JP2010151800A - Diagnostic agent for lifestyle-related diseases and/or cancers - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diagnostic agent and detecting method of lifestyle-related diseases and/or cancers utilizing a marker which enables diagnostic of lifestyle-habit diseases and/or cancers, especially, prescience of prognosis and recurrence. <P>SOLUTION: This is the diagnostic agent of lifestyle-related diseases and/or cancers containing antibody to human angiopoietin-like factor-2 (ANGPTL2), wherein these lifestyle-related diseases include adipositas, insulin resistant syndrome, ischemic heart disease or inflammatory disease, diabetic retinopathy, abdominal aneurysm, angina, or any other chronic disease, the status of which is adversely affected by lifestyle including connective tissue diseases, including dermatomyositis, rheumatism, etc. In addition, the cancers include breast cancer, ovarian cancer, kidney cancer, liver cancer, uterine cancer or skin cancer. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a diagnostic agent for lifestyle-related diseases and / or cancer, and a method for detecting lifestyle-related diseases and / or cancer.

  Angiogenesis is the process by which new capillaries are formed from blood vessels, and it is known that numerous factors (such as vascular endothelial growth factor (VEGF) and angiopoietin) are involved. Angiopoietin has been suggested to have angiogenic activity, epidermal cell proliferation activity, chondrocyte proliferation activity, wound healing promotion activity, tissue regeneration activity, and the like.

  Since all known markers for cancer are tumor antigens, they reflect the size (amount) of tumor antigen-positive cancer, and the cancer status is estimated as the size (amount). However, it is known that cancer actually progresses and metastasizes regardless of the size (amount) of the primary lesion. That is, none of the existing markers for cancer is a marker related to the pathogenesis and progression mechanism. Furthermore, regarding markers for lifestyle-related diseases (for example, obesity, insulin resistance disease, ischemic heart disease, or inflammatory disease), it is desired to identify novel markers capable of early diagnosis and prognosis.

J. Biol. Chem. 274, 26523-26528 1999 Proc. Natl. Acad. Sci. 102: 13502-13507 2005 Cancer Res. 68: 5067-5075 2008

  The present invention finds a marker that enables diagnosis of lifestyle-related diseases and / or cancer, in particular, prognosis and recurrence, and provides a diagnostic agent and detection method for lifestyle-related diseases and / or cancer using the same. It was set as a problem to be solved.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors detected human angiopoietin-like factor 2 (ANGPTL2) in human serum or plasma by using an antibody against human angiopoietin-like factor 2 (ANGPTL2). The inventors have found that it is possible to diagnose habitual diseases and / or cancers, and have completed the present invention.

That is, according to the present invention, the following inventions are provided.
(1) A diagnostic agent for lifestyle-related diseases and / or cancer comprising an antibody against human angiopoietin-like factor 2 (ANGPTL2).
(2) The diagnostic agent according to (1), wherein the lifestyle-related disease is obesity, insulin resistance syndrome, ischemic heart disease, or inflammatory disease.
(3) The lifestyle-related diseases are chronic diseases including diabetic retinopathy, abdominal aortic aneurysm, angina pectoris, or collagen diseases such as dermatomyositis and rheumatism whose lifestyle is involved in pathological deterioration, (1) or ( The diagnostic agent according to 2).
(4) The diagnostic agent according to (1), wherein the cancer is breast cancer, ovarian cancer, kidney cancer, liver cancer, uterine cancer or skin cancer.
(5) The diagnostic agent according to any one of (1) to (4), which is used for prediction of prognosis of lifestyle-related diseases and / or cancer and / or prediction of recurrence.
(6) A method for detecting lifestyle-related diseases and / or cancer, comprising contacting a sample with an antibody against human angiopoietin-like factor 2 (ANGPTL2) and detecting or measuring human angiopoietin-like factor 2 (ANGPTL2) in the sample.
(7) The method according to (6), wherein prediction of prognosis and / or prediction of recurrence of lifestyle-related diseases and / or cancer is performed.
(8) The method according to (6) or (7), wherein the sample is serum or plasma.

  In considering the growth, progression, and metastasis of cancer, it is necessary to consider both the problem of the cancer cell itself (species; Seed) and the problem of the environment in which the cancer cells grow (soil). In other words, factors secreted from the cancer cells themselves (Seed) induce angiogenesis and leukocyte invocation, and are favorable for cancer cells important for cancer growth, progression, and metastasis (Soil). It contributes to. In fact, an attempt to prevent tumor progression by angiogenesis control using a neutralizing antibody against the angiogenic factor VEGF has been started in the clinical setting, and a significant improvement in life prognosis has been observed. However, new problems have emerged in vascular control in cancer, such as drug resistance and increased expression of angiogenic factors other than VEGF. The molecular mechanism of vascular / lymphangiogenesis in cancer has been further elucidated, and the tumor circulation mechanism It is considered to be important to clarify the mechanism of the soil. The usefulness of many biomarkers is used for diagnosis and treatment of diseases other than cancer. CK-MB for acute myocardial infarction, natriuretic peptide and related peptides are widely used as biomarkers for heart failure, and high-sensitivity CRP is widely used as a risk factor for arteriosclerosis and acute coronary syndrome. The fact that these biomarkers are closely related to the pathogenesis of each disease confirms their significance as biomarkers. In that regard, all known markers for cancer are tumor antigens and therefore reflect the size (amount) of tumor antigen-positive cancer, but ANGPTL2 is an important environment for cancer growth, invasion, and metastasis. Serum ANGPTL2 concentration measurement is a novel marker for cancer onset, progression and recurrence diagnosis and prognosis based on the onset and progression mechanism of cancer.

FIG. 1 shows the molecular cloning of an angiopoietin-like protein. FIG. 2 shows the relationship between serum ANGPTL2 values and plasma ANGPTL2 values. FIG. 3 shows the relationship between serum ANGPTL2 concentration, high-sensitivity CRP value, insulin value, and body mass index (BMI). FIG. 4 shows the relationship between serum ANGPTL2 concentration, healthy subjects, obesity groups showing insulin resistance, and ischemic heart disease. FIG. 5 shows the relationship between serum ANGPTL2 concentration, high sensitivity CRP value, insulin resistance index (HOMA-R), visceral fat area, and insulin sensitivity index (M value). FIG. 6 shows expression of ANGPTL2 in mouse chemical-induced skin cancer. FIG. 7 shows the relationship between serum ANGPTL2 concentration value, stage and prognostic days in ovarian cancer. FIG. 8 shows the relationship between tumor size, malignancy, presence / absence of metastasis and serum ANGPTL2 concentration value in primary breast cancer. FIG. 9 shows the relationship between serum ANGPTL2 concentration values of healthy individuals and breast cancer patients. FIG. 10 shows the positive rates of serum Angptl 2, CEA, CA15-3 and NCCST439 in non-invasive cancer (DCIS), invasive breast cancer without distant metastasis (Invasive BC), and breast cancer with distant metastasis (Metastatic BC). FIG. 11 shows the relationship between serum Angptl 2 and CEA in metastatic breast cancer. FIG. 12 shows the relationship between serum Angptl 2 and CA15-3 in metastatic breast cancer. FIG. 13 shows the prognosis by type of breast cancer. FIG. 14 shows the prognosis of triple negative (TN) type breast cancer. FIG. 15 shows the Angptl2 value before treatment start, prognosis, recurrence and outcome in TN type primary breast cancer. FIG. 16 shows the relationship between serum Angptl 2 and tumor characteristics in primary breast cancer. FIG. 17 shows changes in serum Angptl 2 values during surgery and recurrence in the same case. FIG. 18 shows changes in serum Angptl 2 in the same case. FIG. 19 shows gene targeting of the Angptl2 locus. FIG. 20 shows serum Angptl2 concentration and rheumatoid factors. FIG. 21 shows the presence or absence of diabetic retinopathy and the concentration of Angptl2 in the vitreous humor. FIG. 22 shows immunostaining of ANGPTL2 in human abdominal aortic aneurysm lesions. FIG. 23 shows the expression of Angptl2 in macrophages at the lesion of human aortic aneurysm FIG. 24 shows the expression of Angptl2 in a part of smooth muscle of a lesion site of human aortic aneurysm. FIG. 25 shows the creation of a mouse aneurysm model by applying calcium chloride for gene expression analysis. FIG. 26 shows the expression of Angptl2 in the course of aneurysm formation in a mouse aneurysm model by calcium chloride application. FIG. 27 shows Angptl2 protein expression in a mouse aneurysm model by calcium chloride application. FIG. 28 shows a patient background of vasospastic angina. FIG. 29 shows coronary spastic angina and serum Angptl2. FIG. 30 shows serum Angptl2 values and high-sensitivity CRP values in patients with coronary spastic angina. FIG. 31 shows the correlation between coronary artery endothelial function and serum Angptl2 value in patients with coronary spastic angina. FIG. 32 shows the correlation between coronary artery endothelial function and high-sensitivity CRP value in patients with coronary spastic angina. FIG. 33 shows skin cancer and Angptl2. FIG. 34 shows the creation of a skin cancer model using a chemical carcinogenic agent. FIG. 35 shows enhanced sensitivity to carcinogenesis by chemical carcinogens in skin in which Angptl2 is persistently expressed. FIG. 36 shows the expression of Angptl2 in human dermatomyositis.

Embodiments of the present invention will be described below.
(1) Development of concentration measurement system for human ANGPTL2 (Fig. 1) A monoclonal antibody against human angiopoietin-like factor 2 (ANGPTL2) was prepared, an ELISA system was constructed, and a system capable of measuring human ANGPTL2 concentration was developed.

(2) Application of the human ANGPTL2 concentration measurement system to lifestyle-related diseases, especially obesity, insulin resistance, ischemic heart disease, inflammatory disease, and early diagnosis and prognosis in 100 healthy people using the system developed above Human ANGPTL2 concentrations were measured in serum and plasma, and it was found that serum ANGPTL2 values and plasma ANGPTL2 values showed a positive correlation (FIG. 2). It was found that the serum ANGPTL2 concentration value of 100 healthy people positively correlated with high-sensitivity CRP value, insulin value, and body mass index (BMI) (FIG. 3). Moreover, it was found that when serum human ANGPTL2 concentration was measured in an obese disease state group exhibiting insulin resistance, it was clearly higher than that in healthy individuals (FIG. 4). In addition, serum human ANGPTL2 concentrations in obese disease groups that show insulin resistance are highly sensitive CRP values, insulin resistance index (HOMA-R), positive correlation with visceral fat area, and negative correlation with insulin sensitivity index (M value). It was found to be shown (FIG. 5). Furthermore, when the serum human ANGPTL2 concentration was measured in the group having ischemic heart disease, it was found that the serum human ANGPTL2 concentration was clearly higher than that in healthy individuals (FIG. 4). From these results, it was found that measuring serum and plasma human ANGPTL2 concentrations would be an early diagnosis and prognostic factor for lifestyle-related diseases, particularly obesity, insulin resistance syndrome, ischemic heart disease, and inflammatory diseases. It was.

(3) Application of human ANGPTL2 concentration measurement system to cancer onset, progression and recurrence diagnosis and prognosis prediction ANGPTL2 is a prostate cancer, colon cancer, ovarian cancer, pancreas in immunostaining analysis using human tumor tissue It was found to be expressed in cancer, kidney cancer, breast cancer, hepatocellular carcinoma, lung cancer, and brain tumor. In addition, we found that expression of ANGPTL2 appeared and increased in mouse chemical substance-induced skin cancer (FIG. 6). We found that serum ANGPTL2 levels were clearly higher in the affected groups of breast cancer, ovarian cancer, kidney cancer, hepatocellular carcinoma, and uterine cancer compared to 100 healthy individuals. In ovarian cancer, the serum ANGPTL2 concentration value was higher as the stage progressed, and the survival time was found to be negatively correlated (FIG. 7). In breast cancer, the tumor size, malignancy, presence of metastasis, and serum ANGPTL2 concentration values were correlated with primary breast cancer, and serum ANGPTL2 concentration values were clearly high in recurrent cases (Fig. 8, 9 and 10). Although the positive rate was inferior to the existing tumor marker, primary breast cancer showed no association with any existing marker, indicating that it was a new marker (FIG. 10). Metastatic cancer showed association with CA-15-3 but not with CEA (FIGS. 11 and 12).

  Estrogen receptor (ER) negative, progesterone receptor (PgR) negative and HER2 negative Triple negative (TN) type breast cancers are known to have a poor prognosis (FIGS. 13 and 14). The ANGPTL2 concentration before the start of treatment in TN type primary breast cancer was correlated with prognosis, recurrence and outcome (FIG. 15). In addition, it was found that the serum ANGPTL2 concentration value is clearly high in TN type breast cancer with poor prognosis due to breast cancer that has no effect of hormone therapy or the molecular target drug Herceptin (FIG. 16). In fact, in breast cancer, there are cases in which the value of serum ANGPTL2 concentration increases when recurrence occurs (FIGS. 17 and 18).

  The usefulness of many biomarkers is used for diagnosis and treatment of diseases other than cancer. CK-MB for acute myocardial infarction, natriuretic peptide and related peptides are widely used as biomarkers for heart failure, and high-sensitivity CRP is widely used as a risk factor for arteriosclerosis and acute coronary syndrome. The fact that these biomarkers are closely related to the pathogenesis of each disease confirms their significance as biomarkers. In that regard, all known markers for cancer are tumor antigens and therefore reflect the size (amount) of tumor antigen-positive cancer, but ANGPTL2 is an important environment for cancer growth, invasion, and metastasis. Serum ANGPTL2 concentration measurement is based on the pathogenesis of cancer pathology, such as prostate cancer, colon cancer, ovarian cancer, pancreatic cancer, kidney cancer, breast cancer, and hepatocytes. It is a novel marker for the onset, progression and recurrence diagnosis and prognosis of cancer such as cancer, lung cancer, brain tumor and skin cancer.

  The present invention relates to a diagnostic agent for lifestyle-related diseases and / or cancer comprising an antibody against human angiopoietin-like factor 2 (ANGPTL2); and contacting the sample with an antibody against human angiopoietin-like factor 2 (ANGPTL2), and human angiopoietin in the sample The present invention relates to a method for detecting lifestyle-related diseases and / or cancer, comprising detecting or measuring like factor 2 (ANGPTL2).

The amino acid sequence and base sequence of human angiopoietin-like factor 2 (ANGPTL2) are known, for example, registered in the GenBank ^TM database under the registration number AF125175, and J. Biol. Chem. 274 (37), 26523-26528 (1999). Therefore, those skilled in the art can easily obtain ANGPTL2 protein and the gene encoding it.

  The present invention provides a diagnostic agent for lifestyle-related diseases and / or cancer comprising an antibody against human angiopoietin-like factor 2 (ANGPTL2). The antibody against ANGPTL2 used in the present invention may be a polyclonal antibody or a monoclonal antibody, and they are prepared by methods known to those skilled in the art (see, for example, “Shinsei Kagaku Koza 1, Protein I, 389-406, Tokyo Kagaku Dojin”). Is possible. ANGPTL2 has a known amino acid sequence as described above, and can be produced using a normal protein expression technique based on the amino acid sequence. The partial peptide of ANGPTL2 can be produced by selecting an appropriate partial sequence from the amino acid sequence of ANGPTL2 and using a normal peptide synthesis technique.

  For preparation of a polyclonal antibody against ANGPTL2, for example, an appropriate amount of ANGPTL2 protein or a partial peptide thereof is administered to animals such as rabbits, guinea pigs, mice and chickens. Administration may be performed with an adjuvant (FIA or FCA) that promotes antibody production. Administration is usually every few weeks. The antibody titer can be increased by performing immunization multiple times. After the final immunization, antiserum is obtained by collecting blood from the immunized animal. Polyclonal antibodies can be prepared by subjecting this antiserum to, for example, ammonium sulfate precipitation, fractionation by anion chromatography, or affinity purification using protein A or immobilized antigen.

  On the other hand, for preparation of a monoclonal antibody against ANGPTL2, for example, ANGPTL2 protein or a partial peptide thereof is immunized to the animal in the same manner as described above, and the spleen or lymph node is collected from this immunized animal after the final immunization. The antibody-producing cells and myeloma cells contained in the spleen or lymph node are fused using polyethylene glycol or the like to prepare a hybridoma. The target hybridoma is screened, cultured, and a monoclonal antibody can be prepared from the culture supernatant. The monoclonal antibody can be purified by, for example, ammonium sulfate precipitation, fractionation by anion chromatography, or affinity purification using protein A or immobilized antigen. For the purposes of the present invention, the antibody may recognize any epitope of ANGPTL2.

  In the present invention, the above-mentioned antibody fragments may be used. Examples of the antibody fragment include F (ab ') 2 fragment, Fab' fragment and the like.

  For accuracy as a diagnostic agent, the antibody against ANGPTL2 may be a human antibody or a human antibody. For example, if the human antibody is a mouse-human chimeric antibody, an antibody gene is isolated from a mouse cell that produces an antibody against ANGPTL2 protein, and its H chain constant region is recombined with a human IgE H chain constant region gene. It can be prepared by introduction into tumor cells. In addition, human antibodies can be prepared by immunizing mice in which the immune system is replaced with humans with ANGPTL2 protein.

  In addition to the antibody against ANGPTL2, the diagnostic agent of the present invention can appropriately contain a pharmaceutically acceptable carrier or the like as necessary.

  The types of lifestyle-related diseases that can be diagnosed with the diagnostic agent of the present invention are not particularly limited, and examples thereof include obesity, insulin resistance syndrome, ischemic heart disease, and inflammatory diseases. Specific examples of lifestyle-related diseases include diabetic retinopathy, abdominal aortic aneurysm, angina pectoris, or chronic diseases including collagen diseases such as dermatomyositis and rheumatism whose lifestyle is involved in pathological deterioration.

  The type of cancer that can be diagnosed with the diagnostic agent of the present invention is not particularly limited, and includes all benign tumors and malignant tumors. Specific examples of cancers that are malignant tumors are malignant melanoma, malignant lymphoma, digestive organ cancer, lung cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, colon cancer, ureteral tumor, gallbladder cancer, bile duct cancer, biliary tract cancer. Breast cancer, liver cancer (liver cancer), pancreatic cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, kidney cancer, ovarian cancer, uterine cancer, prostate cancer, thyroid cancer, brain tumor, Kaposi's sarcoma, hemangioma, leukemia, polycythemia vera, neuroblastoma, retinoblastoma, myeloma, cystoma, sarcoma, osteosarcoma, myoma, skin cancer, basal cell carcinoma, skin appendage cancer, skin metastasis cancer Examples include, but are not limited to, cutaneous melanoma. Preferred is breast cancer, ovarian cancer, kidney cancer, liver cancer, uterine cancer or skin cancer, more preferred is breast cancer or ovarian cancer, and particularly preferred is breast cancer.

  The present invention further provides a method for detecting lifestyle-related diseases and / or cancer, comprising contacting a sample with an antibody against human angiopoietin-like factor 2 (ANGPTL2). In the present invention, examples of the sample include tissue, serum, plasma, saliva, urine and the like obtained from a subject who may suffer from lifestyle-related diseases and / or cancer, and particularly preferably serum or plasma. The contact between the sample and the antibody may be performed based on a method commonly used in the art, and is not particularly limited. In the diagnosis, for example, after the contact between the sample and the above antibody, the specific binding of the antibody against ANGPTL2 that may be present in the sample is quantified using a secondary antibody labeled with a fluorescent substance, a luminescent substance, an enzyme, or the like. This can be done by detecting automatically. Moreover, the reaction for diagnosis may be performed in a liquid phase such as a well, or may be performed on a solid support on which an antibody against ANGPTL2 is immobilized. In this case, by comparing with a standard value prepared in advance using a normal sample not suffering from lifestyle-related disease and / or cancer, or a sample known to be lifestyle-related disease and / or cancer, Whether the measured value is positive as a lifestyle-related disease and / or cancer can be determined. In diagnosis, the cut-off value can be set by measuring the amount of ANGPTL2 in the serum or plasma of many lifestyle-related diseases and / or cancer patients and healthy individuals.

Specific examples of a method for immunologically detecting ANGPTL2 in a sample using an antibody against ANGPTL2 include an enzyme immunoassay (EIA) such as a sandwich ELISA, or a radioimmunoassay (RIA). This technique is well known to those skilled in the art. For example, in sandwich ELISA, two types of antibodies having different antigen recognition sites are prepared, and one antibody is adsorbed on a plate. The sample is brought into contact with the plate for reaction, the other antibody having a different antigen recognition site is reacted, and an anti-immunoglobulin antibody labeled with an enzyme such as peroxidase or alkaline phosphatase is further reacted. After adding a substrate that develops color with an enzyme to cause a color reaction, ANGPTL2 in the sample can be detected or measured by measuring the color intensity with a spectrophotometer. In the radioimmunoassay, an antibody labeled with a radioactive substance such as ¹²⁵ I is used instead of an enzyme, and the same operation as in the enzyme immunoassay is performed, and radiation is measured with a scintillation counter.

  The detection method of the present invention can be used for diagnosing whether or not the patient has lifestyle-related diseases and / or cancers, and is performed over time to confirm the effects of treatments on lifestyle-related diseases and / or cancers. You can also. For example, the detection method of the present invention can be used for prognosis of lifestyle-related diseases and / or cancer and / or for recurrence.

The present invention further provides a kit for diagnosis of lifestyle-related diseases and / or cancer comprising an antibody against ANGPTL2. The kit of the present invention contains at least the above-mentioned antibody against ANGPTL2, and additionally contains reagents necessary for detecting ANGPTL2 in the sample (for example, secondary antibody, coloring reagent, buffer, etc.) as appropriate. Can do.
EXAMPLES Hereinafter, although an Example is given and this invention is further demonstrated, this invention is not limited to these Examples.

Example 1:
(experimental method)
(1-1) Purification of Recombinant Angptl2 Protein Recombinant mouse and human Angptl2 protein can be obtained from Cancer Res 63: 6651-6657 2003, Blood 103: 3760-3766 2004, and Proc. Natl. Acad. Sci. 102: 13502-13507 2005. Purified as described. That is, the Angptl2 coding region fused to the N-terminus of the FLAG epitope was subcloned into pCEP4 (Invitrogen, Groningen, the Netherlands). HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (FBS) at 37 ° C. with 5% CO ₂ and Fugene-6 (Roche Diagnostics, Mannheim, Germany) was used to transfect pCEP4-Angptl2. Cells were selected using 300 mg / mL hygromycin B (GIBCO-BRL, Gaithersburg, MD) for 5 days, conditioned media was collected and filtered using a 0.22 mm filter (Millipore, Bedford, Mass.). To purify Angptl2 protein, the conditioned medium was transferred to an anti-FLAG antibody (M2) affinity gel (Sigma). After washing the gel with phosphate buffer (PBS), the protein was eluted by adding Gly-HCl (pH 3.0) and immediately neutralized with Tris (Tris (hydroxymethyl) aminomethane) hydrochloric acid (pH 8.0). . Proteins were analyzed by Coomassie Brilliant Blue Staining (CBB) (Wako, Osaka, Japan) on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE), and horseradish peroxidase labeled anti-FLAG antibody (M2) Visualization was by Western blot using (1: 200) (Sigma) and anti-Angptl2 antibodies. An antibody was prepared by immunizing a rabbit with a synthetic peptide (SFRLEPESEYYKLRLGRY) (SEQ ID NO: 1) corresponding to amino acids 383 to 400 of human Angptl2, which has the same sequence as mouse Angptl2.

(1-2) Gene targeting of Angptl2 locus (FIG. 19)
(a) shows the wild-type Angptl2 locus, the targeting vector, and the mutant allele after homologous recombination. Exons and hybridization probes are indicated by white squares and black squares, respectively. E, S and H represent EcoRI, SpeI and HindIII restriction enzyme sites, respectively. (B) shows the result of Southern blot analysis using the probe shown in (a) after SpeI digestion of genomic cDNA (b) from ES cells having homologous recombination. The results of Southern blot analysis of progeny tail genomic DNA after SpeI digestion using the probe shown in (a) are shown. The 9.0 and 12.5 kb bands corresponding to the wild type and mutant alleles are shown, respectively. (D) shows a Northern blot of adipose tissue. Angptl2 mRNA signal was detected in Angptl2 +/- and wild type control mice.

(1-3) Anti-human Angptl2 monoclonal antibody A mouse monoclonal antibody against recombinant human Angptl2 was prepared as described in Blood 105: 4649-4656 2005. Briefly, 6-week-old Angptl2 knockout mice were immunized subcutaneously weekly for 4 weeks with 100 mg human Angptl2 protein in Freund's complete adjuvant (Difco, Detroit, MI). Four days later, spleen cells were collected and fused with mouse myeloma X63 Ag8 6.5.3 cells (ATCC, Manassas, VA). Undiluted hybridoma supernatants were screened using microplates coated with 50 ng / ml human Angptl2 protein. Positive hybridomas were cloned by limiting dilution. The microplate was first incubated with undiluted conditioned medium from a monoclonal hybridoma. To select for hybridomas that react positively with human Angptl2 protein, binding was detected with horseradish peroxidase (HRP) -labeled goat anti-rat IgG antibody (Biosource, Camarillo, Calif.). Enzyme activity bound to the plate was detected by o-phenylenediamine (OPD) chromogen (Sigma), and the absorbance of the sample in each well was measured using a microplate reader (Benchmark Plus; Bio-Rad, Hercules, CA) Measured. The selected clone was mass-cultured, and the monoclonal antibody was purified using agarose beads coated with anti-mouse IgG antibody.

(1-4) ELISA against Angptl2 in human serum
Two types of anti-Angptl2 monoclonal antibodies were used in a double antibody sandwich ELISA to detect human Angptl2. K2-1A1 mouse monoclonal antibody was immobilized on a 96-well plate. A serum sample diluted 10-fold was fixed on a plate at 37 ° C. for 1 hour, washed with PBS containing 0.05% Tween 20 (PBST), and horseradish peroxidase (HRP) -labeled K1-12A4 mouse monoclonal antibody was added. . After 1 hour incubation at 4 ° C, the plate was washed with PBST and HRP
Detection reagent TMB plus for was added. After 30 minutes, an equal amount of 1N H ₂ SO ₄ was added to stop the reaction, and the absorbance at 450 nm was measured.

(result)
(2-1) Diagnosis / detection of lifestyle-related diseases by ANGPTL2 value FIG. 2 shows the results of measurement of human ANGPTL2 concentration in serum and plasma in 100 healthy individuals by ELISA described in (1-4) above. As a result, it was found that serum ANGPTL2 value and plasma ANGPTL2 value were positively correlated (FIG. 2).

  Moreover, the result of having investigated the relationship between the value of the serum ANGPTL2 density | concentration of 100 healthy persons, a highly sensitive CRP value, an insulin value, and a body mass index (BMI) is shown in FIG. As a result, it was found that the serum ANGPTL2 concentration value, high sensitivity CRP value, insulin value, and body mass index (BMI) showed a positive correlation.

  Moreover, the result of having measured the serum human ANGPTL2 density | concentration in the obesity condition group which shows insulin resistance, and the group which has an ischemic heart disease is shown in FIG. As a result, it was found that the serum human ANGPTL2 concentration was clearly higher than that of healthy individuals in any group. Further, in the obesity condition group showing insulin resistance, the results of examining serum human ANGPTL2 concentration, high sensitivity CRP value, insulin resistance index (HOMA-R), visceral fat area and insulin sensitivity index (M value) are shown in FIG. Shown in As a result, it was found that serum human ANGPTL2 concentration showed a high correlation with high sensitivity CRP value, insulin resistance index (HOMA-R), positive correlation with visceral fat area, and negative correlation with insulin sensitivity index (M value). .

  From these results, it was found that measuring serum and plasma human ANGPTL2 concentrations would be an early diagnosis and prognostic factor for lifestyle-related diseases, particularly obesity, insulin resistance syndrome, ischemic heart disease, and inflammatory diseases. It was.

(2-2) Diagnosis of m cancer onset, progression and recurrence by ANGPTL2 value and prognosis prediction ANGPTL2 is a prostate cancer, colon cancer, ovarian cancer, pancreatic cancer, kidney cancer, breast cancer, by immunostaining analysis using human tumor tissue, It was found to be expressed in hepatocellular carcinoma, lung cancer and brain tumor.

  In addition, FIG. 6 shows the results of examining the expression of ANGPTL2 at the mRNA and protein levels for chemical substance-induced skin cancer in normal skin and mice. As a result, it was found that the expression of ANGPTL2 increased in mouse chemical-induced skin cancer.

  As a result of comparison with 100 healthy people, it was found that the serum ANGPTL2 concentration was clearly high in the affected groups of breast cancer, ovarian cancer, kidney cancer, hepatocellular carcinoma, and uterine cancer.

  Moreover, the result of having investigated the relationship between the serum ANGPTL2 concentration value in ovarian cancer, stage, and prognostic days is shown in FIG. As a result, it was found that the serum ANGPTL2 concentration value increased as the stage progressed, and the survival time showed a negative correlation in ovarian cancer.

  Furthermore, the results of examining the relationship between tumor size, malignancy, presence / absence of metastasis and serum ANGPTL2 concentration value in primary breast cancer are shown in FIG. 8, and the relationship between serum ANGPTL2 concentration values in healthy individuals and breast cancer patients was examined. The results are shown in FIG. 9, and the positive rates of serum Angptl 2, CEA, CA15-3, NCCST439 in non-invasive cancer (DCIS), invasive breast cancer without distant metastasis (Invasive BC), and breast cancer with metastasis (Metastatic BC) The result of measuring is shown in FIG. As a result, it was found that the tumor size, malignancy, metastasis, and serum ANGPTL2 concentration value were correlated with the primary breast cancer, and the serum ANGPTL2 concentration value was clearly high in recurrent cases. Although the positive rate was inferior to the existing tumor marker, primary breast cancer showed no association with any existing marker, indicating that it was a new marker (FIG. 10). FIG. 11 shows the relationship between serum Angptl 2 and CEA in metastatic breast cancer, and FIG. 12 shows the relationship between serum Angptl 2 and CA15-3 in metastatic breast cancer. As a result, metastatic cancer showed association with CA-15-3 but not with CEA.

  The prognosis by type of breast cancer is shown in FIG. 13, and the prognosis of triple negative (TN) type breast cancer is shown in FIG. As a result, estrogen receptor (ER) negative, progesterone receptor (PgR) negative and HER2 negative triple negative (TN) type breast cancers were found to have a poor prognosis.

  Moreover, the result of having investigated the relationship between the ANGPTL2 density | concentration before the treatment start in TN type primary breast cancer, prognosis, recurrence, and an outcome is shown in FIG. As a result, the ANGPTL2 concentration before treatment initiation in TN type primary breast cancer was correlated with prognosis, recurrence and outcome.

  FIG. 16 shows the relationship between serum Angptl 2 and tumor characteristics in primary breast cancer. As a result, it was found that the serum ANGPTL2 concentration was clearly higher in TN type breast cancer with poor prognosis and breast cancer that had no effect of hormone therapy or the molecular target drug Herceptin.

  In breast cancer, as a case where the value of serum ANGPTL2 concentration increases when recurrence, the change in serum Angptl 2 value at the time of surgery and recurrence in the same case is shown in FIG. 17, and the change of serum Angptl 2 in the same case is shown. As shown in FIG.

Example 2: Rheumatoid arthritis and angptl2
Serum was collected from a rheumatoid arthritis (RA) patient who had undergone knee replacement, and the concentration of Angptl2 was measured by ELISA using a human angiopoietin like2 measurement kit manufactured by IBL. Clinical data (rheumatoid factor; RF, CRP, blood sedimentation rate; ESR) were investigated from the patients who were the subjects, and the correlation with the measured Angptl2 was analyzed. In addition, Disease Activity Score 28 (DAS28) was calculated from the number of pressure joints (TJC), swollen joint count (SJC), and Visual Analogue Scale (VAS) for each patient. The correlation was analyzed. The formula for DAS28 is as follows.
DAS28-ESR = 0.555 × √ (TJC) + 0.284 × √ (SJC) + 0.7 × LN (ESR) + 0.0142 × (VAS)
DAS28-CRP = 0.555 × √ (TJC) + 0.284 × √ (SJC) + 0.36 × LN ((CRP) × 10 + 1) + 0.0142 × (VAS) +0.96
TJC: tender joints count
SJC: swollen joints count
E: ESR (mm / hr)
C: CRP (mg / dl)
VAS: Visual analogue scale (General Health: 0-100mm by patient)
LN: natural logarithm

  The results are shown in FIG. Serum Angptl2 values tend to be positively correlated with RA-related factors (DAS28-ESR, DAS28-CRP) (N = 12). From these results, serum Angptl2 levels tend to correlate with RA-related factors, and are considered to reflect the pathology of RA, and can be used for diagnosis and follow-up of RA.

Example 3: Presence of diabetic retinopathy and concentration of Angptl2 in vitreous humor 1 μl each of vitreous humor collected from 8 patients with diabetic retinopathy was diluted 10 times and Angptl 2 was measured by ELISA.
The results are shown in FIG. Patients with diabetic retinopathy have higher levels of Angptl2 in the vitreous humor than those without. Angptl2 in the vitreous humor is thought to reflect diabetic retinopathy and may be used for diagnosis and follow-up of diabetic retinopathy.

Example 4: Abdominal aortic aneurysm and Angptl2
FIG. 22 shows the result of immunostaining the pathological specimen of the aneurysm wall of an abdominal aortic aneurysm patient with ANGPTL2. Expression of Angptl2 was observed in inflammatory cells that had infiltrated the aneurysm and in parts that seemed to be degenerated smooth muscle. In normal vascular wall, expression of Angptl2 was not observed except in the vascular endothelium. FIG. 23 shows the result of fluorescent double immunostaining with CD68 and ANGPTL2, which are macrophage markers, in the pathological specimen of the aneurysm wall of an abdominal aortic aneurysm patient. . Both results were consistent.

FIG. 24 shows the result of fluorescent double immunostaining with α-SMA and ANGPTL2, which are markers of vascular smooth muscle. Both results were consistent.
A mouse abdominal aorta model was created using the calcium chloride method. FIG. 25 shows the preparation protocol.

FIG. 26 shows the results of changes over time in gene expression using a real-time PCR method in a mouse abdominal aortic aneurysm model. Genes showing acute inflammation, such as TNFα, IL1-β, and IL-6, are expressed early after surgery and are rapidly disappearing, whereas Angptl2 is persistently (chronically) high, and Mφ (CD68 ) Is similar to the movement.
FIG. 27 shows the results of confirming protein expression using Western blotting in a mouse abdominal aortic aneurysm model.

  It has been conventionally pointed out that chronic inflammation is a key factor in the formation of abdominal aortic aneurysms, but Angptl2 is thought to be involved in the formation of abdominal aortic aneurysms. Therefore, it is considered that serum Angptl2 measurement can be used for diagnosis of abdominal aortic aneurysm and follow-up of the size of the aneurysm.

Example 5: Coronary spasm angina and serum Angptl2
FIG. 28 shows the patient background. There are many women in the control group, but there are no significant differences between the two groups in terms of coronary risk factors such as impaired glucose tolerance, blood pressure, lipid metabolism, and renal function. Smoking tends to be more common in the CSA group, although there is no significant difference. High-sensitivity CRP, an indicator of chronic inflammation, tends to be higher in the coronary vasospastic angina group (CSA).
FIG. 29 shows the measurement results of serum Angptl2 values. Serum Angptl2 levels were elevated in the coronary spastic angina group.

FIG. 30 shows serum Angptl2 values and high-sensitivity CRP values in patients with coronary spastic angina. There is a tendency to be positively correlated.
FIG. 31 shows the results of examining the correlation between coronary artery endothelial function and serum Angptl2 value in patients with coronary spastic angina. When coronary endothelial function is low, serum Angptl2 tends to be high.
FIG. 32 shows the results of examining the correlation between coronary artery endothelial function and high-sensitivity CRP value in patients with coronary spastic angina. There is no correlation between coronary endothelial function and high sensitivity CRP value.

  In patients with coronary spastic angina or patients with vascular endothelial disorder, the serum Angptl2 value is considered to reflect the disease state. Therefore, it is considered that serum Angptl2 can be used for diagnosis and follow-up of coronary spasm angina and vascular endothelial disorder.

Example 6: Skin cancer and Angptl2
FIG. 33 shows skin cancer and Angptl2. From the top, SCC (squamous cell carcinoma) tumor part, resection stump 1 cm away from the tumor part, and normal part 3 cm or more away from the tumor are shown. HE staining on the left and Angptl2 immunostaining on the right. The antibody used is Angptl2 polyclonal antibody (383). Autoclave activation. Imaging condition x100, Angptl2 is positive at the tumor site.

  Carcinogenicity experiments were performed using a chemical carcinogenic agent using a transgenic mouse (K14) overexpressing angptl2 in keratinocytes. FIG. 34 shows the protocol.

FIG. 35 shows the results of examining the increased sensitivity to carcinogenesis by chemical carcinogens in skin in which Angptl2 is continuously expressed. In the skin of mice overexpressing angptl2 in keratinocytes, carcinogenesis by chemical carcinogenic agents was enhanced compared to normal mice.
It is thought that induction of Angptl2 is involved in the onset of skin cancer, and Angptl2 is considered to be used for diagnosis and follow-up of skin cancer.

Example 7: Dermatomyositis and Angptl2
FIG. 36 shows the results of Angptl2 immunostaining (polyclonal antibody (383): 100-fold dilution) in the skin and striated muscle of patients with dermatomyositis. Angptl2 is expressed in the skin of patients with dermatomyositis, and Angptl2 may be used for diagnosis and follow-up of dermatomyositis.

  Cancer is the leading cause of death from Japanese diseases. By developing a human ANGPTL2 concentration measurement system, FOLLOW UP ANGPTL2 concentration in cancer patients can quickly predict the progression, metastasis, and recurrence of cancer, which can contribute to the reduction of medical costs.

Claims (8)

A diagnostic agent for lifestyle-related diseases and / or cancer, comprising an antibody against human angiopoietin-like factor 2 (ANGPTL2). The diagnostic agent according to claim 1, wherein the lifestyle-related disease is obesity, insulin resistance syndrome, ischemic heart disease, or inflammatory disease. The lifestyle-related disease is a chronic disease including collagen diseases such as diabetic retinopathy, abdominal aortic aneurysm, angina pectoris, or dermatomyositis whose lifestyle is involved in pathological deterioration, rheumatism and the like. Diagnostic agent. The diagnostic agent according to claim 1, wherein the cancer is breast cancer, ovarian cancer, kidney cancer, liver cancer, uterine cancer or skin cancer. The diagnostic agent according to any one of claims 1 to 4, which is used for prognosis of lifestyle-related diseases and / or cancer and / or for recurrence. A method for detecting lifestyle-related diseases and / or cancer, comprising contacting a sample with an antibody against human angiopoietin-like factor 2 (ANGPTL2) and detecting or measuring human angiopoietin-like factor 2 (ANGPTL2) in the sample. The method according to claim 6, wherein a prognosis and / or a recurrence is predicted for lifestyle-related diseases and / or cancer. The method according to claim 6 or 7, wherein the sample is serum or plasma.