JP2021117117A - Biomarker of prostate cancer, method of detecting prostate cancer using the biomarker, and diagnostic kit - Google Patents

Abstract

To provide a novel biomarker for detecting prostate cancer, and a method of detecting prostate cancer using the same.SOLUTION: The method is a method of determining prostate cancer onset or risk of developing prostate cancer by detecting lipocalin 2 in a sample derived from a human subject, in which the method detects lipocalin 2 that has a sugar chain different from lipocalin 2 which is detected in normal humans with no prostate cancer.SELECTED DRAWING: Figure 3

Description

The present invention relates to a prostate cancer-specific marker and a method for detecting prostate cancer by detecting the biomarker.

Prostate cancer is the most common malignant tumor in men and has the highest morbidity and mortality. Prostate cancer is cancer that begins in the prostate of men and often grows slowly. However, there are cases of invasive prostate cancer, in which cancer cells can metastasize from the prostate to other parts of the body, especially bones and lymph nodes. Prostate cancer can cause pain, difficulty urinating, poor sexual intercourse, or erectile dysfunction. Other symptoms can occur later in the disease.

Prostate cancer detection rates vary widely across regions of the world, with detection rates in Southeast Asia lower than in Europe and the United States. Prostate cancer is more likely to occur in men over the age of 50 and is a common cancer in men. Patients with prostate cancer are often asymptomatic and often do not receive treatment for prostate cancer.

Early detection and diagnosis of prostate cancer currently relies on rectal finger examination (DRE), prostate-specific antigen (PSA) measurement, transrectal ultrasound (TRUS) and transrectal needle biopsy (TRNB). The primary diagnostic approach uses a combination of DRE and serum PSA measurements. However, while the detection of an increase in PSA level can be detected with high sensitivity, there is a problem that the specificity is low. PAS levels can also be high in benign prostatic hyperplasia and prostatitis, so if a value above the reference value is detected, a detailed examination to see a specialist to see if you have prostate cancer, such as , Will have a biopsy. As a result, PSA screening has resulted in wasteful needle biopsy. PSA screening is said to have a positive predictive value of about 25-40% and, despite its limitations, is still the only widely used biomarker for prostate cancer.

Prostate cancer is divided into early stage cancer that can be cured by surgery and advanced cancer for which hormone therapy is selected. It is no exaggeration to say that the 5-year survival rate after surgical treatment exceeds 95% and the threat has disappeared, but it still causes more than 17,000 deaths from prostate cancer annually, mainly in advanced cancer.

Androgen receptor (AR) signaling is an essential survival pathway for prostate cancer cells. Hormone therapy, androgen ablation therapy (ADT), is a treatment that reduces circulating androgen levels, thereby inhibiting AR activity, and eliminating hormone-sensitive androgen-dependent prostate cancer, for advanced prostate cancer. It is a first-line treatment strategy. The majority of patients initially respond to ADT, but most patients eventually develop castration resistance in which the disease progresses even at castration levels of testosterone. The average duration of effect of ADT is said to be about 3-4 years. Prostate cancer that develops or becomes resistant to treatment with antiandrogens is called castration-resistant prostate cancer (CRPC). CRPC is defined as the case where serum testosterone is less than 50 ng / dL but the disease is exacerbated and PSA is elevated.

The PSA efficacy rate (a decrease of 50% or more in the PSA value) is about 100% at the start of ADT, but by the time CRPC appears and treatment with antiandrogens is discontinued, the efficacy rate is about 30 to 30 to It has dropped to about 40%. Thus, the appearance of CRPC is closely associated with poor prognosis.

When CRPC appears and it becomes impossible to suppress the growth of cancer even by changing the internal medicine, chemotherapy (anticancer drug treatment) becomes a candidate for the next treatment strategy. Common chemotherapeutic agents such as docetaxel are given to prostate cancer that has become refractory to hormone therapy, but the disease progresses due to the sequential use of new hormones that more strongly inhibit the androgen receptor. The response rate is low. Therefore, the tertiary prevention of prostate cancer is the early detection of recurrence due to the acquisition of hormone therapy resistance.

Lipocalin 2 is also known in humans as oncogene 24p3 or neutrophil gelatinase-related lipocalin (NGAL), a protein encoded by the LCN2 gene. NGAL is involved in innate immunity, which inhibits bacterial growth by blocking iron ions. NGAL is expressed in neutrophils. NGAL is also expressed at low levels in the kidney and prostate.

NGAL is a biomarker of kidney injury, with elevated blood and urinary levels in acute kidney injury. Therefore, its use as a biomarker for renal damage by measuring NGAL in urine has been proposed. In addition, it has been proposed to use it as a prognostic diagnosis and a diagnostic marker for the risk of heart and stroke by measuring lipocalin 2 in blood and urine (Patent Document 1: Japanese Patent Application Laid-Open No. 2011-519037). It has been reported that the expression of LCN2 in prostate cancer correlates with the progression of lymph node metastasis (Non-Patent Document 1: Ding et al., Prostate 75: 957-968, 2015). In addition, it has been reported that LCN2 is expressed in the prostate cancer cell lines PC-3 and DU145 (Non-Patent Document 2: Chappell et al., Advances in Biological Regulation 69, 2018 43-62). Furthermore, it has been reported that the expression of CXCL1 and LCN2 is associated with cell biological malignancy (Non-Patent Document 3: Lu et al., Cell Comm. Signal. 2019, 17: 118).

Ding et al., Prostate 75: 957-968, 2015 Chappell et al., Advances in Biological Regulation 69, 2018 43-62 Lu et al., Cell Comm. Signal. 2019, 17: 118

Special table 2011-519037

It is an object of the present invention to provide a new biomarker for detecting prostate cancer and to provide a method for detecting prostate cancer using the biomarker.
It is also an object of the present invention to provide a biomarker for detecting castration-resistant prostate cancer and to provide a method for detecting castration-resistant prostate cancer using the biomarker.
Another object of the present invention is to provide a method for determining when to start chemotherapy by detecting castration-resistant prostate cancer at an early stage.

Since PSA, which is a tumor marker for prostate cancer, is a molecule whose expression is regulated by the androgen receptor, its expression itself is significantly suppressed by hormone therapy. In view of these characteristics of PSA molecules, despite the existence of prostate cancer that has acquired castration resistance and is re-growing, hormone therapy is continued without noticing this, and the disease progresses. The present inventors thought that they were in a situation of castration.
Therefore, as a result of diligent studies, the present inventors have discovered that lipocalin 2 having a specific sugar chain structure is expressed in patients who develop prostate cancer, and completed the present invention. The present invention includes the following aspects.

[1] A method for determining that prostate cancer has developed or is at risk of developing prostate cancer by detecting lipocalin 2 in a sample derived from a human being as a subject, and detection of lipocalin 2. However, a method characterized by detecting and measuring the amount of lipocalin 2 having a sugar chain different from that of lipocalin 2 detected in normal humans who do not have prostate cancer.
[2] The method according to the above [1], wherein the lipocalin 2 detected in a normal human not suffering from the prostate cancer is a neutrophil zeratinase-binding lipocalin expressed in neutrophils.
[3] The method according to the above [1] or [2], wherein the sugar chain of the lipocalin 2 to be detected is modified with GalNAcβ1,4GlcNAc (LacdiNAc).
[4] The method according to any one of the above [1] to [3], wherein the lipocalin 2 is detected by using a lectin or an antibody that specifically binds to LacdiNAc.
[5] The method according to the above [4], wherein the lectin is natsufuji lectin (WJA).
[6] The above-mentioned lipocalin 2 is detected by a sandwich method using an anti-lipocalin 2 antibody that recognizes an epitope other than a sugar chain and a lectin (preferably WJA) that specifically binds to LacdiNAc. 1] The method according to any one of [5].
[7] The method according to [6] above, wherein in the sandwich method, WJA is reacted with lipocalin 2 and then the lipocalin 2 bound to WJA is reacted with an anti-lipocalin 2 antibody.
[8] The method according to any one of the above [1] to [7], wherein the sample is blood or urine.
[9] The method according to any one of the above [1] to [8], wherein the prostate cancer is castration-resistant prostate cancer.
[10] Select from the group consisting of normal prostate and prostate cancer, androgen-dependent and non-androgen-dependent prostate cancer, advanced and non-advanced prostate cancer, and metastatic and non-metastatic prostate cancer. The method according to any one of [1] to [8] above, further comprising distinguishing between the two states to be performed or providing information for it.
[11] Any one of [1] to [8] above, further comprising selecting or providing information for selecting a treatment regimen for the subject based on the measured amount of lipocalin 2. The method described in the second section.
[12] The treatment regimen is selected from the group consisting of needle biopsy reassessment, surgical therapy, radiation therapy, hormone therapy, chemotherapy, radiopharmaceutical administration (preferably radium), antibody therapy, proliferative factor therapy, and cytokine therapy. The method according to [11] above, which is one or more treatments to be performed.

[13] The following steps:
Step a. The step of detecting and measuring the amount of lipocalin 2 in a sample taken from a prostate cancer patient receiving or receiving hormone therapy, where the detection of lipocalin 2 is a normal human without prostate cancer. To detect lipocalin 2 having a sugar chain different from that detected in step b. A step of determining the risk of developing or exacerbating castration-resistant prostate cancer based on the measured values,
Methods of determining the risk of developing or exacerbating castration-resistant prostate cancer, including.
[14] The method according to [13] above, wherein the lipocalin 2 detected in a normal human not suffering from the prostate cancer is a neutrophil zeratinase-binding lipocalin expressed in neutrophils.
[15] The method according to the above [13] or [14], wherein the sugar chain of the lipocalin 2 to be detected is modified with GalNAcβ1,4GlcNAc (LacdiNAc).
[16] The method according to any one of the above [13] to [15], wherein the lipocalin 2 is detected by using a lectin or an antibody that specifically binds to LacdiNAc.
[17] The method according to the above [16], wherein the lectin is natsufuji lectin (WJA).
[18] The above-mentioned [13] to [17], wherein the lipocalin 2 is detected by a sandwich method using an anti-lipocalin 2 antibody that recognizes an epitope other than a sugar chain and WJA. the method of.
[19] The method according to [18] above, wherein in the sandwich method, WJA is reacted with lipocalin 2 and then the lipocalin 2 bound to WJA is reacted with an anti-lipocalin 2 antibody. [20] The sample. The method according to any one of the above [13] to [19], wherein is blood or urine.
[21] Any one of the above [13] to [20], further comprising providing information for selecting or selecting a treatment regimen for the patient based on the measured amount of the lipocalin 2. The method described in the second section.
[22] The treatment regimen is selected from the group consisting of needle biopsy reassessment, surgical therapy, radiation therapy, hormone therapy, chemotherapy, radiopharmaceutical administration (preferably radium), antibody therapy, proliferative factor therapy, and cytokine therapy. The method according to [21] above, which is one or more treatments to be performed.

[23] A method for monitoring the pathology of prostate cancer in a subject.
(1) A step of detecting and measuring the amount of lipocalin 2 in the first sample obtained at the first time derived from a subject suffering from prostate cancer;
(2) A step of detecting and measuring the amount of lipocalin 2 in the second sample obtained in the second time after the first time; and (3) of the lipocalin 2 in the second sample. The step of comparing the measured amount with the measured amount of lipocalin 2 in the first sample;
Here, the detection of lipocalin 2 is to detect lipocalin 2 having a sugar chain different from that of lipocalin 2 detected in normal humans who do not have prostate cancer.
If the measured amount of lipocalin 2 is increased in the second sample compared to the first sample, it indicates that the disease of prostate cancer is increased, while the second sample is compared with the first sample. A method of indicating that the disease of prostate cancer is reduced if it is reduced in the sample.
[24] The method according to the above [23], wherein the sugar chain of the lipocalin 2 to be detected is modified with GalNAcβ1,4GlcNAc (LacdiNAc).
[25] The method according to [23] or [24] above, wherein the lipocalin 2 is detected using a lectin or antibody that specifically binds to LacdiNAc.
[26] The method according to the above [25], wherein the lectin is natsufuji lectin (WJA).
[27] The above-mentioned [23] to [26], wherein the lipocalin 2 is detected by a sandwich method using an anti-lipocalin 2 antibody that recognizes an epitope other than a sugar chain and WJA. the method of.
[28] The method according to any one of the above [23] to [27], wherein the sample is blood or urine.

[29] A kit for detecting prostate cancer, detecting castration-resistant prostate cancer, or monitoring the progression of prostate cancer, which differs from lipocalin 2 detected in normal humans who do not have prostate cancer. A kit containing a reagent for specifically detecting lipocalin 2 having a sugar chain.
[30] The kit according to the above [29], wherein the reagent for specifically detecting lipocalin 2 is a reagent that recognizes a sugar chain modified with GalNAcβ1,4GlcNAc (LacdiNAc).
[31] The kit according to the above [29], wherein the reagent for specifically detecting the lipocalin 2 is a lectin or an antibody that specifically binds to LacdiNAc.
[32] The kit according to the above [29], wherein the reagent for specifically detecting the lipocalin 2 is natsufujirectin (WJA).
[33] The kit according to any one of the above [29] to [32], further comprising an anti-lipocalin 2 antibody that recognizes an epitope other than a sugar chain.

[34] A biomarker for determining whether or not a human being a subject has prostate cancer, which is a sugar chain different from lipocalin 2 detected in normal humans who do not have prostate cancer. A prostate cancer biomarker that is lipocalin 2 with.
[35] The biomarker according to the above [34], wherein the sugar chain of the lipocalin 2 is modified with GalNAcβ1,4GlcNAc (LacdiNAc).
[36] The biomarker according to the above [34] or [35], wherein the prostate cancer is castration-resistant prostate cancer.

The present invention provides a biomarker for the detection of new prostate cancers. The present invention also provides a method for detecting prostate cancer using the prostate cancer biomarker.
The present invention also provides a biomarker for detecting castration-resistant prostate cancer and a method of using it to detect castration-resistant prostate cancer.
The present invention further provides a method of determining when to initiate a treatment regimen other than hormonal therapy (eg, chemotherapy) by early detection of castration-resistant prostate cancer.

FIG. 1 shows the time course of expression of PSA and the abnormal sugar chain lipocalin 2 of the present invention during treatment with hormone therapy for prostate cancer. In FIG. 2, the C4-2 cell line and the LNCap cell line were cultured in a normal medium (FBS only added: non-castrated environment) and a CSS-added medium (castrated environment), respectively, and genes whose expression was enhanced in the castrated environment were confirmed. The result is shown. FIG. 3 shows the results of confirming the expression of lipocalin 2 (LCN2) protein by Western blot using the cell lysates of the androgen-independent cell line C4-2 and the androgen-dependent cell line LNCaP derived from prostate cancer. WJA is a lectin that specifically recognizes LacdiNAc. The anti-LCN2 antibody (Cloud-clone, MAB388Hu21) is a monoclonal antibody that reacts with the N-terminus of lipocalin 2 (LCN2). Lanes 1 and 3 are the results of using cells cultured in a normal medium, and lanes 2 and 4 are the results of using cells cultured in a CSS medium (androgen removal environment). FIG. 4 shows the results of tissue staining of prostate cancer tissue from a patient obtained by pathological autopsy with lipocalin 2 using WFA or WJA. FIG. 5 shows the results of subculturing the C4-2 cell line, which is a castration-resistant cell line, in a castration environment (CSS-added medium) and confirming changes in the expression of lipocalin 2.

Hereinafter, the present invention will be described together with preferred methods and materials that can be used in the practice of the present invention, using exemplary embodiments as examples. Unless otherwise specified in the text, all technical terms and scientific terms used in the present specification have the same meanings as generally understood by those skilled in the art to which the present invention belongs. Also, any material and method equivalent to or similar to that described herein can be used in the practice of the present invention as well. In addition, all publications and patents cited herein in connection with the invention described herein are described herein, for example, as indicating methods, materials, etc. that can be used in the present invention. It constitutes a part.

The present invention is a sugar chain different from lipocalin 2 (hereinafter referred to as "normal lipocalin 2") detected in normal humans who do not have prostate cancer as a useful molecule in the diagnosis and treatment of prostate cancer (PC). It relates to the detection of lipocalin 2 having the above (hereinafter, may be referred to as “abnormal sugar chain lipocalin 2”). Normal lipocalin 2, also called neutrophil gelatinase-related lipocalin (NGAL), is known as an inflammatory marker and is used in urinalysis as an initial marker for various renal diseases such as acute kidney injury. The present invention is characterized in that abnormal sugar chain lipocalin 2 is specifically detected.

One aspect of the present invention is to detect the presence of abnormal sugar chain lipocalin 2 using a sample derived from a subject such as blood, urine, or prostate tissue, thereby causing or developing prostate cancer in the subject. It is a method for determining whether or not a person has a risk. If abnormal sugar chain lipocalin 2 is detected in a subject, it indicates that the subject has or is at risk of developing prostate cancer.

Another aspect of the present invention is normal prostate and prostate cancer, androgen-dependent and androgen-independent prostate cancer, advanced prostate cancer and non-prostate cancer, based on the results of detecting abnormal sugar chain lipocalin 2 by the above method. It is a method for distinguishing between the states of advanced prostate cancer or metastatic prostate cancer and non-metastatic prostate cancer, or for providing information for that purpose. When abnormal sugar chain lipocalin 2 is detected in a subject, it is indicated which state the subject is in.

In another aspect of the present invention, a subject develops castration-resistant prostate cancer (CRPC) by detecting the presence of abnormal sugar chain lipocalin 2 using a sample derived from a subject such as blood, urine, or prostate tissue. It is a method for determining whether or not the patient is at risk of developing the disease. If abnormal sugar chain lipocalin 2 is detected in a subject, it indicates that the subject has or is at risk of developing CRPC in the future.

Another aspect of the present invention is to detect the presence of abnormal sugar chain lipocalin 2 in a subject who has developed prostate cancer and is undergoing hormone therapy using a sample derived from the subject such as blood, urine, or prostate tissue. This is a method for determining whether a subject has or is at risk of developing CRPC. If abnormal sugar chain lipocalin 2 is detected, it indicates that the subject has or is at risk of developing CRPC. In addition, when abnormal sugar chain lipocaine 2 is detected at a high level, it is judged that the onset of CRPC is suspected or the risk of developing CRPC is high.

Another aspect of the present invention is to measure the level of abnormal sugar chain lipocalin 2 over time in a subject who has developed prostate cancer using a sample derived from the subject such as blood, urine, or prostate tissue. This is a method for determining whether prostate cancer is advanced or non-advanced. Increased levels of lipocalin 2 over time indicate progression of prostate cancer, while decreased levels indicate non-progression or shrinkage of prostate cancer.

Another aspect of the present invention provides information for selecting or selecting a treatment regimen of interest based on the results of detecting abnormal sugar chain lipocalin 2 or measuring its level by any of the above methods. It is a method that further includes that. Treatment regimens include, but are not limited to, needle biopsy reassessment, surgical therapy, radiation therapy, hormone therapy, chemotherapy, antibody therapy, proliferative factor therapy, or cytokine therapy.

Yet another aspect of the invention is a kit for detecting prostate cancer, detecting castration-resistant prostate cancer, or monitoring the progression of prostate cancer, comprising a reagent for specifically detecting abnormal sugar chain lipocalin 2. Is.

Examples of the sample derived from the subject include a body fluid sample and a tissue sample derived from the subject. Body fluid samples include, but are not limited to, blood (eg, serum, plasma), urine, saliva, tears and the like, preferably blood, especially peripheral blood. The tissue sample is preferably prostate tissue.

Abnormal sugar chain lipocalin 2 has a sugar chain structure not found in normal lipocalin 2. Normal lipocalin 2 is also known as oncogene 24p3 or neutrophil gelatinase-related lipocalin (NGAL). Although there is no report on the details of the sugar chain structure of normal lipocain 2, the existence of the NVT sequence is confirmed from the sequence structure of all proteins, so that it is possible that one N-type sugar chain is present. Examples of the sugar chain structure found in the abnormal sugar chain lipocalin 2 include, but are not limited to, a sugar chain structure modified with GalNAcβ1,4GlcNAc (LacdiNAc). LacdiNAc is a disaccharide shown by the following structure, and LacdiNAc is a sugar chain structure found in the α subunits of leuropin (LH) and thyrotropin (TSH), and is β4GalNAc-T3 (Sato et al., J. Biol. It is synthesized by Chem. 2003, 28; 278 (48), 47534-44) or T4 (Gotoh, FEBS Lett, 562 (2004) 134-40).

Neutrophil lipocalin 2 expressed in neutrophils, which is normal lipocalin 2, is also abundant in blood. Therefore, if lipocalin 2 in blood is detected using an antibody that recognizes lipocalin 2 in an epitope other than the sugar chain structure, it becomes difficult to detect the abnormal sugar chain lipocalin 2. However, neutrophils, which are the source of blood lipocalin 2, do not express the sugar chain synthase having the above structure, and neutrophil lipocalin 2 does not have LacdiNAc modification. Therefore, the abnormal sugar chain lipocalin 2 expressed in prostate cancer (that is, lipocalin 2 modified with the LacdiNAc structure) can be distinguished from neutrophil lipocalin 2. It was confirmed for the first time by the present inventors that the abnormal sugar chain lipocalin 2 expressed in prostate cancer has a sugar chain modified with a LacdiNAc structure.

LacdiNAc is also known as a sugar chain structure detected on the molecule of prostate cancer specific antigen (PSA). In prostate cancer, it is known that the sugar chain structural change on the PSA molecule, that is, LacdiNAc is increased. Therefore, a method for detecting LacdiNAc-PSA with high sensitivity has been attempted.

(Detection and measurement of abnormal sugar chain lipocalin 2)
The abnormal sugar chain lipocalin 2 has the above-mentioned sugar chain structure that is not present in the normal lipocalin 2. Abnormal sugar chain lipocalin 2 can be detected using a lectin or antibody that specifically binds to the above sugar chain structure.
Examples of the lectin that specifically binds to the lectin include nodafuji lectin (WFA: Wisteria Floribunda Agglutinin) and natsufuji lectin (WJA: Wisteria Japonica lectin), and WJA is preferable.

WFA is known as one of the lectins that recognizes GalNac at the non-reducing end, and is commercially available. However, there are problems such as wide sugar chain recognition characteristics and weak binding force. Although WFA can be used in the present invention, it detects lipocalin 2 modified with LacdiNAc because it causes a non-specific reaction such as recognizing a sugar chain structure other than the LacdiNAc structure, for example, the terminal GalNAc of chondroitin sulfate. Although it is possible to use it as a lectin for this purpose, it is not preferable. Therefore, WFA with improved specificity for LacdiNAc has been reported (for example, WO2914 / 098112). These modified WFAs can also be used in the present invention.

WJA has been reported by Soga et al. (PLOS ONE Vol. 8, Issue 12, e83886, 2013). WJA specifically binds to sugar chains having GalNAcβ1-3GlcNAc and GalNAcβ1-4GlcNAc (LacdiNAc) structures. WJA is preferably used in the present invention because it specifically recognizes the LacdiNAc structure, has narrow sugar chain recognition characteristics, and has a higher binding property to LacdiNAc than WFA. In addition, WJA and its variants can also be used in the present invention as long as they exhibit sufficient specificity and binding to the LacdiNAc structure. For example, but not limited to this, when looking at the binding property to a sugar having a LacdiNAc structure, the modification is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 100, as compared with WJA. It is possible to give a variant showing a binding activity of more than%, and when the binding specificity to a sugar having a LacdiNAc structure is observed, a variant having an excellent binding specificity as compared with WFA can be given. can. The WJA variant used in the present invention can be prepared by using a known genetic engineering technique. For example, it can be prepared by inserting a mutation into an isolated gene of WJA using a known technique, expressing it in a microorganism, and then recovering and purifying the protein. The specificity and binding property to LacdiNAc can be confirmed by using a plate on which LacdiNAc is immobilized and detecting the binding of the modified lectin to the plate using a known method.

Examples of the antibody that specifically binds to the abnormal sugar chain lipocalin 2 include an antibody that recognizes the LacdiNAc structure. Since an antibody that recognizes the LacdiNAc structure is commercially available, it can be used in the present invention, but an antibody that does not show cross-reactivity with other sugar chain structures is preferable.

The detection of abnormal sugar chain lipocalin 2 can be performed by, for example, an immunological method. Examples of the immunological method include a solid phase immunoassay method (RIA, EIA, FIA, CLIA, etc.), a dot blotting method, a latex agglutination method (LA: Latex Aggregation-Turbidimmunoassay), an immunochromatography method, and the like. Among these, the ELISA method, which is one of the EIA methods, is preferable from the viewpoint of quantitativeness. It can also be detected using the avidin-biotin system.

Hereinafter, examples using the sandwich ELISA method by the enzyme antibody method and the immunohistochemical staining method will be described, but the detection of the abnormal sugar chain lipocalin 2 is not limited to these, and can be carried out by appropriately using a known technique.
Detection by the sandwich ELISA method using an antibody that specifically recognizes the sugar chain structure of abnormal sugar chain lipocalin 2 can be performed, for example, as follows. Prepare an antibody (referred to as antibody A) that recognizes the sugar chain structure of the abnormal sugar chain lipocalin 2 and an anti-lipocalin 2 antibody (referred to as antibody B) that recognizes an epitope other than the sugar chain. Each antibody may be prepared by using a commercially available antibody, or may be prepared by using a known antibody preparation method. The antibody may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable as the antibody that recognizes the sugar chain structure of the abnormal sugar chain lipocalin 2. A sample (for example, blood of a subject, preferably serum) is added to a microtiter plate on which antibody B is immobilized, and an antigen-antibody reaction is carried out. After washing, enzyme-labeled antibody A is added to cause an antigen-antibody reaction, and after washing, the enzyme substrate is reacted and colored, and the absorbance is measured to detect abnormal sugar chain lipocalin 2 in the sample, and the measurement thereof. From the value, the amount of abnormal sugar chain lipocalin 2 in the sample can be calculated. The immobilized antibody and the enzyme-labeled antibody may be reversed, but from the viewpoint of reducing noise, the unlabeled antibody A is immobilized (used as the primary antibody) and the antibody B is labeled as the secondary antibody. Is preferably used. When the two antibodies are derived from different species, neither antibody A nor antibody B is enzyme-labeled and is an enzyme-labeled antibody that binds to a (secondary) antibody to be used later (for example, the secondary antibody is a mouse). Labeled anti-mouse IgG antibody) may be used if it is derived.

Detection by the sandwich ELISA method using a lectin that specifically recognizes the sugar chain structure of abnormal sugar chain lipocalin 2 can be performed, for example, as follows. An anti-lipocalin 2 antibody that recognizes an epitope other than the sugar chain of lipocalin 2 and a lectin that specifically recognizes the sugar chain structure of the abnormal sugar chain lipocalin 2 are prepared. The antibody may be a commercially available antibody, or may be prepared by using a known antibody preparation method. The antibody may be either a polyclonal antibody or a monoclonal antibody. A sample (for example, blood of a subject, preferably serum) is added to a microtiter plate on which an antibody is immobilized, and an antigen-antibody reaction is carried out. After washing, biotinylated lectin is added and reacted, and after washing, abnormal sugar chain lipocalin 2 in the sample is detected using the biotin-avidin system, and the amount of abnormal sugar chain lipocalin 2 in the sample is detected from the measured value. Can be calculated. Various methods of the biotin-avidin system have been reported and are commercially available, and they can be used as appropriate. Alternatively, the order of using the lectin and the antibody may be reversed. In such a case, for example, the lectin is fixed to the microtiter plate by a known method. A sample (eg, subject's blood, preferably serum) is added to a lectin-immobilized microtiter plate to allow the lectin to react with a sugar chain. After washing, anti-lipocalin 2 antibody is added and reacted. After washing, a labeled anti-IgG antibody that reacts with anti-lipocalin antibody is added and reacted, and after washing, it reacts with an enzyme substrate and develops color, and the absorbance is measured to detect abnormal sugar chain lipocalin 2 in the sample, and the abnormal sugar chain lipocalin 2 is detected. The amount of abnormal sugar chain lipocalin 2 in the sample can be calculated from the measured value. A labeled anti-lipocalin 2 antibody may be used. In such a case, after adding an antibody and washing, a color reaction can be carried out to detect it. From the viewpoint of reducing noise, a method of immobilizing a lectin is preferable.

Detection in tissues using an antibody or lectin that specifically recognizes the sugar chain structure of abnormal sugar chain lipocalin 2 can be performed by using a known tissue immunostaining method after preparing a tissue section. When using an antibody (primary antibody) that specifically recognizes the sugar chain structure of abnormal sugar chain lipocalin 2, the antibody is reacted with the tissue, washed, and then specifically stained with a secondary antibody that recognizes the primary antibody. I do. Specific examples of the specific staining include a fluorescent antibody multiple staining method using a fluorescently labeled secondary antibody and an enzyme antibody multiple staining method using an enzyme-labeled secondary antibody. Alternatively, when a lectin that specifically recognizes the sugar chain structure of the abnormal sugar chain lipocalin 2 is used, for example, a biotinylated lectin (preferably WJA) is reacted with the tissue, washed, and then the tissue is used with the biotin-avidin system. Abnormal sugar chain lipocaine 2 in is detected.

Further, the detection of the abnormal sugar chain lipocalin 2 in the method of the present invention can be carried out by using other known methods. Not limited to this, for example, a fluorescent antibody method, a method using a radiation-labeled antibody, or the like can be mentioned.

When abnormal sugar chain lipocalin 2 is detected or measured in a sample using the method of the present invention, it can be determined whether the subject (human) from which the sample is derived has or is at risk of developing prostate cancer. The comparison can be made by measuring the amount of abnormal sugar chain lipocalin 2 in the sample derived from the test subject and also measuring the amount of abnormal sugar chain lipocalin 2 in the sample derived from a normal person and comparing the two. Based on the results, it can be determined that the subject has or is at risk of developing prostate cancer. Alternatively, in a normal person, since abnormal sugar chain lipocalin 2 is not normally present in a sample, for example, in serum, the amount of abnormal sugar chain lipocalin 2 in a sample derived from the measurement target can be measured to cause prostate cancer. It can be determined that the affected or at risk of developing prostate cancer.

In addition, based on the measured amount of abnormal sugar chain lipocalin 2, it can be determined that the measurement target has or may have castration-resistant prostate cancer. In addition, information on whether or not the measurement target already has prostate cancer and is receiving hormone therapy can be used for judgment.

In addition, the therapeutic treatment regimen of the subsequent measurement target (patient) is determined based on the measured amount of abnormal sugar chain lipocalin 2. In addition, information on whether or not the measurement target already has prostate cancer and is receiving hormone therapy can be used for judgment. Therapeutic treatment regimens can include, but are not limited to, reassessment by needle biopsy, surgical therapy, radiation therapy, hormone therapy, chemotherapy, antibody therapy, proliferative factor therapy, or cytokine therapy.

The amount of abnormal sugar chain lipocalin 2 according to the present invention can be measured a plurality of times or even over time. This makes it possible to monitor the pathology of prostate cancer in the subject and further confirm the therapeutic effect. Samples are obtained from a subject who has or is likely to have prostate cancer at a first time and a second time after the first time, but not limited to this. For each of the sample (Sample-1) obtained in the first time and the sample (Sample-2) obtained in the second time, the amount of abnormal sugar chain lipocalin 2 is measured according to the present invention, and they are compared. Therefore, it can be confirmed whether the disease has progressed in the second time as compared with the first time. If the treatment was performed between the first time and the second time, and if the amount of abnormal sugar chain lipocalin 2 decreased in the second time, the disease condition was attenuated or there was a therapeutic effect. It can be determined that it was.

The interval between the first time and the second time is not particularly limited and may be, for example, days, weeks, months, a year or more, but is a number in the sense of monitoring the progression of the disease. It is preferably months to one year, and several weeks to one year in order to confirm the effect of the treatment. By doing so, it becomes easy to monitor the medical condition and confirm the treatment effect, and it is possible to provide useful information for determining the treatment policy.

The abnormal sugar chain lipocalin 2 to be measured by the method of the present invention is also a biomarker for prostate cancer. PSA is known as a tumor marker for prostate cancer, and is actually used in the diagnostic field of prostate cancer. However, since PSA is a molecule whose expression is regulated by the androgen receptor, its expression itself is significantly suppressed when prostate cancer is diagnosed and hormone therapy is performed. The onset of castration-resistant prostate cancer is also diagnosed using PSA as a marker, but the problem is that the onset of castration-resistant prostate cancer cannot be detected early because the expression of PSA is suppressed during hormone therapy. Exists. On the other hand, the abnormal sugar chain lipocalin 2 used in the method of the present invention is more suitable as a tumor marker for prostate cancer because the influence of hormone therapy on its expression does not need to be considered or is small, and castration-resistant prostate cancer. The onset of the disease can be detected earlier. Abnormal sugar chain lipocalin 2, which is a tumor marker for prostate cancer, and its utilization are also one of the aspects of the present invention. FIG. 1 shows the time course of the expression of PSA and the abnormal sugar chain lipocalin 2 of the present invention during the treatment of hormone therapy for prostate cancer.

One aspect of the present invention is a kit for detecting prostate cancer, which comprises a reagent for specifically detecting abnormal sugar chain lipocalin 2. Examples of the reagent for specifically detecting the abnormal sugar chain lipocalin 2 include a reagent that specifically binds to LacdiNAc, and examples thereof include a lectin or an antibody that specifically binds to LacdiNAc. The lectin used in the kit is not limited to this, and examples thereof include WFA or a variant thereof, or WJA or a variant thereof, preferably WJA or a variant thereof. Kits for the detection of prostate cancer can include anti-lipocalin 2 antibodies that recognize epitopes other than sugar chains, in addition to lectins or antibodies that specifically bind to LacdiNAc.

The detection method of the present invention may be used in combination with another detection method for prostate cancer. For example, it is also possible to detect prostate cancer by applying the present invention to a sample that is positive in PAS detection by using a combination of prostate cancer detection methods that utilize PAS detection. By such a method, it is possible to make a more accurate judgment on a sample that is falsely positive in PAS detection. However, according to the method of the present invention, prostate cancer can be detected with higher accuracy than PAS detection, so that the detection method of the present invention is preferably the primary screening.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following Examples.
(Example 1) Identification of an expressed gene in castration-resistant prostate cancer A cell line derived from prostate cancer (C4-2) that has acquired castration resistance and a cell line derived from human prostate cancer (LNCaP) of a patient with lymph node metastasis. The gene expression was analyzed using the gene, and the gene whose expression was upregulated was identified as follows. The C4-2 cell line is androgen-independent and the LNCaP cell line is androgen-dependent.
C4-2 cells and LNCaP cells were cultured in a cell culture medium containing FBS (RPMI-1640, FUJIFILM) and a cell culture medium containing charcoal (charcoal-added cell culture solution: CSS). In the culture in CSS, since steroids are adsorbed by charcoal, androgen in the culture medium is also reduced, and the survival and proliferation of androgen-independent cells can be confirmed. Therefore, it is considered that C4-2 cells proliferate faster than LNCaP cells when cultured in CSS.

Each cell line is cultured in a normal medium (FBS only added: hereinafter, sometimes referred to as a non-casting environment) and a CSS-added medium (hereinafter, sometimes referred to as a castering environment), and gene expression under each environment is performed. We searched for genes whose expression does not decrease due to the absence of androgen. For gene expression, total RNA was extracted and the amount of RNA in each gene was measured. As a result, in the LNCap cell line, 138 genes were identified in which the gene expression was increased in the castrated environment as compared with the non-castrated environment, while in the C4-2 cell line, the gene expression was found in the castrated environment. Below, 37 genes were identified, which increased more than three times as compared to the non-castrated environment. In addition, 21 genes were identified as common genes that satisfy these conditions in each cell line. The results are shown in FIG. Of the 21 genes, we focused on lipocalin 2 and conducted the following experiments. Lipocalin 2 is a protein expressed in neutrophils and has been known as a marker for acute renal failure, but it has been reported that its expression in the prostate is low. When the amino acid sequence of lipocalin 2 was examined, it was found to be a protein having an NVT sequence (Nos. 85 to 87) and having one site capable of modifying an N-type sugar chain.

(Example 2) Detection of lipocalin 2 in prostate cancer-derived cells The expression of lipocalin 2 (LCN2) protein was confirmed using the above-mentioned androgen-independent cell line C4-2 and androgen-dependent cell line LNCaP. The experiment was carried out as follows.
The C4-2 cell line and LNCaP cell line were cultured in normal medium or CSS medium. All cell lines were cultured in normal medium for 10 days and cells were collected. In the CSS medium, the LNCaP cell line hardly grew and died in 3 days, so the cells were recovered after culturing for 2 days. On the other hand, the C4-2 cell line was subcultured for 2 generations in 10 days, and then the cells were collected. Cells recovered were lysed with Lysis buffer (20 mM This-HCl, 40 mM NaCl, 5 mM EDTA, 1% Triton X100, protease inhibitor (complete Mini, EDTA-free, Roche). After removing the non-specific binding to Sepharose, it was used for the next immunoprecipitation operation. A specific antibody (anti-LCN2 polyclonal IgG antibody: Cloud-Cone, product number PAB388Hu01) was added to the cell lysate, and 4 The reaction was carried out at ° C. overnight. A mixture of cell lysate and antibody was added to protein G Sepharose, reacted in a rotator at room temperature for 3 hours, and then centrifuged to separate Sepharose beads and the supernatant. After adding Wash buffer and mixing,

A sample that had been heat-treated and modified by adding SDS-PAGE sample buffer was applied to an SDS-PAGE gel and electrophoresed. Using an anti-LCN2 monoclonal antibody (Cloud-clone, product number: MAB388Hu21) or biotinylated WJA (provided by Professor Yamamoto of the University of Tokyo) that is transferred to a PVDF membrane according to a conventional method and reacts with the N-terminal of LCN2. Lipocalin 2 was detected using a chemical luminescent reagent according to a conventional method. The results are shown in FIG. The band around 30KD is LCN2 bound to WJA, and the band around 65KD is an IP antibody band (anti-LCN2 antibody band). From this result, it was shown that lipocalin 2 derived from the prostate cancer cell line responds to WJA.
From the above results, it was found that the sugar chain structure of lipocalin 2 expressed in the prostate cancer cell line has a LacdiNAc structure.

(Example 3) Detection of lipocalin 2 in tissues derived from prostate cancer patients Using tissues obtained by pathological autopsy of prostate cancer patients in accordance with the ethical regulations of Chiba University Hospital, the sugar chain structure of LacdiNAc in prostate cancer tissues The expression of lipocalin 2 having the above was confirmed as follows.
A sliced specimen of prostate cancer tissue was prepared from a pathological autopsy specimen of a prostate cancer patient. This was deparaffinized, antigen activated, and blocked with Ventana Benchmark Ultra (Roche Diagnostics), and WFA (provided by Professor Yamamoto of the University of Tokyo) and WJA were reacted to form a biotin-actin bond. Staining was performed with a solution (A + B solution: VECTASTAIN).
The results are shown in FIG. The detection efficiency of the sugar chain structure of LacdiNAc was remarkably better when WJA was used than when WFA was used.

(Example 4) Subculture of castration-resistant prostate cancer cell line C4-2 in a castration environment A castration-resistant prostate cancer cell line C4-2 cell line was subcultured in a CSS-added medium, and lipocalin 2 was subcultured. Expression was confirmed by immunostaining with an anti-LCN antibody. The results are shown in FIG. The results of immunostaining are shown in the left figure, and the graphs are shown in the right figure. It was confirmed that the proportion of cells expressing lipocalin 2 increased with each passage.

(Example 5) Detection of LDN-modified lipocalin 2 in serum by sandwich ELISA Abnormal sugar chain lipocalin 2 in serum of a patient suffering from prostate cancer was measured by the sandwich ELISA method using WJA and an anti-lipocalin antibody. The sera of a patient with castration-resistant prostate cancer (patient 1-11) and the culture supernatant of a 4-generation C4-2 cell line as a positive control were used, and the sera of 4 healthy researchers were used as a negative control. , I did as follows.
A well to which the anti-LCN2 antibody (polyclonal) attached to the CircuLex Human NGAL / Lipocalin-2 ELISA Kit was attached was prepared. Each serum was added thereto, incubated, washed with the attached wash buffer, and then reacted with WJA. After washing, the attached HRP-SA (streptavidin) was reacted as a labeled antibody, and after washing, the cells were stained with TMB under shading. After the incubation, a stop solution was added, and the absorbance was measured at a wavelength of 450 nm with a micro plate reader.
As a result, significant color development was confirmed in the patient serum and C4-2 cell line, and the presence of abnormal sugar chain lipocalin 2 was confirmed in the patient serum and in the castration environment culture supernatant of C4-2 cells, which is a positive control. ..

The above detailed description merely describes the object and object of the present invention, and does not limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein, without departing from the appended claims.

The detection method of the present invention utilizing a new biomarker for prostate cancer is useful in the detection of prostate cancer.

Claims (21)

It is a method of determining that prostate cancer has developed or is at risk of developing prostate cancer by detecting lipocalin 2 in a sample derived from a human being as a subject, and detection of lipocalin 2 is the prostate. A method comprising detecting lipocalin 2 having a sugar chain different from that of lipocalin 2 detected in normal humans who do not have cancer. The method according to claim 1, wherein the sugar chain of the lipocalin 2 to be detected is modified with GalNAcβ1,4GlcNAc (LacdiNAc). The method according to claim 1 or 2, wherein the lipocalin 2 is detected by using a lectin or an antibody that specifically binds to LacdiNAc. The method according to claim 3, wherein the lectin is natsufuji lectin (WJA). One of claims 1 to 4, wherein the lipocalin 2 is detected by a sandwich method using an anti-lipocalin 2 antibody that recognizes an epitope other than a sugar chain and a lectin that specifically binds to LacdiNAc. The method described. The method according to any one of claims 1 to 5, wherein the sample is blood. The method according to any one of claims 1 to 6, wherein the prostate cancer is castration-resistant prostate cancer. The method of any one of claims 1-7, further comprising providing information for selecting or selecting a treatment regimen for the subject based on the measured amount of lipocalin 2. .. The method of claim 8, wherein the treatment regimen is one or more treatments selected from the group consisting of surgery, radiation therapy, hormone therapy, antibody therapy, growth factor therapy, cytokine therapy, and chemotherapy. The following steps:
Step a. The step of detecting and measuring the amount of lipocalin 2 in a sample taken from a prostate cancer patient receiving or receiving hormone therapy, where the detection of lipocalin 2 is a normal human without prostate cancer. It has a different sugar chain than lipocalin 2 detected in; and step b. Steps to determine the risk of developing or exacerbating castration-resistant prostate cancer based on measurements,
Methods of determining the risk of developing or exacerbating castration-resistant prostate cancer, including. The method according to claim 10, wherein the sugar chain of the lipocalin 2 to be detected is modified with GalNAcβ1,4GlcNAc (LacdiNAc). The method of claim 10 or 11, wherein the lipocalin 2 is detected using a lectin or antibody that specifically binds to LacdiNAc. The method according to claim 12, wherein the lectin is natsufuji lectin (WJA). One of claims 10 to 13, wherein the lipocalin 2 is detected by a sandwich method using an anti-lipocalin 2 antibody that recognizes an epitope other than a sugar chain and a lectin that specifically binds to LacdiNAc. The method described. The method according to any one of claims 10 to 14, wherein the sample is blood. The method of any one of claims 10-15, further comprising providing information for selecting or selecting a treatment regimen for the subject based on the measured amount of lipocalin 2. .. 16. The method of claim 16, wherein the treatment regimen is one or more treatments selected from the group consisting of surgery, radiation therapy, hormone therapy, antibody therapy, growth factor therapy, cytokine therapy, and chemotherapy. A kit for detecting prostate cancer, detecting castration-resistant prostate cancer, or monitoring the progression of prostate cancer, which contains a sugar chain different from lipocalin 2 detected in normal humans who do not have prostate cancer. A kit containing a reagent for specifically detecting lipocalin 2 having. The kit according to claim 18, wherein the reagent for specifically detecting lipocalin 2 is a reagent that recognizes a sugar chain modified with GalNAcβ1,4GlcNAc (LacdiNAc). A biomarker for determining whether or not a human subject has prostate cancer, and a lipocalin having a sugar chain different from that of lipocalin 2 detected in normal humans who do not have prostate cancer. Prostate cancer biomarker that is 2. The biomarker according to claim 20, wherein the sugar chain of lipocalin 2 is modified with GalNAcβ1,4GlcNAc (LacdiNAc).

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