Methods for detecting prostate cancer, associated biomarkers, and kits for detecting biomarkers

Abstract

The present invention relates to the non-invasive detection of prostate cancer based on altered glycosylation patterns, the use of biomarkers in this detection, and kits containing reagents for determining the levels of said biomarkers in a biological sample.

Description

[Technical Field]

[0001] The present disclosure relates to the non-invasive detection of prostate cancer based on altered glycosylation patterns, particularly the glycosylation patterns of CA19-9-containing entities and / or free prostate-specific antigen, and kits comprising said glycosylation patterns as biomarkers for use in the detection, as well as reagents for determining the levels of said biomarkers in biological samples. [Background technology]

[0002] Prostate cancer is the second most common cancer in men worldwide. An estimated 1.4 million men were diagnosed with prostate cancer in 2020, accounting for 14.1% of all cancers diagnosed in men, and with an estimated 375,304 deaths in 2020, prostate cancer is the fifth leading cause of cancer deaths in men worldwide (6.8% of all male deaths).

[0003] Early detection and treatment can significantly improve prostate cancer survival. Prostate-specific antigen (PSA) is the most frequently used biomarker for screening for prostate cancer. While PSA is effective in detecting early-stage prostate cancer, it can miss potentially harmful cancers. However, PSA levels are widely elevated in benign conditions and non-invasive cancers, leading to serious overdiagnosis and overtreatment of prostate cancer.

[0004] To improve the diagnostic sensitivity and accuracy of total PSA in the early detection of prostate cancer, modified assays have been developed to detect alternative molecular forms of PSA, including intact PSA (iPSA) and free PSA (fPSA). In addition to these three PSA-based biomarkers, a fourth prostate biomarker, human kallikrein-related peptidase 2 (hK2), is included in OPKO Health's in vitro test, the 4Kscore Test, recently approved by the U.S. Food and Drug Administration (FDA) to identify risk for aggressive prostate cancer. The improved performance of this multikallikrein test over conventional PSA has been widely reported.

[0005] Despite advances in prostate cancer diagnosis, there remains a recognized need for biomarkers that can distinguish between prostate cancers that are likely to progress to aggressive or fatal disease if left untreated, and that cause the majority of disease-related morbidity and mortality, and those that are clinically insignificant. Such markers would be extremely useful in avoiding over-treatment of less aggressive and indolent forms of prostate cancer and in specifically targeting more intensive treatments to clinically significant prostate cancers.

[0006] Glycosylation is one of the most important post-translational protein modifications. The glycosylation patterns of many proteins have been shown to change during the initiation and progression of cancer. Indeed, several serum glycan structures have been recognized as biomarkers for various types of cancer for many years.

[0007] CA19-9 (sialylated Lex a) is one of several blood group glycans identified by monoclonal antibodies and has been widely used as a marker for cancer, especially pancreatic cancer. CA19-9 is a glycan, a sialylated lacto-N-fucopentose II ganglioside, and is a Le a Sialylated derivatives of blood group antigens, including Le xa Expression of the CA19-9 antigen requires the Lewis gene product, 1,4-fucosyltransferase. CA19-9 is synthesized by normal human pancreatic and biliary duct cells, as well as epithelial cells of the stomach, colon, endometrium, and salivary glands. In the circulation, it has been reported to be present on several high molecular weight glycoprotein complexes and other proteins. Genetically inherited Le a-b-Patients with pancreatic cancer (approximately 5-10% in Caucasians) do not express CA19-9. A monoclonal antibody against CA19-9 was developed from the human colon cancer cell line SW-1116. Several diagnostic companies produce CA19-9 immunoassays. Because significant differences exist between assays due to differences in the antibodies used, individual patient results between different assays are not interchangeable. Typically, CA19-9-reactive antibodies are used as both capture and detection antibodies, i.e., the assay is based on a sandwich format. The established upper reference limit for CA19-9 is 37 kU / L, based on the 99th percentile of normal subjects. This cutoff value distinguishes between pancreatic cancer and benign pancreatic disease, with a clinical sensitivity of 69-93% and a clinical specificity of 76-99%. Elevated CA19-9 concentrations (>37 kU / L) are also seen in patients with hepatobiliary (67%), gastric (40-50%), hepatocellular (30-50%), colorectal (30%), and breast (15%) cancers. CA19-9 has no diagnostic or prognostic value in the detection and management of prostate cancer. Summary of the Invention

[0008] In one embodiment, provided herein are biomarkers for prostate cancer, particularly clinically significant prostate cancer. The biomarkers are glycan structures contained in carbohydrate antigen 19-9 (CA19-9)-containing molecular entities that are capable of specific binding to mannose-binding lectin (MBL). In another embodiment, the biomarkers are glycovariants of free prostate-specific antigen (fPSA) that are capable of specific binding to macrophage galactose-type lectin (MGL).

[0009] In a further aspect, a method for determining the disease state of prostate cancer in a subject is provided. The method includes: a) assaying a sample obtained from the subject for the level of a carbohydrate antigen 19-9 (CA19-9)-containing entity comprising a glycan structure capable of specifically binding to MBL; b) comparing the assay level obtained in step a) with a control sample or a predetermined threshold; and c) determining the disease state of prostate cancer based on the comparison. Alternatively or additionally, step a) may include assaying a sample obtained from the subject for the level of a glycovariant of fPSA capable of specifically binding to macrophage galactose-type lectin (MGL). Accordingly, step b) includes comparing the assay level of the glycovariant of fPSA with a control sample or a predetermined threshold, and c) determining the disease state of prostate cancer based on the comparison.

[0010] In a further embodiment, a kit is provided for use in determining the disease state of prostate cancer in a subject. The kit comprises: i) a CA19-9 binding agent, and ii) a binding molecule specific for a glycan structure capable of specifically binding to MBL. Either the CA19-9 binding agent or the binding molecule comprises a detectable label. Alternatively or additionally, the kit comprises: i) a multi-well plate on which an fPSA binding agent is immobilized in an area having a size smaller than the size of the bottom surface, and ii) a binding molecule specific for a glycan structure capable of specifically binding to MGL. The use of these kits for determining the disease state of prostate cancer in a subject is also provided.

[0011] In yet another aspect, there is provided the use of a binding molecule selected from the group consisting of a binding molecule specific for a glycan structure capable of specifically binding to MBL, a binding molecule specific for a glycan structure capable of specifically binding to WGA, and a binding molecule specific for a glycan structure capable of specifically binding to MGL, for determining the presence or absence of a prostate cancer-associated CA19-9-containing entity in a sample.

[0012] Also provided is the use of a binding molecule specific for MBL or a glycan structure capable of specifically binding to MBL for determining the disease state of prostate cancer in a subject.

[0013] Further aspects, embodiments, and details are set forth in the following drawings, detailed description, examples, and dependent claims. [Brief explanation of the drawings]

[0014] The accompanying drawings illustrate several embodiments of the disclosed subject matter and, together with the description, serve to explain the principles of the disclosed compositions and methods.

[0015] [Figure 1-1] 1A-1D show the calibration curves for the conventional CA19-9 ELISA immunoassay (EIA) and the CA19-9MGL, CA19-9WGA, and CA19-9MBL glycovariant assays of the present invention, respectively. [Figure 1-2] Same as above.

[0016] [Figure 2] Figures 2A and 2B show boxplots for the conventional CA19-9 ELISA immunoassay and the median and mean CA19-9 concentrations (U / mL) for both clinically significant prostate cancer (PCa) and indolent disease, as well as similar data for individual patient groups. Clinically significant PCa (Gleason ≥ 7) could not be distinguished from indolent disease (benign cases and Gleason grade 6 cancers), nor was separation of different cancer groups from benign samples achieved.

[0017] [Figure 3]Figures 3A and 3B show boxplots for the CA19-9MBL glycovariant assay and median and mean values ​​of CA19-9MBL (U / mL) for both clinically significant PCa (Gleason ≥ 7) and indolent disease (benign cases and Gleason grade 6 cancers), as well as similar data in individual patient groups, respectively.

[0018] [Figure 4] Figures 4A and 4B show boxplots for the CA19-9WGA glycovariant assay and median and mean CA19-9WGA (U / mL) values ​​for both clinically significant PCa and indolent disease, respectively, as well as similar data in separate patient groups.

[0019] [Figure 5] Figures 5A and 5B show boxplots for the CA19-9MGL glycovariant assay and median and mean values ​​of CA19-9MGL (U / mL) for both clinically significant PCa and indolent disease, as well as similar data in separate patient groups, respectively.

[0020] [Figure 6] Figure 6 shows the receiver operating characteristic curves for the conventional ELISA assay and the three CA19-9 glycovariant assays. All measured CA19-9 glycovariants (CA19-9WGA, CA19-9MBL, and CA19-9MGL) significantly improved the discrimination of patients with more aggressive prostate cancer (Gleason total 7-10) compared with patients with benign or clinically insignificant Gleason 6 cancers, compared with the reference ELISA.

[0021] [Figure 7]Figure 7 shows receiver operating characteristic curves and area under the curve (AUC) values ​​for the multikallikrein model (total PSA, free PSA, intact PSA, and hK2), three CA19-9 glycovariants (CA19-9MBL, CA19-9WGA, and CA19-9MGL), the combination of these three CA19-9 glycovariants with the multikallikrein marker, or the free PSA MGL glycovariant. GV = glycovariant.

[0022] [Figure 8-1] Figures 8A-8D show the concentrations of conventional CA19-9 EIA, CA19-9 MBL, total PSA, and free PSAMGL glycovariants in CA19-9-negative patients (n = 20) and CA19-9-positive patients (n = 229). Higher elevations of total PSA and free PSAMGL glycovariants were observed in CA19-9-negative patients with aggressive PCa compared with CA19-9-positive patients. [Figure 8-2] Same as above.

[0023] [Figure 9] Figures 9A and 9B show box plots for the CA19-9DC-SIGN glycovariant assay for clinically significant PCa and indolent disease, and for individual patient groups, respectively. NS = not statistically significant.

[0024] [Figure 10-1] Figure 10A shows the dose-response curves (mean fluorescent signals from triplicate measurements) obtained from LNCaP PSA used as a standard in fPSAMGL assays performed in either spot or whole-well format. Figure 10B shows the dose-response curves for healthy seminal plasma PSA (□) and cancerous LNCaP PSA (◇) obtained by the PSAMGL glycovariant assay in spot format, while Figure 10C shows similar dose-response curves obtained by a conventional free PSA immunoassay. [Figure 10-2] Same as above.

[0025] [Figure 11] Figures 11A and 11B show box plots for the conventional free PSA immunoassay and the free PSAMGL glycovariant assay of the present invention, respectively. As shown in Figure 11B, free PSAMGL discriminated both Gle7 and Gle8-10 cancers compared with the benign + Gle6 cancer group with high statistical significance. In contrast, Figure 11B shows that conventional free PSA did not discriminate. Free PSAMGL concentration (ng / mL)* refers to values ​​based on calibration results using PSA purified from LNCaP cell cultures. The relative content of free PSAMGL glycovariants in LNCaP PSA preparations is unknown.

[0026] [Figure 12] FIG. 12 shows ROC plots showing the AUC of free PSAMGL glycovariants (black solid line) and free PSA (black dashed line) from PCa patients with Gle≧7 (n=107) and benign+Gle6 (n=142).

[0027] [Figure 13] Figure 13 shows ROC plots showing the AUCs for free PSAMGL glycovariants (solid black line), free PSAAAL glycovariants (dashed black line), and both combined (dashed gray line) from patients with Gle≧7 (n=107) and benign + Gle6 (n=142) PCa. DETAILED DESCRIPTION OF THE INVENTION

[0028] Unless otherwise defined, the terms and phrases used in this specification and claims have the meanings commonly applied in the field of cancer diagnostics. Some terms and phrases used in this specification have the meanings defined below.

[0029] As used herein, the singular references include the plural references unless otherwise specified, and thus singular terms can also include the plural references. In other words, the terms "a" or "an" can mean one or more.

[0030] As used herein, the term "or" in the claims is intended to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the present disclosure supports both the alternatives and the "and / or" definition.

[0031] As used herein, the terms "biological sample" and "sample" are used interchangeably, and refer to samples obtained from subjects, particularly those consisting of bodily fluids such as ascites, seminal plasma, urine, blood (e.g., plasma or serum), and peritoneal fluid. Generally, obtaining a sample to be analyzed from a subject is not included in the method for determining the disease state of a subject's cancer. In this method and all of its embodiments, urine, blood, serum, or plasma samples are the most preferred sample types for use. In some embodiments, the sample is an EDTA plasma sample.

[0032] In embodiments involving assessment of the level of more than one biomarker, the same or different samples obtained from the subject whose cancer disease status is being determined may be used for each assessment, and the different samples may be of the same or different types.

[0033] As used herein, the terms "biomarker" and "marker" are used interchangeably and refer broadly to molecular entities that are differentially present in samples taken from subjects with cancer or a particular grade of that cancer compared to comparable samples taken from control subjects (e.g., apparently healthy subjects or subjects with a different grade of cancer).

[0034] As used herein, the terms "glycoform" and "glycovariant" are interchangeable and refer to a specific form of a glycosylated biomarker. That is, when the same molecular backbone that is part of a biomarker can bind to different glycans or sets of glycans, each different version of the biomarker is called a "glycoform" or "glycovariant."

[0035] As used herein, the term "CA19-9" refers to "carbohydrate antigen 19-9," also known as "cancer antigen CA19-9." a CA19-9 is a derivative of the blood group antigen (sialyl-Lewis A; sLeA) and is present in many secretory glycolipids and glycoproteins, such as mucins and carcinoembryonic antigen (CEA). CA19-9 is a tumor marker primarily associated with gastrointestinal malignancies, including cancers of the pancreas, gallbladder, stomach, and colon. Of note, CA19-9 is a marker of sialyl-Lewis A, which accounts for approximately 5% of the general population. a It is unable to detect blood type negative subjects. As used herein, the terms "CA19-9" and "CA19.9" are interchangeable.

[0036] CA19-9 belongs to the Lewis antigen glycan family. The common feature is a core N-acetyllactosamine (LacNAc), a disaccharide consisting of galactose linked to N-acetylglucosamine. The monosaccharides fucose and sialic acid can be attached to LacNAc in various linkages. Sulfate groups can also be attached to the galactose or N-acetylglucosamine.

[0037] As used herein, "CA19-9 MBL The term "CA19-9" refers to a glycovariant of a CA19-9-containing molecular entity capable of specifically binding to MBL, without being limited to any detection technique. MBL and "CA19-9-containing entity comprising a glycan structure capable of specifically binding to MBL" are interchangeable with the term "MBL-binding glycovariant of a CA19-9-containing entity".

[0038] Similarly, "CA19-9 WGA " and "CA19-9 MGL " refers to a glycovariant of a CA19-9-containing entity that can specifically bind to WGA and MGL, respectively, without being limited to any detection method. These terms are used interchangeably with the terms "CA19-9-containing entity comprising a glycan structure that can specifically bind to WGA," "WGA-binding glycovariant of a CA19-9-containing entity," "CA19-9-containing entity comprising a glycan structure that can specifically bind to MGL," and "MGL-binding glycovariant of a CA19-9-containing entity," respectively.

[0039] As used herein, the term "multikallikrein" refers to a panel of four prostate kallikrein protein biomarkers: total prostate-specific antigen (PSA), free PSA, and intact PSA, and kallikrein-related peptidase 2 (hK2).

[0040] As used herein, "fPSA" refers to MGL " refers to a glycovariant of free PSA that can specifically bind to MGL, without being limited to any detection method. This term is interchangeable with the term "MGL-binding glycovariant of free PSA."

[0041] As used herein, the term "binding molecule" refers broadly to any molecule capable of binding to a biomarker or its glycan moiety. Non-limiting examples of binding molecules include lectins, antibodies, antibody mimetics, and oligonucleotide and peptide aptamers.

[0042] As used herein, the terms "CA19-9 conjugate" and "CA19-9 binding agent" refer to a binding molecule specific for CA19-9. Preferably, such a binding molecule is an anti-CA19-9 antibody, more preferably a monoclonal anti-CA19-9 antibody.

[0043] As used herein, the terms "fPSA binder" and "fPSA binder" refer to a binding molecule specific for free PSA. Preferably, such a binding molecule is an anti-fPSA antibody, preferably a monoclonal anti-fPSA antibody, or an antigen-binding fragment thereof (e.g., a Fab fragment, a F(ab)2 fragment, or a F(ab')2 fragment), or a single-chain variant thereof. Preferably, the antigen-binding fragment is site-specifically biotinylated.

[0044] The term "glycan binder" as used herein refers to a binding molecule that specifically binds to a specific glycan structure contained in a molecular entity. Preferably, such a binding molecule is a lectin, particularly a lectin selected from the group consisting of MBL, WGA, and MGL, or an anti-glycan antibody, particularly an anti-glycan antibody specific for a glycan structure that can be recognized by MBL, WGA, or MGL. It should be noted that because CA19-9 is a glycan, CA19-9 binders also specifically bind to glycan structures, but the term "glycan binder" does not encompass CA19-9 binders. In other words, as used herein, "CA19-9 binder" and "glycan binder" refer to different binding molecules, and the latter refers to a binding molecule specific for a glycan structure other than CA19-9 contained in a CA19-9-containing molecular entity.

[0045] Lectins are a group of carbohydrate-binding proteins present in many plants, especially seeds, as well as in fungi, bacteria, and animals.

[0046] As used herein, the term "antibody" generally refers to an immunoglobulin structure consisting of two heavy chains and two light chains interconnected by disulfide bonds. Antibodies can exist as intact immunoglobulins or as well-characterized antigen-binding fragments or single-chain variants, all of which are encompassed by the term "antibody." Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)2 fragments, F(ab')2 fragments, and Fv fragments. These fragments and variants can be produced by recombinant DNA technology or by isolating intact immunoglobulins by enzymatic or chemical means, as is well known in the art. Thus, the term "antigen-binding fragment" refers to any fragment of a monoclonal antibody, regardless of the method of production (including the enzymes used, e.g., pepsin, papain, bromelain), as long as it retains the antigen-binding specificity derived from the monoclonal antibody.

[0047] The term "subject" as used herein refers to an animal, preferably a mammal, more preferably a human, most preferably a male. Depending on the embodiment in question, the subject may have cancer, whether diagnosed or not, may be suspected of having cancer, may be at risk of cancer, or may already be undergoing cancer treatment. As used herein, the terms "human subject," "patient," and "individual" are interchangeable.

[0048] As used herein, the term "benign condition" refers to a non-cancerous urological disease, including, but not limited to, benign prostatic hyperplasia (BPH), a common non-cancerous prostate adenoma in aging men. BPH is generally not considered to be a pre-cancerous or precursor to cancer.

[0049] As used herein, the term "prostate cancer" refers to any cancer of the prostate gland. Prostate cancer is usually slow-growing and initially localized within the prostate gland, where it may not cause serious clinical consequences. While slow-growing types of prostate cancer may require minimal or no treatment, some other types of prostate cancer are more aggressive and may metastasize to other parts of the body. The Gleason scoring system is the most commonly used grading system for prostate cancer. The scores are summed to derive a total score of 6 to 10. Gleason scores below 5 are not used, so the lowest Gleason score is 6, which indicates low-grade or clinically insignificant cancer. A Gleason score of 7 indicates intermediate-grade cancer, and scores of 8 to 10 indicate high-grade cancer. Gleason scores of 7 to 10 are collectively referred to herein as "clinically significant prostate cancer."

[0050] As used herein, the term "indolent disease" refers collectively to benign urological disease and clinically insignificant Gleason grade 6 prostate cancer.

[0051] As used herein, the term "indicative of cancer," when applied to a biomarker, refers to a level that allows for the diagnosis of cancer or a stage of cancer using routine statistical methods with a confidence level of at least 95%, meaning that the detected level is significantly more frequent in subjects with cancer or a stage thereof compared to subjects without the cancer or subjects with another stage.

[0052] As used herein, the term "level" is interchangeable with the terms "amount" and "concentration," unless otherwise specified.

[0053] To determine whether a biomarker indicates the presence or risk of cancer associated with the biomarker, the detected level of the biomarker must be compared with the level in a relevant control.Once the control level is known, the measured marker level can be compared therewith, and the significance of the difference can be assessed using standard statistical methods.In some embodiments, a statistically significant difference between the measured biomarker level and the control level is an indicator of clinically significant prostate cancer.In some further embodiments, before comparing with the control, the biomarker level is normalized using standard methods.

[0054] Comparing the assay level of a biomarker in the sample being analyzed to an associated control or predetermined threshold may, in some embodiments, be performed by a processor of a computing device.

[0055] Whether or not the comparison is performed by a processor of a computing device, in some embodiments, the assay level of a biomarker in the sample being analyzed is determined to be "increased" or "high" if it is at least about 1.5-fold, at least about 1.75-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold the level of the biomarker in a predetermined threshold level or control sample. In some embodiments, the difference between the level of the biomarker in the sample being analyzed and the predetermined threshold level or the level of the biomarker in a control sample must be statistically significant to provide an appropriate diagnostic, prognostic, or predictive result.

[0056] The concentration of a biomarker in a sample obtained from a subject being determined to have a cancer disease status or being screened, diagnosed, prognosed, or monitored for cancer is considered "not elevated" or "normal" if the detected concentration is lower, substantially the same, or substantially unchanged compared to a relevant control sample or a predetermined threshold.

[0057] The term "control" used herein may refer to the control sample obtained from an apparently healthy individual or a pool of apparently healthy individuals, or may refer to the control sample obtained from an individual or a pool of individuals with benign conditions, such as benign urological diseases such as benign prostatic hyperplasia, or may refer to a cut-off value, i.e., a predetermined threshold, that indicates the presence or absence of clinically significant prostate cancer.For example, a predetermined threshold may refer to a level where the level of a biomarker is higher than that level, indicating a high probability of having clinically significant prostate cancer, or where the level is lower than that level, indicating a low probability of having clinically significant prostate cancer.The statistical method for determining an appropriate threshold is obvious to those skilled in the art.If necessary, the threshold may be determined based on the sample of subjects with the same age, demographic characteristics, and / or disease state, etc.The threshold may be a value based on a single individual who does not have clinically significant prostate cancer, or may be a value pooled from such multiple individuals.

[0058] The term "control" used herein may refer to the control sample obtained from an apparently healthy individual or a group of apparently healthy individuals, or may refer to the control sample obtained from an individual or group of individuals with benign conditions, such as benign urological diseases such as benign prostatic hyperplasia, or may refer to a cut-off value, i.e., a predetermined threshold, that indicates the presence or absence of clinically significant prostate cancer.For example, a predetermined threshold may refer to the level of a biomarker that, when above this level, is likely to have clinically significant prostate cancer, or when below this value, is unlikely to have clinically significant prostate cancer.The statistical method for determining an appropriate threshold is obvious to those skilled in the art.If necessary, the threshold may be determined based on the sample of subjects with the same age, demographic characteristics, and / or disease state, etc.The threshold may be a value based on a single individual that does not have clinically significant prostate cancer, or may be a value pooled from more than one such individual.

[0059] As used herein, the term "cancer disease state" refers to any distinguishable manifestation of cancer, including non-cancerous manifestations. For example, this term includes, but is not limited to, the presence or absence of cancer, the presence or absence of preclinical stages of cancer, the risk of having or developing cancer, the stage or grade of cancer, and information regarding the progression of cancer.

[0060] The term "spot assay" as used herein refers to an assay format in which a binding molecule, more specifically a capture agent, is immobilized in a certain area (i.e., a spot) on a solid surface, and the area is smaller than the rest of the surface. In other words, in a multi-well assay plate such as a microtiter plate, the size of the spot, more specifically its diameter, is smaller than the size (diameter) of the well containing the spot. An assay format in which a single well contains multiple spots, each with a different binding molecule immobilized thereon, is referred to herein as an "array-in-well assay." An assay format in which the area in which the binding molecules are immobilized covers at least the entire bottom surface is referred to as a "whole-well assay." Immobilization techniques suitable for each assay format are widely known to those skilled in the art.

[0061] Some of the additional terms and phrases used in this disclosure are explained below in the detailed description of the invention.

[0062] Detailed Description Biomarkers and clinical methods The present disclosure is based on research aimed at identifying cancer-associated glycovariants of molecular entities as cancer biomarkers with improved sensitivity over other variants of the same molecular entity. In accordance with this objective, the present disclosure provides means and methods for determining the disease status of prostate cancer in subjects suspected of having or at risk of having prostate cancer. The means and methods are provided particularly for the screening, diagnosis, prognosis, or monitoring of prostate cancer.

[0063] Surprisingly, it has now been shown that several glycovariants of CA19-9-containing molecular entities can serve as biomarkers for prostate cancer. Traditionally, CA19-9 has been primarily associated with gastrointestinal malignancies, such as pancreatic, gallbladder, gastric, and colon cancers.

[0064] Conventional CA19-9 immunoassays are based on the recognition of the CA19-9 antigen in a sample, such as serum or plasma, using the same (monoclonal) antibody, or alternatively, two different (monoclonal) antibodies that both recognize the CA19-9 antigen. The actual glycoprotein that carries the CA19-9 antigen and is therefore determined by the CA19-9 assay has not been precisely characterized, but is likely one of several proteins belonging to the mucin family. However, other proteins may also carry the CA19-9 antigen.

[0065] It is shown herein that detection of specific glycovariants of CA19-9-containing molecular entities, instead of using conventional CA19-9 immunoassays, makes it possible to distinguish between subjects with indolent urological disease and those with clinically significant prostate cancer among subjects suspected of having prostate cancer.

[0066] In experiments leading to some embodiments of the present invention, mannose-binding lectin (MBL) demonstrated excellent discrimination (p=0.000003) between CA19-9-containing entities from subjects with benign urological disease or Gleason 6 prostate cancer and those from subjects with clinically significant prostate cancer. As known to those skilled in the art, MBL has excellent affinity for fucose and mannose / mannan. Interestingly, another lectin with affinity for high-mannose structures, namely dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), did not show corresponding discrimination between different patient groups among subjects suspected of having prostate cancer.

[0067] In accordance with the above, by assaying biological samples obtained from subjects suspected of having prostate cancer for CA19-9-containing molecular entities capable of specifically binding to MBL, subjects with indolent urological disease can be distinguished from subjects with clinically significant prostate cancer, a distinction not possible with conventional CA19-9 immunoassays.

[0068] Thus, in one embodiment, MBL-binding glycovariants of CA19-9-containing entities are provided as biomarkers for prostate cancer, particularly clinically significant prostate cancer. Notably, the prostate cancer biomarkers of the present invention are independent of prostate volume, as shown in Example 4 below. In other embodiments, the use of MBL-binding glycovariants of CA19-9-containing entities in the detection of clinically significant prostate cancer, as well as various clinical methods using the biomarkers, are provided, as described in more detail below.

[0069] The clear discrimination between subjects with indolent disease and those with clinically significant prostate cancer was also confirmed in experiments using wheat germ agglutinin (WGA) and macrophage galactose-type lectin (MGL). More specifically, among the lectins tested, WGA and MGL also showed excellent discrimination between CA19-9-containing entities derived from subjects with benign urological disease or Gleason 6 prostate cancer and those derived from subjects with clinically significant prostate cancer.

[0070] Therefore, herein, WGA binding and MGL binding glycovariants of CA19-9 containing entities are provided as independent biomarkers for prostate cancer, particularly clinically significant prostate cancer.Also provided are the detection of clinically significant prostate cancer using these biomarkers, and various clinical methods using these biomarkers.

[0071] The biomarkers of the present invention can be used in different combinations. Some embodiments may involve only one of the three biomarkers disclosed above, while other embodiments may involve any two of the three biomarkers. That is, a combination of MBL-binding glycovariants and WGA-binding glycovariants of CA19-9-containing entities, a combination of MBL-binding glycovariants and MGL-binding glycovariants, or a combination of WGA-binding glycovariants and MGL-binding glycovariants. Furthermore, there are also configurations involving all three biomarkers. Notably, as demonstrated in the Examples, the WGA-binding glycovariants of CA19-9-containing entities and the MGL-binding glycovariants of CA19-9-containing entities complemented the diagnostic performance of the MBL-binding glycovariants of CA19-9-containing entities.

[0072] In some embodiments, the biomarkers of the present invention are total PSA, free PSA, intact PSA, hK2 and / or fPSA. MGL It may be used in combination with one or more known prostate cancer biomarkers, such as CA19-9. Of note, as shown in the Examples, MBL , CA19-9 WGA and CA19-9 MGL Each biomarker was superior to free PSA, intact PSA, total hK2, and free hK2 in distinguishing between clinically significant cancer and indolent disease. MBL , CA19-9 WGA and CA19-9 MGL The combination outperformed the multikallikrein panel biomarker for this purpose. Furthermore, the performance of CA19-9 glycovariants was improved when used in combination with the multikallikrein panel.

[0073] Interestingly, CA19-9 negativity was associated with progression to higher-grade prostate cancer, as well as with progression to higher-grade total PSA and fPSA. MGLCA19-9 negative subjects account for 8% of the entire cohort, which is consistent with the general understanding that 5-10% of Caucasian individuals are Lewis antigen negative and therefore lack CA19-9.Therefore, in one embodiment, the present invention provides CA19-9 negativity as a new marker for more aggressive prostate cancer in subjects already diagnosed with prostate cancer.

[0074] Of note, despite being determined to be CA19-9 negative by conventional CA19-9 immunoassay, MBL were able to distinguish between subjects with indolent disease and those with clinically significant prostate cancer in a study cohort classified as CA19-9 negative.

[0075] In one embodiment of the present disclosure, fPSA MGL However, fPSA is provided as a blood-based prostate cancer biomarker, particularly a clinically significant prostate cancer biomarker, which may or may not be used in conjunction with glycovariants of CA19-9-containing entities and / or known prostate cancer biomarkers. MGL fPSA has been suggested as a biomarker for prostate cancer (WO2018011474), but while it performed well in urine samples and lysates of cancerous prostate tissue, it did not perform well in blood samples. However, using a spot assay format, fPSA MGL It has been unexpectedly found that the assay also works well in blood samples. Indeed, the assay herein demonstrates the ability to measure fPSA in blood samples using the above assay format. MGL Detection of fPSA has been shown to distinguish between subjects with indolent urological disease and those with clinically significant prostate cancer among subjects suspected of having prostate cancer. MGL showed no gland volume dependency and showed similar performance across different gland volume groups.

[0076] As mentioned above, the biomarkers of the present invention or their combinations can be used in various prostate cancer-related clinical methods, with or without the addition of known prostate cancer biomarkers. Such methods can be defined or expressed in different ways. For example, in some forms, this method is a method for detecting prostate cancer, particularly clinically significant prostate cancer, or a method for determining the prostate cancer disease state in a subject. This method can also be expressed as a method for identifying a subject who has clinically significant prostate cancer or is at risk of having or developing clinically significant prostate cancer. Furthermore, this method can also be expressed as a diagnostic method for prostate cancer, i.e., determining whether a subject has or is at risk of prostate cancer, particularly clinically significant prostate cancer. Additional ways of expressing the method will be apparent to those skilled in the art. Usually, the subject whose prostate cancer disease state is to be determined is suspected of having prostate cancer. In short, even if the purpose of the method is expressed differently, the steps of the method itself are the same.

[0077] The clinical method discussed above is intended to include cases where the presence or risk of prostate cancer is not finally determined, but additional diagnostic tests are warranted.In such embodiments, this method itself does not determine the presence or risk of prostate cancer in a subject, but may indicate that additional diagnostic tests are required or will be beneficial.Therefore, this method can be used in combination with one or more other diagnostic methods to finally determine the presence or risk of prostate cancer in a subject.Such other diagnostic methods are well known to those skilled in the art.

[0078] Because it is non-invasive and suitable for analyzing body fluid samples, this clinical method and its various embodiments can be easily incorporated into population screening protocols to identify subjects who have prostate cancer or who are at risk of having or developing prostate cancer. This will enable not only early diagnosis but also active surveillance for the development of clinically significant prostate cancer in subjects who are determined to be at high risk of developing prostate cancer in the future. Furthermore, early detection of clinically significant cancer will allow for early treatment of the disease at the stage when it is most likely to be cured.

[0079] It is envisioned that the present method and its various embodiments may be used not only for diagnostic purposes but also for various monitoring purposes. Such monitoring purposes include monitoring the onset of clinically significant prostate cancer in subjects suspected of having prostate cancer or at risk of having or developing prostate cancer, and monitoring the progression of a subject's prostate cancer disease state, including, but not limited to, recovery or survival from cancer, likelihood of disease recurrence or relapse, or response to treatment. In such embodiments, the method is a method of monitoring prostate cancer in a subject, comprising the step of detecting one or more biomarkers of the invention (more specifically, one or more CA19-9-containing entities comprising glycan structures capable of specifically binding to MBL, WGA, and / or MGL, and / or fPSA). MGLThe method includes comparing the level of a biomarker (or biomarkers) in one or more other samples obtained from the same subject at different time points. Samples that can be used for monitoring include, but are not limited to, samples taken at different time points after cancer diagnosis, and / or samples taken before, during, or after a therapeutic intervention (e.g., surgery, radiation therapy, chemotherapy, other appropriate therapeutic treatment, or any combination thereof). In some embodiments, the monitoring is performed by repeating the assay step at least two times at different time points independently selected from the above time points. In some embodiments, the monitoring is performed during or after treatment for cancer, and / or the method includes determining that the subject has or is at risk for cancer recurrence or relapse if the level of at least one of the biomarkers is elevated above the level in one or more samples previously obtained from the same subject or above the level in a relevant control, or above a predetermined threshold.

[0080] In some embodiments, the method may further comprise therapeutic intervention. If a subject is determined to have clinically significant prostate cancer or to be at risk of having or developing clinically significant prostate cancer, the subject may be administered a cancer treatment that is predicted to be effective. Such a method may be formulated in various ways. For example, in some embodiments, the present invention provides a method for determining whether a subject has clinically significant prostate cancer or is at risk of having or developing clinically significant prostate cancer, the method comprising: a) assaying a sample obtained from the subject for the level of CA19-9-containing entities that comprise glycan structures that can specifically bind to a specific lectin; b) comparing the determined level of the entity with the level of the entity in a control sample or a predetermined threshold; c) determining the presence or risk of clinically significant prostate cancer in the subject according to the comparison, wherein an increase in the level of the entity in the sample obtained from the subject compared with the level of the corresponding entity in the control or a predetermined threshold indicates that the subject has clinically significant prostate cancer or is at risk of having or developing clinically significant prostate cancer; and d) administering cancer treatment to the subject. Alternatively, or in addition, step a) may comprise assaying a sample obtained from the subject for a level of a glycovariant of fPSA capable of specifically binding to MGL, in which case step b) comprises comparing the assayed level of the glycovariant of fPSA with a control sample or a predetermined threshold, step c) determining a prostate cancer disease state based on the comparison, and step d) administering a cancer treatment to the subject.

[0081] In this context, the term "treatment" refers to providing cancer therapy to a subject in need thereof, with the goal of which may include ameliorating, alleviating, inhibiting, or curing the cancer. Cancer treatment may involve one or more therapies selected from surgery, chemotherapy, radiation therapy, immunotherapy, or targeted therapy such as treatment with small molecule inhibitors.

[0082] Furthermore, the development of new pharmaceuticals and therapies for subjects with prostate cancer is complicated by the highly variable morbidity and mortality associated with cancer, and therefore markers that allow for more accurate selection of subjects with clinically significant forms of prostate cancer for therapeutic trials are critically needed to facilitate the development of new or alternative treatment strategies, particularly for aggressive forms of the disease. It is envisioned that the biomarkers of the present invention are suitable for this purpose.

[0083] As will be readily understood by those skilled in the art, the details and embodiments of the present biomarkers and clinical methods disclosed herein above also apply to the assays, assay formats, and kits disclosed below, even if the details and embodiments are not repeated. Thus, the details and embodiments disclosed below with respect to the present assays, assay formats, and kits also apply to the biomarkers and clinical methods disclosed above, as appropriate and in a manner that will be readily understood by those skilled in the art, even if the details and embodiments are not repeated.

[0084] Assay Format As mentioned above, the biomarkers of the present invention or their combinations can be used in various prostate cancer-related clinical methods. Such methods can be defined or expressed in different ways. For example, in some embodiments, a method for determining a subject's prostate cancer disease state is provided, in which a sample obtained from the subject is first analyzed for the level of CA19-9-containing entities, which contain glycan structures capable of specific binding to a specific lectin, and / or fPSA. MGL In a next step, the assayed entity or said fPSA is assayed for a level of MGLThe level of the entity is compared with that of a control sample or a predetermined threshold level. The subject's prostate cancer disease status is then determined based on the comparison. In some embodiments, an elevated level of the entity is indicative of clinically significant prostate cancer. On the other hand, a non-elevated level of the entity compared with that of a control sample or a predetermined threshold level is indicative of benign urological disease or clinically insignificant (Gleason 6) prostate cancer. In accordance with the above description, the lectin of interest for the CA19-9-containing entity is either MBL, WGA, or MGL, preferably MBL. In some embodiments, assaying a sample for multiple CA19-9-containing entities capable of specifically binding to different lectins may be included. In some preferred embodiments, a sample is assayed for a CA19-9-containing entity capable of specifically binding to MBL, and additionally for a CA19-9-containing entity capable of specifically binding to either WGA or MGL. In yet other embodiments, a sample is assayed for a CA19-9-containing entity capable of specifically binding to each of MBL, WGA, and MGL. Assaying for multiple glycovariants of a CA19-9-containing entity may be performed simultaneously or sequentially in the same assay (i.e., simultaneously) or in separate assays (i.e., in parallel). MGLThe assay may include assaying for one or more CA19-9-containing entities in any combination of the above, or not. Again, when the method involves assaying for multiple biomarkers, these may be performed simultaneously or sequentially in the same assay, in different arrays, or in different assays. When performed in the same assay, this may be achieved by multiplexing or by using different wells or arrays in a multiwell plate, preferably a microtiter plate. In some embodiments, the assay may be performed in an array-in-well format, in which a single well contains distinct spots on which a CA19-9-binding agent and an fPSA-binding agent, preferably an antigen-binding fragment of an anti-fPSA antibody (e.g., Fab, F(ab')2, or F(ab)2 fragment), more preferably a site-specifically biotinylated antigen-binding fragment of an anti-fPSA antibody, are immobilized. In some other embodiments, the assay may be performed on a multiwell plate containing one or more wells in which a CA19-9 binding agent is immobilized and one or more different wells in which an fPSA binding agent, preferably an antigen-binding fragment of an anti-fPSA antibody (e.g., a Fab, F(ab')2, or F(ab)2 fragment), more preferably a site-specifically biotinylated antigen-binding fragment of an anti-fPSA antibody, is immobilized.

[0085] Before detailing the steps for assaying a sample for levels of CA19-9-containing entities containing glycan structures capable of specific binding to specific lectins, the following should be noted. Extracellular vesicles (EVs) are lipid bilayer membrane-enclosed particles naturally secreted by cells and present in biological fluids, including, but not limited to, blood, urine, and cerebrospinal fluid. EVs carry contents including proteins, nucleic acids, lipids, and metabolites derived from parent cells and may present various antigens, such as CA19-9, on their surface. However, CA19-9 can also exist in biological fluids as soluble glycoconjugates unbound to vesicles. Therefore, the present method encompasses determining the levels of specific glycan structures in a biological sample, regardless of whether they are present as glycovariants of soluble glycoconjugates or contained in EVs. That is, assaying a sample for CA19-9-containing entities includes not only assaying a sample for the presence of soluble glycoconjugates of CA19-9 containing glycan structures capable of specific binding to a particular lectin, but also assaying a sample for EVs that display both CA19-9 and glycan structures capable of binding to the lectin on their surface. Such an assay can be achieved by assaying a sample for CA19-9-containing entities (soluble glycoconjugates or EVs) that are captured by anti-CA19-9 antibodies and contain glycan structures capable of specific binding to a particular lectin. However, in some embodiments, the assay may specifically target glycovariants of soluble glycoconjugates containing CA19-9, and in other embodiments, the assay may specifically target glycan structures displayed by EVs displaying CA19-9.

[0086] Assaying the level of a CA19-9-containing entity containing a glycan structure capable of specifically binding to a particular lectin can be performed by a variety of means and methods readily available in the art. In some embodiments, a glycan binder, such as a lectin or an anti-glycan antibody, can be used. Such embodiments can include contacting a sample obtained from a subject suspected of having prostate cancer with a CA19-9 binder, such as an anti-CA19-9 antibody, and a glycan binder specific for a glycan structure capable of specifically binding to a particular lectin, i.e., MBL, WGA, or MGL, and detecting the resulting binding reaction. The degree of the binding reaction is indicative of the level of the CA19-9-containing entity of interest in the sample. In some embodiments, the glycan binder is a lectin, more specifically, MBL, WGA, or MGL, or an antibody capable of specifically binding to the same glycan structure as MBL, WGA, or MGL. Corresponding steps may also be applied to a control sample to allow comparison of the level of the CA19-9-containing entity in a sample obtained from a subject suspected of having prostate cancer with the level of the same entity in a related control sample.

[0087] Therefore, CA19-9-containing entities containing glycan structures capable of specifically binding to MBL (CA19-9 MBL The assay of the level of CA19-9 can be carried out by using an anti-CA19-9 antibody and MBL. However, the expression "capable of specifically binding to MBL" is not limited to the use of MBL for detection. In other words, MBL can be detected or assayed not only by techniques using MBL, but also by techniques using other lectins with binding specificities similar to or overlapping with MBL. The specificity of each lectin and its suitability for this purpose can be easily determined by those skilled in the art. In addition, anti-glycan antibodies specific to glycan structures that can be specifically recognized by MBL have been reported to be effective against CA19-9. MBL It may also be used as a binding molecule for use in the detection of

[0088] Similarly, CA19-9-containing entities (CA19-9) containing glycan structures capable of specifically binding to WGA WGA The assay of the level of CA19-9 can be carried out by using an anti-CA19-9 antibody and WGA. However, the expression "capable of specifically binding to WGA" does not limit the detection to the use of WGA. In other words, the level of CA19-9 can be measured by using an anti-CA19-9 antibody and WGA. WGA CA19-9 can be detected or assayed not only by techniques using WGA, but also by techniques using other lectins with binding specificities similar to or overlapping with WGA. The specificity of each lectin and its suitability for this purpose can be easily determined by those skilled in the art. In addition, anti-glycan antibodies specific for glycan structures that can be specifically recognized by WGA have been reported. WGA It may also be used as a binding molecule for use in the detection of

[0089] Similarly, CA19-9-containing entities (CA19-9) containing glycan structures capable of specifically binding to MGL MGL The assay of the level of CA19-9 can be carried out by using an anti-CA19-9 antibody and MGL. However, the expression "capable of specifically binding to MGL" is not limited to the use of MGL for detection. In other words, MGL CA19-9 can be detected or assayed not only by techniques using MGL, but also by techniques using other lectins with binding specificities similar to or overlapping with MGL. The specificity of each lectin and its suitability for this purpose can be easily determined by those skilled in the art. In addition, anti-glycan antibodies specific for glycan structures that can be specifically recognized by MGL can also be used for CA19-9. MGL It may also be used as a binding molecule for use in the detection of

[0090] The level of CA19-9-containing entities containing glycan structures capable of specifically binding to a specific lectin can be assayed, for example, by using a sandwich assay. In this sandwich assay, a CA19-9-binding molecule, such as an anti-CA19-9 antibody, preferably a monoclonal antibody, is used as a capture agent, and a glycan conjugate with the desired binding specificity (e.g., a lectin or a corresponding anti-glycan antibody) is used as a tracer. When used as a tracer, the glycan conjugate may be attached with a directly or indirectly detectable label.

[0091] In some other embodiments, the sandwich assay may be performed in the reverse manner. In such cases, a glycan binder, such as a lectin or a corresponding anti-glycan antibody, is used as a capture agent, and a CA19-9-specific binding molecule, such as an antibody, preferably a monoclonal antibody, is used as a tracer, which is directly or indirectly labeled with a detectable label. Because urine contains fewer interfering glycosylated molecules than blood, it is expected that the reverse sandwich assay will work better with urine samples than with blood samples.

[0092] Of note, sandwich assays may be used regardless of whether the glycan structures being assayed are on glycoconjugates with soluble CA19-9 or on vesicles displaying CA19-9.

[0093] In some embodiments, the sandwich assay may include one or more washing steps after the capture step to remove all non-target and unbound molecular entities that are not specific to the capture agent. Suitable washing solutions and conditions (e.g., time and temperature) are known to those skilled in the art. After the capture step and, optionally, one or more washing steps, the captured molecular entities are subjected to a detection step using an appropriate tracer.

[0094] Sandwich assays according to various embodiments of the present invention may be performed on a solid surface, such as a microtiter plate, or in a lateral flow format. Means and methods for attaching capture agents to solid surfaces (e.g., via streptavidin-biotin complexes) and incorporating capture agents into lateral flow assays are known in the art and readily apparent to those skilled in the art.

[0095] Suitable substrates for use in this solid-phase sandwich assay include, but are not limited to, glass, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, gold, various clays, nitrocellulose, or nylon. As noted above, in some embodiments, the substrate may be coated with a suitable compound, such as streptavidin, to facilitate binding of the capture agent (i.e., either the CA19-9 conjugate or the glycan conjugate) to the substrate. In yet other embodiments, one or more control conjugates, such as a control antibody or control lectin, may be attached to the substrate.

[0096] In some embodiments, the solid-phase sandwich assay may be provided in a spot format. That is, a CA19-9 capture agent, preferably an antibody, more preferably a monoclonal antibody or its antigen-binding fragment or single-chain variant, is immobilized as a spot on a multiwell plate (preferably a microtiter plate) rather than immobilized over the entire surface of the multiwell plate (preferably a microtiter plate) as in conventional full-well assays. Typically, the diameter of the spot area is 1-5 mm, 1-3 mm, or 2.5-3 mm. The density of the capture agent may vary depending on the capture agent used, but the concentration of the immobilized capture agent is typically in the range of 25-150 μg / mL, preferably 50-125 μg / mL, or 75-100 μg / mL.

[0097] In some embodiments, the solid phase sandwich assay may be provided in a conventional whole-well assay format.

[0098] Anti-CA19-9 antibodies are commercially available from multiple sources and can be used as either capture agents or tracers in the above-mentioned sandwich assay.In addition, CA19-9-specific monoclonal antibodies can be produced by methods well known in the art.For use as a tracer, anti-CA19-9 antibodies can be labeled with suitable labels known in the art, including but not limited to fluorescent labels, bioluminescent labels, chemiluminescent labels, and enzyme labels such as alkaline phosphatase.Depending on the type of detectable label used, suitable detection techniques can be easily selected in the art.

[0099] Lectins are commercially available from several sources. Anti-glycan antibodies with the desired specificity for carbohydrate antigens are also commercially available from several sources or can be produced by methods known in the art. For use as tracers, glycan conjugates, such as lectins and corresponding anti-glycan antibodies, can be labeled with a detectable label using methods well known in the art. In some embodiments, the glycan conjugates used can be directly labeled with any detectable label using standard techniques. In other embodiments, the glycan conjugates used can be indirectly labeled with a detectable label, for example, by immobilization to a detectable particle such as a nanoparticle. Furthermore, in embodiments using multiple different types of glycan conjugates, the molecules can be directly or indirectly labeled with the same or different labels. In some embodiments, multiplexing can be achieved by using different labeled nanoparticles carrying different glycan conjugate species in a single assay.

[0100] As used herein, the term "nanoparticle (NP)" refers to a synthetic or natural particle having one or more dimensions, e.g., a diameter of less than about 1000 nm, e.g., about 500 nm or less, about 100 nm or less, or about 50 nm or less. As used herein, the term "about" refers to a range of ±10% of the specified value. For example, the expression "about 100 nm" includes ±10% of 100 nm, i.e., a range of 90 nm to 110 nm. Nanoparticles may generally have a spherical shape, although non-spherical shapes, such as ellipsoidal shapes, are also possible. In some embodiments, all dimensions of the nanoparticle are less than about 1000 nm, about 500 nm or less, about 100 nm or less, or about 50 nm or less.

[0101] Various materials can be used for the nanoparticles of the present invention. Non-limiting examples of suitable polymeric materials include polyethylene glycol (PEG), polystyrene, polyethylene, polyacrylic acid, polymethyl methacrylate (PMMA), polysaccharides, and copolymers or combinations thereof. Other suitable nanoparticle materials include, but are not limited to, colloidal gold, silver, quantum dots, carbon, porous silicon, and liposomes. In addition, suitable nanoparticle materials include protein nanoparticles, inorganic nanoparticles, glass nanoparticles, nanocrystals, metal nanoparticles, and plastic nanoparticles.

[0102] Nanoparticles that may be used in various embodiments of the present invention may be directly or indirectly detectable, qualitatively or quantitatively, by any known means. For example, nanoparticles may be detectable by their intrinsic properties, such as upconverting (UCP) nanoparticles, resonance particles, quantum dots, gold particles, etc. UCP particles are particularly suitable for use as tracers in lateral flow formats. In other embodiments, nanoparticles may be made detectable by fluorescent, bioluminescent, chemiluminescent, or other labeling. In still other embodiments, labeling or addition with lanthanides, i.e., luminescent lanthanide ions that emit light in the visible, near-infrared, or infrared wavelength ranges and exhibit long fluorescence decays (e.g., europium(III), terbium(III), samarium(III), dysprosium(III), ytterbium(III), erbium(III), and neodymium(III), is a preferred means for making the nanoparticles of the present invention detectable.

[0103] Glycan conjugates, such as lectins or anti-glycan antibodies, can be immobilized on nanoparticles by any suitable method known in the art, including, but not limited to, the methods disclosed in the Examples herein. In embodiments including multiple different glycan conjugates (e.g., multiple different lectins and / or multiple different anti-glycan antibodies), these different glycan conjugates may be immobilized on the same or different nanoparticles in any ratio. Notably, these different glycan conjugates may be of different types (e.g., lectins, anti-glycan antibodies, antibody mimetics, or aptamers), may have different glycan specificities, or both.

[0104] In some non-limiting embodiments, the most preferred nanoparticles are polystyrene nanoparticles having a diameter of about 97 nm or about 107 nm. These nanoparticles are commercially available from at least Thermo Scientific Seradyn Inc. Further preferred nanoparticles include nanoparticles loaded with europium chelates.

[0105] Advantages associated with the use of nanoparticles include: i) signal amplification due to the inclusion of multiple chelates per particle; ii) enhanced functional affinity (avidity) of the lectin or anti-glycan antibody for the target glycan structure epitope, achieved by high density immobilization of the lectin or anti-glycan antibody on the particle; and iii) glycan structure specificity of the lectin or anti-glycan antibody used, made possible by the creation of multivalent nanoparticles. However, nanoparticles are merely one preferred means of providing sufficient avidity and signal amplification for practicing the present invention or disclosure and its various embodiments.

[0106] The detectable signal can be generated using any available sensor technology. For example, a solid surface may incorporate a recognition element (transducer) that converts the binding reaction into a detectable signal, with or without the use of a labeling moiety. Various types of transducers can be used, including those based on electrochemical or optical detection. Detection can be based on either homogeneous or heterogeneous detection techniques, as will be apparent to those skilled in the art.

[0107] Furthermore, assays of samples for CA19-9-containing entities containing glycan structures capable of specific binding to specific lectins can be performed by methods and techniques such as nuclear magnetic resonance (NMR), electrophoresis, chromatography, mass spectrometry, or suitable combinations thereof. Suitable NMR methods include, for example, correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame Overhauser enhanced spectroscopy (ROESY), all of which can be used in 1D or 2D. Suitable electrophoretic methods include, for example, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), capillary gel electrophoresis (CGE), and capillary zone electrophoresis (CZE). Non-limiting examples of mass spectrometry include fast atom bombardment mass spectrometry (FAB-MS), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), liquid chromatography mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS / MS), and matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS).

[0108] A biological sample obtained from a subject suspected of having prostate cancer was analyzed using fPSA. MGLIn clinical methods of the present invention that involve assaying for CA19-9, the assay can be performed in a manner substantially similar to the method described above for assaying glycovariants of CA19-9-containing entities. As will be apparent to those skilled in the art, instead of a CA19-9-binding agent, an fPSA-binding agent, such as an anti-fPSA antibody, preferably a monoclonal anti-fPSA antibody, more preferably an antigen-binding fragment thereof, more preferably a site-specifically biotinylated antigen-binding fragment thereof, is used in a binding-based assay format. The glycan-binding agent is preferably MGL or another lectin or anti-glycan antibody capable of recognizing the same glycan structure as MGL. Either the fPSA-binding agent or the glycan-binding agent is immobilized on a solid surface, and the other is directly or indirectly labeled with a detectable label.

[0109] However, particularly when the biological sample is a blood sample, such as plasma (e.g., EDTA plasma) or serum, fPSA MGL A spot assay format should be applied to assay for fPSA antibodies. In such embodiments, the capture agent is an antigen-binding fragment of an anti-fPSA antibody, such as a Fab, F(ab')2, or F(ab)2 fragment, which is preferably immobilized as a high-density spot in a small area of ​​a multiwell plate, such as a microtiter well. In some embodiments, the diameter of the spotted area is 1-5 mm, 1-3 mm, or 2.5-3 mm, and / or the concentration of the immobilized capture agent is 25-150 μg / mL, preferably 50-125 μg / mL, or 75-100 μg / mL. In some embodiments, the capture agent is preferably a site-specifically biotinylated antigen-binding fragment of an anti-fPSA antibody, which allows for orderly immobilization in the desired orientation determined by the selected biotinylation site. Means and methods for site-specific biotinylation are readily available to those skilled in the art.

[0110] Anti-fPSA antibodies and antigen-binding fragments and single-chain variants thereof are available from several sources, and further, such molecules can be produced by methods well known to those skilled in the art.

[0111] Additionally, fPSA was performed on the samples. MGL Assaying can be performed by nuclear magnetic resonance (NMR), electrophoresis, chromatography, and mass spectrometry, or a suitable combination thereof.

[0112] kit The present disclosure also provides kits for use in the clinical method and various embodiments thereof. The kits include reagents for assaying CA19-9-containing entities capable of specifically binding to at least one lectin selected from MBL, WGA, and MGL in a biological sample. In some preferred embodiments, the kits include reagents for assaying CA19-9-containing entities capable of specifically binding to MBL, and optionally reagents for assaying CA19-9-containing entities capable of specifically binding to either WGA or MGL. In yet other embodiments, the kits may include reagents for assaying three different glycovariant CA19-9-containing entities, each capable of specifically binding to a different lectin selected from MBL, WGA, and MGL.

[0113] For each glycovariant of a CA19-9-containing entity to be assayed, at least one reagent is a CA19-9-binding agent specific for CA19-9 (e.g., a monoclonal anti-CA19-9 antibody), and at least one other reagent is a glycan-binding substance (e.g., a lectin or anti-glycan antibody) specific for the glycan structure of interest (i.e., a glycan structure capable of specifically binding to either MBL, WGA, or MGL). The same or different CA19-9-binding agents may be used for each glycovariant. Preferably, either the CA19-9-binding agent or the glycan-binding substance is immobilized on a solid surface, such as a microtiter plate. In yet another embodiment, streptavidin coating on the solid surface and biotinylation of the binding molecule of interest may be used for immobilization. Other means for achieving the same purpose are readily available to those skilled in the art. When the CA19-9-binding agent is immobilized on a solid surface to be used as a capture agent, the glycan-binding substance is labeled with a directly or indirectly detectable label to be used as a tracer. In some embodiments, the glycan conjugate may be indirectly labeled via the detectable nanoparticle to which it is immobilized. In other embodiments, the CA19-9 binder is used as a tracer, while the glycan conjugate is used as a capture agent.

[0114] To assay CA19-9-containing entities containing glycan structures capable of specific binding to MBL, MBL may be used as a glycan binder. Alternatively, an anti-glycan antibody specific for the same glycan structure may be used. It is also contemplated that other lectins may be used, as long as they can specifically recognize the same glycan structure in the same way as MBL.

[0115] Similarly, WGA can be used as a glycan binder to assay CA19-9-containing entities that contain glycan structures that can specifically bind to WGA. Alternatively, an anti-glycan antibody specific for the same glycan structure can be used. It is also contemplated that other lectins can be used, as long as they can specifically recognize the same glycan structure in the same way as WGA.

[0116] Similarly, MGL can be used as a glycan binder to assay CA19-9-containing entities containing glycan structures capable of specifically binding to MGL. Alternatively, an anti-glycan antibody specific for the same glycan structure can be used. It is also contemplated that other lectins can be used as long as they can specifically recognize the same glycan structure in the same way as MGL.

[0117] Optionally, the kit may also include a control for comparison with the assayed level of the glycovariant of the CA19-9-containing entity of interest. In some embodiments, the control is a threshold value for comparison with the assayed level.

[0118] In some embodiments, the kit may further include reagents for assaying the sample for one or more known prostate biomarkers, such as one or more kallikrein biomarkers, including total PSA, free PSA, intact PSA, total hK2, and free hK2. Reagents suitable for such assays are known to those skilled in the art and are readily available.

[0119] In some embodiments, the kit comprises fPSA MGLAlternatively, or in addition, the sample may include reagents for assaying the sample for MGL. Preferably, such reagents include an fPSA-specific fPSA binder, such as an anti-fPSA antibody, preferably a monoclonal anti-fPSA antibody, more preferably an antigen-binding fragment thereof (e.g., a Fab, F(ab')2, or F(ab)2 fragment), even more preferably a site-specifically biotinylated antigen-binding fragment (e.g., a Fab, F(ab')2, or F(ab)2 fragment), and a glycan binder (e.g., a lectin or an anti-glycan antibody) specific for a glycan structure contained in free PSA and capable of specifically binding to MGL. In some preferred embodiments, the glycan binder is MGL. In some embodiments, particularly those involving urine samples or tissue lysates, either the fPSA binder or the glycan binder is used as a capture agent and is immobilized on a solid surface, and the other is used as a tracer and is labeled with a directly or indirectly detectable label. In another preferred embodiment, particularly for the analysis of blood samples, the capture agent is an antigen-binding fragment (e.g., Fab, F(ab')2, or F(ab)2 fragment) of an anti-fPSA antibody, preferably a site-specifically biotinylated antigen-binding fragment (e.g., Fab, F(ab')2, or F(ab)2 fragment), immobilized on a solid surface in an area having a diameter smaller than that of the solid support. Meanwhile, the tracer is a glycan binder (e.g., an antibody or lectin) specific for the glycan structure contained in free PSA and capable of specifically binding to MGL, preferably MGL, more preferably MGL-nanoparticles labeled with a directly or indirectly detectable label. Thus, a kit is provided, comprising: i) a multiwell plate (preferably a microtiter plate) on which the above-mentioned capture agent is immobilized, and ii) the tracer agent.

[0120] In some embodiments, the kit includes a multi-well plate, such as a microtiter plate, with one or more wells immobilized with a CA19-9 binder and one or more different wells immobilized with an fPSA binder. The kit further includes at least two glycan conjugates labeled with directly or indirectly detectable labels, each specific for a glycan structure capable of specifically binding to MBL and a glycan structure capable of specifically binding to MGL. Optionally, additional glycan conjugates may be included in the kit, according to the above disclosure. In some embodiments, the glycan conjugates may be provided immobilized on nanoparticles.

[0121] In some embodiments, the kit includes a multiwell plate, such as a microtiter plate, with one or more wells having at least two distinct regions or spots on the bottom surface, one of which has a CA19-9 binder immobilized thereon and the other of which has an fPSA binder immobilized thereon, as described above. The kit also includes at least two glycan conjugates labeled with directly or indirectly detectable labels, one specific for a glycan structure capable of specifically binding to MBL and the other specific for a glycan structure capable of specifically binding to MGL, respectively. In accordance with the above disclosure, the kit may optionally include additional glycan conjugates. In some embodiments, the glycan conjugates may be provided immobilized on the same or different nanoparticles.

[0122] In yet other embodiments, the kit may include instructions for practicing any of the methods of the present disclosure. In yet other embodiments, the kit may include a computer-readable medium containing computer-executable instructions for practicing any of the methods of the present disclosure.

[0123] Various details and embodiments of the clinical methods herein also apply to the kits, as will be readily understood by those skilled in the art, and thus, for example, the properties and characteristics of suitable nanoparticles will not be repeated herein with respect to the kits.

[0124] Also provided is the use of biomarkers and kits disclosed herein for determining the disease state of prostate cancer in subjects.Therefore, provided is the use of a binding molecule selected from the group consisting of a binding molecule specific for the glycan structure that can be specifically bound to MBL, a binding molecule specific for the glycan structure that can be specifically bound to WGA, and a binding molecule specific for the glycan structure that can be specifically bound to MGL for determining the presence or absence of prostate cancer-related CA19-9-containing entities in samples.Also provided is the use of a binding molecule specific for MBL or a glycan structure that can be specifically bound to MBL for diagnosing and determining the disease state of prostate cancer in subjects.All details and special mentions disclosed for the present method and kit and their various embodiments apply to the various uses of the biomarkers and kits of the present invention, even if not repeated here.

[0125] Non-Exhaustive List of Numbered Embodiments Some embodiments of the present invention are numbered as follows: [1] Glycan structures contained in carbohydrate antigen 19-9 (CA19-9)-containing molecular entities and capable of specifically binding to mannose-binding lectin (MBL) as biomarkers for prostate cancer. [2] Glycan structures contained in carbohydrate antigen 19-9 (CA19-9)-containing molecular entities and capable of specifically binding to wheat germ agglutinin (WGA) as biomarkers for prostate cancer. [3] Glycan structures contained in carbohydrate antigen 19-9 (CA19-9)-containing molecular entities and capable of specifically binding to macrophage galactose-type lectin (MGL) as biomarkers for prostate cancer. [4] 1. A method for determining prostate cancer disease status in a subject, comprising: a) assaying a sample obtained from said subject for the level of a CA19-9-containing entity comprising a glycan structure capable of specifically binding to MBL, WGA or MGL; b) comparing the assay level obtained in step a) with a control sample or a predetermined threshold value; and c) determining the disease state of prostate cancer based on said comparison. A method comprising: [5] 5. The method of embodiment 4, wherein the sample is assayed for the level of at least two different CA19-9-containing entities comprising glycan structures capable of specific binding to different lectins selected from the group consisting of MBL, WGA, and MGL. [6] The method of embodiment 3 or 4, wherein an increased level of the CA19-9-containing entity in the sample compared to the level of the CA19-9-containing entity in a control sample or a predetermined threshold indicates that the subject has prostate cancer or is at risk of having or developing prostate cancer. [7] 7. The method of any one of embodiments 2 to 6, wherein the assay is carried out by using a binding molecule specific for the glycan structure, or by using mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, electrophoresis, chromatography, or a combination thereof. [8] 8. The method of embodiment 7, wherein the binding molecule is, depending on the biomarker, MBL, WGA, MGL, or an antibody specific for a glycan structure capable of specifically binding to MBL, WGA, or MGL. [9] The sample was subjected to the following procedures: total prostate-specific antigen (PSA), free PSA, free PSA capable of specifically binding to MGL (fPSA MGL9. The method of any one of embodiments 2-8, further comprising assaying for one or more biomarkers selected from the group consisting of: intact PSA, total human kallikrein 2 (hK2), and free hK2.

[10] A kit for use in determining the disease status of prostate cancer in a subject, comprising a CA19-9 binding agent and at least one binding molecule specific for a glycan structure capable of specifically binding to MBL, WGA, or MGL, wherein either the CA19-9 binding agent or the binding molecule comprises a detectable label.

[11] 11. The kit of embodiment 10, wherein the binding molecule is selected from the group consisting of MBL, WGA, or MGL, or an antibody specific for a glycan structure capable of specifically binding to MBL, WGA, or MGL.

[12] Total prostate-specific antigen (PSA), free PSA, free PSA that can specifically bind to MGL (fPSA MGL 12. The kit of embodiment 10 or 11, further comprising reagents for assaying the sample for one or more biomarkers selected from the group consisting of: intact PSA, total human kallikrein 2 (hK2), and free hK2.

[13] Use of the kit according to any one of embodiments 10 to 12 for determining the disease state of prostate cancer in a subject.

[14] Use of a binding molecule selected from the group consisting of a binding molecule specific for a glycan structure capable of specifically binding to MBL, a binding molecule specific for a glycan structure capable of specifically binding to WGA, and a binding molecule specific for a glycan structure capable of specifically binding to MGL, for determining the presence or absence of a prostate cancer-associated CA19-9-containing entity in a sample.

[15] Use of a binding molecule specific for MBL, or a glycan structure capable of specifically binding to MBL, for determining the disease state of prostate cancer in a subject.

[16] CA19-9 negativity as a biomarker for aggressive prostate cancer.

[17] 1. A method for determining prostate cancer disease status in a subject, comprising: a) assaying a blood sample obtained from the subject for the level of free PSA (fPSA) capable of specifically binding to MGL using a spot assay, in which an fPSA binder immobilized on a solid surface in an area having a diameter smaller than the diameter of a solid support is used to capture fPSA present in the sample, and a detectably labeled conjugate capable of specifically binding to MGL is used to detect MGL-binding glycovariants of the captured fPSA; b) comparing the assay level obtained in step a) with a control sample or a predetermined threshold value; and c) determining the disease state of prostate cancer based on said comparison. A method comprising:

[18] The method according to embodiment 17, wherein the level of free PSA that can specifically bind to MGL in the sample is increased compared with the level of free PSA that can specifically bind to MGL in a control sample or a predetermined threshold, indicates that the subject has prostate cancer or is at risk of having or developing prostate cancer.

[19] The method of embodiment 17 or 18, wherein the fPSA binding agent is an anti-fPSA antibody, preferably an antigen-binding fragment thereof, more preferably a Fab fragment, F(ab)2 fragment, or F(ab')2 fragment, and even more preferably a site-specifically biotinylated Fab fragment, F(ab)2 fragment, or F(ab')2 fragment.

[20] The method of any one of embodiments 17 to 19, further comprising assaying the sample for one or more biomarkers selected from the group consisting of total prostate-specific antigen (PSA), free PSA, intact PSA, total human kallikrein 2 (hK2) and free hK2. [twenty one] 21. The method of any one of embodiments 17 to 20, further comprising assaying the sample for the level of at least one CA19-9-containing entity comprising a glycan structure capable of specifically binding to a lectin selected from the group consisting of MBL, WGA, and MGL. [twenty two] i) a multiwell plate comprising one or more wells having a region on the bottom surface thereof where an fPSA binder is immobilized, said region having a diameter smaller than that of the bottom surface; and ii) A binder specific for a glycan structure capable of specifically binding to MGL 10. A kit for use in determining the disease state of prostate cancer in a subject, comprising: [twenty three] 23. The kit of embodiment 22, wherein the fPSA binding agent is an anti-fPSA antibody, preferably an antigen-binding fragment thereof, more preferably a Fab fragment, F(ab)2 fragment, or F(ab')2 fragment, and even more preferably a site-specifically biotinylated Fab fragment, F(ab)2 fragment, or F(ab')2 fragment. [twenty four] 24. The kit according to embodiment 22 or 23, wherein the binder is an antibody specific for MGL or a glycan structure capable of specifically binding to MGL. [twenty five] 25. The kit according to any one of embodiments 22 to 24, wherein the conjugate is labeled with a detectable label.

[26] 26. The kit of any one of embodiments 22 to 25, wherein the conjugate is immobilized on nanoparticles.

[27] The samples were analyzed using a 100-well platelet count (1000µL) of total prostate-specific antigen (PSA), free PSA (fPSA), and free PSA capable of specifically binding to MGL (fPSA MGL 27. The kit of any one of embodiments 22-26, further comprising reagents for assaying for one or more biomarkers selected from the group consisting of: intact PSA, total human kallikrein 2 (hK2), and free hK2.

[28] A kit according to any one of embodiments 22 to 27, wherein the multiwell plate is further immobilized with a CA19-9 binding agent in one or more wells different from the fPSA binding agent, or in an area of ​​the bottom of one or more different wells, each area having a diameter smaller than the diameter of the well and distinct from the area in which the fPSA binding agent is immobilized, and further comprises a binder specific for a glycan structure capable of specifically binding to MBL.

[29] i) a multi-well plate comprising one or more wells having at least two distinct regions on the bottom surface, one region having a CA19-9 binding agent immobilized thereon and one region having a fPSA binding agent immobilized thereon; and ii) at least two conjugates, one of which is specific for a glycan structure capable of specifically binding to MBL and the other of which is capable of specifically binding to MGL; 29. The kit according to any one of embodiments 22 to 28, comprising: [Example]

[0126] Example 1. CA19-9 Glycovariant Assay Anti-CA19-9 antibody The anti-CA19-9 antibody c192 was obtained from Fujirebio Diagnostics (Gothenburg, Sweden). To obtain c192 F(ab')2 fragments, monoclonal antibody (mAb) c192 was digested with ID-Dilution 1 (50 μL per mg of mAb) in reaction buffer (50 mM Tris-HCl, pH 7.0, 1 mM NaCl, 3 mM EDTA) at +37°C for 2 hours. The digestion was stopped by adding 0.2 M N-ethylmaleimide (NEM) to a final concentration of 0.02 M. The c192 F(ab')2 antibody fragments were purified using protein G affinity purification.

[0127] For use as a solid-phase capture agent, c192 F(ab')2 fragments were biotinylated using standard techniques known in the art with a 40-fold molar excess of biotin isothiocyanate for 4 hours at room temperature (RT). Biotinylated antibodies were purified on NAP-5 and NAP-10 gel filtration columns (GE Healthcare, Schenectady, NY, USA) using 50 mmol / L Tris-HCl (pH 7.75) containing 150 mmol / L NaCl and 0.5 g / L NaN3. Labeled antibodies were stabilized with 1 g / L BSA (Bioreba, Nyon, Switzerland) and stored at +4°C.

[0128] Lectins The lectin wheat germ agglutinin (WGA) was purchased from Vector Laboratories (UK). Mannose-binding lectin (MBL) and macrophage galactose-type lectin (MGL) were purchased from R&D Systems (Minneapolis, MN, USA).

[0129] The lectin was immobilized on monodisperse, carboxylated, modified Fluoro-Max™ polystyrene nanoparticles (97 nm diameter) loaded with europium chelate, obtained from Thermo Scientific Seradyn Inc. (Indianapolis, IN). The nanoparticles used emit long-lived fluorescence equivalent to 30,000 chelated ions per particle.

[0130] The primary amino groups of the lectin were covalently coupled to the activated carboxyl groups of the nanoparticles using a procedure previously described (Soukka et al., Anal. Chem. 2001, 73, 2254-2260) with slight modifications. 12 The lectin particles were suspended in 10 mmol / L phosphate buffer (pH 7.0) and surface-activated with 0.75 mmol / L N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (Sigma-Aldrich, St. Louis, MO, USA) and 10 mmol / L N-hydroxysulfosuccinimide sodium salt (Sigma-Aldrich). The WGA concentration was 0.4 mg / mL, and the MGL and MBL concentrations were 0.25 mg / mL. The reaction contained 100 mmol / L NaCl. The activated particles and lectin were mixed. The coupling reaction was incubated at 23 °C for 2 hours with vigorous agitation. Final washing and blocking of remaining active groups were performed in a Tris-based buffer (10 mmol / L Tris, 0.5 g / L NaN3, pH 8.5), and the nanoparticle-lectin conjugates were stored at 4 °C in the same buffer supplemented with 2 g / L BSA. Before first use, the particles were thoroughly mixed, sonicated, and briefly centrifuged (350 g, 5 min) to separate non-colloidal aggregates from the monodisperse suspension.

[0131] The major glycan-binding specificities of the lectins used are shown in Table 1 below. [Table 1]

[0132] Other materials, reagents, and equipment used Streptavidin-coated 96-well microtiter plates (SA plates, product number 41-07TY), assay wash buffer (product number 42-01TY), and RED assay buffer (product number 42-02TY) were obtained from Kaivogen Oy (Turku, Finland). A plate washer (Delfia PlateWash 1296-026) and plate shaker (Delfia PlateWash 1296-026) were obtained from Wallac Oy (Turku, Finland). Time-resolved fluorescence signals from europium nanoparticles were measured using a Hidex Microplate Reader (Hidex, Finland).

[0133] Bromelain solution ID-Diluent 1 was obtained from DiaMed (Créssier FR, Switzerland). NAP-5 and NAP-10 buffer exchange columns and phosphate-buffered saline were purchased from GE Healthcare (Chicago, IL, USA).

[0134] CA19-9 glycovariant assay All incubations were carried out at room temperature with gentle agitation. All assays were performed in yellow streptavidin-coated 96-well microtiter plates.

[0135] CA19-9 MBL CA19-9 standards for the glycovariant assay were prepared using equal amounts of CA19-9 antigen from Fujirebio and Meridian Bioscience, diluted to concentrations of 5, 10, 50, and 100 U / mL in TSA-BSA buffer (50 mM Tris-HCl, pH 7.75, 150 mM NaCl, 0.05% NaN3, 0.5% BSA).

[0136] CA19-9 WGA and CA19-9 MGLThe standards used in the glycovariant assay were Meridian CA19-9 antigen at 10, 50, 100, and 250 U / mL and Fujirebio CA19-9 antigen at 30, 100, 300, and 600 U / mL in TSA-BSA buffer (50 mM Tris-HCl, pH 7.75, 150 mM NaCl, 0.05% NaN3, 0.5% BSA).

[0137] RED assay buffer (Kaivogen Oy, Turku, Finland) in the incubation steps of standards and samples was used. MBL For glycovariant assays, additional 50 mM CaCl2 and 100 mM NaCl were added, and for CA19-9 WGA For glycovariant assays, 80 mM CaCl2 was used. MGL The glycovariant assay contained 50 mM CaCl2 and 150 mM NaCl.

[0138] To immobilize the capture agent in the whole-well assay format, the SA plate was washed once and CA19-9 MBL , CA19-9 WGA , and CA19-9 MGL For the glycovariant assay, 35 ng, 40 ng, or 50 ng of biotinylated anti-CA19-9 antibody, bio-c192F(ab')2, was added to 40 μL of RED assay buffer. After 1 hour of incubation, each well was washed twice, and 35 μL, 38 μL, or 30 μL of assay buffer was added, followed by CA19-9. MBL , CA19-9 WGA , and CA19-9 MGLFor glycovariant assays, 5 μL, 2 μL, or 10 μL of clinical plasma sample was added. Calibration curves for each assay type were prepared in triplicate using 10 μL of each standard solution listed above and 30 μL of assay buffer. After 1 hour of incubation, each well was washed four times, and the glycan-binding tracer molecule, lectin nanoparticles, were added to CA19-9 in 40 μL of RED assay buffer supplemented with 10 mM CaCl2. MBL , CA19-9 WGA , and CA19-9 MGL 2.5 x 10 in each assay 7 , 2.8×10 7 , and 1.2 × 10 7 After 90 min of incubation, each well was washed six times and the time-resolved fluorescence of Eu was measured using a Hidex Microplate Reader.

[0139] CA19-9 MGL , CA19-9 WGA , and CA19-9 MBL The standard curves for the glycovariant assay are shown in Figures 1B-1D. The standard curve for the commercially available CA19-9 ELISA immunoassay (EIA) (Fujirebio), which represents a conventional CA19-9 immunoassay, is shown in Figure 1A.

[0140] Of note, concentrations calculated using the CA19-9 ELISA and the three CA19-9 glycovariants are not comparable to each other due to the unknown glycovariant composition of the calibration curve used.

[0141] Example 2. Analysis of clinical samples with the CA19-9 glycovariant assay and comparison with conventional CA19-9 immunoassays A cohort of clinical samples was analyzed in parallel using the disclosed CA19-9 glycovariant assay and a conventional CA19-9 immunoassay.

[0142] Clinical samples A study cohort of 249 consecutive men with clinically suspected prostate cancer (PCa) was used in the prospective, registered multi-IMPROD trial (ClinicalTrials.gov Identifier: NCT02241122). EDTA plasma samples were obtained from the University Hospitals of Turku, Helsinki, Tampere, and Pori with appropriate authorization and informed consent.

[0143] In the analysis, patients' Gleason scores (Gle) were used as "ground truth" and the cohort was divided into three groups: patients with non-cancerous benign conditions (n ​​= 99), patients with clinically insignificant prostate cancer, i.e., PCa patients with a Gleason score of 6 (n = 43), and patients with clinically significant cancer, i.e., PCa patients with a Gleason score of 7 or higher (n = 107). Table 2 summarizes the clinical characteristics of the patient groups.

[0144] Patients with benign PCa or a Gleason score of 6 ranged in age from 29 to 76 years, with a median age of 62 years, while patients with significant PCa ranged in age from 40 to 81 years, with a median age of 67 years.

[0145] [Table 2]

[0146] CA19-9 glycovariant assay Clinically derived EDTA plasma samples were analyzed for CA19-9 glycovariants according to the method described in Example 1. Glycovariant concentrations were determined using the calibration curves also described in Example 1 and shown in Figures 1B-1D.

[0147] Conventional CA19-9 immunoassay CA19-9 concentrations in clinically derived EDTA plasma samples were analyzed by ELISA using the CanAg CA19-9 EIA kit (Fujirebio Diagnostics) according to the manufacturer's instructions, utilizing the calibration curve shown in Figure 1A .

[0148] Boxplot analysis As shown in Figures 2A and 2B, the conventional CA19-9 ELISA immunoassay was unable to separate clinically significant cancers from benign and Gle6 cancers, nor could it separate individual cancers. Therefore, the conventional CA19-9 assay has no diagnostic value in the testing of PCa.

[0149] As shown in Figure 3A, CA19-9 MBL The assay was able to significantly separate the intermediate or high-grade cancer group from the benign and Gle6 cancer groups, whereas it was unable to separate the benign cases from the Gle6 cancer groups, as shown in Figure 3B. In conclusion, the CA19-9 of the present invention MBL The assay can be used to distinguish patients with clinically significant PCa from those with indolent disease.

[0150] As shown in Figure 4A, CA19-9 WGA The assay also significantly separated the intermediate or high-grade cancer group from the benign and Gle6 cancer groups, whereas it failed to separate the benign cases from the Gle6 cancer groups, as shown in Figure 4B. WGA The assay can be used to distinguish patients with clinically significant PCa from those with indolent disease.

[0151] Furthermore, as shown in Figure 5A, CA19-9 MGL The assay also significantly separated the group of intermediate or high-grade cancers from the group of benign and Gle6 cancers. Again, as shown in Figure 5B, it was not possible to separate the benign cases from the Gle6 cancers. In conclusion, the CA19-9 of the present invention MGLThe assay can be used to distinguish patients with clinically significant PCa from those with indolent disease.

[0152] ROC curve and AUC value To assess the detection ability of each variable for GS ≥ 7, the area under the receiver operating characteristic (ROC) curve (AUC) with the corresponding 95% confidence interval (CI) was calculated.

[0153] All CA19-9 glycovariants separated clinically significant cancers from the combined group of benign cases and Gleason grade 6 cancers, whereas the conventional reference CA19-9 immunoassay did not (AUC 0.495). Based on the AUCs shown in Table 3 below, CA19-9 MBL showed the best performance, followed by CA19-9 WGA and CA19-9 MGL In particular, CA19-9 MBL demonstrated excellent performance with a specificity of 80–90% (Fig. 6).

[0154] ROC curves were also constructed for different combinations of biomarkers, and AUC values ​​were calculated. The results showed that the CA19-9 glycovariants complemented each other's diagnostic performance. Furthermore, as described in Example 3 below, samples were analyzed using PSA MGL Further complementary effects were obtained by also assaying for AUC values ​​shown in Table 3 below.

[0155] [Table 3]

[0156] Example 3. Comparison of CA19-9 glycovariants with other PCa markers The performance of the CA19-9 glycovariant assay of the present invention was compared to that of other PCa markers in distinguishing between unmarked and marked cases.

[0157] Conventional kallikrein immunoassay A multikallikrein model consisting of tPSA (total PSA), fPSA (free PSA), iPSA (intact PSA), and hK2 (total hK2) was used as a reference. This multikallikrein model is included in the recently FDA-approved 4Kscore test (OPKO Health Inc.), which is designed to assess patients' likelihood of aggressive PCa. The concentrations of these markers were measured using an in-house immunoassay according to the method described by Vickers et al., Clin Cancer Res; 16(12), 2010.

[0158] Free PSA MGL Assay Purified PSA from the prostate cancer cell line LNCaP and the anti-PSA antibody 5A10 Fab were obtained from the Department of Biotechnology, University of Turku, Finland. Human MGL was obtained from R&D Systems.

[0159] LNCaP PSA was used as a calibrator, and dilutions (1–40 ng / mL LNCaP PSA) were prepared in Tris-saline-azide buffer (50 mM / L, 150 mmol / L NaCl, 0.5 g / L NaN, pH 7.75) containing 5 g / L BSA and run in triplicate.

[0160] Biotinylated 5A10 Fab (100 μg / mL) was printed in a spot assay format onto a yellow streptavidin-coated 96-well microtiter plate (Kaivogen Oy, Turku, Finland) using a Nano-Plotter non-contact microdispensing device (GeSiM, Germany) with the following settings: 70% humidity, 50 μs pulse, 90 V voltage, 250 μs delay, and 100 Hz frequency. The printing buffer contained phosphate-buffered saline (pH 7.4) with 10% (v / v) glycerol. The diameter of the printed spots was 2.5–3 mm.

[0161] Biotinylated 5A10 Fab (100 μg / ml) was spotted onto a streptavidin-coated microtiter plate, and fPSA was detected. MGL MGL nanoparticles were used as capture agents in glycovariant assays. After two washes, 15 μL of sample (EDTA plasma) or calibrator and 25 μL of RED assay buffer containing 5 μg / ml native mouse IgG, 5 μg / ml HBR-2, and 5 μg / ml MAK-33 were added in triplicate and incubated for 1 hour. MGL nanoparticles were diluted in RED assay buffer containing 6 mM CaCl. After four washes, the concentration of MGL nanoparticles added was 3 × 10 per well. 7 The plates were incubated for 90 min and washed six times, after which time-resolved fluorescence of Eu was measured.

[0162] Univariate analysis Each variable (CA19-9 MBL , CA19-9 WGA , CA19-9 MGL , CA19-9, tPSA, fPSA, iPSA, hK2 and fPSA MGL To evaluate the ability of CA19-9 glycovariant assays to detect Gle≥7, the area under the receiver operating characteristic (ROC) curve (AUC) with the corresponding 95% confidence interval (CI) was calculated using the DeLong method, as known to those skilled in the art. In univariate analysis, all three different CA19-9 glycovariant assays were able to distinguish Gle≥7 cancers from the combined group of benign and Gle6 cases with high statistical significance. In contrast, conventional CA19-9 could not separate the two groups. Of the four conventional prostate kallikreins (total PSA, free PSA, intact PSA, and hK2), total PSA separated the two groups with high statistical significance. A subform of free PSA, namely free PSA glycovariants (fPSA), was also able to distinguish Gle≥7 cancers from the combined group of benign and Gle6 cases with high statistical significance. MGL ) provided the best discrimination between the two groups (AUC 0.70), which was not possible using conventional free PSA (Table 4).

[0163] [Table 4]

[0164] Logistic Regression Analysis - Different Combinations Five logistic regression models (models a, b, c, d, and e) were fitted to analyze the combinations of variables. The analysis results are shown in Tables 5-9, along with odds ratios (ORs), 95% confidence intervals (CIs), and P values. The predictive performance of each model was evaluated by calculating the mean area under the curve (AUC) and standard deviation (SD) using holdout cross-validation with 10,000 replicates. The holdout cross-validation consisted of randomly splitting the data into 70% training and 30% testing groups.

[0165] [Table 5]

[0166] [Table 6] The three parameters that performed best in this model were total PSA, free PSA, and CA19-9. MBL and CA19-9 WGA In contrast, intact PSA and CA19-9 MGL contributed slightly to the model performance.

[0167] [Table 7] In this model, the three parameters that contributed most significantly were fPSA MGL , CA19-9 MBL , and CA19-9 WGA It was CA19-9 MGL did not contribute to the model.

[0168] [Table 8] In this model, all three CA19-9 glycovariants significantly contributed to the model, with CA19-9 MBL showed the highest contribution.

[0169] [Table 9] In this model, the three parameters that performed best were total PSA, free PSA, and free PSA. MGL In contrast, intact PSA and hK2 contributed slightly to model performance.

[0170] The various models presented here are examples of how biomarker combinations can be constructed, and the P-values ​​of each individual parameter specifically indicate its contribution to the model. The final choice of parameters in the model to use will depend on multiple factors, including the availability of biological samples, budgetary and logistical constraints for performing the tests, and importantly, the type of patient population included in the study, which may differ in terms of age, general health, and prostate-related clinical conditions.

[0171] Model evaluation was performed using 10,000 iterations of standard cross-validation (30% test data, 70% training data). The use of CA19-9 glycovariants resulted in significant improvement. CA19-9 glycovariants were compared with free PSA glycovariants (PSA MGL) and four prostate kallikreins significantly improved discrimination performance, reaching particularly high specificity (80-90%) (Figure 7, Table 10). A multikallikrein model consisting of total PSA, free PSA, intact PSA, and hK2 was used as a reference. Adding the three CA19-9 glycovariants to the multikallikrein model improved performance, increasing the AUC from 0.73 to 0.83. Each of the three CA19-9 glycovariants independently outperformed the multikallikrein model, with AUCs of 0.77 and 0.73, respectively. Furthermore, including all four glycovariants increased the AUC to 0.79, significantly improving the performance of free PSA. MGL Complementation was provided by glycovariants.

[0172] [Table 10]

[0173] Example 4. Gland volume dependence The dependence of CA19-9 glycovariants on prostate volume was analyzed. The median prostate volume in the entire cohort (n = 249) was 40 mL (range 14 to 129 mL). Table 11 shows the results of the ROC analysis of benign / Gle6 vs. Gle≥7 for the single parameters in the entire cohort (n = 249) and in subgroups with gland volumes below (n = 133) and above (n = 116) the median (40 mL).

[0174] CA19-9 MBL showed comparable performance in different glandular volume groups, whereas CA19-9 WGA performed best in groups with a prostate volume of 40 mL or less. In contrast, total PSA, free PSA, intact PSA, and hK2 performed best in groups with a gland volume below the median and performed poorly in groups with a larger gland volume. Unlike other prostate kallikreins, free PSA MGL The glycovariants showed no dependence on glandular volume and performed equally well across different glandular volume groups. MBLperformed better than all other parameters without showing any clear gland volume dependency.

[0175] [Table 11] Despite the relatively low total AUC (0.702 and 0.703), CA19-9 MBL and CA19-9 WGA Glycovariants performed better than total PSA (AUC 0.724) even in the low prostate volume group, as both markers showed improved sensitivity to high specificity (80–90%) compared with total PSA.

[0176] Example 5. CA19-9 negative cases A particularly interesting finding was that patients with CA19-9-negative cancer (8% of the cohort) showed signs of more aggressive disease (total and free PSA compared with the benign / Gle6 group). MGL (This suggests a more pronounced increase in Gleason grades.) The proportion of cancers with higher Gleason grades also increased in the CA19-9-negative group. The proportions of Gle8-10 cancers in the CA19-9-positive and -negative groups were 12% and 25%, respectively. The results are shown in Figures 8A-8D.

[0177] Based on these results, total and free PSA MGL It is thought that Lewis antigen-negative patients tend to constitute a more aggressive group, due to the higher concentrations of .

[0178] Therefore, CA19-9 negativity is suggested to be a marker for aggressive PCa.

[0179] Example 6. DC-SIGN assay (comparative example) Human DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3 complementary non-integrin) was purchased from R&D Systems. Anti-CA19-9 monoclonal antibody M66106M and CA19-9 antigen were purchased from Meridian Bioscience.

[0180] CA19-9 DC-SIGN As a standard in the glycovariant assay, CA19-9 antigen from Meridian Bioscience was used, diluted to concentrations of 10, 50, 200, and 1000 U / mL in TSA-BSA buffer (50 mM Tris-HCl, pH 7.75, 150 mM NaCl, 0.05% NaN3, 0.5% BSA).

[0181] The RED assay buffer (Kaivogen Oy, Turku, Finland) used in the incubation steps of standards and samples additionally contained 50 mM CaCl2 and 90 mM NaCl.

[0182] SA plates were washed once and 60 ng of biotinylated monoclonal anti-CA19-9 antibody, bio-M66106M, was added in 40 μL of RED assay buffer. After 1 hour of incubation, wells were washed twice and 30 μL of assay buffer was added, followed by 10 μL of either clinically derived EDTA plasma sample or standard dilution. After 1 hour of incubation, wells were washed four times and 3.0 × 10 glycan-binding reporter molecule, DC-SIGN nanoparticles (prepared essentially as described in Example 1 for other lectins), was added in 40 μL of RED assay buffer supplemented with 10 mM CaCl. 7 After 90 minutes of incubation, wells were washed six times and Eu time-resolved fluorescence (TRF) was measured using Hidex. DC-SIGN The concentrations were determined using a calibration curve constructed based on the TRF signals measured in standard dilutions.

[0183] As a result, CA19-9 DC-SIGN Glycovariants did not discriminate between clinically significant PCa and indolent disease (benign and Gle6), as well as between benign and various cancer groups (Figure 9).

[0184] Example 7. Free PSA in spot and whole-well assay formats MGL Assay Comparison Free PSA in a spot assay format MGL The assay was performed according to the method described in Example 3. In the conventional whole-well glycovariant assay format, 75 ng of biotinylated 5A10 Fab was added to each well in 40 μL of RED assay buffer and incubated for 1 hour with gentle agitation. The wells were washed twice to remove unbound capture molecules, and the subsequent procedure was carried out with the addition of calibrators according to the array-in-well format protocol described in Example 3.

[0185] Both fPSAs MGL In the assay format, LNCaP PSA was used as a standard. As shown in Figure 10A, the signal from 3 mm spots coated with 5A10 Fab (spot assay format) was significantly higher than that of free PSA. MGL This was approximately 8-10 times higher compared to the signal from a conventional whole-well assay format in which glycovariants were spread over a 15-fold larger area.

[0186] Free PSA in a spot assay format MGL The glycovariant assay was able to distinguish between semen PSA from healthy donors and PSA from the prostate cancer cell line LNCaP. As expected, the assay detected only cancerous PSA (Figure 10B). In contrast, the conventional free PSA immunoassay was unable to distinguish between noncancerous semen-derived PSA standards and cancerous LNCaP-derived PSA (Figure 10C).

[0187] Example 8. Free PSA MGLComparison of the assay with conventional free PSA immunoassays In addition to the comparative analysis described in Example 3, free PSA in an array-in-well format MGL Box plot analysis was performed for the assay and conventional free PSA immunoassay. MGL The glycovariant assay was able to statistically significantly distinguish clinically significant PCa (Gle ≥ 7) from benign controls and indolent disease (Gle 6). This discrimination ability was observed in both the Gle 7 patient group and the Gle 8-10 patient group (Figure 11B). In contrast, the conventional free PSA assay was unable to distinguish between clinically significant PCa patients and PCa with benign status or graded as Gle 6 (Figure 11A). In addition, as shown in Table 12 below, there was a significant difference in free PSA between benign controls and Gle 6 cancers. MGL No significant separation was observed for glycovariants, tPSA, and fPSA. In contrast, iPSA and the hK2 kallikrein form were also able to distinguish between these two groups.

[0188] Free PSA MGL Although glycovariants are subforms of the total free PSA assay, their calibration using LNCaP PSA yielded apparently over-high concentrations that were very similar to the reference free PSA assay. This is clearly due to the high free PSA content in the LNCaP PSA formulation. MGL This is due to the lack of information on the relative abundance of glycovariants.

[0189] [Table 12]

[0190] Figure 12 shows the free PSA MGLThe ROC curves for the assay and the conventional free PSA immunoassay are shown, with the respective AUC values ​​and corresponding 95% confidence intervals (CI) shown in Table 4 of Example 3. The results show that fPSA MGL showed that it distinguished clinically significant cancers from the combined group of benign cases and Gleason grade 6 cancers with a mean AUC (95% CI) of 0.70 (0.63, 0.77), whereas conventional free PSA failed to discriminate with a mean AUC (95% CI) of 0.52 (0.45, 0.59).

[0191] Example 9. PSA AAL Assay (comparison) Purified PSA from the prostate cancer cell line LNCaP and anti-PSA antibody H50 Fab were obtained from the Department of Biotechnology, University of Turku, Finland. AAL (Aleuria aurantialectin) was purchased from Vector Laboratories (UK).

[0192] LNCaP PSA was used as a calibrator, and dilutions of LNCaP PSA from 10 to 200 ng / mL were prepared in Tris-saline-azide buffer (50 mM / L, 150 mmol / L NaCl, 0.5 g / L NaN3, pH 7.75) containing 5 g / L BSA and used in triplicate.

[0193] Biotinylated H50 Fab (100 μg / mL) was added to the free PSA in Example 3. MGL Similarly, the samples were printed in spot format onto a yellow streptavidin-coated 96-well microtiter plate (Kaivogen Oy, Turku, Finland) using a Nano-Plotter non-contact microdispensing device (GeSiM, Germany).

[0194] Biotinylated H50 Fab (100 μg / mL) spotted on streptavidin-coated microtiter plates was used to detect PSA. AALThe plates were used as capture agents in glycovariant assays. After two washes, 15 μL of calibrator or 5 μL of EDTA plasma sample and 25 μL or 35 μL of RED assay buffer containing 5 μg / mL native mouse IgG, 5 μg / mL HBR-2, and 5 μg / mL MAK-33 were added in triplicate and incubated for 1 hour. After four washes, 3.3 × 10 cells were added in 40 μL of RED assay buffer (supplemented with 8 mM CaCl) per well. 7 AAL nanoparticles prepared at a concentration of 1000 μg / ml were added to the plate. After incubation for 90 min, the plate was washed six times and the time-resolved fluorescence of Eu was measured.

[0195] PSA AAL Glycovariants and free PSA MGL The results are shown in Figure 13. AAL Glycovariants did not discriminate between clinically significant PCa and indolent disease (benign and Gle6), and free PSA MGL There was no improvement when combined with (Figure 13).

Claims

1. A glycan structure containing the glycan antigen 19-9 (CA19-9) and capable of specifically binding to mannose-binding lectin (MBL), serving as a biomarker for prostate cancer.

2. A method for determining the disease status of prostate cancer in a subject, the following: a) Assay the samples obtained from the subject to determine the level of CA19-9-containing entities that include a glycan structure capable of specifically binding to MBL. b) Compare the assay level obtained in step a) with the level of the control sample or a predetermined threshold, and c) Determining the disease state of prostate cancer based on the above comparison. Methods that include...

3. The method according to claim 2, wherein an increase in the level of the CA19-9-containing entity in the sample compared to the level of the CA19-9-containing entity in the control sample, or compared to a predetermined threshold, indicates that the subject has prostate cancer or is at risk of having prostate cancer.

4. The method according to claim 3, wherein the assay is performed by using a binding molecule specific to the glycan structure, or by using mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, electrophoresis, chromatography, or a combination thereof.

5. The method according to claim 4, wherein the binding molecule is mannose-binding lectin (MBL) or an antibody specific to a glycan structure that can specifically bind to MBL.

6. The aforementioned sample is a CA19-9-containing entity (CA19-9) that can specifically bind to wheat germ agglutinin (WGA). ＷＧＡ ), and / or vesicles displaying CA19-9 antigen or CA19-9 that can specifically bind to macrophage galactose-type lectin (MGL) (CA19-9 ＭＧＬ The method according to any one of claims 2 to 5, further comprising assaying for ).

7. The aforementioned sample contains total prostate-specific antigen (PSA), free PSA (fPSA), and free PSA (fPSA) that can specifically bind to MGL. ＭＧＬ The method according to any one of claims 2 to 5, further comprising assaying for one or more biomarkers selected from the group consisting of intact PSA, total human kallikrein 2 (hK2), and free hK2.

8. A kit for determining the disease status of prostate cancer in a subject, comprising a CA19-9 binder and a binding molecule specific to a glycan structure that can specifically bind to MBL, wherein either the CA19-9 binder or the binding molecule is labeled for detection.

9. The kit according to claim 8, wherein the binding molecule is MBL, or an antibody specific to a glycan structure that can specifically bind to MBL.

10. The kit according to claim 8 or 9, further comprising at least one binding molecule specific to a glycan structure that can specifically bind to WGA and / or a glycan structure that can specifically bind to MGL.

11. The kit according to claim 10, wherein the binding molecule is selected from the group consisting of WGA, MGL, an antibody specific to a glycan structure that can specifically bind to WGA, and an antibody specific to a glycan structure that can specifically bind to MGL.

12. The sample contains total prostate-specific antigen (PSA), free PSA (fPSA), and free PSA (fPSA) that can specifically bind to MGL. ＭＧＬ The kit according to claim 8, further comprising reagents for assaying one or more biomarkers selected from the group consisting of intact PSA, total human kallikrein 2 (hK2), and free hK2.

13. i) A multiwell plate comprising one or more wells immobilized with CA19-9 binder and one or more different wells immobilized with fPSA binder; and ii) At least two conjugates, one of which is specific to a glycan structure that can specifically bind to MBL, and the other which can specifically bind to MGL. The kit according to claim 12, including the following:

14. The kit according to claim 13, wherein the CA19-9 binder, the fPSA binder, or both are immobilized in a region having a diameter smaller than the diameter of the well.

15. i) A multiwell plate comprising one or more wells having a bottom surface containing at least two different regions, one of which is immobilized with a CA19-9 binder and the other with an fPSA binder; and ii) At least two conjugates, one of which is specific to a glycan structure that can specifically bind to MBL, and the other which can specifically bind to MGL. The kit according to claim 12, including the following:

16. The fPSA conjugate is an anti-fPSA antibody, preferably an antigen-binding fragment thereof, more preferably a Fab fragment, F(ab) ２ Fragment, or F(ab') ２ The kit according to claim 13 or 15, wherein the fragment is, more preferably, a site-specifically biotinylated fragment.

17. Use of the kit according to claim 8 or 9 for determining the disease status of prostate cancer in a subject.

18. Use of a binding molecule selected from the group consisting of a binding molecule specific to a glycan structure that can specifically bind to MBL, a binding molecule specific to a glycan structure that can specifically bind to WGA, and a binding molecule specific to a glycan structure that can specifically bind to MGL, to determine whether or not a prostate cancer-related CA19-9-containing entity is present in a sample.

19. The use of MBL, or binding molecules specific to glycan structures that can specifically bind to MBL, for determining the disease status of prostate cancer in a subject.

20. A method for determining the disease status of prostate cancer in a subject, the following: a) Assaying a blood sample obtained from the subject using a spot assay to determine the level of free PSA (fPSA) that can specifically bind to MGL, wherein an fPSA binder immobilized on a solid surface in a region having a diameter smaller than the diameter of a solid support is used to capture the fPSA present in the sample, and a detectably labeled conjugate that can specifically bind to MGL is used to detect the MGL-binding glycovariant of the captured fPSA; b) Comparing the assay level obtained in step a) with the level of the control sample or a predetermined threshold; and c) Determining the disease state of prostate cancer based on the above comparison. Methods that include...

21. The method according to claim 20, wherein an increase in the level of free PSA that can specifically bind to MGL in a sample compared to the level of free PSA that can specifically bind to MGL in a control sample, or compared to a predetermined threshold, indicates that the subject has prostate cancer or is at risk of having prostate cancer.

22. The fPSA conjugate is an anti-fPSA antibody, preferably an antigen-binding fragment thereof, more preferably a Fab fragment, F(ab) ２ Fragment, or F(ab') ２ The method according to claim 20 or 21, wherein the fragment is, more preferably, a site-specifically biotinylated fragment.

23. The method according to claim 20, further comprising assaying the sample for one or more biomarkers selected from the group consisting of total prostate-specific antigen (PSA), free PSA, intact PSA, total human kallikrein 2 (hK2), and free hK2.

24. The method according to claim 20, further comprising assaying the sample for the level of at least one CA19-9-containing entity comprising a glycan structure capable of specifically binding to a lectin selected from the group consisting of MBL, WGA, and MGL.

25. A kit for determining the disease status of prostate cancer in subjects, the following: i) A multiwell plate comprising one or more wells, each having a bottom surface including a region on which a PSA binder is immobilized, wherein the region has a diameter smaller than the bottom surface; and ii) A specific conjugate for glycan structures that can specifically bind to MGL. A kit that includes this.

26. The fPSA binding agent is an anti-fPSA antibody, preferably an antigen-binding fragment thereof, more preferably a Fab fragment, F(ab) ２ fragment, or F(ab')[ ２ fragment, and more preferably the fragment is a site-specifically biotinylated fragment. The kit according to claim 25.

27. The kit according to claim 25 or 26, wherein the conjugate is an antibody specific to MGL or a glycan structure that can specifically bind to MGL.

28. The kit according to claim 25, wherein the conjugate is detectably labeled.

29. The kit according to claim 25, wherein the conjugate is immobilized on nanoparticles.

30. The sample contains total prostate-specific antigen (PSA), free PSA (fPSA), and free PSA (fPSA) that can specifically bind to MGL. ＭＧＬ The kit according to claim 25, further comprising reagents for assaying one or more biomarkers selected from the group consisting of intact PSA, total human kallikrein 2 (hK2), and free hK2.

31. The multiwell plate is further comprising a CA19-9 binder immobilized in one or more wells different from the fPSA binder, or in regions on the bottom surface of one or more different wells, where each region has a diameter smaller than the diameter of the well and each region is different from the region on which the fPSA binder is immobilized; and the kit further comprises a conjugate specific to a glycan structure that can specifically bind to MBL, according to claim 25.

32. i) A multiwell plate comprising one or more wells having a bottom surface containing at least two different regions, one of which is immobilized with a CA19-9 binder and the other with an fPSA binder; and ii) At least two conjugates, one of which is specific to a glycan structure that can specifically bind to MBL, and the other which can specifically bind to MGL. The kit according to claim 31, including the following:

Images