make a 50% PEG solution.

(4) Cell fusion Rats 14 days after immunization were euthanized, dissected, and bilateral iliac lymph nodes were aseptically removed. The following operations were performed aseptically in a clean bench. The lymph nodes were injected with a small amount of DMEM, swollen, and then chopped with scissors. Lymph node tissue was crushed and filtered on a 70 μm mesh cell strainer (BD Falcon). And collected lymphocytes were mixed for about 10 ⁷ mouse multiple myeloma cell line SP2, and the supernatant was removed by centrifugation for 10 minutes at 1280 rpm. After thoroughly loosening the cell mass of the sediment, 1 mL of a 50% PEG solution kept at 37 ° C was removed while mixing well over 1 minute to fuse the lymphocyte cells and SP2 cells. Then, in order to dilute and remove PEG, 9 mL of DMEM kept at 37 ° C. was slowly added dropwise over 5 minutes, and the supernatant was removed by centrifugation at 900 rpm for 5 minutes. Thoroughly loosen the cell mass of the sediment, add 40 mL of hybridoma medium (78% GIT medium (WAKO), 2% HAT supplement (Thermo Fisher Scientific), 10% BM Condimed H1 Hybridoma cloning supplement, 10% fetal bovine serum), and add 40 mL. The cells were dispensed into 4 96-well plates at 100 μL / well and cultured in a _{37 ° C CO 2 incubator.}

(5) Screening by ELISA After 4 to 5 days of culturing, medium was exchanged with 200 μL of hybridoma medium. Hybridoma clones that produce antibodies that react with antigenic peptides by ELISA (Enzyme-linked immunosorbent assay) were screened using the culture 2 days after the exchange according to the following procedure.
The day before, a 3 μg / mL solution of antigenic peptide PBS was added to a 96-well ELISA plate at 50 μL / well and allowed to stand overnight at 4 ° C. to coat the plate. As a negative control, PBS containing no peptide was used. Each well was treated with a blocking solution (PBS containing 1% bovine serum albumin) at 37 ° C. for 1 hour to block, and then reacted with a culture supernatant of 50 μL / well at 37 ° C. for 1 hour. After washing each well with PBS three times, secondary antibody solution (ECL Rat IgG, HRP-linked whole antibody (from goat) (GE Healthcare; NA935), diluted 1000 times) was added at 50 μL / well at 37 ° C. for 1 hour. It was reacted. After washing 3 times with PBS, the color was developed using TMB substrate (BioLegend), the reaction was stopped with 1M phosphoric acid, and the absorbance was quantified.
As a result of screening, 3 clones showed strong positive.

(6) Selection of Clone by Immunohistochemistry As a result of performing immunohistochemistry on the cell mass paraffin section described in Example 3 described later and the malignant pleural mesothelioma tissue paraffin section derived from the patient, both are good. One clone (2C11) was obtained to obtain a stained image. In the following examples, 2C11 clones were used as anti-CLDN15 antibody.

<Example 2: Immunohistochemistry of CLDN15 protein in malignant pleural mesothelioma tissue>
(Purpose)
To examine whether anti-CLDN15 antibody can be used to distinguish malignant mesothelioma.

(Method)
(1) Collection of tissue specimens
33 patients diagnosed with malignant pleural mesothelioma by respiratory surgery at Fukushima Medical University Hospital from 2003 to 2019, and malignant pleural mesothelioma diagnosed at Shirakawa Health and Welfare General Hospital from 2009 to 2018. Tissue specimens were collected and clinicopathological factors were analyzed in 9 patients. The collection of tissue materials was approved by the Fukushima Medical University Ethics Committee (approval number 2512) and was carried out in compliance with the ethical guidelines related to clinical research.

(2) Immunohistochemical staining of CLDN15 protein and carretinine protein in mesothelioma tissue of 42 cases (epithelial type 28 cases, sarcoma type 5 cases, biphasic type 9 cases) collected by surgical excision or biopsy. Expression was verified.
All collected pleural and / or lung tissues including malignant pleural mesothelioma were fixed with 10% formalin and then embedded in paraffin, and hematoxylin-Eosin (HE) staining and immunohistochemical staining were performed. The sliced sections were deparaffinized and dexylene-treated, and then treated with methanol containing 0.3% hydrogen peroxide for 20 minutes at room temperature to remove endogenous peroxidase. The antigen was activated by treating the anti-CLDN15 antibody with 0.1% semicarbazide hydrochloric acid aqueous solution at room temperature for 1 hour and then with Immunosaver (Nisshin EM) at 70 ° C. for 16 hours. The anti-calretinin antibody (Life Technologies; product number 081211) was heated to 100 ° C. for 10 minutes in 10 mM citrate buffer using a microwave oven and cooled to room temperature. Blocking was performed with 5% skim milk (Morinaga Milk Industry; # 0646869), and the culture supernatant containing anti-CLDN15 antibody or anti-calretinin antibody was reacted with a primary antibody solution diluted 10-fold with PBS containing 2% BSA overnight at 4 degrees. It was. For the secondary antibody reaction, the anti-CLDN15 antibody was reacted with a secondary antibody solution diluted 400-fold with an anti-rat IgG-HRP antibody for 30 minutes at room temperature. The anti-calretinin antibody was reacted with a biotin-labeled anti-rabbit IgG antibody for 20 minutes at room temperature, and then reacted with HRP-labeled streptavidin for 10 minutes at room temperature. Then, the color reaction was carried out in 3,3'-Diaminobenzidine (DAB) solution (50 mM Tris buffer 100 mL, DAB 0.02 g, 30% hydrogen peroxide solution 17 μL) at room temperature for 20 minutes, and nuclear staining was performed with hematoxylin to dehydrate.・ Transparent and enclosed. Observation was performed using an optical phase contrast microscope (OLYMPUS BX61, OLYMPUS), and images were taken with a DP controller (OLYMPUS).

(3) Histological evaluation From the stained image obtained in (2), the expression levels of CLDN15 protein and carretinin protein are evaluated by the following method.
After masking the patient background, two pathologists and one respiratory surgeon used the Immunoreactive score (IRS; Remmele et al., 1986) method to obtain the CLDN15 protein in the cell membrane and cytoplasm. The expression of carretinin protein in the nucleus was semi-quantified. Specifically, the staining intensity (Signal intensity; I) of CLDN15 protein or carretinine protein is I = 0 (negative), I = 1 (weak), I = 2 (moderate), I = 3 (strong). It is classified into 4 stages. Furthermore, the percentage of positive staining cells (P) in the tumor area was determined by P = 0 (r <1%) and P = 1 (1% ≤ r) based on the percentage of the staining range (r). It is classified into 5 stages: <10%), P = 2 (10% ≤ r <30%), P = 3 (30% ≤ r <50%), and P = 4 (r ≥ 50%). Next, IRS is calculated as IRS (Immunoreactive score) = S × P, and based on the obtained IRS, score 0 (IRS = 0), score 1+ (IRS = 1, 2, 3), score 2+ Classify into (IRS = 4, 6) and score 3+ (IRS = 8, 9, 12). Based on the obtained scores, a score of 0 was determined to be negative, a score of 1+, a score of 2+, and a score of 3+ were determined to be positive.

(result)
Representative examples of immunohistochemical staining with HE, anti-CLDN15 monoclonal antibody, and anti-calretinin antibody are shown in FIG. 1 (epithelial malignant pleural mesothelioma), FIG. 2 (biphasic malignant pleural mesothelioma), and FIG. 3 (sarcoma). Type malignant pleural mesothelioma).

Table 2 below also shows anti-CLDN15 monoclonal antibody and / or anti-carretinine antibody for each of all histological types of malignant pleural mesothelioma, or epithelial, biphasic, or sarcomatous malignant pleural mesothelioma. The result when the discrimination was performed using. When both the anti-CLDN15 antibody and the anti-calretinin antibody were used for differentiation (“CLDN15 or carretinin” in Table 2), if at least one type of antibody was positive, the case was counted as a positive case.

The sensitivity of anti-CLDN15 antibody alone to epithelial and biphasic malignant pleural mesothelioma was 86% and 67% (“CLDN15” in Table 2), and the sensitivity of anti-calretinin antibody alone (93% and 78%). It was slightly inferior to that (“Carretinin” in Table 2). However, there were cases in which only one of CLDN15 protein and carretinin was positive, and there were cases in which CLDN15 protein was highly expressed even if carretinin was negative (Figs. 1 to 3). Therefore, the sensitivity of the combined use of both markers was higher than that of each marker alone, reaching 100% in epithelial and biphasic malignant pleural mesothelioma (“CLDN15 or carretinin” in Table 2).

On the other hand, the sensitivity of anti-CLDN15 antibody alone, the sensitivity of anti-carretinin antibody alone, and the sensitivity of both markers to sarcoma-type malignant pleural mesothelioma were 40%, 20%, and 40%, respectively. Although the number of cases of sarcoma-type malignant pleural mesothelioma was small, the sensitivity of the anti-CLDN15 antibody alone and the sensitivity of the combined use of both markers were slightly higher than those of the anti-calretinin antibody alone.

The sensitivity of anti-CLDN15 antibody alone, the sensitivity of anti-carretinine antibody alone, and the sensitivity of both markers to 42 cases of malignant pleural mesothelioma of epithelial type, biphasic type, and sarcoma type were 76% and 81%, respectively. , And 93%. Therefore, the sensitivity of the combined use of both markers was the highest.

From the above results, it was clarified that the CLDN15 protein is useful as a positive marker for malignant pleural mesothelioma, especially sarcoma-type and biphasic-type malignant pleural mesothelioma. In particular, it was revealed that malignant pleural mesothelioma can be differentiated with an extremely high sensitivity of 93% when anti-CLDN15 antibody and anti-calretinin antibody are used in combination.

<Example 3: Antigen specificity of anti-CLDN15 monoclonal antibody (1)>
(Purpose)
The antigen specificity and cross-reactivity of the anti-CLDN15 monoclonal antibody (2C11) will be verified.
(Method)
A plasmid vector expressing the human CLDN protein (any of CLDN1, CLDN4, CLDN5, CLDN6, CLDN9, and CLDN15 protein) and the fluorescent protein Venus under the control of the EF1α promoter was prepared, and PEI-max (GE Healthcare) was used to prepare a plasmid vector. It was introduced into the HEK293T cell line. In addition, as a negative control, a plasmid vector containing no CLDN gene was introduced into the HEK293T cell line (vector control). Forty-eight hours after gene transfer, cells were fixed with 10% formalin and then embedded in paraffin to prepare paraffin sections of CLDN-expressing cell clusters, and immunohistochemistry was performed by a method similar to Example 2.
(result)
As shown in FIG. 4, the rat anti-CLDN15 monoclonal antibody (2C11) specifically reacted only with CLDN15-expressing cells and did not react with other CLDNs at all. On the other hand, when 2C11 was not added as the primary antibody, staining of CLDN15-expressing cells was not observed (Fig. 4, αCLDN15 (-)). The results showed that the rat anti-CLDN15 monoclonal antibody (2C11) had high antigen specificity for the CLDN15 protein and no non-specific cross-reactivity for other CLDN proteins.

<Example 4: Antigen specificity of anti-CLDN15 monoclonal antibody (2)>
(Purpose)
The antigen specificity of the anti-CLDN15 monoclonal antibody (2C11) is further verified to determine the epitope.
(Method and result)
In the amino acid sequence of CLDN15 protein, mutants (mut1 to mut20 shown in FIG. 5) in which the region corresponding to the antigen peptide described in Example 1 was replaced with alanine or threonine were prepared by PCR and introduced into the HEK293T cell line. .. Cells 48 hours after gene transfer were lysed with SDS sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 10% Glycerol, 0.25% Bromophenol blue, 5% 2-mercaptoehanol). This solution was run on a 5-20% gradient polyacrylamide gel and transferred to a PVDF membrane. After blocking with 5% skim milk, the 2C11 culture supernatant was used as the primary antibody and reacted at 4 degrees overnight. After washing the PVDF membrane, ECL Rat IgG, HRP-linked whole antibody (from goat) (GE Healthcare; NA935) diluted 4000 times was used as a secondary antibody solution, reacted at room temperature for 1 hour, and then emitted with ECL Prime. , Image Quant LAS 4000 was used for detection. Venus was detected using anti-GFP antibody (MBL) as a loading control.
As shown in FIG. 5, mutants 1 to 8 (mut1 to mut8) and mutant 20 (mut20) reacted with the anti-CLDN15 monoclonal antibody (2C11) (αCLDN15) in the same manner as the wild type (WT). , Mutants 9-19 (mut9-mut19) did not react at all. From this, it was clarified that among the antigenic peptides (C-SDQEGDSSFGKYGRNA) used for immunization, the latter 8 residues (FGKYGRNA) are necessary and sufficient for the reaction with the anti-CLDN15 monoclonal antibody (2C11). From the above results, it was proved that the anti-CLDN15 monoclonal antibody (2C11) uses FGKYGRNA as an epitope and is highly specific for this epitope sequence.

<Example 5: CDR sequencing of anti-CLDN15 monoclonal antibody (2C11)>
(Purpose)
Determine the CDR (complementarity determining region) sequences that determine the antigen specificity of the anti-CLDN15 monoclonal antibody (2C11).
(Method and result)
From hybridomas about 10 ⁷ to produce 2C11, total RNA was extracted using TRIzol (Thermo Fisher Scientific). Immunoglobulin G heavy chain and immunoglobulin light chain (κ, λ) were reverse transcribed using 5'-Full RACE Core Set (Takara Bio; 6122) and specific phosphorylation primers using 700 ng of Total RNA as a template. The reverse transcriptase was concatemated with T4 RNA ligase and nested PCR was performed with specific primers to amplify the CDR-encoding regions of the heavy and light chains. The band confirmed by agarose gel electrophoresis was purified and TA cloned using the pGEM-T Easy vector system (Promega; A1360). The sequences of the resulting plasmids were macrogenized using M13R primers and the CDR sequences were determined according to Kabat's antibody numbering system (Table 1).

Claims (10)

A biomarker consisting of the claudin 15 (CLDN15) gene or CLDN15 protein for differentiating malignant mesothelioma. Anti-CLDN15 antibody or fragment thereof for differentiating malignant mesothelioma. The anti-CLDN15 antibody or fragment thereof according to claim 2, wherein the anti-CLDN15 antibody or fragment thereof recognizes an epitope contained in the intracellular domain on the C-terminal side of the CLDN15 protein. The anti-CLDN15 antibody or fragment thereof according to claim 3, wherein the epitope comprises the amino acid sequence shown in SEQ ID NO: 1. The anti-CLDN15 antibody or a fragment thereof
CDR1, consisting of the amino acid sequence shown in SEQ ID NO: 2,
CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 3 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 4.
CDR1, consisting of a heavy chain variable region containing, and the amino acid sequence shown in SEQ ID NO: 5.
CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 6 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 7.
The anti-CLDN15 antibody or fragment thereof according to any one of claims 2 to 4, which comprises a light chain variable region comprising. The anti-CLDN15 antibody or a fragment thereof
The anti-CLDN15 antibody or fragment thereof according to claim 5, which comprises a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 9. A malignant mesothelioma differential kit comprising the anti-CLDN15 antibody according to any one of claims 2 to 6 or a fragment thereof for detecting the biomarker according to claim 1. A method for differentiating malignant mesothelioma
A detection step for detecting a transcript of the CLDN15 gene or CLDN15 protein in a sample derived from a region suspected to be a malignant tumor in and around the inner skin of a subject.
A determination step for determining whether or not a malignant tumor is contained in the sample based on any one or more of cell atypia, structural atypia, infiltration, and metastasis, and a determination step in which the sample contains a malignant tumor and is malignant thereof. The method comprising a differentiation step of differentiating a subject from malignant mesothelioma when a transcript of the CLDN15 gene or CLDN15 protein is detected in the tumor. 8. The step of detecting the CLDN15 protein, wherein the CLDN15 protein is detected using the anti-CLDN15 antibody according to any one of claims 2 to 6 or a fragment thereof, according to claim 8. the method of. The detection step is a step of further detecting another positive marker protein for differentiating malignant mesothelioma and / or other negative marker protein for differentiating malignant mesothelioma in the sample.
In the differentiation step, when CLDN15 protein is detected in the malignant tumor, the other positive marker protein for differentiating malignant mesothelioma is detected and / or other negative marker protein for differentiating malignant mesothelioma is detected. If not, the subject is in the process of distinguishing it from having malignant mesothelioma.
The method according to claim 9.