阅读说明：本技术 一种从血液诊断癌症的方法 (A method for diagnosing cancer from blood ) 是由 金圣镇 梁境珉 于 2018-10-24 设计创作，主要内容包括：公开了一种诊断癌症的方法以及用于诊断癌症的试剂盒,该方法包括以下步骤：将从个体分离的试料与特异性结合于BAG2多肽或其片段的抗体、多肽或其组合接触；从通过接触所述特异性结合于BAG2多肽或其片段的抗体、多肽或其组合来形成的复合体中测量所述试料中BAG2的存在或水平；以及将从所述试料测量的BAG2的存在或水平与从对照组测量的BAG2的水平进行比较。(Disclosed are a method for diagnosing cancer and a kit for diagnosing cancer, the method comprising the steps of: contacting a sample isolated from an individual with an antibody, polypeptide, or combination thereof that specifically binds to a BAG2 polypeptide or fragment thereof; measuring the presence or level of BAG2 in said sample from a complex formed by contacting said antibody, polypeptide, or combination thereof that specifically binds to a BAG2 polypeptide or fragment thereof; and comparing the presence or level of BAG2 measured from the sample with the level of BAG2 measured from a control group.)

1. A method of diagnosing cancer, comprising:

contacting a sample isolated from an individual with an antibody, polypeptide, or combination thereof that specifically binds to a BAG2 polypeptide or fragment thereof;

measuring the presence or level of BAG2 in said sample from a complex formed by contacting said antibody, polypeptide, or combination thereof that specifically binds to a BAG2 polypeptide or fragment thereof; and

the presence or level of BAG2 measured from the sample is compared to the level of BAG2 measured from a control group.

2. The method of claim 1, wherein: the sample is blood, serum, plasma or a combination thereof.

3. The method of claim 1, wherein the BAG2 is soluble in blood, serum, plasma, or a combination thereof.

4. The method of claim 1, wherein the amino acid sequence of BAG2 polypeptide has the amino acid sequence of seq id No. 1 or 2.

5. The method of claim 1, further comprising:

contacting a sample isolated from said individual with an antibody, polypeptide, or combination thereof that specifically binds to a polypeptide of cathepsin B or a fragment thereof;

measuring the presence or level of cathepsin B from a complex formed by contacting the antibody, polypeptide or combination thereof that specifically binds to a polypeptide of cathepsin B; and

comparing the presence or level of cathepsin B measured from the sample with the level of cathepsin B measured from a control group.

6. The method of claim 5, wherein said cathepsin B is cathepsin B.

7. The method of claim 5, wherein the amino acid sequence of the cathepsin B polypeptide has an amino acid sequence selected from any one of SEQ ID NOs 3 to 8.

8. The method of claim 5, wherein the step of measuring the presence or level of BAG2 or cathepsin B from the sample is performed by immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, radioimmunodiffusion, two-way immunodiffusion, rocket immunoelectrophoresis, histoimmunostaining, immunoprecipitation analysis, complement fixation analysis, flow cytometry, protein chips, or a combination thereof.

9. The method of claim 1, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, sarcoma, melanoma, head and neck cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, and neuroblastoma.

10. The method of claim 9, wherein the breast cancer is metastatic breast cancer.

11. The method of claim 9, wherein the breast cancer is triple negative breast cancer.

12. A kit for diagnosing cancer, which detects the presence of BAG2 in blood, serum, plasma or a combination thereof,

and includes such antibodies, polypeptides, or combinations thereof that specifically bind to a BAG2 polypeptide or fragment thereof.

13. The kit of claim 12, further comprising: an antibody, polypeptide, or combination thereof that specifically binds to a polypeptide of cathepsin B or a fragment thereof.

14. The kit of claim 12, wherein,

the cancer is selected from breast cancer, carcinoma of large intestine, lung cancer, sarcoma, melanoma, head and neck cancer, cervical cancer, uterine cancer, liver cancer, renal cancer, pancreatic cancer and neuroblastoma.

15. The kit of claim 12, wherein the breast cancer is metastatic breast cancer.

16. The kit of claim 12, wherein the breast cancer is triple negative breast cancer.

Technical Field

The present invention relates to a method for diagnosing cancer from BAG2, tissue zymogen B or a combination thereof present in blood, and more particularly, to a method for diagnosing triple-negative breast cancer (triple-negative breast cancer).

Background

The BAG (co-chaperone Bcl-2-associated alkane) protein family mediates a variety of physiological processes including intracellular protein folding, stress response, neural differentiation, apoptosis, cell proliferation, and the like, and functionally binds to a variety of synergistic proteins (Takayama and Reed., 2001; Doong et al, 2002). BAG2 is one of the members of the BAG domain family with anti-apoptotic activity, called negative regulator of CHIP (C-terminal of Hsc70-interacting protein) as a chaperone-related ubiquitin ligase (Arndt et al, 2005; Dai et al, 2005). The major role of BAG2 in the regulation of proteins by inhibition of CHIP activity is associated with neurodegenerative and autosomal recessive genetic diseases stabilized by chaperone-associated proteins such as PINK1 and CFTR (quetal, 2015; Arndt et al, 2005). Expression of BAG2 has been shown to increase proteasome inhibitor-induced apoptosis, and BAG2 knockdown partially inhibits apoptosis when thyroid cancer cells are exposed to the proteasome inhibitor MG132, so BAG2 may have pro-apoptotic activity (Wang et al, 2008). On the other hand, it has been shown that overexpression of BAG2 can promote stabilization of the strong tumor gene STK33 protein in various mutated K-Ras-induced tumors, leading to promotion of tumorigenesis (azoiti et al, 2012). However, despite these findings, the role of BAG2 in cancer progression and metastasis, particularly in breast cancer, is still unclear.

Cathepsin B (Cathepsin B, CTSB) is a lysosomal cysteine protease with endogenous and exogenous peptidase activity, which is thought to play a role in protein circulation (mott and button, 1997). CTSB is synthesized as an inert/immature pro-form (pro-form) enzyme (41/43kDa) and converted to the active short-chain form (31kDa) or double-chain form (heavy chain), 25/26 kDa; light chain (light chain), 5kDa) by proteolytic processing of the N-terminal 62 amino acid propeptide. Aberrant (aborrant) expression/activity of CTSB is often associated with malignancy (joynet al., 2004; Withans, 2012). However, it is well recognized that mature CTSB are usually located in lysosomes and function as inhibitor proteases in the protein cycle during lysosome-mediated autophagy/apoptosis, thereby exhibiting the anti-apoptotic effect of cytoplasmic CTSB (Stoka et al, 2001; Foghsgaardet al, 2001; Bhopathiet al, 2010). On the other hand, it is not clear that this dual function of CTSB is a regulator in cancer progression.

Moreover, the position, distribution, positional shift according to cell functions, and the like of BAG2 and cathepsin B present in cells have not been studied in detail.

Disclosure of Invention

Technical problem

In one aspect, the present invention provides a method for diagnosing cancer, comprising: contacting a sample isolated from an individual with an antibody, polypeptide, or combination thereof that specifically binds to a BAG2 polypeptide or fragment thereof; measuring the presence or level of BAG2 in said sample from a complex formed by contacting said antibody, polypeptide, or combination thereof that specifically binds to a BAG2 polypeptide or fragment thereof; and comparing the presence or level of BAG2 measured from the sample with the level of BAG2 measured from a control group.

Another aspect of the present invention provides a kit for diagnosing cancer, which detects BAG2 present in blood, serum (serum), plasma (plasma), or a combination thereof, and includes the antibody, polypeptide, or a combination thereof, which specifically binds to BAG2 polypeptide or a fragment thereof.

Technical scheme

The present invention will be described in more detail below.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In addition, although the present specification describes preferred methods or samples, methods or samples similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications cited in this specification as references are incorporated herein by reference in their entirety.

In one aspect, the present invention provides a method for diagnosing cancer, comprising: contacting a sample isolated from an individual with an antibody, polypeptide, or combination thereof that specifically binds to a BAG2 polypeptide or fragment thereof; measuring the presence or level of BAG2 in said sample from a complex formed by contacting said antibody, polypeptide, or combination thereof that specifically binds to a BAG2 polypeptide or fragment thereof; and comparing the presence or level of BAG2 measured from the sample with the level of BAG2 measured from a control group.

In another embodiment, as a method of providing information for diagnosing cancer, the method is for diagnosing cancer present in an individual by detecting the presence of cancer in the individual.

When BAG2 is present in a sample isolated from an individual, an antibody, polypeptide, or combination thereof that specifically binds to a BAG2 polypeptide or fragment thereof can bind to BAG2 in the sample. The antibody or polypeptide may be labeled with, for example, a fluorophore (fluorophore), a chromophore (chromophore), or an enzyme that converts a substrate to a chromophore to visualize the presence of BAG2 in a sample.

Due to the binding reaction, BAG2 in the sample forms a complex with the antibody, polypeptide, or combination thereof that specifically binds to BAG2 polypeptide or fragment thereof, and the presence of BAG2 can be confirmed from the complex using methods known to those skilled in the art, or its level can be measured as needed. In a specific embodiment, the step of measuring the complex may be performed by immunoblotting, enzyme linked immunosorbent Assay (ELISA), Radioimmunoassay (RIA), radioimmunodiffusion (radioimmunodiffusion), two-way (okhterlong) immunodiffusion, rocket (rock) immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay (Immunoprecipitation Assay), Complement Fixation Assay (Complement Fixation Assay), flow cytometry (FACS), protein chip (protein chip), or a combination thereof.

The presence or level of BAG2 measured from the sample can be compared to the level of BAG2 measured from a control group to diagnose the presence or absence of cancer in the individual. The control group may be a sample obtained from a healthy individual, or may be a sample obtained from various types of patients with average breast cancer, non-metastatic breast cancer or non-TNBC type breast cancer, as necessary. Thus, the level of BAG2 measured from the control group may refer to the average of the BAG2 concentration in samples taken from healthy individuals at the BAG2 level, or samples taken from various types of average breast cancer patients, non-metastatic breast cancer patients, or non-TNBC type breast cancer patients as needed. The sample used as the control group may be of the same type as a sample collected at the same anatomical position as the sample used for the diagnosis. For example, if the sample is blood collected from the Median Cubital Vein (Median Vein) of an individual, the control group may be blood collected from the Median Cubital Vein of the control group. The healthy individual does not suffer from any acute or chronic disease, at least does not suffer from cancer, and preferably may not suffer from breast cancer.

The level of BAG2 in a sample taken from said healthy individual may be such that BAG2 is substantially absent. Thus, when the control group is set to a value obtained from a healthy individual, the subject may be suspected of having cancer if the presence of BAG2, or a significantly high level thereof, in the diagnosed individual is measured. On the other hand, as shown in the examples of the present invention, the presence of BAG2 was detected with a high probability and a significantly high mean value in the blood of metastatic breast cancer patients among breast cancer patients, particularly TNBC type patients. Thus, when the control group is set as a sample isolated from various types of mean breast cancer patients, non-metastatic breast cancer patients or non-TNBC type breast cancer patients, an individual diagnosed with a cancer may be suspected of having metastatic breast cancer or TNBC type breast cancer if the level of BAG2 is measured to be higher or significantly higher than the control group.

In the present specification, the "polypeptide" and "protein" are used interchangeably.

The sample is isolated from an individual to be diagnosed and may be a cell, organ, cell lysate, whole blood, serum, plasma, lymph, extracellular fluid, bodily fluid, urine, stool, tissue, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof.

In one embodiment, the sample can be blood, serum (serum), plasma (plasma), or a combination thereof. The present inventors have demonstrated that BAG2 can be secreted extracellularly and that BAG2 was actually detected in the blood of breast cancer patients. Thus, in a specific embodiment, the BAG2 is soluble in blood, serum (serum), plasma (plasma), or a combination thereof.

In a specific embodiment, the diagnostic method of the present invention may further comprise: contacting a sample isolated from said subject with an antibody, polypeptide, or combination thereof that specifically binds to a polypeptide of cathepsin b (cathepsin b), or a fragment thereof; measuring the presence or level of cathepsin B from a complex formed by contacting the antibody, polypeptide or combination thereof that specifically binds to a polypeptide of cathepsin B; and comparing the presence or level of cathepsin B measured from the sample with the level of cathepsin B measured from a control group.

The present inventors have demonstrated that BAG2 prevents cathepsin B from being converted into a mature form and thus can ultimately inhibit apoptosis (apoptosis) of cancer cells, and in line therewith, it was observed that both BAG2 and the immature form of cathepsin B are secreted extracellularly. Therefore, the diagnostic method of the present invention can further improve the accuracy of diagnosis by simultaneously detecting BAG2 and cathepsin B.

Cathepsin B can be divided into cathepsin B and mature forms of single-chain (single-chain) or double-chain (double-chain) cathepsin B, which the inventors have demonstrated is secreted extracellularly in an immature form due to BAG 2. Thus, according to a specific embodiment, in the diagnostic method of the invention, the cathepsin B may be pro-cathepsin B. The cathepsin B may be used in the same sense as "immature cathepsin B" or "immature CTSB".

In a specific example, the step of measuring a complex formed by contacting an antibody, a polypeptide, or a combination thereof that specifically binds to cathepsin B and a polypeptide of cathepsin B or a fragment thereof in the sample can be performed by immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, radioimmunodiffusion, bidimensional immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry, protein chips, or a combination thereof.

The term "antibody" refers to a specific immunoglobulin directed against an antigenic site. The antibody refers to a polypeptide or a combination of polypeptides that specifically binds to BAG2 and cathepsin B, respectively, and BAG2 or cathepsin B gene is cloned into an expression vector to obtain a protein encoded by each gene, and an antibody specific to each protein can be prepared from the obtained BAG2 or cathepsin B protein according to a conventional method in the art. The form of the antibody includes a polyclonal antibody, a monoclonal antibody or a recombinant antibody (e.g., ScFv fragment, diabody, single chain antibody, etc.), and includes all immunoglobulin antibodies. The antibodies also include functional fragments of antibody molecules that do not have the complete morphology with two full-length light chains and two full-length heavy chains, but also include structures of complete antibodies that do not have the complete morphology with two light chains and two heavy chains, but have specific antigen-binding sites (domains) for antigenic sites to retain antigen-binding function.

The BAG2 and the cathepsin B polypeptide may be derived from humans (Homo sapiens) or mice (mususculus), respectively, and may be derived from other mammals, such as monkeys, cows, horses, etc. The amino acid sequence of BAG2 may include seq id No. 1 or2 (GenBank accession nos. NP _004273.1 and NP _663367.1, respectively), and the amino acid sequence of tissue zymogen B may include seq id nos 3 to 8 (GenBank accession nos. NP _001899.1, NP _680090.1, NP _680091.1, NP _680092.1, NP _680093.1, or NP _031824.1, respectively). Even if some of the amino acid sequences of the sequence numbers 1 to 8 are not identical, so that an amino acid sequence having a biologically equivalent activity can be regarded as BAG2 or a cathepsin B polypeptide. The BAG2 or cathepsin B polypeptide may comprise an amino acid sequence having at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% sequence homology with any of seq id nos 1 or2 and 3 or 8, respectively. In addition, the BAG2 or the cathepsin B polypeptide may be a polypeptide having a sequence which differs by at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, or at least 7 amino acid residues in any one of the sequences of seq id nos 1 or2 and 3 or 8, respectively.

The subject to be subjected to the diagnostic method may be a mammal, such as a human, mouse (mouse), rat (rat), cow, horse, pig, dog, sheep, goat or cat, preferably a human.

In a specific embodiment, the cancer that can be diagnosed by the diagnostic method of the present invention may be selected from the group consisting of breast cancer, colorectal cancer, lung cancer, sarcoma, melanoma, head and neck cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, and neuroblastoma.

The present inventors have demonstrated that BAG2 is overexpressed in breast cancer patients, and particularly breast cancer patients with higher measured levels of BAG2 have a higher probability of having metastatic breast cancer. Thus, in a specific embodiment, the breast cancer that can be diagnosed by the diagnostic method of the present invention can be metastatic breast cancer.

Among the molecular subtypes of breast cancer, triple-negative breast cancer (TNBC) is a very aggressive type, and not only does the prognosis become poor, but also the mortality rate becomes high despite systemic treatment. TNBC is a heterogeneous type (Foulkeset al., 2010; Dent et al., 2007; Masuda et al., 2013) compared to either luminal (luminal) or HER2 enriched (HER 2-enriched) types. Most targeted breast cancer treatments show positive therapeutic effects on hormone receptor and HER2 positive breast cancers, but because TNBC patients do not have three targeted receptors (ER, PR and HER2) or other well-defined molecular targets, their options for effective treatment (e.g., poly ADP-ribose polymerase (PARP), Epidermal Growth Factor Receptor (EGFR), Src tyrosine kinase, etc.) are limited. Therefore, in order to prevent unnecessary treatment of TNBC patients and select an effective treatment method, it is necessary to quickly and accurately distinguish TNBC patients among various types of breast cancer patients.

The present inventors have demonstrated that BAG2 is overexpressed in breast cancer patients, and in particular breast cancer patients with higher measured levels of BAG2 have a higher probability of having TNBC-type breast cancer. Thus, in a specific embodiment, the breast cancer that can be diagnosed by the diagnostic method of the present invention can be triple negative breast cancer (or TNBC-type breast cancer).

Another aspect of the present invention provides a kit for diagnosing cancer, comprising the antibody, polypeptide or combination thereof specifically binding to BAG2 polypeptide or a fragment thereof. The kit detects BAG2 present in blood, serum, plasma, or a combination thereof, and a sample suitable for use in the kit provides blood, serum, plasma, or a combination thereof isolated from an individual.

In a specific embodiment, the kit may further comprise an antibody, polypeptide or group thereof that specifically binds to a polypeptide of cathepsin B or fragment thereof.

The kit can further include at least one other component composition, solution, or device suitable for an analytical method used with the kit (e.g., immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, radioimmunodiffusion, bidimensional immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation analysis, complement fixation analysis, flow cytometry, protein chips, or combinations thereof). For example, in order to detect an immunocomplex of BAG2 or cathepsin B and its specific antibody in a sample, a secondary antibody or a chromogenic substrate labeled with a substrate, an appropriate buffer solution, a chromogenic enzyme or a fluorophore may be further included. The substrate may be a 96-well plate synthesized from nitrocellulose membrane, polyethylene resin, a 96-well plate synthesized from polystyrene resin, a glass slide, or the like, peroxidase (peroxidase) and alkaline phosphatase (alkaline phosphatase) may be used as chromophoric enzymes, FITC, RITC, or the like may be used as fluorophores, and 2,2' -diaza bis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS) or o-phenylenediamine (OPD), Tetramethylbenzidine (TMB), or the like may be used as chromophoric substrates.

In terms or elements mentioned in the kit, the same as mentioned in the description of the claimed diagnostic method is to be understood as the same as mentioned in the description of the claimed diagnostic method.

Advantageous effects

According to an aspect of the present invention, a method for diagnosing cancer by measuring the presence or level of BAG2 polypeptide in blood can diagnose cancer more easily and accurately than the existing method of collecting tissue to diagnose cancer.

According to another aspect of the present invention, the method for diagnosing cancer can be easily used for diagnosing cancer in various diagnostic mechanisms by measuring the presence or level of BAG2 polypeptide in blood from the blood using a kit for diagnosing cancer.

Drawings

FIG. 1 shows a volcanic plot (volcano plot) of the difference in BAG family expression levels in luminal and TNBC cell lines analyzed using RNA sequencing.

FIG. 2 shows mRNA expression levels of BAG2 in luminal and TNBC cell lines analyzed from volcano plots.

FIG. 3 shows a scatter plot (scatter dot-plot) of the expression level of BAG2 analyzed from 52 breast cancer cell lines.

FIG. 4 shows microarray (4A) and RNA sequencing results (4B) for analysis of the amount of BAG2 expression in normal and individual breast cancer cell types.

FIG. 5 shows the results of RT-PCR quantification of the expression of BAG2 in luminal and TNBC tissues.

Fig. 6 shows the results of analyzing the effect of BAG2 on the form and activity of CTSB according to immunoblot (6A) and degree of CTSB activity (6B).

FIG. 7 shows the results of detection by immunoblotting of BAG2 and CTSB from conditioned medium cultured with TNBC.

FIG. 8 is a graph showing the level of cathepsin B detected from conditioned media cultured with TNBC.

FIG. 9 shows the results of detection by immunoblotting of BAG2 and CTSB detected from conditioned medium cultured with a strain of BAG2 overexpressing cells.

Fig. 10 shows the results of detecting the presence of BAG2 from the sera of healthy humans and breast cancer patients.

Detailed Description