JP7522412B2 - Methods for aiding in the diagnosis of prostate cancer - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Abstracts of the 79th Annual Meeting of the Japanese Cancer Association, posted on the website on September 28, 2020, at https://www.meeting-schedule.com/jca2020/author. html (2) The 79th Annual Meeting of the Japanese Cancer Association, Title: "Development of a serum core-type fucosylated PSA measurement method for high Gleason prostate cancer diagnosis by automated microcapillary electrophoresis (Automated immunoassay system for serum core-type fucosylated PSA to predict high Gleason prostate cancer) (Program number PE14-7-3), Date of website posting: October 1, 2020, Website address: https://site2.convention.co.jp/jca2020/

The present invention relates to a novel method for determining prostate cancer.

When a man has prostate cancer, the level of prostate specific antigen (PSA) in the blood increases, and the PSA level is recognized as the most important tumor marker for diagnosing prostate cancer.

The currently widely used method for diagnosing prostate cancer is to use the total PSA amount in serum (i.e., the total amount of free PSA and bound PSA, hereafter abbreviated as "total PSA amount") as an indicator. However, it is known that the total PSA amount is often high in cases of prostate diseases other than prostate cancer, such as benign prostatic hyperplasia and prostatitis. Therefore, even if the total PSA amount is high, it is difficult to distinguish between prostate cancer and benign prostatic hyperplasia based on the total PSA amount.

Therefore, patients with abnormally high total PSA levels undergo a tissue test (biopsy) to make a definitive diagnosis of whether or not they are affected by prostate cancer.
In a biopsy, a portion of the collected prostate tissue is examined under a microscope, and the tissue condition (histological type) of the cells is classified into a grade and given a score. To determine the degree of malignancy, the tissue increase in the largest and second largest areas is assigned to a tissue classification of 1 to 5. The sum of these scores is the Gleason score, which is classified into 9 levels, from 2, the lowest malignancy, to 10, the highest. For diagnosis, the condition is determined as "low malignancy (low risk, 2-6),""intermediate (intermediate risk, 7)," or "high malignancy (high risk, 8-10)."

However, in many cases, the biopsy results indicated that the patient did not have prostate cancer. In other words, there were many cases of excessive testing, with biopsies being performed on patients who did not actually need them. Moreover, biopsies carry the risk of infection and bleeding, and are a major physical and financial burden on patients.
Furthermore, when the biopsy result shows a Gleason score of 6 or less, surveillance therapy, in which the patient is observed without undergoing active treatment, can be selected, taking into consideration other indicators such as the stage of the disease. However, although the currently used markers can determine whether or not a patient has prostate cancer, they cannot distinguish or predict the degree of risk (low risk to high risk).

Therefore, there is currently a need to establish a new diagnostic method that has higher diagnostic specificity and can avoid unnecessary biopsies.

On the other hand, fucosylation is one of the important glycosylation modifications in cancer and inflammation, and there are three types: core type, Lewis type, and H type. It has become clear that core type fucosylated proteins are highly expressed in high-risk prostate cancer and play an important role in the invasion and metastasis of prostate cancer.

For example, measurements using lectin antibody ELISA using P. japonica lectin or P. sugitake lectin revealed that the amount of free PSA with core fucosylated glycans in the urine of patients decreased as the malignancy of prostate cancer progressed (Patent Document 1).

Patent Document 2 discloses a method for detecting prostate cancer, which comprises reacting fucosylated PSA in a serum sample from a subject with a lectin specific to fucose α1→6 sugar chain and detecting the reacted lectin, and adjusts the pH of at least one step of the group of steps consisting of the reaction step of fucosylated PSA with the lectin and the subsequent treatment steps to a value higher than 8.5 and lower than 11.0. The value of the complex of fucosylated PSA-fucose α1→6 specific lectin in blood measured by this method increases with the risk (malignancy) of prostate cancer (Patent Document 2).

WO2017/138457 Brochure WO2019/065527 Brochure

However, none of the above-mentioned methods of assessment can correlate with the Gleason score, and it is difficult to improve cases where patients end up undergoing unnecessary biopsies. Therefore, in order to obtain a definitive diagnosis of prostate cancer, patients still have no choice but to undergo excessive testing.

The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a new method for diagnosing prostate cancer while avoiding unnecessary biopsies.

As a result of intensive research conducted by the present inventors to solve the above problems, they found that the ratio of the amount of core fucosylated PSA to the amount of free PSA in the blood is significantly higher in prostate cancer patients, and that this ratio can be used as a new marker for diagnosing prostate cancer.
The present inventors also found that the marker is elevated in prostate cancer patients with a Gleason score of more than 6, which is deemed necessary for a biopsy, and found that the use of this marker makes it possible to determine the malignancy of prostate cancer with higher specificity, and that the marker is useful as a marker for determining the necessity of a biopsy.
Based on the above findings, the present inventors have completed the present invention.

That is, the present invention comprises the following:
[1] A method for assisting in the diagnosis of prostate cancer, comprising determining the amount of free PSA (FucfPSA) having a fucosylated glycan, fucose α1→6 glycan, and the amount of free PSA (fPSA) in a sample derived from a subject, calculating the obtained values by applying the obtained values to the following formula, and determining prostate cancer based on the obtained value, %FucfPSA.
%FucfPSA = (FucfPSA amount/fPSA amount) x 100
[2] A method for assisting in the diagnosis of prostate cancer described in [1] above, in which the amount of FucfPSA is determined by measuring the amount of FucfPSA using an anti-fPSA antibody, which is an antibody that binds to fPSA, and a lectin having affinity for the fucose α1→6 glycan.
[3] A method for assisting in the diagnosis of prostate cancer described in [2] above, in which the amount of FucfPSA is determined based on the measurement results by contacting a subject-derived sample with the antibody, separating complex 1 between FucfPSA and the antibody and complex 2 between free nonfucosylated prostate-specific antigen and the antibody in the obtained subject-derived sample based on their affinity for the lectin, measuring the amount of the separated complex 1.
[4] The method for assisting in the diagnosis of prostate cancer described in [2] or [3] above, wherein the lectin having affinity for the fucose α1→6 glycan is Sugitake lectin or Lens lectin.
[5] The method for assisting prostate diagnosis described in any one of [1] to [4] above, wherein the subject-derived sample is serum, plasma or blood.
[6] The method for assisting in the diagnosis of prostate cancer described in [1] above, further comprising determining the total PSA amount, which is the total amount of PSA in a sample derived from a subject, calculating the obtained value by applying the obtained value to the following formula, and diagnosing prostate cancer based on the obtained value, FPI.
FPI = total PSA amount/(101-%FucfPSA)

The method of the present invention for assisting in the diagnosis of prostate cancer can determine prostate cancer with high specificity. In addition, it can determine the degree of malignancy correlated with the Gleason classification and determine whether or not a biopsy is required, thereby avoiding unnecessary biopsies for those who do not actually need one.

FIG. 2 is a schematic diagram of an example of PSA having a fucose α1→6 sugar chain. FIG. 1 is a schematic diagram of a microchip used in Example 1. FIG. 2 is a schematic diagram of a flow path in the microchip used in Example 1. The results obtained in Example 1 are shown below, comparing % Fuc-PSA between prostate cancer patients (Pca) and non-cancer subjects (Negative). 1 shows the results obtained in Example 1, comparing FPI between prostate cancer patients (Pca) and non-cancer subjects (Negative). The results obtained in Example 1 are shown, comparing % FucPSA between prostate cancer patients with a Gleason score of GS7-9 (GS7-9) and non-cancer subjects and prostate cancer patients with a Gleason score of GS6 or lower (Negative-GS6). 1 shows the results obtained in Example 1, comparing FPI between prostate cancer patients with a Gleason score of GS7-9 (GS7-9) and non-cancer subjects and prostate cancer patients with a Gleason score of GS6 or lower (Negative-GS6). 1 shows the results of ROC analysis performed based on the measurement results of FPI, %FucPSA, total PSA amount, and FucfPSA value obtained in Example 1.

<<PSA according to the present invention>>
The term "bound PSA" according to the present invention refers to PSA that has bound to a binding protein such as α1-antichymotrypsin or α2-macroglobulin to form a complex.

In the present invention, "free PSA" refers to PSA that is not bound to binding proteins such as α1-antichymotrypsin or α2-macroglobulin. Hereinafter, it may be abbreviated as "fPSA."

There are three types of fucosylation (addition of fucose) to sugar chains: core type, Lewis type, and H type. The fucosylation in the present invention is the addition of a core type "fucose α1→6 sugar chain."

An example of the structure of the glycan of PSA having the "fucose α1→6 glycan" according to the present invention (fucosylated PSA) is shown in the following formula (I).

An example of PSA having a glycan of formula (I) is shown in the schematic diagram in Figure 1. That is, the glycan is bound to an asparagine residue (N) of the PSA protein.

The "free PSA having a fucose α1→6 glycan" of the present invention may be abbreviated as "FucfPSA" below.

Methods for aiding in the diagnosis of prostate cancer
The method for assisting in the diagnosis of prostate cancer of the present invention comprises:
"A method for assisting in the diagnosis of prostate cancer, comprising determining the amount of FucfPSA and the amount of fPSA in a sample derived from a subject, calculating the obtained values by applying the formula [%FucfPSA = (amount of FucfPSA / amount of fPSA) x 100], and determining prostate cancer based on the obtained value of %FucfPSA" or "A method for assisting in the diagnosis of prostate cancer, comprising determining the amount of total PSA, which is the total amount of prostate specific antigen in a sample derived from a subject, calculating the obtained value by applying the formula [FPI = total PSA amount / (101 - %FucfPSA)], and determining prostate cancer based on the obtained value of FPI"
It is.

1. Samples derived from subjects
The subject-derived sample according to the present invention (hereinafter also referred to as "subject sample" or simply "sample") is a sample derived from a human subject, and examples thereof include blood, plasma, serum, semen, bladder washings, urine, tissue extracts, prostate tissue slices, prostate tissue biopsy samples, and the like, or samples prepared from these. Among these, serum, plasma, and the like are preferred. Serum is particularly preferred.

2. Method for determining %Fuc-fPSA
"% FucfPSA" in the method for assisting in the diagnosis of prostate cancer of the present invention means the ratio of the amount of FucfPSA to the amount of fPSA in a sample derived from a subject [(amount of FucfPSA/amount of fPSA) x 100].
A method for measuring the amount of fPSA related to %FucfPSA and a method for measuring the amount of FucfPSA will be described below.

(1) How to measure fPSA levels

The method for measuring the amount of fPSA in a sample according to the present invention includes the following steps:
(1)-1. A method of directly measuring the amount of fPSA in a sample, or (1)-2. A method of measuring the amount of FucfPSA and the amount of non-fucosylated fPSA (non-FucfPSA) in a sample and calculating the amount of fPSA from the sum of these amounts.

(1)-1. Method for directly measuring the amount of fPSA in a sample
Methods for directly measuring the amount of fPSA include known methods for measuring the amount of fPSA, such as known immunological measurement methods using anti-fPSA antibodies.

The anti-PSA antibody according to the present invention may be any antibody that binds to PSA, and includes antibodies capable of binding to both fPSA and bound PSA, and antibodies that specifically bind to fPSA (anti-fPSA antibodies).
When the anti-PSA antibody is an antibody that specifically binds to fPSA, it is referred to as an "anti-fPSA antibody." When the anti-PSA antibody includes an antibody that can bind to fPSA and bound PSA, and an anti-fPSA antibody, it is simply referred to as an "anti-PSA antibody."

The anti-PSA antibodies of the present invention may be any antibodies having the above-mentioned properties, and may be either commercially available or prepared by conventional methods. They may be either monoclonal or polyclonal antibodies. They may be used alone or in combination.

The origin of the anti-PSA antibody according to the present invention is not particularly limited, and any commercially available product or product produced by a method known per se using cell fusion technology, gene recombination technology, or the like and having the above-mentioned properties can be used.

In addition, the anti-PSA antibody of the present invention may be an antibody Fab, Fab', F(ab') ₂ , Fv, Fd, single-chain Fv (scFv), disulfide-linked Fv (sdFv), _VL , _VH , diabody ((VL- _{VH ) 2} or ( _VH - _VL ) ₂ ), triabody (trivalent antibody), tetrabody (tetravalent antibody), minibody (( _scFV - _CH3 ) ₂ ), IgG-delta-CH2, scFv-Fc, (scFv) ₂ -Fc fragment, etc.

Among the anti-PSA antibodies according to the present invention, commercially available antibodies capable of binding to fPSA and bound PSA include, for example, Anti PSA monoclonal antibody PSA10 (Anti PSA monoclonal antibody clone No. PSA10, Fujifilm Wako Pure Chemical Industries, Ltd.), Anti PSA monoclonal antibody (5A6) (HyTest), Anti PSA monoclonal antibody (5G6) (HyTest), Anti PSA monoclonal antibody (PS6) (HyTest), Anti PSA monoclonal antibody (PSA14) (Fujifilm Wako Pure Chemical Industries, Ltd.), Anti-Prostate Specific Antigen antibody (EP1588Y) (Abcam), Anti-Prostate Specific Antigen antibody (A67-B/E3) (Abcam), Anti-Prostate Specific Antigen antibody (35H9) (Abcam), Anti-Prostate Specific Antigen Examples of such antibodies include anti-prostate specific antigen antibody (KLK3/801) (Abcam), anti-prostate specific antigen antibody (3E6) (Abcam), anti-prostate specific antigen antibody (8301) (Abcam), anti-prostate specific antigen antibody (A5D5) (Abcam), anti-prostate specific antigen antibody (PSA 28/A4) (Abcam), and anti-prostate specific antigen antibody (1H12) (Abcam).

Among the anti-PSA antibodies according to the present invention, commercially available anti-fPSA antibodies include, for example, anti-PSA monoclonal antibody PSA12 (anti-PSA monoclonal antibody clone No. PSA12, Fujifilm Wako Pure Chemical Industries, Ltd.), anti-PSA monoclonal antibody (8A6) (HyTest), anti-PSA monoclonal antibody (PS1) (HyTest), anti-PSA monoclonal antibody (clone 108) (Anogen), anti-prostate specific antigen antibody (PS2) (Abcam), and anti-prostate specific antigen antibody (2H9) (Abcam).

The anti-PSA antibody of the present invention may be labeled with a detectable labeling substance.

Examples of labeling substances used to label the antibody include labeling substances commonly used in publicly known EIA, RIA, FIA, etc., and the antibody labeling method is appropriately selected from publicly known labeling methods depending on the labeling substance.

Specific examples of methods for directly measuring the amount of fPSA in a sample according to the present invention include the following methods.

The sample is reacted with a first antibody, which is an anti-PSA antibody, and a labeled second antibody labeled with a labeling substance that can detect the anti-PSA antibody, to generate a complex of the first antibody, fPSA, and the labeled second antibody. The amount of the complex is then measured by measuring the signal derived from the labeling substance of the labeled second antibody that constitutes the complex. The amount of fPSA in the sample is determined by a standard method based on the obtained measurement value. Before measuring the amount of the complex, an appropriate operation may be performed to remove the first antibody and the labeled second antibody that have not bound to fPSA.

In the above method, at least one of the first antibody and the second antibody is an anti-fPSA antibody. In addition, it is preferable that the epitopes of the first antibody and the second antibody are different.

In the above method, the amount of fPSA can be determined by converting the results of a similar measurement using an fPSA standard of known concentration in advance into a quantitative value using the usual method. That is, using fPSA of known concentration, the signal is measured by carrying out the same procedure using the same reagents as when the amount of fPSA in the sample was measured. A calibration curve is created between the measured value and the concentration of the fPSA standard used. The amount of fPSA in the sample is determined by applying the signal measurement value obtained in the measurement using the sample to the calibration curve.

The first antibody used in the above method is preferably immobilized on a solid phase. The type of solid phase may be, for example, an insoluble carrier used in conventional immunological assays, and the antibody may be immobilized on the solid phase according to a known method for immobilization that is commonly used in this field.

The method for measuring the signal derived from the labeling substance of the labeled anti-PSA antibody described above varies depending on the type of labeling substance, but may be appropriately selected depending on the properties of the labeling substance that can be detected by some method.

The amount of fPSA may be measured using a commercially available kit for measuring fPSA, such as Human Circulating Cancer BioMarker Panel 1 Select Kit (manufactured by LUMINEX), Abbott Free PSA (Abbott), Lumipulse Free PSA (Fujirebio), Vitros Free PSA (Ortho Clinical Diagnostics), ST AIA-PACK free PSA (Tosoh), and Eclusis ^™ Reagent free PSA (Roche Diagnostics).

In the measurement methods in this specification, including the method of (1)-1 above, unless otherwise specified, all separation/measurement devices and various reagents used in the field of immunological measurement methods known per se can be used in the methods. In addition, the type of lectin and solid phase used in the measurement according to the present invention described below can be appropriately selected according to the measurement device used and the measurement method to be performed, and the concentrations of the antibody and reagents used in the measurement can be appropriately selected from the concentration ranges usually used in this field. The labeling substance used to label the antibody can be appropriately selected depending on the measurement method and measurement device of the labeling substance. In addition, the measurement conditions when performing the measurement (reaction temperature, reaction time, pH during reaction, measurement wavelength, measurement device, etc.) can be appropriately selected according to the method known per se.

(1)-2. A method of measuring the amount of FucfPSA and non-FucfPSA in a sample and calculating the amount of fPSA from the sum of the amounts. For example, a method including using a lectin having affinity for fucosylated glycans to separate FucfPSA and non-FucfPSA based on their affinity for the lectin can be mentioned.

[Lectin according to the present invention]
Examples of lectins having affinity for fucosylated sugar chains include lectins having affinity for the Fucose α1→6 sugar chain. Lectins specific to the Fucose α1→6 sugar chain, which have affinity for the Fucose α1→6 sugar chain but do not have affinity for other sugar chains, are preferred.

Examples of lectins with such properties include Pholiota squarrosa lectin (hereafter abbreviated as "PhoSL") and Lens culimaris agglutinin (hereafter abbreviated as "LCA").

In the methods described below, "separating based on affinity" means, for example, separating the objects to be separated "based on differences in binding strength." For example, "separating the first complex and the second complex based on affinity for the lectin of the present invention" means "separating the first complex and the second complex based on the difference in binding strength between the first complex and the second complex to the lectin of the present invention."

As an example of the method (1)-2 using the lectin according to the present invention, the lectin according to the present invention is used to separately measure the amount of FucfPSA and the amount of non-FucfPSA in a sample, and the amount of fPSA is calculated from the sum of the measured amounts. In addition, the lectin according to the present invention may be used to separately measure the amount of FucfPSA and the amount of non-FucfPSA in a sample in a single step, and the amount of fPSA may be calculated from the sum of the measured amounts.

The following method can be used, for example, to measure the amount of FucfPSA and non-FucfPSA in a sample separately using the lectin of the present invention and calculate the amount of fPSA from the sum of the measurements.

"The sample is reacted with the lectin of the present invention to generate a complex between FucfPSA and the lectin of the present invention. The complex and non-FucfPSA that did not bind to the lectin of the present invention are then separated, for example, by B/F separation. The amount of FucfPSA is determined by measuring the amount of fPSA that bound to the separated lectin. The amount of non-FucfPSA is also determined by determining the amount of fPSA that did not bind to the separated lectin. The amount of fPSA is obtained by calculating the sum of the obtained amount of FucfPSA and the amount of non-FucfPSA."

Examples of such methods include the following [Method 1] and [Method 2], with [Method 2] being preferred in terms of yield.

[Method 1]
A sample is passed through a column packed with a packing material in which the lectin according to the present invention is immobilized on a solid phase such as agarose beads. FucfPSA binds to the lectin according to the present invention on the packing material, while other substances including non-FucfPSA are eluted from the column without binding to the lectin according to the present invention. If the amount of fPSA in the eluate is measured, the "amount of non-FucfPSA" can be obtained. Next, the column is washed with an appropriate buffer, and then about 2 to 5 column volumes of lactose-containing buffer (0.4 M) are passed through the column to elute FucfPSA. If the amount of fPSA in the eluate is measured, the "amount of FucfPSA" can be obtained.
The "amount of fPSA" is obtained by adding the obtained "amount of non-FucfPSA" and "amount of FucfPSA".

The amount of FucfPSA and non-FucfPSA in the above [Method 1] may be measured in the same manner as described in the above section "(1)-1. Method for directly measuring the amount of fPSA in a sample."

[Method 2]
A sample is contacted with a solid phase, such as a microtiter plate or polystyrene beads, on which the lectin according to the present invention is immobilized. FucfPSA binds to the lectin according to the present invention on the solid phase. Non-FucfPSA does not bind to the lectin according to the present invention and exists in the liquid phase. The solid phase and the liquid phase are separated, and the amount of fPSA in the liquid phase is measured to obtain the "amount of non-FucfPSA." The amount of fPSA bound to the solid phase is then measured to obtain the amount of FucfPSA.

The amount of non-FucfPSA in the liquid phase in the above [Method 2] may be measured in the same manner as the method for measuring the amount of fPSA described in (1)-1 above.

In the above [Method 2], the amount of FucfPSA bound to the solid phase can be measured, for example, by the following method.

The FucfPSA bound to the solid phase is reacted with a labeled anti-fPSA antibody to form a complex of the lectin of the present invention, FucfPSA, and labeled anti-fPSA antibody on the solid phase. The amount of the complex is then measured by measuring the signal derived from the labeling substance of the labeled anti-fPSA antibody that constitutes the complex. The amount of FucfPSA can be calculated by converting the quantitative value in the usual manner using the results obtained by performing a similar measurement using an fPSA standard with a known concentration.

The "fPSA amount" is obtained by adding the obtained "non-FucfPSA amount" and "FucfPSA amount".

In addition, the following method can be used as a method for separately measuring the amount of FucfPSA and non-FucfPSA in a sample in a single step using the lectin according to the present invention, and calculating the amount of fPSA from the sum of the measured amounts.

"The sample is reacted with anti-fPSA antibody to generate a complex (first complex) between FucfPSA and the anti-fPSA antibody in the sample, and a complex (second complex) between non-FucfPSA and the anti-fPSA antibody. Both complexes are separated (fractionated) based on their affinity for the lectin in the presence of the lectin according to the present invention, and then the amount of the first complex and the amount of the second complex are measured, thereby separating and measuring both FucfPSA and non-FucfPSA in a single step. The amount of fPSA (fPSA amount) is obtained by calculating the sum of the amount of the first complex and the amount of the second complex obtained."

Specific examples of this method include the following steps [Method 3] or [Method 4].

[Method 3]
1) contacting a sample with a labeled anti-fPSA antibody labeled with a labeling substance to form a complex between the labeled anti-fPSA antibody and FucfPSA (first complex) and a complex between the labeled anti-fPSA antibody and non-FucfPSA (second complex);
2) separating (differentiating) the first complex and the second complex obtained in the above step 1) in the presence of the lectin according to the present invention based on their affinity for the lectin according to the present invention;
3) measuring the amount of the first complex and the amount of the second complex separated in the above step 2) by measuring a signal derived from the labeling substance of the labeled anti-fPSA antibody constituting the first complex and the second complex;
4) A step of calculating the sum of the amount of the first complex and the amount of the second complex obtained in the above step 3) and defining this sum as the amount of fPSA.

[Method 4]
1) contacting a sample with a labeled first antibody, which is an anti-PSA antibody labeled with a labeling substance, and a second antibody which is an anti-PSA antibody, to form a complex (first complex) between the labeled first antibody, FucfPSA, and the second antibody, and a complex (second complex) between the labeled first antibody, non-FucfPSA, and the second antibody;
2) separating (differentiating) the first complex and the second complex obtained in the above step 1) in the presence of the lectin according to the present invention based on their affinity for the lectin according to the present invention;
3) measuring the amount of the first complex and the amount of the second complex separated in the above step 2) by measuring a signal derived from the labeling substance of the labeled first antibody constituting the first complex and the second complex;
4) A step of calculating the sum of the amount of the first complex and the amount of the second complex obtained in the above step 3) and defining this sum as the amount of fPSA.

At least one of the first and second antibodies used in the above [Method 4] is an anti-fPSA antibody. It is preferable that the epitopes of the first and second antibodies are different.

In the above [Method 3] and [Method 4], the anti-PSA antibody or the lectin of the present invention may be immobilized on a solid phase depending on the measurement method.

In the above [Method 3] and [Method 4], a specific example of a step of separating (fractionating) based on affinity for the lectin of the present invention in the presence of the lectin of the present invention will be described later in "(3) Method for determining %FucfPSA".

As for "(1) a method for measuring the amount of fPSA", method (1)-2 is preferred, and a method for measuring FucfPSA and non-FucfPSA in one step is more preferred in consideration of the complexity of the measurement procedure. Among these, [Method 4] is preferred.

(2) Method for measuring the amount of FucfPSA The method includes a method for determining the amount of FucfPSA by measuring the amount of FucfPSA using an anti-fPSA antibody and a lectin having affinity for the fucose α1→6 glycan, for example, the following method.
(2)-1. A method for directly measuring the amount of FucfPSA in a sample,
(2)-2. The amount of FucfPSA is calculated by subtracting the amount of non-FucfPSA from the amount of fPSA in the sample.

(2)-1. Method for directly measuring the amount of FucfPSA in a sample For example, there can be mentioned a method for directly measuring the amount of FucfPSA in a sample by contacting a sample derived from a subject with an anti-fPSA antibody, separating a complex of FucfPSA and the anti-fPSA antibody (first complex) from a complex of non-FucfPSA and the anti-fPSA antibody (second complex) in the obtained sample derived from the subject based on the affinity for the lectin according to the present invention, measuring the amount of the separated first complex, and calculating the amount of FucfPSA based on the measurement result obtained.

Specifically, for example, FucfPSA and non-FucfPSA can be separated and the amount of FucfPSA measured according to the method described in (1)-2 above. In this case, it is not necessary to measure the amount of non-FucfPSA.

More specifically, for example, FucfPSA can be measured according to the above-mentioned [Method 1] or [Method 2], with [Method 2] being more preferred.

Furthermore, the amount of FucfPSA may be measured by measuring FucfPSA and non-FucfPSA in one step according to the method described in (1)-2 above. In this case, there is no need to measure the amount of non-FucfPSA. There is also no need to calculate the sum of the amount of FucfPSA and the amount of non-FucfPSA.

Specific examples include the following methods:

[Method 3']
1) contacting a sample with a labeled anti-fPSA antibody labeled with a labeling substance to form a complex between the labeled anti-fPSA antibody and FucfPSA (first complex) and a complex between the labeled anti-fPSA antibody and non-FucfPSA (second complex);
2) separating (differentiating) the first complex and the second complex obtained in the above step 1) in the presence of the lectin according to the present invention based on their affinity for the lectin according to the present invention;
3) A step of measuring the amount of the first complex separated in the above step 2) by measuring a signal derived from the label of the labeled anti-fPSA antibody that constitutes the first complex.

[Method 4']
1) contacting a sample with a labeled first antibody, which is an anti-PSA antibody labeled with a labeling substance, and a second antibody which is an anti-PSA antibody, to form a complex (first complex) between the labeled first antibody, FucfPSA, and the second antibody, and a complex (second complex) between the labeled first antibody, non-FucfPSA, and the second antibody;
2) separating (differentiating) the first complex and the second complex obtained in the above step 1) in the presence of the lectin according to the present invention based on their affinity for the lectin according to the present invention;
3) A step of measuring the amount of the first complex separated in the above step 2) by measuring a signal derived from the label of the labeled first antibody that constitutes the first complex.

At least one of the first and second antibodies used in the above [Method 4'] is an anti-fPSA antibody. It is preferable that the epitopes of the first and second antibodies are different.

In the above [Method 3'] and [Method 4'], the anti-PSA antibody, the anti-fPSA antibody, and the lectin according to the present invention may be immobilized on a solid phase depending on the measurement method.

(2)-2. The amount of FucfPSA is calculated by subtracting the amount of non-FucfPSA from the amount of fPSA in the sample.

In the method of (2)-2, the "fPSA amount" may be determined by the method described in the above section "(1) Method for measuring fPSA amount."

In the method of (2)-2, the "amount of non-FucfPSA" is determined by separating FucfPSA and non-FucfPSA, for example, according to the method described in (1)-2 above. The amount of the separated non-FucfPSA can then be measured. In this case, it is not necessary to measure the amount of FucfPSA. It is preferable to use a method that measures both the amount of FucfPSA and the amount of non-FucfPSA in the sample in one step. Of these, a method that measures only the amount of non-FucfPSA separated according to the method of [Method 3] or [Method 4] is more preferable. A method that measures only the amount of non-FucfPSA separated according to the method of [Method 4] is particularly preferable.

To determine the amount of FucfPSA, measure the amount of non-FucfPSA using the method described above, and subtract the amount of non-FucfPSA obtained from the amount of fPSA in the same sample measured separately.

As the "(2) method for measuring the amount of FucfPSA", method (2)-1 is preferred. Among the methods (2)-1, [Method 3'] and [Method 4'] are preferred. [Method 4'] is more preferred.

The amount of the lectin according to the present invention to be used may be determined, for example, taking into consideration the binding constant of the lectin according to the present invention to the fucose α1→6 glycan and the amount of FucfPSA in the sample.

For example, when using a lectin according to the present invention that has a strong affinity for the fucose α1→6 glycan and does not dissociate once bound to FucfPSA, a sufficient amount of the lectin according to the present invention is used during measurement so that 80% or more of the FucfPSA in the sample forms a complex with the lectin according to the present invention.

For example, when using a lectin according to the present invention that does not have sufficient affinity for the fucose α1→6 glycan fPSA and dissociates once it binds to FucfPSA, it is necessary to use a sufficient amount of the lectin according to the present invention so that at least 80% of the FucfPSA in the sample forms a complex with the lectin according to the present invention at the time of measurement, even after re-dissociation.

In order to satisfy the above conditions, it is preferable to use an excess (saturating amount) of the lectin according to the present invention relative to the FucfPSA in the sample.

(3) Method for Determining %FucfPSA Examples of a method for measuring the amount of fPSA used in the method for determining %FucfPSA according to the present invention include the methods described in the above section "(1) Method for Measuring the Amount of fPSA."

Methods for measuring the amount of FucfPSA used in the method for determining %FucfPSA according to the present invention include the methods described in the above section "(2) Method for measuring the amount of FucfPSA."

Specific examples of methods for calculating %FucfPSA include the following [Method A] and [Method B].

[Method A]
The amount of FucfPSA is measured by [Method 1] or [Method 2] described in the above section "(2) Method for measuring the amount of FucfPSA." Separately, the amount of fPSA is measured by the method described in the above section "(1) Method for measuring the amount of fPSA." The ratio of the amount of FucfPSA to the amount of fPSA thus obtained [(amount of FucfPSA/amount of fPSA) x 100] is calculated to determine %FucfPSA.

[Method B]
Both the amount of FucfPSA and the amount of non-FucfPSA are measured separately in one step using [Method 3] and [Method 3'], or [Method 4] and [Method 4'] described in the above section "(2) Method for measuring the amount of FucfPSA". Based on the obtained values, the value of [amount of FucfPSA/(sum of amount of FucfPSA and amount of non-FucfPSA)]×100 is calculated to determine %FucfPSA.

The method for calculating %FucfPSA is preferably [Method B], and it is more preferable to perform measurements using [Method 4] and [Method 4'] to calculate %Fc-fPSA.

In addition, when determining %FucfPSA in the present invention, the amount of FucfPSA and the amount of fPSA may be the amount of fPSA or the amount of FucfPSA (PSA protein amount), or their concentrations, or the actual measured value (signal value such as fluorescence intensity or absorbance, peak area, or peak height, etc.) as long as the units or types of actual measured values are the same.

For example, in the method of (1)-2 or (2)-1 above, when FucfPSA and non-FucfPSA are separated (fractionated) by capillary electrophoresis described below, the peak area of each separated fraction is determined, and the value of [peak area of FucfPSA fraction/(peak area of FucfPSA fraction+peak area of non-FucfPSA fraction)] x 100 is taken as %FucfPSA.

For example, when FucfPSA and non-FucfPSA are separated (fractionated) and each is detected using a fluorescently labeled anti-PSA antibody, the value of [fluorescence amount of FucfPSA fraction/(fluorescence amount of FucfPSA fraction+fluorescence amount of non-FucfPSA fraction)]×100 can be used as %FucfPSA.

As an example of one embodiment of the method for calculating %FucfPSA, the method of measuring the amount of fPSA and the amount of FucfPSA using [Method 4] and [Method 4'] in [Method B] and determining %FucfPSA is described below.

1) A sample is contacted with a labeled first antibody in which the anti-PSA antibody is labeled with a labeling substance, and a second antibody which is an anti-PSA antibody to form a complex of the labeled first antibody and FucfPSA (first complex) and a complex of the labeled first antibody, non-FucfPSA, and the second antibody (second complex).
2) The first complex and the second complex obtained in the above step 1) are separated (differentiated) in the presence of the lectin according to the present invention based on their affinity for the lectin according to the present invention.
3) The amount of the first complex and the amount of the second complex separated in the above step 2) are measured by measuring a signal derived from the labeling substance of the labeled first antibody constituting the first complex and the second complex.
4) The sum (fPSA amount) of the amount of the first complex and the amount of the second complex obtained in step 3) above is calculated, and %FucfPSA is calculated by calculating the ratio of the amount of the first complex obtained in step 3) above to said sum.

At least one of the first antibody and the second antibody is an anti-fPSA antibody. The first antibody is preferably an anti-fPSA antibody.

For example, in the above-mentioned methods [Method 3], [Method 4], [Method 3'], [Method 4'], and other measurements using the lectin according to the present invention, the step of separating (fractionating) a complex (first complex) of a (labeled) first antibody and FucfPSA and a complex (second complex) of a (labeled) first antibody, non-FucfPSA, and a second antibody in the presence of the lectin according to the present invention based on their affinity for the lectin according to the present invention can be carried out, for example, by high performance liquid chromatography (HPLC) using an HPLC-lectin column or by capillary electrophoresis. Capillary electrophoresis is more preferred.

HPLC using an HPLC-lectin column may be performed according to a conventional method. For example, FucfPSA and non-FucfPSA may be separated and measured by HPLC using a packing material carrying the lectin according to the present invention.

A specific example of a method for measuring the amount of FucfPSA and fPSA and determining %FucfPSA using the lectin of the present invention by capillary electrophoresis is described below.

For example, first, a sample containing PSA is contacted and reacted with a labeled anti-fPSA antibody obtained by labeling the anti-fPSA antibody of the present invention with a labeling substance, and the [labeled anti-fPSA antibody-FucfPSA] complex and the [labeled anti-fPSA antibody-non-FucfPSA] complex in the resulting reaction solution are separated by capillary electrophoresis in the presence of the lectin of the present invention, and the amount of the labeled substance derived from the [labeled anti-fPSA antibody-FucfPSA] (first complex) and the amount of the labeled substance derived from the [labeled anti-fPSA antibody-non-FucfPSA] (second complex) are measured. The amount of fPSA in the sample is the sum of the amounts of the labeled substances in the first complex and the second complex. Then, the ratio of the amount of FucfPSA to the amount of fPSA obtained (%FucfPSA) is calculated.

The amount of labeled substance derived from the first complex and the amount of labeled substance derived from the second complex correspond to the respective peak area values obtained by capillary electrophoresis, so the ratio can be calculated using the respective peak area values.

Specific examples of the anti-fPSA antibody and its labeling substance used in the above method are as described above.

In the present invention, among capillary electrophoresis, it is preferable to perform electrophoresis using a capillary chip or a microcapillary chip.

Capillary electrophoresis is classified into capillary zone electrophoresis and capillary gel electrophoresis depending on the electrophoretic solution used, but the method of the present invention can be applied to either. Considering the accuracy of separation, capillary gel electrophoresis is preferred among the above.

The lectin according to the present invention may be contained in the electrophoretic solution. However, during separation by capillary electrophoresis, the lectin according to the present invention is preferably present at a concentration higher than the amount at which FucfPSA and the lectin according to the present invention can completely bind to each other.

The solution for dissolving the sample or antibody to be subjected to capillary electrophoresis, the type of electrophoretic solution, additives, and specific conditions for capillary electrophoresis may be in accordance with methods known per se.

fPSA having a fucose α1→6 glycan that has affinity for the lectin of the present invention interacts with the lectin of the present invention in the carrier, resulting in a decrease in electrophoretic mobility. On the other hand, PSA that does not have a fucose α1→6 glycan has a reduced interaction with the lectin of the present invention, resulting in a smaller degree of attenuation in electrophoretic mobility. Therefore, the electrophoretic fraction of FucfPSA and the electrophoretic fraction of non-FucfPSA can be identified based on the difference in mobility.

As a specific example of a method for measuring FucfPSA by capillary electrophoresis, the following method is shown: using PhoSL as the lectin according to the present invention, a first antibody in which an anti-fPSA antibody is labeled with a fluorescent substance, and a second antibody in which an anti-PSA antibody is labeled with DNA, microchip capillary electrophoresis is performed using lectin affinity, and PSA is separated based on the degree of affinity of the lectin for the glycan of PSA, and then measured using a fluorescence detector.

That is, 1 to 50 μL of a sample containing PSA is reacted with a test solution containing fluorescently labeled anti-fPSA antibody, usually at 0.001 to 10 μM, and preferably 0.01 to 1 μM.

The sample used in capillary electrophoresis may be a sample collected from a living body, or a sample prepared by subjecting a sample collected from a living body to desalting or various purification processes.

The resulting reaction solution, 2 to 50 μL of a test solution containing usually 0.001 to 10 μM, preferably 0.01 to 1 μM, DNA-labeled anti-PSA antibody, a buffer solution for electrophoresis, and an internal standard (e.g., a fluorescent substance: HiLyte647 (AnaSpec) etc.) are introduced into a capillary having an inner diameter of, for example, 5 to 500 μm, preferably 50 to 200 μm, more preferably 50 to 100 μm, and a length of 1 to 10 cm, by a pressurization method at 1 to 10 psi for 30 to 60 seconds. The reaction is allowed to proceed for 5 to 30 minutes, preferably 10 to 15 minutes, while maintaining the temperature at 20 to 40°C. The resulting complex of [fluorescently labeled anti-fPSA antibody-FucfPSA-DNA labeled anti-PSA antibody] and the complex of [fluorescently labeled anti-fPSA antibody-non-FucfPSA-DNA labeled anti-PSA antibody] are separated by electrophoresis in the presence of PhoSL (0.1 mg/mL-20 mg/mL) by applying a voltage of 1000-5000 V for 10 seconds to 60 minutes. The migration state of the complex is then measured using a detector such as a fluorescence detector or UV detector to obtain an electropherogram.

The FucfPSA peak (including the complex of [fluorescently labeled anti-fPSA antibody] - [FucfPSA] - [DNA-labeled anti-PSA antibody]) and other fPSA peaks (including the complex of [fluorescently labeled anti-fPSA antibody] - [non-FucfPSA] - [DNA-labeled anti-PSA antibody]) can be distinguished from their electrophoretic positions. Therefore, the amount of FucfPSA in the sample is taken as the peak area of that peak. The sum of the obtained FucfPSA peak area and non-FucfPSA peak area is taken as the amount of free PSA.

As described above, capillary electrophoresis can be used to measure the amounts of fPSA and FucfPSA simultaneously in the same sample.

%FucfPSA is obtained by calculating the ratio of the peak areas obtained, i.e., [peak area of FucfPSA fraction/(peak area of FucfPSA fraction+peak area of non-FucfPSA fraction)] × 100.

Capillary electrophoresis may be performed using a commercially available fully automated measuring device, such as the Mutas Wako i30 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

<3. Method for determining FPI>
The FPI according to the present invention calculates the total amount of PSA in a sample derived from a subject, and calculates the obtained value by the following formula:
FPI = total PSA amount/(101-% FucfPSA amount)
This can be obtained by applying the formula:

The %FucfPSA amount used to obtain FPI can be determined by the method described in the above section "2. Method for determining the %FucfPSA amount."

(1) Method for measuring total PSA amount
The total amount of PSA in a subject-derived sample used to obtain FPI is the total amount of PSA in the test sample derived from the same subject from whom the sample used to calculate the %FucfPSA amount was collected. The total amount of PSA may be the amount (PSA protein amount), the concentration, or the actual measurement value (signal value such as fluorescence intensity or absorbance, peak area, or peak height, etc.).

The amount of total PSA can be determined by measuring the amount of total PSA using an anti-PSA antibody or a fragment thereof by a publicly known immunological method such as ELISA, etc. Alternatively, the amount of total PSA may be measured using a commercially available total PSA measuring kit.
Commercially available kits for measuring total PSA levels include, but are not limited to, AIA-Pack CL ^™ PSA (Tosoh Corporation), Total PSA Abbott (Abbott Japan LLC), and DELfIA ^™ PSA Kit (PerkinElmer).

(2) Method for determining FPI The FPI can be obtained by applying the total PSA amount and %FucfPSA obtained by the above method to the above formula.

For example, the method for determining FPI when the %FucfPSA and total PSA amounts are as follows:
%FucfPSA＝41(%)
Total PSA amount = 30 (ng/mL)
FPI = 30/(101-41) = 0.5

4. Methods for assisting in the diagnosis of prostate cancer
The method of the present invention for assisting in the diagnosis of prostate cancer includes diagnosing prostate cancer based on the %FucfPSA obtained in "<2. Method for determining %FucfPSA>" above, or the FPI obtained in "<3. Method for determining FPI>" above.

That is, %FucfPSA is determined by the method described in the above section "<2. Method for determining %FucfPSA>". Based on the result, data (e.g., information on %FucfPSA, comparison of %FucfPSA with a cutoff value, degree of increase in %FucfPSA, etc.) for diagnosing prostate cancer using %FucfPSA as an index is obtained.
Alternatively, FPI is determined by the method described in the above section "3. Method for determining FPI". Based on the result, data (e.g., information on FPI, comparison of FPI with a cutoff value, degree of increase in FPI, etc.) for diagnosing prostate cancer using FPI as an index is obtained.

(1) Diagnosis of Prostate Cancer Using the obtained data, prostate cancer is diagnosed (diagnosed/tested), for example, by the following method.

If the %FucfPSA or FPI obtained using a sample from a subject is equal to or higher than a predetermined cutoff value (reference value), it is possible to determine that the subject who provided the sample is suffering from prostate cancer (prostate cancer positive) or has a high possibility of having prostate cancer.

Another method is to set multiple assessment categories corresponding to the cutoff value or range of the %FucfPSA or FPI and make a judgment. For example, the following assessment categories are set: (1) no risk of prostate cancer, (2) low risk of prostate cancer, (3) symptoms of prostate cancer, (4) high risk of prostate cancer, etc. Then, it is possible to judge whether or not a patient has prostate cancer by determining which assessment category the %FucfPSA or FPI of the subject-derived sample falls into.

In addition, by comparing the %FucfPSA or FPI of a subject-derived sample determined at a certain time point with the same %FucfPSA or FPI determined at a different time point for the same subject, and evaluating whether or not the %FucfPSA or FPI has increased or decreased and/or the degree of increase or decrease, it is possible to diagnose the progression or malignancy of prostate cancer, or to diagnose the post-operative prognosis.
In other words, if the test results show an increase in the %FucfPSA or FPI, it can be determined that the disease has progressed to prostate cancer (or the malignancy of prostate cancer has increased), or that there are signs of the disease progressing to prostate cancer (or there are signs of an increase in the malignancy of prostate cancer).
Furthermore, if the test results show no change in the %FucfPSA or FPI, it can be determined that there is no change in the pathological condition of prostate cancer.

The cutoff value (reference value) used in the method of assisting the diagnosis of prostate cancer according to the present invention is obtained by using a sample from a person confirmed to have prostate cancer (prostate cancer patient) and a sample (reference sample) from a person confirmed not to have prostate cancer (cancer-free person) by the above-mentioned method, respectively.Then, the cutoff value may be set based on the boundary value between the %FucfPSA or FPI obtained using the test sample from the prostate cancer patient and the %FucfPSA or FPI obtained using the reference sample.The average value of the measured value obtained using the reference sample may be set as the reference value.
The "non-cancer subject" may be a healthy person or a patient with prostatic hyperplasia or the like.

The cutoff value may also be determined, for example, by analysis using a relative operating characteristic curve (ROC curve) in a conventional manner.

(2) Determination of high-risk prostate cancer According to the method for assisting in the determination of prostate cancer of the present invention, it is possible to determine high-risk prostate cancer in subjects suspected of having prostate cancer based on the results of clinical tests such as total PSA amount.
When making this determination, it is preferable to use FPI as a marker.

In the present invention, patients with a Gleason score of 7 (GS7) or higher are considered to be high-risk prostate cancer patients.

For example, a sample from an individual confirmed by biopsy to be GS7 or higher (GS7 sample) is used to obtain %FucfPSA or FPI by the above-mentioned method, and a cutoff value for determining high-risk prostate cancer patients is obtained.
Then, %FucfPSA or FPI is obtained using a test sample from a subject suspected of having prostate cancer. The %FucfPSA or FPI obtained using the test sample from the subject is compared with a cutoff value obtained from the value obtained using the GS7 sample. If the %FucfPSA or FPI obtained using the test sample from the subject suspected of having prostate cancer is equal to or higher than the cutoff value obtained using the GS7 sample, the subject is determined to be a high-risk prostate cancer patient and is to be subjected to detailed examination such as a biopsy. On the other hand, if the %FucfPSA or FPI obtained using the test sample from the subject suspected of having prostate cancer is less than these values obtained using the GS7 sample, the subject is to be subjected to surveillance therapy without performing a biopsy.

Alternatively, a sample from an individual whose biopsy has confirmed that their prostate cancer level is GS6 or lower (GS6 sample) is used to obtain %FucfPSA or FPI using the above-mentioned method, and a cutoff value for determining whether the patient is high-risk for prostate cancer is obtained.
Then, %FucfPSA or FPI is obtained using a test sample from a subject suspected of having prostate cancer. The %FucfPSA or FPI obtained using the test sample from the subject is compared with a cutoff value obtained from the value obtained using the GS6 sample. If the %FucfPSA or FPI obtained using the test sample from the subject suspected of having prostate cancer is higher than the cutoff value obtained using the GS6 sample, the subject is determined to be a high-risk prostate cancer patient and is to be subjected to detailed examination such as biopsy. On the other hand, if the %FucfPSA or FPI obtained using the test sample from the subject suspected of having prostate cancer is equal to or lower than these values obtained using the GS6 sample, the subject is to be subjected to surveillance therapy without performing a biopsy.

A specific example of how the present invention uses FPI to determine prostate cancer is shown below.

[Example 1]
(1) Determine FPI using samples from non-cancer individuals.
(2) FPI' is determined using a sample derived from the subject.
(3) The FPI' is compared with the FPI, and if the FPI' is the same as or higher than the FPI, the subject who provided the sample is determined to be suffering from prostate cancer (prostate cancer positive) or to have a high possibility of having prostate cancer.

[Example 2]
(1) The FPI is determined using a sample derived from a non-cancer subject, and a cutoff value of the FPI is set for diagnosing prostate cancer based on the FPI.
(2) The FPI is determined using a sample derived from the subject.
(3) The FPI is compared with the cutoff value set in (1) above, and if the FPI is equal to or higher than the cutoff value, the subject who provided the sample is determined to be suffering from prostate cancer (prostate cancer positive) or to have a high possibility of having prostate cancer.
If an appropriate cutoff value is determined, there is no need to set the cutoff value again each time a subject is evaluated.

[Example 3]
(1) Determine the FPI using samples from prostate cancer patients confirmed to be GS7 or higher.
(2) FPI' is determined using a sample from a subject suspected of having prostate cancer.
(3) The FPI' is compared with the FPI, and if the FPI' is the same as or higher than the FPI, it is determined that the subject who provided the sample is suffering from high-risk prostate cancer of GS7 or higher, or is highly likely to be suffering from such cancer.
On the other hand, when the FPI' is lower than the FPI, the subject who provided the sample is judged to be suffering from low-risk prostate cancer of GS6 or less, or to be highly likely to be suffering from such cancer.
Patients who are determined to have high-risk prostate cancer are candidates for detailed examinations such as biopsy, whereas patients who are determined to have low-risk prostate cancer are candidates for active surveillance.

Conventional markers for determining prostate cancer were insufficiently specific. As a result, even patients with negative biopsy findings had to undergo repeated biopsies to rule out the possibility of prostate cancer. As a result, there was a problem in that many unnecessary biopsies were performed, despite the risks of infection and bleeding.

In contrast, the method of the present invention for assisting in the diagnosis of prostate cancer makes it possible to determine high-risk prostate cancer that requires a biopsy with high specificity, high sensitivity, and by non-invasive means.

<<Diagnostic aid kit for prostate cancer>>
The kit for determining prostate cancer according to the present invention comprises:
"(1) A lectin according to the present invention,
(2) A kit for determining prostate cancer, including an instruction manual describing a procedure for determining %FucfPSA or FPI of a sample derived from a subject and determining prostate cancer or high-risk prostate cancer based on the value.
It is.

Details of the lectin according to the present invention and preferred embodiments and specific examples thereof are as described in the above section (1)-2 "Lectin according to the present invention."
The lectin according to the present invention may be in the form of a test liquid in a solution state, such as a suspension in an appropriate buffer solution, or may be a frozen product or a lyophilized product.

The kit may further contain an anti-PSA antibody according to the present invention (an antibody capable of binding to fPSA and bound PSA and/or an anti-fPSA antibody). Details of preferred embodiments and specific examples thereof are as described in the explanation of the PSA antibody according to the present invention in "<2. Method for determining %FucfPSA>" above.

The anti-PSA antibody of the present invention may be in the form of a test solution in a solution state, such as a suspension in an appropriate buffer solution, or may be a frozen product or a lyophilized product.
When the lectin of the present invention is in the form of a test solution, the anti-PSA antibody of the present invention may be coexistent in a test solution containing the lectin of the present invention, or may be included as a test solution, frozen product, or lyophilized product separate from the lectin of the present invention.

The concentrations of the lectin according to the present invention and the anti-PSA antibody according to the present invention in the test solution may be any concentration that initiates the reaction of the intended measurement when the test solutions are mixed. Specifically, the concentrations are as described in the above section "Method for assisting in the diagnosis of prostate cancer." Specific examples of the solvent that constitutes the test solution are also as described in the above section "Method for assisting in the diagnosis of prostate cancer."

The test solution containing the lectin of the present invention and/or the anti-PSA antibody of the present invention constituting the kit of the present invention may contain additives that are usually used in this field, such as reagents, such as buffers, reaction promoters, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc., that do not inhibit the reaction between fPSA and the lectin of the present invention. The concentrations of these reagents may also be appropriately selected from the concentration ranges usually used in this field.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

Example 1.

(1) Samples Serum samples were collected from 165 prostate cancer patients who had been diagnosed with prostate cancer by prostate biopsy and had undergone a histopathological diagnosis (determination of Gleason score) and from 87 non-cancer subjects who had been confirmed to be negative for prostate cancer by prostate biopsy.
Patient demographics are summarized in Table 1.

(2) Measurement of total PSA amount The total PSA amount in each sample was determined using a total PSA measurement kit, AIA-Pack CL ^™ PSA reaction reagent (Tosoh Corporation), and a fully automated chemiluminescent enzyme immunoassay device, AIA-CL2400 (Tosoh Corporation), according to the protocol attached to the kit.

(3) Measurement by Capillary Electrophoresis (i) Preparation of DNA-Labeled Anti-PSA Antibody A DNA-bound PSA antibody Fab' fragment was prepared according to the following procedure.

That is, anti-human PSA mouse monoclonal antibody PSA10 (Anti PSA monoclonal antibody clone No. PSA10, Fujifilm Wako Pure Chemical Industries, Ltd.) was reacted with a linker-bound 250 bp DNA fragment in a conventional manner using an anti-PSA antibody PSA10 Fab' fragment prepared in a conventional manner. The resulting reaction product was purified to prepare an anti-PSA antibody PSA10 Fab' fragment bound to the 250 bp DNA fragment (hereinafter abbreviated as "DNA-labeled anti-PSA antibody").

The anti-human PSA mouse monoclonal antibody used (Anti PSA monoclonal antibody clone No. PSA10) has affinity for human PSA and binds to bound PSA and fPSA. In other words, the antibody binds to FucfPSA and non-FucfPSA.

(ii) Preparation of fluorescently labeled anti-fPSA antibody
Anti-human PSA monoclonal antibody PSA12 (Anti PSA monoclonal antibody clone No. PSA12, Fujifilm Wako Pure Chemical Industries, Ltd.), which recognizes an epitope of PSA different from that of anti-PSA monoclonal antibody PSA10 and specifically binds only to fPSA, was treated by a conventional method to obtain anti-PSA antibody PSA12 Fab' fragment. The fluorescent substance HiLyte647 (AnaSpec, Inc.) was introduced to the amino group of the obtained fragment by a conventional method to obtain HiLyte647-labeled anti-fPSA antibody PSA12 Fab' fragment (hereinafter abbreviated as "fluorescently labeled anti-fPSA antibody").

(iii) Preparation of Samples and Test Solutions - Preparation of Sample A for Electrophoresis 2 μL of the sample, 1 μL of the 1 μM fluorescently labeled anti-fPSA antibody prepared in (ii) above, and 7 μL of electrophoresis buffer 1 [containing 5% (w/v) polyethylene glycol (PEG20000), 3% (w/v) glycerol, 150 mM NaCl, 0.01% BSA, 75 mM Tris-HCl (pH 7.5), and 10 mM MES] were added to a 0.5 mL tube and mixed to react, preparing 10 μL of a reaction solution.
The reaction solution (10 μL) containing the [fluorescence-labeled anti-fPSA antibody-fPSA] complex obtained in the above reaction was used as sample A for electrophoresis.
The final concentration of the fluorescently labeled anti-fPSA antibody in this reaction solution was 100 nM.

Preparation of running buffer 2 (containing PhoSL)
A 75 mM Tris-HCl buffer (pH 7.5) containing 4.5% (w/v) polyethylene glycol (PEG8000), 3% (w/v) glycerol, 10 mM NaCl, and 0.01% BSA was prepared. PhoSL (VECTOR) was added to this buffer to a final concentration of 4 mg/mL and mixed to prepare electrophoresis buffer 2.
Preparation of running buffer 3
A buffer containing 2% (w/v) polyethylene glycol (PEG20000), 3% (w/v) glycerol, 0.01% BSA, 125 mM HEPES, and 75 mM Tris-HCl (pH not adjusted) was used as electrophoresis buffer 3.
Preparation of running buffer 4
The electrophoresis buffer 4 was 75 mM Tris-HCl buffer (pH 7.5) containing 2% (w/v) polyethylene glycol (PEG20000), 3% (w/v) glycerol, and 0.01% BSA.
Preparation of DNA-labeled antibody solution (containing DNA-labeled anti-PSA antibody) A buffer [containing 2% (w/v) polyethylene glycol (PEG20000), 0.5 mM EDTA (2Na), 3% (w/v) glycerol, 50 mM NaCl, 0.01% BSA, and 75 mM BisTris (pH 6.0)] containing 100 nM of the DNA-labeled anti-PSA antibody obtained in (i) 1) above was prepared as a DNA-labeled antibody solution.
・Preparation of fluorescent solution
50 mM BisTris (pH 6.0) containing 30 nM HiLyte647 and 20% (w/v) glycerol was used as the fluorescent solution. The fluorescent solution was used for adjustments such as confirming the position in the detection part of the measurement device (MUTASU Wako i30).

(iv) Electrophoresis Microchip capillary electrophoresis was performed using a fully automated fluorescent immunoassay device Mutasu Wako i30 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the procedure described below in accordance with the device's instruction manual.
That is, 5.4 μL of the electrophoresis sample A prepared in (iii) above was dispensed into a designated well (SP well) of the MUTASU Wako i30 dedicated microchip. Then, each of the test solutions prepared in (iii) above was dispensed into each well of the microchip as follows.
R2 wells (R2(FLB) wells, R2(LB) wells): 10.0 μL each of electrophoresis buffer 2 (containing PhoSL),
R3 well: 10.0 μL of running buffer 3,
R4 well: 5.4 μL of running buffer 4,
・C1 well: 3.0 μL of DNA-labeled antibody solution,
・FD well: 7.0 μL of fluorescent solution.

A schematic diagram of the microchip used is shown in Figure 2.

In Figure 2, the waste well is used as a waste liquid reservoir (drain well) when the test solution in each well (R2, R3, R4, C1) and electrophoretic sample A are introduced into the analysis flow path.

Next, a pressure of -5 psi was applied between each of the four waste wells (drain wells) for 30 seconds to introduce electrophoresis sample A and each test solution into the analysis flow path of the chip.

Furthermore, after introducing the electrophoresis sample A and each test solution into the analytical flow channel of the chip, separation and detection of PSA were carried out by the following method.
FIG. 3 shows a schematic diagram of the flow path in the microchip used.
In Fig. 3, W indicates the waste well. The R3 well side is the cathode, and the R2 (LB) well side is the anode. In Fig. 3, the electrophoretic sample A and the placement of the test solution in each well are shown in different colors, i.e., dots and white (no dots).

A voltage of 4000V was applied between the R3 well and the R2 (LB) well in Figure 3, and the DNA-labeled anti-PSA antibody in the test solution was brought into contact with the [fluorescently labeled anti-fPSA antibody-fPSA] complex in the electrophoresis sample A at 30°C to form a [fluorescently labeled anti-fPSA antibody-fPSA-DNA-labeled anti-PSA antibody] complex, which was then concentrated by isotachophoresis (ITP). The migration direction of isotachophoresis is indicated by "ITP" and a dotted line in Figure 3.

The immunoreaction time for each labeled antibody to capture fPSA was approximately 200 seconds.

The complexes formed here are specifically a complex of [fluorescently labeled anti-fPSA antibody - FucfPSA - DNA-labeled anti-PSA antibody] (first complex) and a complex of [fluorescently labeled anti-fPSA antibody - non-FucfPSA - DNA-labeled anti-PSA antibody] (second complex).

After isotachophoresis to the R2(FLB) well, the complex was judged to have passed through the R2(FLB) well by a change in voltage, and the negative electrode was switched from R3 to R2(FLB). Capillary gel electrophoresis (CE) was then performed in the presence of PhoSL until a peak for the complex of [fluorescently labeled anti-fPSA antibody-fPSA-DNA labeled anti-PSA antibody] was detected in the detection area (the capillary section 2 cm downstream from the channel crossing section of R2(FLB) and R2(LB)). The position where CE was performed and the direction of electrophoresis are indicated by "CE" and a dotted line in Figure 3.

Detection was performed by measuring the fluorescence intensity over time in the capillary section 2 cm downstream from the channel crossing section of R2(FLB) and R2(LB) using a 635 nm laser excitation with a photodiode (Fuji Film Corporation).

In addition, except for using electrophoresis buffer 2 that does not contain PhoSL, the same electrophoresis sample A, electrophoresis test solution, and measurement device as above were used, and the same method as above was carried out to separate and detect PSA.

The peak of the second complex appears at the same position as the peak that appears when electrophoresis buffer 2 not containing PhoSL is used. On the other hand, the complex of [fluorescently labeled anti-fPSA antibody-FucfPSA-DNA labeled anti-PSA antibody] that reacts with PhoSL (first complex) takes longer to migrate than the complex of [fluorescently labeled anti-fPSA antibody-FucfPSA-DNA labeled anti-PSA antibody] that does not react with PhoSL (second complex), and therefore the appearance of the peak is delayed. In other words, the peak of the first complex appears after the peak of the second complex.

The peak areas of the fractions of the first complex and the second complex were determined using the analysis software provided with the measuring device.

Then, the peak area of the first complex fraction and the peak area of the second complex fraction were used to calculate the ratio (%) of the amount of FucfPSA to the amount of fPSA in the sample (%FucfPSA).

The specific calculation method is as follows.
fPSA amount = [peak area of the first complex fraction] + [peak area of the second complex fraction]
- Ratio of FucfPSA to fPSA (%) (%FucfPSA)
= [peak area of the first complex fraction]/[amount of fPSA]×100

Next, the total PSA amount (D) of each patient and the %FucfPSA of the same patient obtained above were applied to the following formula to calculate the FPI.
FPI = total PSA amount/(101-%FucfPSA)

Based on the above results, further analysis was performed using a Relative Operating Characteristic curve (ROC curve).

(5) Results 1) Distinction from non-cancer patients Figure 4 shows the results of comparing %FucfPSA between non-cancer patients (Negative) and prostate cancer patients (PCa). Figure 5 shows the results of comparing FPI between non-cancer patients (Negative) and prostate cancer patients (PCa). Comparison between the two groups was performed using the Mann Whitney U test.
As is clear from Figures 4 and 5, %FucfPSA and FPI were significantly higher in prostate cancer patients than in non-cancer subjects (both P<0.0001).
From the above results, it is evident that prostate cancer can be diagnosed using %FucfPSA and FPI as markers.

2) Distinguishing between high-risk prostate cancer and others Figure 6 shows the results of comparing %FucfPSA between non-cancer subjects and prostate cancer patients with Gleason score 6 (GS6) or less (Negative-GS6) and high-risk prostate cancer patients with Gleason score 7-9 (GS7-9). Figure 7 also shows the results of comparing FPI between non-cancer subjects and prostate cancer patients with GS6 or less (Negative-GS6) and high-risk prostate cancer patients with GS7-9 (GS7-9). Comparison between the two groups was performed using the Mann Whitney U test.
As is clear from Figures 6 and 7, %FucfPSA and FPI were also associated with the Gleason score, and showed significantly higher values in prostate cancer patients (GS7-9) compared to non-cancer subjects and prostate cancer patients (GS6) (both P<0.0001).
From the above results, it can be seen that %FucfPSA and FPI can be used as markers to determine high-risk prostate cancer patients.Furthermore, from these results, %FucfPSA and FPI can be used as markers to determine whether or not a patient suspected of prostate cancer needs to undergo biopsy.That is, if a patient is determined to be a high-risk prostate cancer patient by the method of the present invention, the patient is selected to undergo biopsy.
3) ROC curve analysis Based on the %FucfPSA, FPI, FucfPSA, and total PSA amount of the prostate cancer patients in GS7-9 obtained above, ROC curve analysis was performed. Statistical analysis was performed using SPSS ver24.
The results obtained are shown in FIG.
The line styles corresponding to each marker in FIG. 8 and their respective AUC (Area under the curve) values are summarized in Table 2 below.

ROC curve analysis showed that FPI had a sensitivity of 90% and a specificity of 36% when the cutoff value was 0.43.
When the %FucfPSA cutoff was 87%, the sensitivity was 91% and the specificity was 38%.
On the other hand, when the amount of FucfPSA, which is a known Pca marker, and the amount of total PSA achieved a sensitivity of 90%, the specificity was 11% and 12%, respectively.

From the above ROC curve analysis results, it can be seen that if the %FucfPSA and FPI of the present invention are used as markers to determine prostate cancer, prostate cancer can be determined with higher sensitivity and specificity than the known prostate cancer markers, i.e., the total PSA amount and serum FucfPSA amount are used.Furthermore, from this, it can be seen that if the marker of the present invention is used to determine prostate cancer, it can be determined with higher diagnostic accuracy than the known prostate cancer markers.
Furthermore, the results of ROC curve analysis showed that FPI and %FucfPSA were significantly associated with Gleason score, and were promising markers for determining high-risk prostate cancer with high specificity. Comparing the AUC values, it was found that FPI in particular is a promising marker for determining high-risk prostate cancer.

For prostate cancer patients with a Gleason score of 6 or less, surveillance therapy, in which the patient is observed without undergoing active treatment, can be selected, taking into consideration other indicators such as the stage of the disease. However, it is difficult to predict the malignancy or Gleason score of prostate cancer with the currently used prostate cancer markers.
However, the values of FPI and %FucfPSA, which are the markers of the present invention, are significantly higher in patients with a Gleason score of 7 or higher than in those with a Gleason score of less than GS6, and as is clear from the ROC curve analysis, patients with a Gleason score of 7 or higher can be identified with high specificity. Therefore, it is possible to determine whether or not to perform a biopsy based on the results. It is also expected that this will prevent unnecessary biopsies from being performed, and thus avoid the burden on the subject and the risks associated with biopsies.

Claims (6)

A method for assisting in the diagnosis of prostate cancer, comprising determining the amount of free prostate-specific antigen (FucfPSA) having a fucosylated glycan, fucose α1→6 glycan, and the amount of free prostate-specific antigen (fPSA) in a sample derived from a subject, calculating the obtained values by applying the obtained values to the following formula, and determining prostate cancer based on the obtained value, %FucfPSA.
%FucfPSA = (FucfPSA amount/fPSA amount) x 100 The method for assisting in the diagnosis of prostate cancer according to claim 1, wherein the amount of FucfPSA is determined by measuring the amount of FucfPSA using an anti-fPSA antibody that binds to fPSA and a lectin that has affinity for the fucose α1→6 glycan. The method for assisting in the diagnosis of prostate cancer according to claim 2, wherein the amount of FucfPSA is determined by contacting a subject-derived sample with the antibody, separating a complex 1 between FucfPSA and the antibody and a complex 2 between free nonfucosylated prostate-specific antigen and the antibody in the obtained subject-derived sample based on their affinity for the lectin, measuring the amount of the separated complex 1, and determining the amount of FucfPSA based on the measurement results obtained. The method for assisting in the diagnosis of prostate cancer according to claim 2, wherein the lectin having affinity for the fucose α1→6 glycan is Sugitake lectin or Lens lectin. The method for assisting in the diagnosis of the prostate according to claim 1, wherein the subject-derived sample is serum, plasma or blood. The method for assisting in the diagnosis of prostate cancer according to claim 1, further comprising determining the total PSA amount, which is the total amount of prostate-specific antigen in a sample derived from a subject, calculating the obtained value by applying it to the following formula, and diagnosing prostate cancer based on the obtained value, FPI.
FPI = total PSA amount/(101-%FucfPSA)

Images