Lectin-based cancer diagnostics

Abstract

To provide methods for non-invasive diagnostics of cancer, in particular, breast cancer, colorectal cancer, pancreatic cancer and prostate cancer based on altered glycosylation patterns of cancer-associated biomarkers.SOLUTION: A method of determining prostate cancer disease state in a subject is provided, the method comprising: assaying a sample obtained from the subject for the level of PSA glycoform binding to macrophage galactose-type lectin (MGL) by determining binding of PSA to MGL; comparing the detected level of the lectin-binding PSA glycoform in the sample with that of a control sample or a predetermined threshold value; and determining the prostate cancer disease state in the subject on the basis of the comparison.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to non-invasive diagnosis of cancers such as breast cancer, colorectal cancer, pancreatic cancer, and prostate cancer based on altered glycosylation patterns of cancer-associated biomarkers, particularly CA15-3, CEA, CA19-9, and PSA. [Background technology]

[0002] Some specific embodiments of the present disclosure relate to breast cancer (BrCa), the second leading cause of cancer death in women worldwide. Incidence is very low among women under 30 years of age, but increases after age 45. Mucin 1 (MUC1, also known as CA15-3), located on the apical surface of human epithelial cells, is a large transmembrane glycoprotein with a molecular weight ranging from 500 to 1000 kDa, with an extracellular portion consisting of a highly conserved 20-amino acid tandem repeat with five potential glycosylation sites. The CA15-3 antigen is secreted by tumor cells and is a well-established serological marker for monitoring the clinical course of breast cancer patients; however, it has poor sensitivity, being positive in only 17% of all breast cancer patients (Fleisher M. et al. 2002). Although the CA15-3 assay is not suitable as a diagnostic test in stage I and II disease due to low sensitivity, in advanced breast cancer, changes in antigen levels provide a useful non-invasive indicator of early recurrence, the presence of residual disease, and sustained remission with poor prognosis. Thus, there is an urgent need for improved BrCa biomarkers, particularly for early detection of primary lesions, and also for earlier and more accurate monitoring of cancer grade, progression, and treatment effectiveness.

[0003] There is also a need for improved biomarkers for cancers such as gastrointestinal cancers (including colorectal and pancreatic cancer) and prostate cancer. Summary of the Invention

[0004] In one aspect, the present invention provides a method for determining the disease status of prostate cancer in a subject. The method includes: i) assaying a sample obtained from the subject for the level of a glycoform of PSA that binds to macrophage galactose-type lectin (MGL) by determining binding of PSA to MGL; ii) comparing the detected level of the lectin-bound glycoform of PSA in the sample with that level in a control sample or with a predetermined threshold; and iii) determining the disease status of prostate cancer in the subject based on the comparison. In some embodiments, the method may further include assaying the sample for conventional PSA protein antigen.

[0005] In another aspect, the present invention provides a method for determining the disease status of colorectal cancer in a subject. The method includes: i) assaying a sample obtained from the subject for the level of CEA glycoforms that bind to one or more lectins selected from the group consisting of mannose-binding lectin (MBL), dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), and MGL by determining binding of CEA to the lectin; ii) comparing the detected level of the lectin-bound CEA glycoform in the sample with the level in a control sample or with a predetermined threshold; and iii) determining the disease status of colorectal cancer in the subject based on the comparison. In some embodiments, the method may further include assaying the sample for CEA binding to MBL and DC-SIGN; MBL and MGL; DC-SIGN and MGL; or MBL, DC-SIGN, and MGL. In some further embodiments, the method may also include assaying the sample for the conventional CEA protein antigen.

[0006] In a further aspect, the present invention provides a method for determining the disease status of pancreatic cancer in a subject. The method includes: i) assaying a sample obtained from the subject for the level of CA19-9 glycoforms that bind to DC-SIGN by determining the binding of CA19-9 to DC-SIGN; ii) comparing the detected level of the lectin-bound CA19-9 glycoform in the sample with that of a control sample or a predetermined threshold; and iii) determining the disease status of prostate cancer in the subject based on the comparison. In some embodiments, the method may further include assaying the sample for conventional CA19-9 protein antigen.

[0007] In one aspect, the present disclosure provides a method for determining breast cancer disease status in a subject, the method comprising: i) assaying a sample obtained from the subject for a level of CA15-3 glycoforms that bind to one or more lectins selected from the group consisting of MGL and WGA; ii) comparing the detected level of the lectin-binding CA15-3 glycoform in the sample with that level in a control sample or a predetermined threshold; and iii) determining the breast cancer disease status in the subject based on the comparison.

[0008] In some embodiments, the method for determining breast cancer disease status comprises testing a sample obtained from said sample for one or more additional lectin-binding glycoforms of a biomarker, wherein said biomarker is CA15-3 and said lectin is selected from the group consisting of DSL and Gal4, and / or for one or more conventional biomarkers selected from the group consisting of CA15-3, CA125 and CEA, and / or for one or more additional lectin-binding glycoforms of a biomarker, wherein said biomarker is selected from CA125 and CEA and said lectin is selected from the group consisting of SBA, SNA, PNA, MAA The method may further comprise assaying for a protein selected from the group consisting of: II, AAL, UEA, PHA-E, RCA, WGA, WFA, PSA, VVL, TJA-II, DSL, HPA, MGL, DC-SIGN, MMR, MBL, Siglec-2, Siglec-3, Siglec-5, Siglec-9, Siglec-10, Siglec-11, Galectin-3, Galectin-4, and E-selectin.

[0009] An increase in the level of any one of the above-described glycoforms or biomarkers in the assayed sample compared to that level in a control sample or a predetermined threshold value may indicate that the subject has or is at risk of having cancer.

[0010] In some embodiments, the above-described methods of determining the disease status of cancer in a subject can be carried out to screen for, diagnose, predict, or monitor the cancer, hi some embodiments, the monitoring is for monitoring the onset of the cancer, for monitoring any change in the risk of having or developing the cancer, for monitoring response to treatment, for monitoring the relapse of the cancer, or for monitoring the recurrence of the cancer.

[0011] In a further aspect, the present disclosure provides kits for use in any one of the above-described methods or embodiments thereof. In some embodiments, the kit comprises a PSA-binding agent and MGL, wherein either the CA15-3-binding agent or the MGL comprises a detectable label. In other embodiments, the kit comprises a CEA-binding agent and one or more lectins selected from the group consisting of MBL, DC-SIGN, and MGL, wherein either the CA15-3-binding agent or the lectins comprise a detectable label. In some still further embodiments, the kit comprises a CA19-9-binding agent and DC-SIGN, wherein either the CA15-3-binding agent or the DC-SIGN comprises a detectable label. In some still further embodiments, the kit comprises a CA15-3-binding agent and one or more lectins selected from the group consisting of MGL and WGA, wherein either the CA15-3-binding agent or the one or more lectins comprise a detectable label. In some embodiments, the lectin in question can be immobilized on a detectable nanoparticle, or it can be labeled with a detectable label.

[0012] In some embodiments, the kit may further comprise at least one reagent that binds to one or more lectins selected from the group consisting of SBA, SNA, PNA, MAA II, AAL, UEA, PHA-E, RCA, WGA, WFA, PSA, VVL, TJA-II, DSL, HPA, MGL, DC-SIGN, MMR, MBL, Siglec-2, Siglec-3, Siglec-5, Siglec-9, Siglec-10, Siglec-11, Galectin-3, Galectin-4, E-selectin, DSL, and Gal4, optionally provided immobilized on nanoparticles, and / or a biomarker selected from the group consisting of CA15-3, CA125, and CEA.

[0013] In some further embodiments, the present invention provides the use of different lectins to determine the disease status of cancer in a subject. More specifically, provided herein are the use of MGL to determine the disease status of prostate cancer in a subject by assaying a sample obtained from the subject for the level of PSA glycoforms that bind MGL; the use of MBL, DC-SIGN and / or MGL to determine the disease status of colorectal cancer in a subject by assaying a sample obtained from the subject for the level of CEA glycoforms that bind MBL, DC-SIGN and / or MGL; the use of DC-SIGN to determine the disease status of pancreatic cancer in a subject by assaying a sample obtained from the subject for the level of CA19-9 glycoforms that bind DC-SIGN; and the use of MGL or WGA, either alone or in combination, optionally in combination with DSL and / or Gal4, to determine the disease status of breast cancer in a subject by assaying a sample obtained from the subject for the level of CA15-3 glycoforms that bind MGL, WGA, DSL, and / or Gal4. In any of these embodiments, the lectin in question may be immobilized on the nanoparticle or may be labeled with a detectable label.

[0014] Other objects, aspects, embodiments, details and advantages of the present invention will become apparent from the following figures, summary, examples and dependent claims. [Brief explanation of the drawings]

[0015] In the following, the present invention will be described in more detail using preferred embodiments with reference to the accompanying drawings.

[0016] [Figure 1]Schematic representations of different assay formats. Figure 1A illustrates a conventional immunoassay for CA15-3, in which a capture monoclonal antibody and a tracer monoclonal antibody detect the protein core and sialylated carbohydrate epitopes of CA15-3, respectively. Figure 1B illustrates a combined immunolectin assay, in which CA15-3 is captured by a specific monoclonal antibody and traced by a lectin coated on Eu+3-nanoparticles, which binds to the glycan moiety of CA15-3. [Figure 2] Figure 1 shows the binding of lectin-nanoparticles (lectin-NPs) to CA15-3 captured on A) biotinylated Ma552 monoclonal antibody or B) biotinylated Ma695 monoclonal antibody. The X-axis indicates the different lectin-NPs used, while the Y-axis indicates the signal-to-background ratio. Only MGL lectin and WGA lectin show significant binding to BrCa-associated CA15-3. [Figure 3] We demonstrate recovery in the CA15-3MGL assay. We compared the specific signal of a parallel CA15-3MGL assay with spiked BrCa-associated CA15-3 in either a simple matrix (TSA-BSA) or a complex matrix (i.e., serum). Excellent recovery was observed (>95%) with both spiked buffer and spiked pooled healthy male serum, with an estimated analytical sensitivity of 1 U / ml. [Figure 4] Dose-response curve for CA15-3 WGA assay in pooled spiked healthy male serum. CA15-3 from BrCa cell lines at 5-250 / ml was captured on biotinylated Ma552 monoclonal antibody. WGA-NP was used as a tracer. The estimated detection limit was 1 U / ml of BrCa-associated CA15-3. [Figure 5]Cross-reactivity of capture monoclonal antibodies is demonstrated. When anti-CA15-3 monoclonal antibodies (either bioMa695 (A) or bioMa552 (B)) are used as capturers, the CA15-3MGL assay shows no cross-reactivity with OvCa-CA125. When anti-CA125 bioOv197 monoclonal antibody is used as capture agent, the CA125MGL assay shows 10-fold lower binding to BrCa-associated CA15-3 compared to OvCa-associated CA125 (C). [Figure 6] Box plot analysis of conventional CA15-3 immunoassay (A) and CA15-3MGL assay (B and C) between breast cancer (BC) patients and healthy controls (HCs) is shown. All longitudinal BC samples (n=199) from 45 different BC patients were compared with samples from 31 HCs. As capture agents, CA15-3MGL assay 1 is based on Ma552 (B), while CA15-3MGL assay 2 is based on Ma696 monoclonal antibody (C). The fold difference between the median concentrations measured in the BC and HC groups is given in the figure. The lines do not represent the fold difference. [Figure 7] Box plots of the traditional CA15-3 immunoassay (A) and the CA15-3MGL assay (B and C) between breast cancer (BC) patients and healthy controls (HCs) are shown. The first samples (n=45) out of a total of 199 longitudinal samples from 45 different BC patients were compared with 31 HCs. As a capture agent, CA15-3MGL assay 1 is based on Ma552 (B), whereas CA15-3MGL assay 2 is based on the Ma696 monoclonal antibody. The fold difference between the median concentrations measured in the BC and HC groups is shown. [Figure 8] Box plots of CA15-3 WGA assays between primary samples obtained from healthy controls (HC, n=18) and breast cancer patients (BC, n=41) show a 3.7-fold difference in median values ​​between HC and BC. [Figure 9]The receiver operating characteristic curves (ROC) for all three CA15-3 assays (the conventional CA15-3 immunoassay "CA153IA1st45," the CA15-3MGL assay 1 "CA153MGL11st45," and the CA15-3MGL assay 2 "CA153MGL21st45") were generated using the first samples from BrCa patients (n=45) and healthy controls (n=31). The AUC was highest for CA15-3MGL assay 1 (0.897), followed by CA15-3MGL assay 2 (0.848), and lowest for the conventional CA15-3 immunoassay (0.805). [Figure 10] Figure 1 shows ROC curves for the conventional CA15-3 immunoassay "CA153IA1st41" and the CA15-3 WGA "CA153WGA1st41" assays generated using plasma samples from BrCa cases (n=41) and healthy female controls (n=18). The AUC is higher for the CA15-3 WGA assay (0.880) than for the conventional CA15-3 immunoassay (0.833). [Figure 11] We demonstrate correlation between the CA15-3MGL and CA15-3WGA assays. Low correlation between the assays was observed in all 41 plasma samples from BrCa patients, indicating that these assays recognize different epitopes of captured CA15-3. [Figure 12] Demonstrating correlation between the traditional CA15-3 IA and CA15-3MGL assays. (A) In all (n=199) longitudinal breast cancer patient samples, as well as (B) in the first samples (n=45), low correlation between the assays was observed, indicating that these assays recognize different epitopes of captured CA15-3. [Figure 13] We demonstrate correlation between the conventional CA15-3 IA and CA15-3 WGA assays. In 41 plasma samples from BrCa patients, low correlation between the assays was observed, indicating that these assays recognize different epitopes of captured CA15-3. [Figure 14]1 shows the ROC curves and AUCs for the three assays of Example 4 (i.e., the conventional CA15-3 assay (AUC=0.833), the CA15-3MGL assay (AUC=0.865), and the CA15-5WGA assay (AUC=0.939)) using plasma samples from metastatic breast cancer cases (n=54) and healthy female controls (n=23). [Figure 15] 1 shows the ROC curves and AUCs for the three assays of Example 4: the conventional CA15-3 assay (AUC=0.826), the CA15-3WGA Eu-chelate labeled assay (AUC=0.890), and the Eu nanoparticle CA15-3WGA assay (0.926). [Figure 16] Comparison of conventional CEA immunoassay (Fig. 16A) and CEADC-Sign glycovariant assay (Fig. 16B) and CEAMGL glycovariant assay (Fig. 16C) from CRC patients (both treatment-naive and treatment-under-treatment at the time of sampling) and healthy controls with plasma CEA <5 ng / ml. [Figure 17] CEA levels measured by conventional and lectin CEA assays in patients with colitis and CRC are shown (Figure 17A). In Figure 17B, CRC patients are divided into two categories: patients who were alive at the end of follow-up and those who died during follow-up. Mann-Whitney U tests indicated p<0.01: **, p<0.05: *, and p>0.05: ·. Short horizontal bars indicate median values. The long horizontal line indicates the 5 ng / ml cutoff for commercial CEA immunoassays, and the dotted lines indicate the expected cutoff levels for different glycovariant CEA assays. [Figure 18] Receiver operating characteristic curves (ROC) for plasma CEA from the MGL lectin assay, MBL lectin assay, and DC-sign lectin assay in comparison to a conventional CEA immunoassay are shown. ROC was performed on EDTA plasma CEA from patients with colorectal cancer (n=23) and colitis (n=14). The dashed line represents the conventional CEA immunoassay, and the solid line represents the lectin CEA assay. [Figure 19]Figure 19A shows plasma CA19-9 concentrations (U / mL) measured by a commercial CA19-9 immunoassay. Only three pancreatic cancer samples had CA19-9 concentrations above the 37 U / mL cutoff. No statistically significant differences in median CA19-9 concentrations were observed between pancreatic cancer patients (n=10) and benign controls (n=27). The short horizontal bar indicates the median CA19-9 value. Figure 19B shows plasma glycovariant CA19-9 (i.e., CA19-9DC-SIGN) concentrations (U / mL) measured by a CA19-9 lectin nanoparticle assay. Plasma glycovariant CA19-9 concentrations are elevated in pancreatic cancer and several benign gastrointestinal diseases. Eight of 10 pancreatic cancer patients had plasma glycovariant CA19-9 concentrations above the 10 U / mL cutoff. There were statistically significant differences in median glycovariant CA19-9 concentrations between pancreatic cancer patients (n=10) and healthy as well as benign controls (n=27). Short horizontal bars indicate median CA19-9 values. [Figure 20] Dose-response curves (measured mean fluorescence signal from triplicate samples) from different PSA forms by the PSAMGL glycovariant assay are shown. Calibration curves for LNCaP PSA (◇) and seminal plasma PSA from healthy donors (□). [Figure 21] Figure 1 shows the fluorescent signal from MGL-conjugated nanoparticles measuring PSA glycovariants in lysates from histologically benign and cancerous prostate tissues. There was a statistically significant increase in PSA glycovariants in cancerous tissues compared to benign tissues. [Figure 22]Figure 1 shows the total PSA-corrected fluorescence signal of the PSAMGL glycan variant assay from urinary PSA from prostate cancer patients (n = 74), BPH patients (n = 69), and healthy young men (n = 11). The tPSA-corrected fluorescence signal was calculated as the fluorescence measured from Eu nanoparticles divided by the tPSA concentration in the sample. The tPSA-corrected fluorescence signal of the MGL-conjugated nanoparticles from urinary PSA was compared between healthy young men (n = 11), men with negative PCA biopsies or Gleason score 6 prostate cancer (n = 69), prostate cancer patients with Gleason scores 7–9 (n = 74), and prostate cancer patients with Gleason score 9 (n = 11). The 10th, 25th, 50th, 75th, and 90th percentiles are marked in the figure. [Figure 23] Fluorescence signals are shown as the average fluorescence signal from three replicate specimens for PSAWGA (Figure 23A), PSAGAl4 (Figure 23B), and PSA Dectin 2 (Figure 23C) glycovariant assays. Fluorescence signals of WGA-conjugated nanoparticles from urinary PSA were compared between young men (n=6), men with negative PCa biopsies or Gleason score 6 PCa (n=14), and men with clinically significant PCa (n=17) (Figure 23A). Fluorescence signals of GAl4-conjugated nanoparticles from urinary PSA were compared between young men (n=9), men with negative PCa biopsies or Gleason score 6 PCa (n=19), and men with clinically significant PCa (n=26) (Figure 23B). The fluorescent signal of GAl4-conjugated nanoparticles from urinary PSA was compared between young men (n=9), men with negative PCa biopsies or PCa with Gleason score 6 (n=9), and men with clinically significant PCa (n=26) (Figure 23C). DETAILED DESCRIPTION OF THE INVENTION

[0017] The present disclosure is based, at least in part, on research aimed at identifying glycoforms of known cancer biomarkers with improved sensitivity over other species of the same biomarker. In accordance with this objective, the present disclosure provides means and methods for determining cancer status in a subject suspected of having or being at risk of having cancer, particularly for screening, diagnosing, predicting, or monitoring said cancer. According to specific embodiments, said cancer is breast cancer, gastrointestinal cancer (such as colorectal cancer or pancreatic cancer), or prostate cancer.

[0018] As used herein, the term "or" has the meaning of both "and" and "or" (i.e., "and / or"). Furthermore, the meaning of the singular includes the meaning of the plural, and thus singular terms may also have their plural meaning unless otherwise specified. In other words, the terms "a" or "an" may mean one or more.

[0019] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, and in some embodiments, such as those associated with breast cancer, most preferably a female, while in other embodiments, such as those associated with prostate cancer, most preferably a male. Depending on the embodiment in question, the subject may have cancer with or without diagnosis, be suspected of having cancer, be at risk for cancer, or may already be treated for cancer. As used herein, the terms "human subject," "patient," and "individual" are interchangeable.

[0020] As used herein, the term "sample" refers to tissue samples (such as a biopsy sample taken from breast tissue) and samples of bodily fluids (such as ascites, urine, blood, plasma, serum, and peritoneal cavity fluid taken from a subject). In some embodiments, the tissue sample may be a formalin-fixed tissue sample or a raffin-embedded tissue sample. Generally, obtaining the sample to be analyzed from a subject is not part of the present method of determining the subject's cancer disease status. Blood, serum, or plasma samples are the most preferred sample types used in the present method and all its embodiments.

[0021] The term "sample" also encompasses samples that have been manipulated or processed in any suitable manner after their procurement, including, but not limited to, centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing, or enrichment for a particular component (such as a cell population) of the sample.

[0022] As used herein, the terms "biomarker" and "marker" are interchangeable and refer to molecules that are differentially present in samples taken from subjects with cancer compared to comparable samples taken from apparently healthy subjects. Thus, a biomarker may be referred to as a cancer biomarker. Preferred cancer biomarkers include CA15-3, CA125, CEA, CA19-9, and PSA, and in particular certain lectin-binding glycoforms thereof. As used herein, the terms "CA15-3," "CA125," "CEA," "CA19-9," and "PSA" refer to the conventional protein antigens of CA15-3, CA125, CEA, CA19-9, and PSA, respectively, which are detectable by conventional immunoassays. As used herein, CA15-3 レクチン , CA125 レクチン , CEA レクチン , CA19-9 レクチン , and PSA レクチン The term "CA15-3" refers to the lectin-binding glycoforms of these biomarkers.WGA The term " refers to the glycoform of CA15-3 that binds to WGA.

[0023] In embodiments that contemplate assessing the levels of two or more biomarkers, the same or different samples obtained from the subject whose cancer disease status is to be determined can be used for each assessment, and the different samples can be of the same type or different types.

[0024] As used herein, the term "level" is interchangeable with "amount" and "concentration" unless otherwise indicated.

[0025] To determine whether a detected level of a biomarker represents the presence, or risk of the presence, of a disease associated with the biomarker, its level in a relevant control must be determined. Once the control level is known, the determined marker level can be compared to it, and the significance of the difference can be assessed using standard statistical methods. In some embodiments, a statistically significant difference between the determined biomarker level and the control level is indicative of the disease in question. In some further embodiments, the biomarker level is normalized using standard methods before being compared to the control.

[0026] Comparing the assay level of a biomarker in a sample to be analyzed with its level in a relevant control or with a predetermined threshold value may, in some embodiments, be performed by a processor of a computing device.

[0027] Regardless of whether a processor of a computing device is used for the comparison, the level of the assay level of a biomarker is determined as "increased" or "higher" in at least some embodiments if the level of the biomarker in the sample is, for example, at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 9-fold, 10-fold, 20-fold, or 30-fold higher than 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 to be analyzed and the level of the biomarker in the predetermined threshold level or control sample must be statistically significant to provide an appropriate diagnostic, prognostic, or predictive result.

[0028] The concentration of a biomarker in a sample obtained from a subject whose cancer disease status is to be determined, or who is to be screened, diagnosed, prognosed, or monitored for cancer, is deemed "not elevated" or "normal" if the detected concentration is lower, essentially the same, or essentially unchanged compared to the concentration in a relevant control sample or a predetermined threshold.

[0029] As used herein, the term "control" may refer to a control sample obtained from an apparently healthy individual or a pool of apparently healthy individuals, or may refer to a predetermined threshold (i.e., cutoff value, which represents the presence or absence of the disease in question). Statistical methods for determining an appropriate threshold will be readily apparent to one of skill in the art. The threshold may, if necessary, be determined from samples of subjects of the same age, demographic characteristics, and / or disease state, etc. The threshold may originate from a single individual not affected by the disease in question, or may be a pooled value from two or more such individuals.

[0030] In some embodiments, the term "control sample" refers to a sample obtained from the same subject whose cancer disease status is to be determined, but obtained at a time point different from the time point of disease status determination. Non-limiting examples of such different time points include one or more time points before diagnosis of the disease, one or more time points after diagnosis of the disease, one or more time points before treatment of the disease, one or more time points during treatment of the disease, and one or more time points after treatment of the disease. Typically, such a control sample obtained from the same subject is used when the purpose of determining the disease status of breast cancer is to monitor the disease, particularly the onset of the disease or risk of disease onset, response to treatment, relapse of the disease, or recurrence of the disease.

[0031] As used herein, the term "apparently healthy" refers to an individual, or pool of individuals, who do not show signs of cancer and are therefore not thought to be cancer-free or predicted not to develop cancer.

[0032] As used herein, the term "disease-indicative" refers to a level that, when applied to a biomarker, aids in the diagnosis of the disease or stage of the disease using routine statistical methods that set a confidence level at least 95%, and where the level detected in subjects with the disease or stage of the disease is found significantly more frequently than in subjects without the disease or at another stage of the disease. Preferably, the disease-indicative level is found in at least 80% of subjects with the disease and less than 10% of subjects without the disease. More preferably, the disease-indicative level is found in at least 90%, at least 95%, at least 98%, or more of subjects with the disease and less than 10%, less than 8%, less than 5%, less than 2.5%, or less than 1% of subjects without the disease.

[0033] Existing CA15-3 immunoassays used in routine diagnostics are based on determining CA15-3 antigen levels in serum or plasma using two different monoclonal antibodies that recognize different epitopes of CA15-3. In some non-limiting embodiments, one of the antibodies may be specific for a protein epitope, while the other antibody may be specific for a carbohydrate epitope (such as a sialylated carbohydrate epitope). Such conventional immunoassays (referred to herein as "assaying a sample for CA15-3 antigen concentration") are commercially available from several different vendors. Thus, the term "CA15-3" refers to a CA15-3 species recognizable by conventional immunoassays. In some embodiments, a cutoff level (such as 20 U / ml, 22 U / ml, 25 U / ml, 30 U / ml, or 35 U / ml) may be used.

[0034] In experiments related to some embodiments of the present invention, among a panel of 28 different plant or human lectins tested, two lectins (i.e., macrophage galactose-type lectin (MGL) and wheat germ agglutinin (WGA)) showed significantly higher reactivity with CA15-3 derived from a breast cancer cell line. Datura stramonium lectin (DSL) and galectin-4 (Gal4) also showed some reactivity with cancerous CA15-3.

[0035] For the sake of brevity, we will refer to "CA15-3 レクチン " is used as a general term to refer to any one or more lectin-binding glycoforms of CA15-3, e.g., CA15-3 MGL , CA15-3 WGA , CA15-3 DSL , or CA15-3 GAL4 (These refer to MGL-binding, WGA-binding, DSL-binding, and Gal4-binding species of CA15-3, respectively).

[0036] The clinical applicability of the results obtained with isolated CA15-3 from breast cancer was validated in a small cohort of samples obtained from patients with metastatic breast cancer. MGL or CA15-3 WGA By assaying samples for CA15-3, breast cancer subjects can be distinguished from healthy controls with greater sensitivity than by using conventional CA15-3 immunoassays. Furthermore, the median fold difference between healthy controls and breast cancer patients is only 2-fold with existing CA15-3 immunoassays, while CA15-3 MGL The median fold difference between healthy controls and breast cancer patients in different cohorts was only 2.8-fold for the existing CA15-3 immunoassay, whereas the CA15-3 MGL In some embodiments, the CA15-3 WGA is the preferred lectin to be used.

[0037] One way to compare the performance of different biomarkers is to draw ROC curves and compare their AUC values, false positive rates, false negative rates, and success rates. As demonstrated in the experimental section, CA15-3 MGL The false-negative rate of the assay was lower than that of the conventional assay. Furthermore, higher AUC values ​​were observed for the CA15-3 assay than for the conventional CA15-3 assay. MGL Assay and CA15-3 WGA was obtained in both assays.

[0038] Receiver operating characteristic (ROC) curves can be used to demonstrate the trade-off between the sensitivity and specificity of a marker, as is well known to those skilled in the art. Sensitivity is a measure of a marker's ability to detect disease, and specificity is a measure of a marker's ability to detect the absence of disease. The horizontal X-axis of the ROC curve represents 1-specificity, which increases with the rate of false positives. The vertical Y-axis of the ROC curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cutoff selected, specificity and sensitivity values ​​can be determined. In other words, the data points on the ROC curve represent the rates of true positive and false positive classifications at various decision boundaries. Optimal results are obtained when the true positive rate approaches 1.0 and the false positive rate approaches 0.0. However, as the cutoff is changed to increase specificity, sensitivity usually decreases, and vice versa.

[0039] As used herein, the term "false positive" refers to a test result that incorrectly classifies a non-diseased subject as a diseased subject. Similarly, a "false negative" refers to a test result that incorrectly classifies a diseased subject as a non-diseased subject.

[0040] As used herein, a "true positive" refers to a test result that correctly classifies a subject with a disease as an affected subject. Similarly, a "true negative" refers to a test result that correctly classifies a non-affected subject as not affected.

[0041] In accordance with the above, the term "success rate" refers to the rate expressed as a percentage of affected individuals who test positive, while the term "false positive rate" refers to the rate expressed as a percentage of unaffected individuals who test positive.

[0042] The area under the ROC curve (often referred to as AUC) is a measure of a marker's usefulness in accurately identifying diseased subjects. Therefore, AUC can be used to determine the effectiveness of a test. An area of ​​1 indicates an ideal test, and an area of ​​0.5 indicates a worthless test. Traditional rough guidelines for classifying the accuracy of a diagnostic or predictive test are as follows: an AUC value of 0.9 to 1 indicates a test with excellent diagnostic or prognostic power, an AUC value of 0.80 to 0.90 indicates a test with good diagnostic or prognostic power, an AUC value of 0.70 to 0.80 indicates a test with fair diagnostic or prognostic power, an AUC value of 0.60 to 0.70 indicates a test with poor diagnostic or prognostic power, and an AUC value of 0.50 to 0.60 indicates a test with poor diagnostic or prognostic power.

[0043] CA15-3 MGL and CA15-3 WGA The assays did not correlate well with each other or with traditional immunoassays, and it is therefore contemplated that in some embodiments it will be advantageous to use these assays in combination to complement their performance.

[0044] In accordance with the above, the present invention provides a method for detecting CA15-3 in a sample obtained from said subject. MGL and / or CA15-3 WGA and determining breast cancer status in a subject by assaying for said CA15-3 in said sample. MGL and / or CA15-3 WGA An increase in the level of CA15-3 in the sample compared to the level in a control sample or a predetermined threshold indicates that the subject has or is at risk of having breast cancer. MGL and CA15-3 WGA A non-elevated or normal level of both CA15-3 and CA15-3 compared to the level in a control sample or a predetermined threshold indicates that the subject is apparently healthy with respect to breast cancer or is not at risk of having or developing breast cancer. MGL Assay and CA15-3WGA CA15-3 was not used because the results of the assay did not correlate well with the clinical analysis performed. MGL and CA15-3 WGA It is contemplated that by performing assays for both, the sensitivity of the method may be improved.

[0045] In some embodiments of the above methods, the sample obtained from the subject for whom breast cancer disease status is to be determined is enriched in CA15-3 to improve the performance of the disease status determination. DSL and CA15-3 GAL4 The sample may further be assayed for one or both of CA15-3 and CA25-3. MGL and CA15-3 DSL ;CA15-3 MGL and CA15-3 GAL4 ;CA15-3 WGA and CA15-3 DSL ;CA15-3 WGA and CA15-3 GAL4 ;CA15-3 MGL , CA15-3 DSL and CA15-3 GAL4 ;CA15-3 WGA , CA15-3 DSL and CA15-3 GAL4 or CA15-3 MGL , CA15-3 WGA , CA15-3 DSL and CA15-3 GAL4 The antibody may be assayed for either

[0046] In any aspect and embodiment disclosed herein, CA15-3 レクチン The default threshold for CA15-3 may be about 2 U / ml to about 3 U / ml (e.g., about 2.5 U / ml). However, depending on the desired sensitivity and specificity, レクチンOther predetermined thresholds for β may be used. Non-limiting examples of other such thresholds include about 2 U / ml to about 7 U / ml, about 2 U / ml to about 6 U / ml, about 2 U / ml to about 5 U / ml, and any value falling within the range of about 2 U / ml to about 4 U / ml. For screening purposes and other uses requiring maximum clinical sensitivity of the assay, a lower threshold such as about 0.5 to 2 U / ml (e.g., about 1 U / ml or 1.5 U / ml) may also be used. Meanwhile, for diagnostic or other embodiments requiring maximum clinical specificity of the assay, a higher threshold such as about 5 to 20 U / ml (e.g., about 10 U / ml or 15 U / ml) may also be used. However, these ranges may also vary depending on the details of the detection technique.

[0047] CA15-3 MGL Assay or CA15-3 WGA Because correlation between the assay and conventional CA15-3 immunoassays is low, it is contemplated that the sensitivity of the method may also be improved by including CA15-3 as an additional biomarker in the method. Thus, in some embodiments, the method for determining a subject's breast cancer disease status comprises: subjecting a sample obtained from said sample to one or more of the above-described CA15-3 immunoassays. レクチン Such methods may include assaying for a specific species and conventionally assaying the same or a different sample obtained from the subject for CA15-3 concentration. レクチン An increased level of at least one of the species, or an increased level of CA15-3, would indicate that the subject has or is at risk of having breast cancer, while an increased level of all assayed CA15-3, in combination with no or normal levels of CA15-3, would indicate that the subject has or is at risk of having breast cancer. レクチン Non-elevated or normal levels of the species would indicate that the subject does not have or is not at risk of having breast cancer (i.e., is apparently healthy). Depending on the desired performance (e.g., in terms of sensitivity and specificity), the method may: CA15-3 MGL and CA15-3; CA15-3 WGA and CA15-3; CA15-3 MGL , CA15-3 WGA , and CA15-3; CA15-3 MGL , CA15-3 DSL , and CA15-3; CA15-3 MGL , CA15-3 GAL4 , and CA15-3; CA15-3 WGA , CA15-3 DSL , and CA15-3; CA15-3 WGA , CA15-3 GAL4 , and CA15-3; CA15-3 MGL , CA15-3 DSL , CA15-3 GAL4 , and CA15-3; CA15-3 WGA , CA15-3 DSL , CA15-3 GAL4 , and CA15-3; or CA15-3 MGL , CA15-3 WGA , CA15-3 DSL , CA15-3 GAL4 , and CA15-3 This may involve assaying the same sample or different samples for either of the above.

[0048] Human cancer antigen 125 (CA125), also known as mucin 16 or MUC16, is a complex transmembrane glycoprotein and is the most widely used biomarker for epithelial ovarian cancer. However, its expression can also be elevated in other cancers, including breast, colorectal, pancreatic, and liver cancers. Thus, one or more lectin-binding glycoforms of CA125 (referred to herein as CA125) are considered to be elevated in other cancers. レクチン(collectively referred to as "macro- and microarrays") may be used in some embodiments of the present method as additional markers for determining the breast cancer disease status of a subject. Depending on the desired performance of the method in determining the breast cancer disease status of a subject (such as sensitivity or specificity), a sample obtained from the subject may be CA15-3 MGL and CA125 レクチン ; CA15-3 WGA and CA125 レクチン ; CA15-3 MGL , CA15-3 WGA , and CA125 レクチン ; CA15-3 MGL , CA15-3 DSL , and CA125 レクチン ; CA15-3 MGL , CA15-3 GAL4 , and CA125 レクチン ; CA15-3 WGA , CA15-3 DSL , and CA125 レクチン ; CA15-3 WGA , CA15-3 GAL4 , and CA125 レクチン ; CA15-3 MGL , CA15-3 DSL , CA15-3 GAL4 , and CA125 レクチン ; CA15-3 WGA , CA15-3 DSL , CA15-3 GAL4 , and CA125 レクチン or CA15-3 MGL , CA15-3 WGA , CA15-3 DSL , CA15-3 GAL4 , and CA125 レクチン The antibody may be assayed for either

[0049] Preferably, CA125 レクチンis CA125 MGL However, it can also be any one or more of the lectin-binding species of CA125. レクチン The general term "lectin" refers to any one or more CA125-binding lectins, wherein the lectin is selected from the group consisting of SBA, SNA, PNA, MAA II, AAL, UEA, PHA-E, RCA, WGA, WFA, PSA, VVL, TJA-II, DSL, HPA, MGL, DC-SIGN, MMR, MBL, Siglec-2, Siglec-3, Siglec-5, Siglec-9, Siglec-10, Siglec-11, Galectin-3, Galectin-4, and E-selectin. レクチン and CA125 レクチン Assaying for any one of the above-disclosed combinations may further include assaying for conventional CA15-3 and / or CA125. In some non-limiting embodiments, a cutoff value of 35 U / ml may be used for CA125.

[0050] Carcinoembryonic antigen (CEA) is a widely used glycoprotein biomarker, particularly for cancer of the large intestine (colorectal cancer). However, its expression is also elevated in many other cancers, including breast cancer, cancer of the gastrointestinal tract, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, and prostate cancer. Therefore, one or more lectin-binding glycoforms of CEA (referred to herein as CEA) are elevated in many cancers. レクチン (collectively referred to as "macro- and microarrays") may be used in some embodiments of the present method as additional markers for determining the breast cancer disease status of a subject. Depending on the desired performance of the method in determining the breast cancer disease status of a subject (such as sensitivity or specificity), a sample obtained from the subject may be CA15-3 MGL and CEA レクチン ; CA15-3 WGA and CEA レクチン ; CA15-3 MGL , CA15-3 WGA , and CEAレクチン ; CA15-3 MGL , CA15-3 DSL , and CEA レクチン ; CA15-3 MGL , CA15-3 GAL4 , and CEA レクチン ; CA15-3 WGA , CA15-3 DSL , and CEA レクチン ; CA15-3 WGA , CA15-3 GAL4 , and CEA レクチン ; CA15-3 MGL , CA15-3 DSL , CA15-3 GAL4 , and CEA レクチン ; CA15-3 WGA , CA15-3 DSL , CA15-3 GAL4 , and CEA レクチン or CA15-3 MGL , CA15-3 WGA , CA15-3 DSL , CA15-3 GAL4 , and CEA レクチン The antibody may be assayed for either

[0051] Preferably, CEA レクチン The CEA MBL , CEA DC-SIGN , and / or CEA MGL However, it may be one or more other lectin-binding species of CEA. レクチンThe general term "lectin" refers to any one or more CEA-binding lectins, wherein the lectin is selected from the group consisting of SBA, SNA, PNA, MAA II, AAL, UEA, PHA-E, RCA, WGA, WFA, PSA, VVL, TJA-II, DSL, HPA, MGL, DC-SIGN, MMR, MBL, Siglec-2, Siglec-3, Siglec-5, Siglec-9, Siglec-10, Siglec-11, Galectin-3, Galectin-4, and E-selectin. レクチン and CEA レクチン Assaying for any one of the above-disclosed combinations can further include assaying for conventional CA15-3 and / or CEA. In some non-limiting embodiments, any cutoff value ranging from about 2.5 μg / L to about 10 μg / L (such as 2.7 μg / L or 5 μg / L) can be used for CEA.

[0052] Additionally, in some embodiments, the present methods for determining breast cancer disease status in a subject include measuring CA15-3 MGL , CA125 レクチン and CEA レクチン ; CA15-3 WGA , CA125 レクチン , and CEA レクチン ; CA15-3 MGL , CA15-3 WGA , CA125 レクチン , and CEA レクチン ; CA15-3 MGL , CA15-3 DSL , CA125 レクチン , and CEA レクチン ; CA15-3 MGL , CA15-3 GAL4 , CA125 レクチン , and CEA レクチン ; CA15-3 WGA , CA15-3 DSL , CA125 レクチン , and CEA レクチン ; CA15-3 WGA , CA15-3 GAL4 , CA125 レクチン , and CEA レクチン ; CA15-3 MGL , CA15-3 DSL , CA15-3 GAL4 , CA125 レクチン , and CEA レクチン ; CA15-3 WGA , CA15-3 DSL , CA15-3 GAL4 , CA125 レクチン , and CEA レクチン or CA15-3 MGL , CA15-3 WGA , CA15-3 DSL , CA15-3 GAL4 , CA125 レクチン , and CEA レクチン The method may include assaying a sample obtained from the subject for any of the following:

[0053] Preferably, CA125 レクチン is CA125 MGL and CEA レクチン CEA MBL , CEA DC-SIGN and / or CEA MGL However, CA125 レクチン and CEA レクチン The "lectin" in the general term "CA125-binding lectin" refers, independently of one another, to any one or more CA125-binding lectins or CEA-binding lectins, wherein the lectins are selected from the group consisting of SBA, SNA, PNA, MAA II, AAL, UEA, PHA-E, RCA, WGA, WFA, PSA, VVL, TJA-II, DSL, HPA, MGL, DC-SIGN, MMR, MBL, Siglec-2, Siglec-3, Siglec-5, Siglec-9, Siglec-10, Siglec-11, Galectin-3, Galectin-4, and E-selectin. レクチン , CA125 レクチン , and CEAレクチン Assaying for any one of the above-disclosed combinations of may further include assaying for conventional CA15-3, CA125, and / or CEA.

[0054] In some embodiments, the present methods for determining breast cancer disease status in a subject include: CEA MBL CEA DC-SIGN , CEA MGL , CEA MBL and CEA DC-SIGN , CEA MBL and CEA MGL , CEA DC-SIGN and CEA MGL , CEA MBL , CEA DC-SIGN , and CEA MGL , CEA MBL and CA15-3 MGL , CEA DC-SIGN and CA15-3 MGL , CEA MGL and CA15-3 MGL , CEA MBL and CA15-3 WGA , CEA DC-SIGN and CA15-3 WGA , CEA MGL and CA15-3 WGA , CEA MBL , CA15-3 MGL , and CA15-3 WGA , CEA MBL , CA15-3 MGL , and CA15-3 DSL , CEA MBL , CA15-3 MGL , and CA15-3 GAL4 , CEA MBL , CA15-3 WGA , and CA15-3 DSL , CEA MBL , CA15-3 WGA , and CA15-3 GAL4 , CEA MBL , CA15-3 MGL , CA15-3 DSL , and CA15-3 GAL4 , CEA MBL , CA15-3 WGA , CA15-3 DSL , and CA15-3 GAL4 , CEA MBL , CA15-3 MGL , CA15-3 WGA , CA15-3 DSL , and CA15-3 GAL4 , CEA DC-SIGN , CA15-3 MGL , and CA15-3 WGA , CEA DC-SIGN , CA15-3 MGL , and CA15-3 DSL , CEA DC-SIGN , CA15-3 MGL , and CA15-3 GAL4 , CEA DC-SIGN , CA15-3 WGA , and CA15-3 DSL , CEA DC-SIGN , CA15-3 WGA , and CA15-3 GAL4 , CEA DC-SIGN , CA15-3 MGL , CA15-3 DSL , and CA15-3 GAL4 , CEA DC-SIGN , CA15-3 WGA , CA15-3 DSL , and CA15-3 GAL4 , CEA DC-SIGN , CA15-3MGL , CA15-3 WGA , CA15-3 DSL , and CA15-3 GAL4 , CEA MGL and CA15-3 MGL , CEA MGL and CA15-3 WGA , CEA MGL , CA15-3 MGL , and CA15-3 WGA , CEA MGL , CA15-3 MGL , and CA15-3 DSL ; CEA MGL , CA15-3 MGL , and CA15-3 GAL4 , CEA MGL , CA15-3 WGA , and CA15-3 DSL , CEA MGL , CA15-3 WGA , and CA15-3 GAL4 , CEA MGL , CA15-3 MGL , CA15-3 DSL , and CA15-3 GAL4 , CEA MGL , CA15-3 WGA , CA15-3 DSL , and CA15-3 GAL4 , CEA MGL , CA15-3 MGL , CA15-3 WGA , CA15-3 DSL , and CA15-3 GAL4 , CEA MBL , CEA DC-SIGN , and CA15-3 MGL , CEA MBL , CEA MGL , and CA15-3 MGL , CEA DC-SIGN, CEA MGL , and CA15-3 MGL , CEA MBL CEA DC-SIGN , and CA15-3 WGA , CEA MBL CEA MGL , and CA15-3 WGA , CEA DC-SIGN CEA MGL , and CA15-3 WGA , CEA DC-SIGN CEA MBL , CA15-3 MGL , and CA15-3 WGA , CEA DC-SIGN CEA MBL , CA15-3 MGL , and CA15-3 DSL , CEA DC-SIGN CEA MBL , CA15-3<000035²>, and CA15-3 GAL4 ; CEA[[ID=¿1]] DC-SIGN CEA % MBL , CA15-3 WGA , and CA15-3 DSL , CEA DC-SIGN CEA MBL , CA15-3 WGA , and CA15-3 GAL4 , CEA MGL CEA MBL , CA15-3 MGL % , and CA15-3 WGA , CEA MGL CEA MBL , CA15-3 MGL , and CA15-3 DSL , CEA MGL CEA MBL , CA15-3 MGL , and CA15-3 GAL4 , CEA MGL CEAMBL 、CA15-3 WGA 、and CA15-3 DSL 、 CEA MGL 、CEA MBL 、CA15-3 WGA 、and CA15-3 GAL4 、 CEA DC-SIGN 、CEA MBL 、CA15-3 MGL 、and CA15-3 WGA 、 CEA DC-SIGN 、CEA MGL 、CA15-3 MGL 、and CA15-3 DSL CEA DC-SIGN 、CEA MGL 、CA15-3 MGL 、and CA15-3 GAL4 ; CEA DC-SIGN 、CEA MGL 、CA15-3 WGA 、and CA15-3 DSL 、 CEA DC-SIGN 、CEA MGL 、CA15-3 WGA 、and CA15-3 GAL4 、 CEA MBL 、CEA DC-SIGN 、CEA MBL 、CA15-3 MGL 、CA15-3 DSL 、and CA15-3 GAL4 、 CEA MBL 、CEA DC-SIGN 、CEA MBL 、CA15-3 WGA 、CA15-3 DSL 、and CA15-3 GAL4 、or CEA MBL 、CEA DC-SIGN 、CEA MBL 、CA15-3 MGL 、CA15-3 WGA 、CA15-3 DSL 、and CA15-3 GAL4 The method may include assaying a sample obtained from the subject for:

[0055] Assaying a sample for any one of the combinations of biomarkers described above may include assaying one or more of CA125 レクチン species (e.g., CA125 MGL ), one or more CA19-9 レクチン (e.g. CA19-9 DC-SIGN ) species, conventional CEA, conventional CA15-3, conventional CA125, and conventional CA19-9.

[0056] More generally, the present methods for determining breast cancer disease status in a subject include, in some embodiments, detecting a sample obtained from the subject by CEA analysis. レクチン (e.g., CEA MBL CEA DC-SIGN and / or CEA MGL ), CA19-9 レクチン (e.g. CA19-9 DC-SIGN ), CA125 レクチン (e.g. CA125 MGL ), and CA15-3 レクチン (e.g. CA15-3 MGL and / or CA15-3 WGA , optionally CA15-3 DSL and / or CA15-3 GAL4 In some embodiments, the method may include assaying for any one or more lectin-binding species of biomarkers selected from the group consisting of: CEA, CA19-9, CA125, and CA15-3, and optionally also assaying the sample for one or more conventional breast cancer-associated biomarkers selected from the group consisting of: CEA, CA19-9, CA125, and CA15-3.

[0057] In any of the above-described embodiments, the method of determining the disease status of breast cancer in a subject may be more specifically defined as a method of screening, diagnosing, prognosing, and / or monitoring breast cancer, whether de novo, presenting as a recurrent disease, or suspected.

[0058] In accordance with the above, in some embodiments, the present method is directed to diagnosing breast cancer, i.e., determining whether a subject has breast cancer or whether the subject is at risk of having or developing breast cancer. This is also intended to encompass cases in which the presence or risk of breast cancer is not conclusively determined, but further diagnostic testing is warranted. In such embodiments, the method itself is not determinative of the presence or absence or risk of breast cancer in a subject, but it may indicate that further diagnostic testing is necessary or beneficial. Thus, the present method can be combined with one or more other diagnostic methods to conclusively determine the presence or absence or risk of breast cancer in a subject. Other such diagnostic methods are well known to those skilled in the art.

[0059] Because it is noninvasive and suitable for analyzing urine and serum samples, the method and its various embodiments can be easily incorporated into population screening protocols to identify subjects who have breast cancer or who are at risk of having or developing breast cancer. This would allow not only for early diagnosis of breast cancer, but also for active surveillance for the onset of breast cancer in subjects with an identified increased risk of developing breast cancer later in life. Furthermore, early detection of breast cancer would allow for early treatment of the disease when the chances of cure are highest.

[0060] The present method and its various embodiments may be used not only for diagnostic purposes, but also for predicting the prognosis or outcome of breast cancer, or for monitoring the onset of breast cancer, the occurrence of any risk of breast cancer, a subject's recovery or survival from breast cancer, any possible relapse or recurrence of the disease, or response to treatment. In some embodiments, the method comprises measuring one or more of the CA15-3 antibodies described above.レクチン Species levels, CA15-3, CA125 レクチン , CA125, CEA レクチン and CEA, with their respective levels in one or more other samples obtained from the same subject at different time points, with or without simultaneous comparison as described above. Samples that can be used in monitoring include, but are not limited to, samples collected at different time points after diagnosis of breast cancer and / or before, during, and after a therapeutic intervention (e.g., surgery, radiation therapy, chemotherapy, other suitable therapeutic treatment, or any combination thereof) that alleviates or cures breast cancer. In some embodiments, the monitoring is performed by repeating the assaying step at different time points at least twice, wherein the time points are selected independently from the time points described above. In some embodiments, monitoring is performed during or after treatment for breast cancer, and / or if the level of at least one of the biomarkers is higher than one or more earlier samples obtained from the same subject, or higher than relevant controls, or above a predetermined threshold, the method comprises determining the subject as having, or at risk of, recurrence or recurrence of breast cancer.

[0061] In some embodiments, the methods are particularly suitable for early diagnosis of breast cancer, as well as early detection of relapse, recurrence, and progression of breast cancer. MGL and / or CA15-3 WGA , optionally CA15-3 DSL , CA15-3 GAL4 , CA15-3, CA125 レクチン , CA125, CEA レクチンand CEA, as described above, can serve as early tumor markers for breast cancer, as well as for relapse, recurrence, and / or progression of breast cancer. Thus, the present methods and any combination of biomarkers disclosed herein can be used not only for diagnostic, prognostic, and monitoring purposes, but also for screening women who are asymptomatic for breast cancer or at risk of developing breast cancer.

[0062] In some embodiments, the present disclosure provides CA15-3 as a biomarker for determining a disease status of a subject other than breast cancer. レクチン (Preferably CA15-3 MGL The inventors also provide for the use of CA15-3 to accurately identify subjects as either epithelial ovarian cancer (EOC) or endometriosis subjects, or as apparently healthy. MGL There is experimental evidence that a biomarker can be used. Thus, the present disclosure provides information about the use of CA15-3 as a biomarker for determining a subject's EOC disease status or for diagnosing, prognosing, or monitoring EOC. レクチン (Preferably CA15-3 MGL In addition to methods for determining the disease status of EOC in a subject, methods are also provided for diagnosing, prognosing, or monitoring EOC in a subject, wherein the method comprises measuring CA15-3 in a sample obtained from the subject. レクチン (preferably CA15-3 MGL The CA15-3 レクチン (Preferably, the CA15-3 MGL An increased level of CA15-3 indicates that the subject has or is at risk of having EOC. レクチン (Preferably, the CA15-3 MGL ) may be expressed as one or more conventional breast cancer-associated biomarkers (including, but not limited to, CA125, CEA, and CA15-3), and / or one or more of their lectin-binding species (e.g., CA125 MGL, CEA DC-SIGN and / or CEA MBL (including, but not limited to, the following:) can be used in any combination with other biomarkers (including, but not limited to, the following: a) a marker for breast cancer ...

[0063] In some further embodiments, any combination of biomarkers disclosed for breast cancer may be used to determine the EOC disease status of a subject, or to screen for, diagnose, prognose, or monitor EOC in a subject.

[0064] In some other aspects, the present disclosure provides multiple uses of various lectin-binding species of biomarkers associated with gastrointestinal cancer (such as colorectal cancer) and pancreatic cancer, as exemplified in more detail below. Additionally, methods for determining the status of gastrointestinal cancer in a subject are provided, as well as methods for screening, diagnosing, prognosing, or monitoring gastrointestinal cancer in a subject. Any details of embodiments disclosed herein with respect to breast cancer apply to embodiments with respect to gastrointestinal cancer, even if not repeated herein.

[0065] In some embodiments, the present disclosure provides methods for detecting CA15-3 as a biomarker for determining colorectal cancer disease status in a subject, or for screening, diagnosing, prognosing, or monitoring colorectal cancer in said subject. レクチン (e.g. CA15-3 MGL and / or CA15-3 WGA , optionally CA15-3 DSL and / or CA15-3 GAL4In addition to the method of determining the disease status of colorectal cancer in a subject, also provided is a method of screening, diagnosing, prognosing, or monitoring colorectal cancer in a subject, wherein the method comprises subjecting a sample obtained from said subject to said CA15-3 レクチン The method includes assaying for CA15-3. レクチン An increased level of the species or an increased level of CA15-3 may indicate that the subject has or is at risk of having colorectal cancer. レクチン The assay may include one or more conventional colorectal cancer-associated biomarkers (including, but not limited to, CEA, CA19-9, CA125, and CA15-3), and / or one or more of their lectin-binding species (e.g., CEA, CA19-9, CA125, and CA15-3). レクチン (e.g., CEA MBL , CEA DC-SIGN and / or CEA MGL ), CA19-9 レクチン (e.g. CA19-9 DC-SIGN ), and CA125 レクチン (e.g. CA125 MGL ) in any combination with other biomarkers, including but not limited to, those described above. Thus, a method of determining the disease status of colorectal cancer in a subject, or screening, diagnosing, prognosing, or monitoring colorectal cancer in a subject may further comprise assaying a sample obtained from the subject for one or more of the additional biomarkers described above.

[0066] Experimental evidence by the present inventors shows that MGL, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), and mannose-binding lectin (MBL) are highly reactive with multiple colorectal cancer-derived CEA preparations. MBL , CEA DC-SIGN and / or CEA MGLis a particularly powerful biomarker for use in the various aspects of colorectal cancer diagnosis described above, and may be used alone or in combination with one or more conventional colorectal cancer-associated biomarkers (including, but not limited to, CEA, CA19-9, CA125, and CA15-3) and / or CA19-9. レクチン (e.g. CA19-9 DC-SIGN ), and CA125 レクチン (e.g. CA125 MGL ), CA15-3 レクチン (e.g. CA15-3 MGL and / or CA15-3 WGA , optionally CA15-3 DSL and / or CA15-3 GAL4 in combination with CEA レクチン The lectin-binding species may be used in any combination with one or more thereof selected from the group consisting of, but not limited to, species.

[0067] As demonstrated in Example 5, different glycovariant CEA assays (i.e., CEA DC-Sign , CEA MGL or CEA MBL The assay for CEA (for CEA) can improve the colorectal cancer specificity of the CEA tumor marker. CRC patients and healthy controls with plasma CEA levels within the normal range were differentially detected by the lectin glycovariant CEA assay, and the amount of CEA glycoforms was increased in CRC. The number of false negatives was significantly higher than that of CEA. DC-Sign , CEA MGL or CEA MBL Thus, some preferred embodiments of the present methods and uses related to colorectal cancer include subjecting a sample obtained from a subject whose disease status for colorectal cancer is to be determined to: i) assaying the sample with or without CEA; MBL ;ii) CEA with or without CEA DC-SIGN ;iii) CEA with or without CEA MGL ;iv) CEA with or without CEA MBL and CEA DC-SIGN;v) CEA with or without CEA MBL and CEA MGL ;vi) CEA with or without CEA DC-SIGN and CEA MGL or vi) CEA with or without CEA. MBL , CEA DC-SIGN and CEA MGL Any embodiments and details disclosed herein with respect to other cancers (particularly breast cancer) apply to embodiments with respect to colorectal cancer even if not repeated herein.

[0068] The carbohydrate antigen (CA19-9) is a widely used biomarker, particularly for pancreatic cancer. However, its expression is also elevated in many other gastrointestinal cancers, including colorectal cancer and liver cancer. Therefore, one or more lectin-binding glycoforms of CA19-9 (collectively referred to herein as CA19-9) are commonly used biomarkers for pancreatic cancer. レクチン ), may be used in some embodiments of the present methods as an additional marker for determining the disease status of a subject with gastrointestinal cancer (such as colorectal cancer and pancreatic cancer). A preferred CA19-9 species is CA19-9 DC-SIGN In some non-limiting embodiments, a cutoff value (such as about 26 U / ml or about 37 U / ml) can be used for CA19-9.

[0069] In some embodiments, the present disclosure provides methods for detecting and / or treating pancreatic cancer, including the use of CA15-3 as a biomarker for determining the disease status of a subject with pancreatic cancer, or for screening, diagnosing, prognosing, or monitoring pancreatic cancer in said subject. レクチン (e.g. CA15-3 MGL and / or CA15-3 WGA , optionally CA15-3 DSL and / or CA15-3 GAL4In addition to the method of determining the disease status of pancreatic cancer in a subject, also provided is a method of screening, diagnosing, prognosing, or monitoring pancreatic cancer in a subject, wherein the method comprises: subjecting a sample obtained from said subject to CA15-3 レクチン The method includes assaying for CA15-3. レクチン An increased level of the species or an increased level of CA15-3 may indicate that the subject has or is at risk of having pancreatic cancer. レクチン The assay may include assays for one or more conventional pancreatic cancer-associated biomarkers (including, but not limited to, CEA, CA19-9, CA125, and CA15-3), and / or one or more lectin-binding species thereof (e.g., CEA, CA19-9, CA125, and CA15-3). レクチン (e.g., CEA DC-SIGN and / or CEA MBL ), CA19-9 レクチン (e.g. CA19-9 DC-SIGN ), and CA125 レクチン (e.g. CA125 MGL ) in any combination with other biomarkers, including, but not limited to, those described above. Thus, a method of determining the disease status of pancreatic cancer in a subject, or screening, diagnosing, prognosing, or monitoring pancreatic cancer in a subject may further comprise assaying a sample obtained from the subject for one or more of the additional biomarkers described above.

[0070] As demonstrated in Example 6, CA19-9 DC-SIGN improved the discrimination of pancreatic cancer patients from healthy controls and benign controls (i.e., patients with benign liver or bile duct disease) compared with conventional CA19-9 immunoassays. DC-SIGN The assay reduced the number of false negatives and significantly improved sensitivity. Thus, some preferred embodiments of the present methods and uses related to pancreatic cancer involve assaying a sample obtained from a subject whose disease status for pancreatic cancer is to be determined for CA19-9, with or without assaying for the conventional CA19-9 antigen.DC-SIGN Any embodiments and details disclosed herein with respect to other cancers (particularly breast cancer) apply to embodiments with respect to pancreatic cancer even if not repeated herein.

[0071] Or CEA レクチン (e.g., CEA MBL , CEA DC-SIGN and / or CEA MGL ), CA19-9 レクチン (e.g. CA19-9 DC-SIGN ), CA125 レクチン (e.g. CA125 MGL ) and CA15-3 レクチン (e.g. CA15-3 MGL and / or CA15-3 WGA , optionally CA15-3 DSL and / or CA15-3 GAL4 Any suitable combination of lectin-binding biomarker species selected from the group consisting of (in combination with any desired combination of conventional CEA, CA19-9, CA125, and CA15-3) may be used in any of the embodiments described above with respect to gastrointestinal cancers (including colorectal and pancreatic cancer). In a non-limiting more specific alternative, CEA MBL , CEA DC-SIGN , and / or CEA MGL may be measured by one or more conventional gastrointestinal cancer-associated biomarkers (including, but not limited to, CEA, CA19-9, CA125, and CA15-3), and / or レクチン (e.g. CA19-9 DC-SIGN ), and CA125 レクチン (e.g. CA125 MGL ), CA15-3 レクチン (e.g. CA15-3 MGL and / or CA15-3 WGA , optionally CA15-3 DSL and / or CA15-3 GAL4 in combination with CEA レクチンIn some further embodiments, any of the biomarker combinations disclosed for breast cancer are used to determine the gastrointestinal cancer status of a subject, or to screen for, diagnose, prognose, or monitor gastrointestinal cancer in a subject, where said gastrointestinal cancer includes, but is not limited to, colorectal cancer and pancreatic cancer.

[0072] Furthermore, in one aspect, the present disclosure provides a method for determining the prostate cancer disease status of a subject, or for screening, diagnosing, prognosing, or monitoring prostate cancer in said subject, using one or more lectin-binding species of prostate specific antigen (i.e., PSA), either alone or in any combination thereof, for use as a biomarker. レクチン , preferably PSA MGL , one or more CEA レクチン species (CEA MBL , CEA DC-SIGN and / or CEA MGL etc.), and / or one or more CA19-9 レクチン Seed (CA19-9 DC-SIGN Optionally, any one or more of the lectin-binding biomarkers described above may be used in any combination with the traditional PSA, CEA, and / or CA19-9 biomarkers. In some specific embodiments, the lectin-binding biomarker species assayed include: PSA MGL ; CEA DC-SIGN ; CEA MBL ; CA19-9 レクチン ; PSA MGL and CEA DC-SIGN ; PSA MGL and CEA MBL ; PSA MGL and CA19-9 レクチン ; CEA DC-SIGN and CEA MBL ; CEA DC-SIGN and CA19-9 レクチン ; CEA MBL and CA19-9 レクチン ; PSA MGL , CEA DC-SIGN , and CEA MBL ; PSA MGL , CEA DC-SIGN , and CA19-9 レクチン ; PSA MGL , CEA MBL , and CA19-9 レクチン ; CEA DC-SIGN , CEA MBL , and CA19-9 レクチン or PSA MGL , CEA DC-SIGN , CEA MBL , and CA19-9 レクチン These include, but are not limited to:

[0073] Assaying the sample for any one of the specific biomarker combinations described above may further include also assaying for one or more conventional biomarkers selected from PSA, CEA, and CA19-9.

[0074] As demonstrated in Example 7, PSA MGL can discriminate between cancerous and non-cancerous PSA antigens. Thus, some preferred embodiments of the present methods and uses related to prostate cancer involve assaying a sample obtained from a subject whose prostate cancer disease status is to be determined for PSA, with or without assaying for conventional PSA antigen. MGLAny embodiments and details disclosed herein with respect to other cancers (particularly breast cancer) apply to embodiments with respect to prostate cancer even if not repeated herein.

[0075] In some further embodiments, PSA WGA , PSA Gal4 , and / or PSA デクチン2 can be used to determine the disease status of prostate cancer in a subject. Accordingly, some preferred embodiments of the present methods and uses related to prostate cancer include subjecting a sample obtained from a subject whose disease status of prostate cancer is to be determined to a PSA level. WGA , PSA Gal4 , or PSA デンチン(Dentin)2 ;PSA MGL and PSA WGA ;PSA MGL and PSA Gal4 ;PSA MGL and PSA デンチン2 ;PSA WGA and PSA Gal4 ;PSA WGA and PSA デンチン2 ;PSA Gal4 and PSA デンチン2 ;PSA MGL , PSA WGA , and PSA Gal4 ;PSA MGL , PSA WGA , and PSA デンチン2 ;PSA WGA , PSA Gal4 , and PSA デンチン2 ;PSA MGL , PSA Gal4 , and PSA デンチン2 ; or PSA MGL , PSA WGA , PSA Gal4 , and PSA デンチン2 Any of these embodiments may also include assaying for antigens of conventional PSA.

[0076] For the sake of brevity, the conventional cancer-associated biomarkers disclosed herein (i.e., CA15-3, CA125, CEA, CA19-9, or PSA) will be collectively referred to by the general term "GlycoProt," while their lectin-binding glycoforms will be referred to as "GlycoProt" レクチン ". Depending on the embodiment in question, this term may refer to one or more biomarkers or glycoforms encompassed by this term, respectively, as would be readily understood by one of skill in the art. Preferred GlycoProt レクチン The species is CA15-3 MGL , CA15-3 WGA , CA15-3 DSL , CA15-3 GAL4 , CA125 MGL , CEA DC-SIGN , CEA MBL , and PSA MGL These include, but are not limited to:

[0077] The sample was analyzed using any of the GlycoProt レクチン Assaying for GlycoProt can be carried out by any means, method, or technique available in the art. A preferred, but non-limiting example, is determining the level of GlycoProt binding to the lectin in question. This can be accomplished, for example, by a sandwich assay, in which an antibody specific for GlycoProt, preferably a monoclonal antibody or a fragment thereof (e.g., a Fab fragment), is used as a capture agent, and the lectin in question is used as a tracer. For use as a tracer, the lectin can be detectably labeled, either directly or indirectly.

[0078] As used herein, the term "antibody" refers to an immunoglobulin structure comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Antibodies can exist as intact immunoglobulins or as any of a number of well-characterized antigen-binding fragments or single-chain variants thereof, all of which are encompassed herein by the term "antibody." Non-limiting examples of such antigen-binding fragments include Fab, Fab', F(ab'), and Fv fragments. Such fragments and variants can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins, as is well known in the art. The term "antibody" also includes, but is not limited to, polyclonal, monoclonal, and recombinant antibodies of the isotype classes IgA, IgD, IgE, IgG, and IgM and their subtypes.

[0079] In some other embodiments, the sandwich assay can be performed using a reversed approach. In such cases, the lectin in question is used as the capture agent, and a GlycoProt-specific antibody (preferably a monoclonal antibody) is used as a directly or indirectly detectably labeled tracer. Because urine contains fewer interfering glycosylated molecules than blood, the reversed sandwich assay may work better with urine samples than with blood samples.

[0080] In some embodiments, the sandwich assay may include one or more wash steps after the capture step to remove any GlycoProt species that are not specific for the capture agent. Suitable wash solutions and conditions (e.g., time and temperature) are known to those of skill in the art.

[0081] The sandwich assays described in various embodiments of the present invention can be performed on a solid surface (microtiter plate) or in a lateral flow format. Means and methods for binding capture agents to solid surfaces, e.g., via streptavidin-biotin complexes, or incorporating capture agents into lateral flow assays, are known in the art and will be readily apparent to one of ordinary skill in the art.

[0082] Suitable substrates for use in the present solid-phase sandwich assays 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 can be coated with a suitable compound to facilitate binding of the capture agent (i.e., either an anti-GlycoProt antibody or a lectin) to the substrate. In some further embodiments, one or more control antibodies or control lectins can also be attached to the substrate.

[0083] Any one or more (preferably monoclonal) anti-GlycoProt antibodies can be used in the above sandwich assays as either capture agents or tracers. Non-limiting examples of suitable commercial anti-CA15-3 antibodies include Ma552 and Ma695, while non-limiting examples of suitable commercial anti-CA125 antibodies include Ov185, Ov197, and OvK95 (available from at least Fujirebio Diagnostics, Sweden). Additional GlycoProt-specific monoclonal antibodies are available in the art or can be produced according to methods well known in the art. For use as a tracer, the anti-GlycoProt antibody can be labeled with any suitable label known in the art, including, but not limited to, fluorescent, bioluminescent, or chemiluminescent labels. In some embodiments, the anti-GlycoProt antibody can be indirectly detectably labeled, for example, via immobilization to a detectable nanoparticle.

[0084] Lectins are commercially available from several sources. Some lectins (such as MGL and DC-SIGN) are available with or without a C-terminal fusion to human IgG1-Fc. Both forms can be used in the present methods. In some embodiments, the Fc-fusion form is preferred.

[0085] For use as a tracer, the lectin can be detectably labeled by a variety of techniques, as is well known in the art. In some embodiments, the sample is subjected to GlycoProt レクチン The lectin or lectins used to assay for levels of GlycoProt can be directly labeled with any available detectable label using standard techniques. レクチン can be directly labeled with a lanthanide chelate (such as a chelate of europium(III), terbium(III), samarium(III), dysprosium(III), ytterbium(III), erbium(III), or neodynium(III)) or can be made detectable via colorimetric detection (e.g., GlycoProt レクチン with horseradish peroxidase (HRP) or alkaline phosphatase (AP). In some other embodiments, the one or more lectins used can be detectably indirectly labeled, for example, by immobilizing the one or more lectins on detectable nanoparticles. Such nanoparticle-immobilized lectins are referred to as lectin-NPs for short.

[0086] As used herein, the term "nanoparticle" (NP) refers to a synthetic or natural particle having one or more dimensions (e.g., 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 values ​​±10% of a given value. For example, the phrase "about 100 nm" encompasses 100 nm ±10% or 90 nm to 110 nm. Nanoparticles generally have a spherical shape, although non-spherical shapes (e.g., ellipsoidal shapes) can also be used. 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.

[0087] A variety of different materials can be utilized in the nanoparticles. Non-limiting examples of suitable polymers include poly(ethylene glycol) (PEG), polystyrene, polyethylene, poly(acrylic acid), poly(methyl 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. Further suitable nanoparticle materials include protein nanoparticles, mineral nanoparticles, glass nanoparticles, nanocrystals, metal nanoparticles, and plastic nanoparticles.

[0088] Nanoparticles suitable for use in various embodiments of the present invention or disclosure may be directly or indirectly detectable, qualitatively or quantitatively, by any known means. For example, nanoparticles may be detectable due to innate properties, such as in the case of upconversion nanoparticles (UCNPs), resonant particles, quantum dots, and gold particles. In some other embodiments, nanoparticles may be made detectable by, for example, fluorescent, bioluminescent, or chemiluminescent labeling. In some further embodiments, labeling or doping with lanthanides (i.e., luminescent lanthanide ions with luminescence emission in the visible, near-infrared, or infrared wavelengths and long fluorescence decays, such as europium(III), terbium(III), samarium(III), dysprosium(III), ytterbium(III), erbium(III), and neodynium(III)) is a preferred means for making the nanoparticles detectable.

[0089] Lectins can be immobilized on nanoparticles by any suitable method known in the art, including but not limited to, those disclosed in Example 1. In those embodiments involving two or more different lectins, the different lectins can be immobilized on either the same nanoparticles or different nanoparticles in any desired ratio.

[0090] In some non-limiting embodiments, the most preferred nanoparticles are polystyrene nanoparticles with a diameter of either about 97 nm or about 107 nm. Further preferred nanoparticles include nanoparticles doped with europium chelates. Advantages of such nanoparticles include: i) signal amplification provided by the large number of chelates per particle; ii) enhanced functional affinity (avidity) of lectins to their target glycan epitopes, enabled by the high density of lectins immobilized on the particles; and iii) glycan specificity of the lectins used, enabled by the creation of multivalent nanoparticles. However, nanoparticles are merely one preferred approach that provides adequate avidity effects and signal amplification for the practice of this invention or disclosure and its various embodiments.

[0091] Furthermore, in those embodiments involving two or more different lectins, the sample may be subjected to the same assay (i.e., simultaneously) or different assays (i.e., in parallel), either simultaneously or sequentially, to different GlycoProt レクチン In any such assay, the different lectins may be detectably labeled, either directly or indirectly, with the same different labels. In some embodiments, multiplexing, e.g., by using differentially labeled nanoparticles carrying different lectin species in a single assay, may be a preferred format for carrying out the methods disclosed herein or any of their embodiments.

[0092] In some specific embodiments, one or more lectins immobilized on the same or different nanoparticles labeled with a detectable label (such as a lanthanide chelate selected from europium(III), terbium(III), samarium(III), and dysprosium(III)) are used as tracers. In some more specific embodiments, europium chelates are used as detectable labels. In a non-limiting preferred embodiment, lectin-NPs are used as tracers and are doped with approximately 30,000 Eu chelates. In other specific embodiments, the lectin in question is attached to upconversion phosphorylation (UCP) particles, which are particularly suitable for use as tracers in lateral flow formats.

[0093] Detectable signals can be generated by using any available sensor technology. For example, a solid surface can incorporate a recognition element (transducer) capable of converting a binding reaction into a detectable signal with or without the use of a labeling moiety. Different types of transducers can be used, including those based on electrochemical or optical detection. As will be apparent to those skilled in the art, detection can also be based on homogeneous or heterogeneous detection techniques.

[0094] Coating lectins on the surface of nanoparticles instead of coating them on solid surfaces (such as microtiter wells, arrays, or sensors) offers significant benefits in assay performance, especially in non-competitive assay formats in which lectins are used in combination with specific antibodies. The typically significantly higher affinity of antibodies compared to the affinity of lectins when such antibodies are bound to a solid phase can be fully utilized as a first step to capture target biomarker molecules onto the solid phase with high efficiency and stability. Given the high affinity of antibodies, even small target molecules with only one copy of the targeting epitope can be captured with high stability. Once the target molecule is captured, the avidity effect of lectin nanoparticles (i.e., multiple adjacent lectins can bind to adjacent captured target molecules, thereby significantly increasing binding strength compared to a single lectin molecule) can also be fully utilized.

[0095] Further coupled with the fact that nanoparticles typically allow for significant enhancement of the measurable signal, the lectin-NP / solid phase-antibody approach optimally combines high specificity of both binding partners, high avidity of a single antibody, high avidity of multiple contiguous lectins on multiple contiguous glycan structures (avidity effect), and high levels of detectable signal for each conjugated nanoparticle, resulting in an optimal combination of both high sensitivity and high specificity with respect to both analytical and clinical attributes.

[0096] Furthermore, while lectins typically have high specificity for target glycan structures (glycoforms), such forms may also exist in molecules other than the targeted one. Therefore, in some preferred embodiments, a washing step is used between the target molecule antibody capture phase and the lectin-NP binding phase to wash away any unbound non-targeted molecules that, in some cases, contain the same glycan structure as the target molecule and thus pose a risk of non-specific detection due to interspecies cross-reactivity. Once a specific target molecule is captured using an antibody and other molecules are washed away before the lectin binding step, the risk of non-specific binding of unwanted targets by the lectin is eliminated. Such a washing step also preferably prevents competition of target molecules for binding to distal sites around the lectin-NP (which in many cases occurs due to low affinity). However, the risks of both cross-reactivity and distal binding increase when the target molecule has multiple reactive glycan structures, as is the case with many molecules with high molecular weights (e.g., CA15-3, where adjacent lectins can bind to adjacent glycan structures in the same target molecule). In such cases, the use of a non-competitive lectin-NP / solid phase-antibody approach with a washing step prior to the lectin-NP binding reaction results in significantly more sensitive and target-specific assays compared to platforms in which lectins are bound to the solid phase or in which lectin-NPs are used without an intermediate washing step. Similarly, due to the almost complete lack of avidity effects, assay platforms in which lectins (instead of antibodies) are bound to the solid phase perform poorly with small target molecules (such as PSA, which has only one carbohydrate moiety per molecule).

[0097] The present disclosure also provides kits for use in the methods and various embodiments thereof. In its broadest form, the kit includes reagents for assaying one or more lectin-binding species of cancer-associated glycoprotein biomarkers selected from the group consisting of CA15-3, CA125, CEA, C19-9, and PSA. In other words, the kit includes reagents for assaying one or more lectin-binding species of cancer-associated glycoprotein biomarkers selected from the group consisting of CA15-3, CA125, CEA, C19-9, and PSA. レクチン , CA125 レクチン , CEAレクチン , CA19-9 レクチン and PSA レクチン Any desired GlycoProt レクチン The assay includes reagents for assaying a sample for each GlycoProt species or combinations thereof. レクチン wherein at least one reagent is a GlycoProt binding agent (such as a monoclonal anti-GlycoProt antibody) specific for the GlycoProt in question, and at least one of the reagents is a lectin in question (preferably immobilized on a nanoparticle). Either the GlycoProt binding agent or the lectin is detectably labeled. The lectin may be indirectly labeled via the detectable nanoparticle to which it is immobilized. The GlycoProt being assayed レクチン The species and therefore the reagents included in the kit will depend on the intended purpose for which the kit is used (particularly screening, diagnosing, prognosing, or monitoring cancer), with preferred combinations being apparent from the present disclosure above.

[0098] Optionally, the kit may also include reagents for assaying one or more conventional GlycoProt antigens, preferably selected from the group consisting of CA15-3, CA125, CA19-9, CEA, and PSA. Non-limiting examples of typical reagents for assaying said conventional GlycoProt antigens include two GlycoProt binding agents (such as two monoclonal anti-GlycoProt antibodies, which bind to different epitopes in said GlycoProt) for each conventional GlycoProt antigen to be assayed. For each specific GlycoProt, one of the binding agents binds to the respective GlycoProt. レクチンThe GlycoProt binding agents may be the same as those provided for the assay of GlycoProt antigens. Nevertheless, one of the GlycoProt binding agents may be immobilized on a solid surface or provided for use as a capture agent in a lateral flow format, while the other GlycoProt binding agent may comprise a detectable label. The conventional GlycoProt antigens to be assayed and therefore the reagents included in the kit will depend on the intended purpose for which the kit is used (particularly for screening, diagnosing, prognosing, or monitoring cancer), with preferred combinations being apparent from the present disclosure above.

[0099] In some embodiments, kits are provided for determining a subject's breast cancer disease status, or for screening, diagnosing, prognosing, or monitoring breast cancer in the subject. In such cases, the kit includes a CA15-3 binding agent (such as a monoclonal anti-CA15-3 antibody) and MGL and / or WGA (optionally immobilized on the same or different nanoparticles). In some further embodiments, DSL and / or Gal4 (optionally immobilized on the same or different nanoparticles) may also be provided. Either the CA15-3 binding agent or the lectin includes a detectable label or is immobilized on a solid surface (such as a microtiter plate). In some further embodiments, streptavidin coating of the plate and biotinylation of the antibody are used for the attachment. Alternative methods for accomplishing the same are readily available to those skilled in the art.

[0100] In some further embodiments, kits are provided for determining the breast cancer disease status of a subject, or for screening, diagnosing, prognosing, or monitoring breast cancer in said subject, comprising: レクチン (e.g. CA125 MGL ), CA125, CEA レクチン (e.g., CEA MBL , CEA DC-SIGN and / or CEA MGLThe composition may also include reagents for assaying additional biomarkers selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,

[0101] Thus, in some embodiments, particularly considering breast cancer, the kit may further include one or more reagents for assaying CA15-3 protein concentration. Non-limiting examples of typical reagents for assaying CA15-3 protein concentration include two CA15-3 binding agents (such as two monoclonal anti-CA15-3 antibodies) that bind to different protein epitopes in CA15-3. One of the CA15-3 binding agents binds to the CA15-3 protein epitope. MGL or CA15-3 WGA The CA15-3 binding agent may be the same as the CA15-3 binding agent provided for the assay of CA125 and / or CEA. One of the two CA15-3 binding agents may be immobilized on a solid surface or provided for use as a capture agent in a lateral flow format, while the other CA15-3 binding agent may contain a detectable label. Alternatively or additionally, corresponding reagents may be included in the kit for assaying samples for CA125 and / or CEA.

[0102] Optionally, the kit may also include a control for comparison to the measured value of CA15-3 binding to MGL and / or WGA. In some embodiments, the control is a threshold value for comparison to the measured value.

[0103] In some embodiments, a kit is provided for determining the disease status of colorectal cancer in a subject, or for screening, diagnosing, prognosing, or monitoring colorectal cancer in said subject. In such cases, the kit comprises a CEA binding agent (such as a monoclonal anti-CEA antibody) and at least one lectin selected from the group consisting of MBL, DC-SIGN, and MGL (optionally immobilized on nanoparticles). If two or more lectins are to be used, they may be immobilized on the same nanoparticles or different nanoparticles. In some embodiments, the lectin used is CEA.MBL , CEA DC-SIGN or CEA MGL While in other embodiments, the lectin CEA MBL and CEA DC-SIGN ;CEA MBL and CEA MGL ;CEA DC-SIGN and CEA MGL ; or CEA; or CEA MBL , CEA DC-SIGN and CEA MGL are used in combination. Either the CEA-binding agent or the lectin contains a detectable label or is immobilized on a solid surface (such as a microtiter plate).

[0104] In some further embodiments, streptavidin coating of the plate and biotinylation of the antibody are used for the attachment. Alternative methods for achieving the same are readily available to those skilled in the art. In some further embodiments, kits provided for determining the colorectal cancer disease status of a subject, or for screening, diagnosing, prognosing, or monitoring colorectal cancer in said subject, may also include reagents for assaying additional biomarkers (such as CEA). Thus, in some embodiments, the kit may further include one or more reagents for assaying CEA protein concentration. Suitable reagents for this purpose are readily available in the art and include, but are not limited to, monoclonal anti-CEA antibodies. In some embodiments, two monoclonal anti-CEA antibodies (which bind to different protein epitopes in CEA) may be used. One of the CEA-binding agents binds to CEA. MBL , CEA DC-SIGN and / or CEA MGL One of the two CEA-binding agents may be immobilized on a solid surface or provided for use as a capture agent in a lateral flow format, while the other CEA-binding agent may contain a detectable label.

[0105] Optionally, the kit may also include a control for comparison to the measured values ​​of CEA binding to MBL, DC-SIGN and / or MGL. In some embodiments, the control is a threshold value for comparison to the measured values.

[0106] In some embodiments, kits are provided for determining the disease status of a subject with pancreatic cancer, or for screening, diagnosing, prognosing, or monitoring pancreatic cancer in the subject. In such cases, the kit includes a CA19-9 binding agent (such as a monoclonal anti-CA19-9 antibody) and at least one lectin (preferably DC-SIGN, optionally immobilized on nanoparticles). Either the CA19-9 binding agent or the lectin includes a detectable label or is immobilized on a solid surface (such as a microtiter plate). In some further embodiments, streptavidin coating of the plate and biotinylation of the antibody are used for the attachment. Alternative methods for achieving the same are readily available to those skilled in the art.

[0107] In some further embodiments, kits provided for determining a subject's pancreatic cancer disease status or for screening, diagnosing, prognosing, or monitoring pancreatic cancer in said subject may also include reagents for assaying additional biomarkers (such as CA19-9). Thus, in some embodiments, the kit may further include one or more reagents for assaying CA19-9 protein concentration. Suitable reagents for this purpose are readily available in the art and include, but are not limited to, monoclonal anti-CA19-9 antibodies. In some embodiments, two monoclonal anti-CA19-9 antibodies (which bind to different protein epitopes in CA19-9) may be used. In such cases, one of the CA19-9 binding agents binds to CA19-9. DC-SIGNOne of the two CA19-9 binding agents may be immobilized on a solid surface or provided for use as a capture agent in a lateral flow format, while the other CA19-9 binding agent may include a detectable label.

[0108] Optionally, the kit may also include a control for comparison to the measured value of CA19-9 binding to DC-SIGN. In some embodiments, the control is a threshold value for comparison to the measured value.

[0109] In some embodiments, a kit is provided for determining the disease state of prostate cancer in a subject, or for screening, diagnosing, prognosing, or monitoring prostate cancer in the subject. In such cases, the kit includes a PSA binder (such as a monoclonal anti-PSA antibody or an antigen-binding fragment thereof) and at least one lectin (preferably MGL, optionally immobilized on nanoparticles). Either the PSA binder or the lectin includes a detectable label or is immobilized on a solid surface (such as a microtiter plate). In some further embodiments, streptavidin coating of the plate and biotinylation of the antibody are used for the attachment. Alternative methods for achieving the same are readily available to those skilled in the art.

[0110] In some further embodiments, kits provided for determining the prostate cancer disease status of a subject, or for screening, diagnosing, prognosing, or monitoring prostate cancer in said subject, may also include reagents for assaying additional biomarkers (such as PSA). Thus, in some embodiments, the kit may further include one or more reagents for assaying PSA protein concentration. Suitable reagents for this purpose are readily available in the art and include, but are not limited to, monoclonal anti-PSA antibodies and antigen-binding fragments thereof. In some embodiments, two monoclonal anti-PSA antibodies (which bind to different protein epitopes in PSA) may be used. In such cases, one of the PSA binding agents may bind to PSA. MGL One of the two PSA binding agents may be immobilized on a solid surface or provided for use as a capture agent in a lateral flow format, while the other PSA binding agent may contain a detectable label.

[0111] Optionally, the kit may also include a control for comparison to the measured value of PSA binding to MGL. In some embodiments, the control is a threshold value for comparison to the measured value.

[0112] In some further embodiments, the kit may also include a computer-readable medium containing computer-executable instructions for the performance of any of the methods of the present disclosure.

[0113] In addition to reagents for assaying a sample for the combination of biomarkers described above, the kit may also include reagents for assaying the sample for other biomarkers, particularly for one or more biomarkers associated with any disease other than the cancer in question, such as other cancers. Thus, the kit may be used not only to screen for, diagnose, prognose, or monitor cancer, but also, for example, to screen for, diagnose, prognose, or monitor other cancers, depending on the specificity and sensitivity of the one or more other biomarkers whose concentrations are assayed.

[0114] As will be readily understood by those skilled in the art, various details and embodiments of the present methods also apply to the present kits, and therefore, the properties and characteristics of suitable nanoparticles, for example, with respect to the kits, will not be repeated herein.

[0115] Use of MGL and / or WGA (optionally in any combination with DSL and / or Gal4), or any composition (such as a nanoparticle composition comprising the same) is also provided for determining the status of breast cancer in a subject, or for screening, diagnosing, prognosing, or monitoring breast cancer in a subject. Any details and particulars disclosed with respect to the methods and embodiments thereof apply to various uses of these biomarkers, even if the details and particulars are not repeated herein.

[0116] Use of MBL, DC-SIGN and / or MGL, or any composition (such as a nanoparticle composition comprising the same) is also provided for determining the status of colorectal cancer in a subject, or for screening, diagnosing, prognosing or monitoring colorectal cancer in a subject. Any details and particulars disclosed with respect to the method and its embodiments apply to various uses of these biomarkers, even if the details and particulars are not repeated herein.

[0117] Use of DC-SIGN, or any composition (such as a nanoparticle composition comprising the same), is also provided for determining the status of, or for screening, diagnosing, prognosing, or monitoring pancreatic cancer in a subject. Any details and particulars disclosed with respect to the present methods and embodiments thereof apply to various uses of these biomarkers, even if the details and particulars are not repeated herein.

[0118] Use of MGL, or any composition (such as a nanoparticle composition comprising the same), is also provided for determining the status of prostate cancer in a subject, or for screening, diagnosing, prognosing, or monitoring prostate cancer in a subject. Any details and particulars disclosed with respect to the method and its embodiments apply to various uses of these biomarkers, even if the details and particulars are not repeated herein.

[0119] The use of various other lectin-binding biomarker species and any combination thereof is also provided, any details and characteristics of such uses being apparent from the present disclosure above.

[0120] It will be obvious to those skilled in the art that as technology advances, the inventive concept can be implemented in various ways. The present invention and its embodiments are not limited to the examples described below, but may vary within the scope of the claims. [Example]

[0121] Breast cancer Example 1: Materials and Methods CA15-3 origin and clinical samples Cancerous CA15-3 from a primary breast cancer (BrCa) cell line was obtained from Fujirebio Diagnostics (Sweden).

[0122] All clinical samples were provided by the University of Tampere (Finland) with appropriate authorization and informed consent in accordance with the ethical guidelines of the Hospital District of Pirkanmaa. The clinical samples included a cohort of longitudinal plasma samples (n = 199) from 45 different BrCa patients and serum / plasma samples from apparently healthy women as controls (HC, n = 31).

[0123] Anti-CA15-3 antibody Two different monoclonal anti-CA15-3 antibodies (i.e., Ma552 and Ma695, which detect the protein core and sialylated carbohydrate epitopes of the MUC-1 (CA15-3) antigen, respectively) were obtained from Fujirebio Diagnostics (Sweden).

[0124] For use as solid-phase capture agents, antibodies were biotinylated with a 40-fold molar excess of biotin isothiocyanate for 4 hours at room temperature (RT) using standard procedures known in the art. 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.

[0125] Lectins A panel of 15 plant lectins was purchased from VECTOR lab, and a panel of 13 human lectins was purchased from RnD System. [Table 1]

[0126] The lectin was immobilized onto monodisperse, carboxyl-modified Fluoro-Max™ polystyrene nanoparticles (97 nm diameter) doped with europium chelates, obtained from Thermo Scientific Seradyn Inc. (Indianapolis, IN). The nanoparticles used produce long-lived fluorescence equivalent to 30,000 chelate ions per particle.

[0127] The primary amino groups of the lectin were covalently coupled to the activated carboxyl groups of the nanoparticles using a previously described procedure with some minor modifications (Soukka et al., Anal. Chem. 2001, 73, 2254-2260). 12 The nanoparticles (particles) were suspended in 10 mmol / L phosphate buffer (pH 7.0), and their surfaces were 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 lectin concentration in the conjugation reaction was 0.625 mg / mL, and the reaction contained 100 mmol / L NaCl. The activated particles were mixed with the lectin. The conjugation reaction was incubated at 23°C for 2 hours with vigorous shaking. 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 the first instance of use, the particles were thoroughly mixed, sonicated and briefly centrifuged (350 g, 5 min) to separate non-colloidal aggregates from the monodisperse suspension.

[0128] Conventional CA15-3 immunoassay Serum CA15-3 concentrations were analyzed by ELISA using the CanAg CA15-3 EIA kit (Fujirebio Diagnostics) according to the manufacturer's instructions. Briefly, a biotinylated capture monoclonal antibody (bioMa695) and an HRP-conjugated tracer monoclonal antibody (Ma552) were added to streptavidin (SAv)-coated microtiter wells along with a 1:41 dilution of calibrators and clinical serum / plasma samples. After a 2-hour incubation, the wells were washed six times, the substrate TMB was added, and the optical density was measured at 450 nm after the addition of a stop solution. The basic principle of this conventional CA15-3 immunoassay is illustrated in Figure 1A.

[0129] Anti-CA15-3 antibody-lectin nanoparticle sandwich assay The Red assay buffer, wash buffer, and streptavidin-coated low-fluorescence microtiter plates used in these experiments were purchased from Kaivogen Oy (Turku, Finland).

[0130] Biotinylated solid-phase antibodies (100 ng) (i.e., bioMa695 or bioMa552) were immobilized onto streptavidin-coated microtiter wells in 40 μL of assay buffer. After 1 h of incubation at RT and shaking at 900 rpm, the wells were washed twice with washing solution and immediately used in the assay.

[0131] Next, 50 μl of diluted sample (1:40 in assay buffer) was added to each well and incubated for 1 hour at RT with shaking. The CA15-3 antigen captured on the well was then subjected to Eu staining to detect lectin-binding glycan epitopes of CA15-3. 3+ Labeled lectin-nanoparticles were used for analysis by time-resolved fluorescence (TRF), which was carried out as follows: 1e coated with various lectins 7 EU 3+25 μl of assay buffer containing NPs was added to each well with an additional 6 mM CaCl for CLRs (DC-SIGN, MGL, MBL, MMR) and incubated for 2 hours at room temperature with shaking. After incubation, wells were washed six times with wash buffer. Time-resolved fluorescence for europium was measured from the dried wells using a Victor3V 1420 Multilabel counter (lex: 340 nm; lem: 615 nm).

[0132] Example 2: Anti-CA15-3 antibody-lectin nanoparticle sandwich assay In these experiments, 5, 10, or 100 U / ml of CA15-3 (Fujirebio Diagnostics) purified from a BrCa cell line was spiked into TSA-BSA (7.5% BSA in Tris-sodium azide). BrCa-associated CA15-3 was first captured with either biotinylated Ma552 or Ma695 anti-CA15-3 antibodies on streptavidin-coated microtiter wells. A panel of plant or human lectins coated on Eu-doped NPs was used as a tracer for screening BrCa-CA15-3-recognizing lectins, as illustrated schematically in Figure 1B and described in more detail in Example 1.

[0133] Among the lectins tested, the human lectin MGL and the plant lectin WGA showed excellent reactivity with the BrCaCA15-3 antigen. The signal-to-background ratio (S / B) was greater than 2 at a rate of 5 U / ml for both lectin NPs (Figure 2). DSL and Gal-4 also showed binding to BrCaCA15-3, but the S / B ratios were lower than those for WGA and MGL. Corresponding results were obtained regardless of whether Ma552 or Ma695 was used as the capture antibody.

[0134] Due to their significantly higher binding levels to the BrCa-CA15-3 antigen, MGL-NPs and WGA-NPs were selected for the development and optimization of the CA15-3 lectin assay.

[0135] Next, we determined the recovery of BrCa-derived CA15-3 in a complex matrix (i.e., human serum). To this end, small amounts of CA15-3 at 5–100 U / ml were spiked in parallel into either pooled serum from healthy men or a simple TSA-BSA buffer. CA15-3 was first captured by biotinylated Ma552 or Ma695 monoclonal antibodies on a streptavidin microtiter plate, and after washing, the recovery rate was finally investigated by tracing with MGL-NPs. Excellent recovery (95–110%) was achieved with an estimated analytical sensitivity of 1 U / ml (Figure 3). The results indicate that inherent serum components do not interfere with the assay.

[0136] Comparably excellent recovery and analytical sensitivity were observed with WGA-NPs (Figure 4). In these experiments, 5–250 U / ml of BrCa-derived CA15-3 was spiked into pooled serum from healthy men, biotinylated Ma552 was used as the capture antibody, and WGA NPs were used as the tracer.

[0137] To confirm the accuracy of the results, CA15-3 MGL Assay and CA125 MGL The cross-reactivity of the antibodies used in the assay was tested (Figure 5). Cross-reactivity with ovarian cancer-derived CA125 was as follows: CA15-3 MGL No cross-reactivity with the BrCa-CA15-3 antigen was found in the assay, in which anti-CA15-3 monoclonal antibody was used as a capture agent (Fig. 5A and 5B). On the other hand, some cross-reactivity with the BrCa-CA15-3 antigen was observed in the CA125 MGL In an assay in which an anti-CA125 monoclonal antibody was used as the capture antibody, a 10-fold difference between the measured concentrations of CA125 and CA15-3 was detected (Figure 5C).

[0138] Example 3: Analysis of clinical samples To test whether the results obtained with the CA15-3 antigen based on breast cancer cell lines could be translated to the clinical situation, a small cohort of clinical samples was analyzed in parallel by the present lectin-NP assay and by a conventional CA15-3 immunoassay.

[0139] Boxplot analysis Their CA15-3 protein content (conventional) and CA15-3 MGL Box plot analysis of clinical breast cancer samples (n=199 from 45 different breast cancer patients) and healthy controls (n=31) for glycoform content is shown in Figures 6A-6C. Results show that the conventional CA15-3 IA also detected CA15-3 in healthy women. While the conventional CA15-3 IA detected a median difference of 2-fold between healthy controls and breast cancer patients, the present CA15-3 MGL The assay detected a >200-fold difference between these clinical groups (Figure 6). Corresponding results were obtained regardless of whether Ma552 or Ma695 was used as the capture antibody. Notably, however, when Ma552 was used as the capture antibody, 77% (24 / 31) of healthy controls had this CA15-3 MGL The assay was undetectable (ie, 0 U / ml).

[0140] Another series of box plot analyses were performed using only the first serum sample for each breast cancer patient (n=45) and all control samples (n=31). In these analyses, the conventional CA15-3 IA detected a median 2.8-fold difference between healthy controls and breast cancer patients, while the present CA15-3 IA did not detect a median difference between healthy controls and breast cancer patients, regardless of whether Ma552 or Ma695 was used as the capture antibody. MGL The assay detected a >400-fold difference between these clinical groups (Fig. 7).

[0141] CA15-3 WGAThe assay was also subjected to box plot analysis. For practical reasons, the first serum samples from 41 breast cancer patients only and 18 control samples only were used in the analysis. The results showed that CA15-3 WGA It is clearly indicated that breast cancer patients can be successfully distinguished from healthy controls using the method (Figure 8).

[0142] ROC curve and AUC analysis ROC curve analysis for discriminating between breast cancer and healthy controls was performed using CA15-3, CA15-3 MGL , and CA15-3 WGA The ROC curves are shown in Figures 9 and 10, while the AUC values ​​obtained are summarized below. [Table 2] [Table 3]

[0143] According to the results, CA15-3 MGL and CA15-3 WGA Both assays were able to discriminate better between breast cancer patients and healthy controls than the conventional CA15-3 immunoassay.

[0144] Assessing success rates Markers CA15-3 and CA15-3 MGL The success rate for CA15-3 was determined for breast cancer cases (n=45, first consecutive sample from each patient). The following cutoff values ​​were used: 25 IU / ml for CA15-3 and 100 IU / ml for CA15-3. MGL A 2 IU / ml (for 1000kJ / mL) was used to classify serum samples as negative or positive for each marker, respectively. [Table 4]

[0145] These results suggest that CA15-3 is a useful tool for accurately identifying breast cancer patients as affected individuals in the clinical cohort used. MGL The success rate of the CA15-3 immunoassay was noted to be superior (71.5%) compared with that of the conventional CA15-3 immunoassay (60%). The combined use of the assays improved the success rate to 77%.

[0146] Correlation between different assays As shown in Figure 2, both MGL and WGA (CA15-3, respectively) when immobilized on Eu(III) nanoparticles MGL Assay and CA15-3 WGA The assay) was able to recognize BrCa-associated CA15-3. However, when the assay was applied to clinical samples, the results did not correlate well (Figure 11). This result suggests that the glycosylation pattern of CA15-3 varies among different breast cancer patients, and that the CA15-3 MGL and CA15-3 WGA It is noted that assaying for both may enhance sensitivity by reducing the false negative rate.

[0147] Furthermore, CA15-3 MGL The correlation between the assay and conventional CA15-3 immunoassay (IA) was very poor in clinical samples from BrCa patients (n=199), as demonstrated in Figure 12 (R 2 =0.26). Again, this result indicates that each of the assays detects CA15-3 differently, but can be used to complement each other.

[0148] Furthermore, CA15-3 WGA The assay correlated poorly with the conventional CA15-3 immunoassay (Figure 13). However, the correlation was not significant with the CA15-3 MGL There was better correlation between the assay and conventional CA15-3 immunoassay.

[0149] Example 4. Further analysis of clinical samples A cohort of metastatic breast cancer patients and healthy controls were screened for CA15-3 lectins by immobilizing the lectin onto detectable nanoparticles or by direct Eu chelate labeling. WGA and CA15-3 MGL We analyzed the following:

[0150] CA15-3 WGA and CA15-3 MGL Eu nanoparticle assay Using baseline plasma samples from metastatic breast cancer patients (n = 54) and healthy women (n = 23) as controls, receiver operating characteristic curves (ROC) curves were generated for both lectin-based biomarker assays and the conventional CA15-3 assay. The AUC (area under the curve) of both lectin-based biomarker assays was superior to that of the conventional assay (AUC = 0.833), and significantly superior to that of CA125. WGA The correlation coefficient (CI) of the CA15-3-lectin (MGL and WGA) nanoparticles was 0.939 (Figure 14). Therefore, this new CA15-3-lectin (MGL and WGA) nanoparticle concept can substantially increase the clinical sensitivity compared to conventional CA15-3 immunoassays without affecting specificity.

[0151] CA15-3 WGA-Eu chelate-based assay Among the numerous lectin nanoparticles investigated, only recombinant human MGL NPs and the plant lectin WGA NPs demonstrated good performance for detecting BCa cell line-associated CA15-3 glycoforms. Because the performance of the WGA NPs assay was superior to that of the MGL NPs assay, and because WGA is a plant lectin and much cheaper than recombinant MGL, WGA was directly labeled with a soluble Eu chelate. The directly labeled WGA performed as well as the WGA nanoparticles (see the table below).

[0152] Lectins are well known to have low affinity. Therefore, Eu-NPs were used to increase their binding affinity via the avidity effect. However, at least WGA works very well with soluble Eu-chelate labeling (without the need for affinity improvement). Without being limited to any theory, this may be due to the good affinity of WGA itself, and also because the WGA-reactive glycan epitope (GlcNAc) exists at multiple positions on CA15-3, a large 200–1000 kDa glycoprotein. Therefore, many molecules of WGA-Eu chelate can bind to a single CA15-3, resulting in sensitivity approaching that of WGA-NPs. [Table 5] [Table 6]

[0153] ROC curves were calculated using baseline plasma samples from metastatic breast cancer patients (n=53) and healthy female control samples (n=20). WGA NPs-based biomarker assay, CA15-3 WGA The WGA-based biomarker assays labeled with either nanoparticles or Eu chelates were superior to the conventional assay (AUC = 0.826). The best performance was observed for the Eu nanoparticle-based CA15-3 assay. WGA (0.926). However, Eu chelate-based CA15-3 WGA The AUC (AUC=0.890) was close to that of the nanoparticle-based assay (AUC=0.926) (Figure 15).

[0154] colorectal cancer Example 5. CEA Glycan Variant Lectin Nanoparticle Assay for Colorectal Cancer Materials and Methods Clinical samples EDTA plasma samples from patients with colitis (n = 14) and colorectal cancer (n = 34) were purchased from the Auria biobank (Turku, Finland). Among colorectal cancer (CRC) patients, 23 EDTA samples were collected before treatment and analyzed quantitatively using a developed CEA glycosylation variant lectin-nanoparticle assay and a commercial CEA immunoassay (Fujirebio Diagnostics Ltd., Gothenburg, Sweden). EDTA plasma samples from CRC patients with CEA < 5 ng / ml (n = 23) were compared to healthy volunteers (n = 11) with CEA in the same range.

[0155] material Human DC-sign (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) fused with human IgG1Fc (immunoglobulin G1 fragment, crystallizable) and human MBL (mannose-binding lectin) were purchased from R&D Systems, Inc. (Minneapolis, Minnesota, USA). Human MGL (macrophage galactose-type lectin) was from Sino Biological, Inc. (Beijing, China). Anti-CEA mAb (monoclonal antibody) 12-140-1 and one CEA preparation were obtained from Fujirebio Diagnostics Ltd. (Goteborg, Sweden). Anti-CEA mAb T84.66 and a CEA preparation were kindly donated by Kjell Nustad of Norwegian Radium Hospital (Oslo, Norway). Another CEA preparation was purchased from Hytest Ltd. (Turku, Finland). Yellow streptavidin-coated 96-well microtiter plates were specially made by Kaivogen Ltd. (Turku, Finland). RED assay buffer was purchased from Kaivogen Ltd. The DELFIA plate shaker, DELFIA plate washer, and Victor™ fluorometer were manufactured by Wallac Oy (Turku, Finland). Bromelain solution ID-dilution 1 was from DiaMed (Cressier FR, Switzerland). NAP-5 and NAP-10 buffer exchange columns were purchased from GE Healthcare (Chicago, Illinois, USA).

[0156] method Fragmentation of captured material To obtain the 12-140-1 Fab2 (fragment, antigen-binding) antibody fragment, 1.15 mg of 12-140-1 mAb was buffer exchanged into 0.9% NaCl using a NAP-5 column and digested for 2 hours at +37°C by incubation with ID-diluted material 1 (50 μl per mg of mAb) in reaction buffer (50 mM Tris-HCl (pH 7.0), 100 mM NaCl, 3 mM EDTA). Digestion was stopped by adding 0.2 M N-ethylmaleimide (NEM) to a final concentration of 0.02 M NEM. The Fab2 antibody fragment was purified using protein G affinity purification.

[0157] Biotinylation The buffer of the fragmented mAb and whole anti-CEA mAb was exchanged into 0.9% NaCl. The antibody was conjugated with biotin by adding a 40-fold molar excess of BITC (biotin isothiocyanate) and 1 / 10 of the total reaction volume in 500 mM Na2CO3 buffer (pH 9.8). The reaction was incubated at RT for 4 hours away from direct light. The mixture was purified by two buffer exchanges into TSA buffer (50 mM Tris-HCl (pH 7.75), 150 mM NaCl, 0.05% NaN3), and 1% DTPA (diethylenetriaminepentaacetic acid)-treated BSA (bovine serum albumin) was added to all mixtures.

[0158] Preparation of biotinylated 12-140-1 Fab2 spots Biotinylated 12-140-1 Fab2 (30 μg / mL) was printed in an array-in-well format onto a yellow streptavidin-coated 96-well microtiter plate (Kaivogen, 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.

[0159] Lectin nanoparticle assay Lectin was coated onto europium-doped nanoparticles as described in Example 1. All assays were performed on yellow streptavidin-coated 96-well microtiter plates. Triplicate samples with a total volume of 25 μl per well were used in all assay incubations, which were performed at room temperature with gentle shaking. CEA standards were prepared by mixing equal amounts of all three CEA preparations together and diluting the mixture to concentrations of 200, 100, 50, 15, 5, 2, and 0 ng / ml in TSA-BSA buffer (50 mM Tris-HCl (pH 7.75), 150 mM NaCl, 0.05% NaN3, 0.1% BSA). Biotinylated mAbs 12-140-1 and T84.66 were added at 50 ng per well in the RED assay and immobilized by 1-hour incubation for use as captures in the MBL and DC-sign assays, respectively. Biotinylated 12-140-1 Fab as spots in streptavidin-coated microtiter plates 2was used as a capture in the human MGL assay. Samples and standards were prepared in buffer A (50 mM Tris-HCl (pH 7.75), 175 mM CaCl, 350 mM NaCl, 37.5 U / ml heparin, 100 μM DTPA, 0.01% Tween 40, 0.5 mg / ml bovine γ-globulin, 5 mg / ml BSA) for the MBL and DC-sign assays, and in buffer B (50 mM Tris-HCl (pH 7.75), 175 mM CaCl, 350 mM NaCl, 37.5 U / ml heparin, 100 μM DTPA, 0.01% Tween 40, 0.5 mg / ml bovine γ-globulin, 5 mg / ml BSA) for the human MGL assay. The lectin nanoparticles were diluted 5-fold in buffer B (50 mM Tris-HCl (pH 7.75), 175 mM CaCl, 350 mM NaCl, 5 μg / ml native mouse IgG, 5 μg / ml HBR-2, 5 μg / ml MAK-33, 105 U / ml heparin, 100 μM DTPA, 0.01% Tween 40, 0.5 mg / ml bovine γ-globulin, 5 mg / ml BSA). After washing the wells twice, samples and standards were added and incubated for 1 hour. The lectin nanoparticles were diluted in RED assay buffer supplemented with 3 mM CaCl for the MBL assay and DC-sign assay, and 12 mM CaCl for the human MGL assay. The lectin nanoparticle concentrations added were 20 × 10 per well for the MBL assay, DC-sign assay, and MGL assay, respectively. 6 , 15×10 6 , and 35 × 10 6 The particles were collected. The wells were washed twice and the nanoparticle solution was added. The plate was incubated for 2 hours and washed six times. The time-resolved fluorescence of europium was measured at a wavelength of 615 nm using an excitation wavelength of 340 nm. The measurement cycle was 1000 μs with a 400 μs delay and a 400 μs measurement window time.

[0160] result EDTA plasma from CRC patients with CEA <5 ng / ml was analyzed by commercial CEA immunoassay as well as CEA DC-Sign Glycosylation variant assay and CEA MGLUsing the glycovariant assay, comparisons were made to healthy volunteers with CEA in the same range (Figure 16). CRC patients and healthy controls with comparable plasma CEA levels were differentially detected by the lectin glycovariant CEA assay, with increased amounts of CEA glycoforms in CRC. Seven of the healthy controls had CEA MGL It was undetectable by the assay.

[0161] Conventional CEA values ​​from patients with colitis or CRC were plotted (Figure 17). The Mann-Whitney U test with 95% confidence intervals was the test used to determine statistical significance. The differences between the two groups in Figure 17A were significant for all assays. When using the commercial CEA immunoassay, only 10 of 23 CRC patients (sampled before treatment) had plasma CEA values ​​above the reference range of 5 ng / ml, and 13 CRC patients had false negatives. The glycosylation variant CEA assay significantly reduced the number of false negatives by 20% compared with the CEA MBL Assay and CEA MGL 6 by assay, as well as CEA DC-Sign The assay could reduce it to 8.

[0162] When the CRC group was divided into subgroups (patients alive after follow-up (n=13) and patients who died after follow-up (n=10)) (Figure 17B), all remained significant except for the differences between the colitis group and the "survival" group by conventional CEA and MGL assays.

[0163] Receiver operating characteristics (ROCs) were calculated for all assays (Figure 18). The area under the curve (AUC) was 0.823 for the conventional CEA assay. The AUCs of the regression models combining the values ​​of the MBL assay and the three lectin assays were higher than that of the conventional assay, 0.837 and 0.848, respectively. The AUCs of the MGL assay and DC-sign assay were lower than that of the conventional assay, 0.758 and 0.817, respectively.

[0164] Among the 32 lectins tested, MBL, DC-SIGN, and MGL showed the highest reactivity and low background for cancer CEA antigen in EDTA plasma.

[0165] Improvements of the lectin CEA assay compared to commercial CEA immunoassays Different glycosylation variants of CEA assay (CEA DC-Sign , CEA MGL , or CEA MBL ) can improve the colorectal cancer specificity of the CEA tumor marker. CRC patients and healthy controls with plasma CEA levels within the normal range were differentially detected by the lectin glycovariant CEA assay, and the amount of CEA glycoforms was increased in CRC. The number of false negatives was significantly higher than that of CEA. DC-Sign , CEA MGL or CEA MBL was significantly reduced using the assay.

[0166] Pancreatic cancer Example 6. CA19-9 Glycan Variant Lectin-Nanoparticle Assay Materials and Methods Clinical and control samples EDTA plasma samples from 16 healthy young volunteers were collected at the Department of Biotechnology, University of Turku with appropriate authorization and informed consent. EDTA plasma samples from 11 control samples from patients diagnosed with gastrointestinal diseases and frequently measured with elevated CA19-9 levels were purchased from Auria Biobank (Turku, Finland). Among these controls, five patients had liver fibrosis and cirrhosis, two patients had chronic hepatitis, one patient was diagnosed with alcoholic liver disease, and three patients were diagnosed with other diseases of the bile duct. Ten samples from pancreatic cancer patients were purchased from Auria Biobank, along with additional EDTA plasma samples from 16 controls with CA19-9 levels >5 U / mL (range up to 4852 U / mL) and no cancer of any kind. Among the pancreatic cancer patients, eight were classified as having adenocarcinoma, one as having neuroendocrine tumor, and two as having metastatic cancer. All 11 patients with gastrointestinal disease and 16 controls with CA19-9 >5 U / mL were judged as benign controls.

[0167] material Anti-CA19-9 mAb (monoclonal antibody) c241 was obtained from Fujirebio Diagnostics Ltd. (Goteborg, Sweden). Human DC-sign (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) fused with human IgG1Fc (immunoglobulin G1 fragment, crystallizable) was purchased from R&D Systems, Inc. (Minneapolis, Minnesota, USA). Yellow streptavidin-coated 96-well microtiter plates were specially made by Kaivogen Ltd. (Turku, Finland). RED assay buffer was purchased from Kaivogen Ltd. The DELFIA plate shaker, DELFIA plate washer, and Victor™ fluorometer were manufactured by Wallac Oy (Turku, Finland).

[0168] Equal amounts of CA19-9 antigen from Fujirebio Diagnostics Ltd. and HyTest Ltd. (Turku, Finland) were pooled and used as calibrators. Dilutions of 1, 5, 10, 20, 50, and 100 U / mL were made in Tris-saline-azide buffer (50 mmol / L Tris, 150 mmol / L NaCl, 0.5 g / L NaN3 (pH 7.7)) containing 5 g / L BSA and used in triplicate.

[0169] method The developed CA19-9 glycovariant lectin-nanoparticle assay was performed in a three-step sandwich format, in which anti-CA19-9 monoclonal antibody was used as the capture and lectin-coated nanoparticles as the tracer. All subsequent steps were performed at room temperature, with incubations performed by slow shaking.

[0170] Biotinylated anti-CA19-9 monoclonal antibody c241 (75 ng / well), used as a capture antibody, was immobilized on streptavidin-coated wells in 25 μL of RED assay buffer with a 1-hour incubation. After two washes, 5 μL of either sample or calibrator and 20 μL of Red assay buffer supplemented with 50 mM CaCl2 were added in triplicate and incubated for 1 hour. The wells were washed twice and Eu(III)-chelate dye nanoparticles (1.0 × 10 7 DC-sign coupled to nanoparticles (particles / well) was added in 30 μl of RED assay buffer supplemented with 12.5 mM CaCl. After a 2-hour incubation of the nanoparticles, the wells were washed six times and the TRF of the bound nanoparticle bioconjugates was measured directly from the well surface.

[0171] A commercial CA19-9 immunoassay kit (Fujirebio Ltd) was used as a reference method to compare the developed CA19-9 glycovariant lectin-nanoparticle assay.

[0172] result The median concentrations of CA19-9 glycosylated variant lectin nanoparticles were 20.8 U / mL (interquartile range: 9.45, 38 U / mL) for pancreatic cancer patients, 5.9 U / mL (interquartile range: 2.6, 16.8 U / mL) for benign patients, and 4.9 U / mL (interquartile range: 2.7, 6.8 U / mL) for healthy volunteers. The CA19-9 glycosylated variant lectin nanoparticle assay improved the discrimination of pancreatic cancer patients from healthy controls (p = 0.0001) and benign controls (p = 0.028) compared with the conventional CA19-9 immunoassay, in which borderline statistically significant differences were observed only between pancreatic cancer patients and healthy controls (p = 0.041) (Figure 1). There was no statistically significant difference between pancreatic cancer patients and benign controls using the conventional CA19-9 immunoassay (p = 0.105).

[0173] Furthermore, only 3 of 10 pancreatic cancer patients had CA19-9 values ​​above the recommended cutoff of 37 U / mL in the conventional CA19-9 immunoassay, resulting in 70% (7 / 10) of the pancreatic cancer patients tested being determined as false negatives.Using the CA19-9 glycosylated variant lectin-nanoparticle assay with a cutoff of 10 U / mL, 8 of 10 pancreatic cancer patients were correctly determined as positive, significantly improving sensitivity.

[0174] Of the 27 lectins tested, DC-SIGN was the most reactive with the cancerous CA19-9 antigen and showed good performance when using either EDTA plasma or serum.

[0175] CA19-9 Glycan Variant Lectin - Improvement by Nanoparticle Assay The CA19-9 glycovariant lectin-nanoparticle assay improved the discrimination of pancreatic cancer patients from healthy controls and benign controls (patients with benign liver or bile duct disease) compared with the conventional CA19-9 immunoassay. Furthermore, the CA19-9 glycovariant assay reduced the number of false negatives and significantly improved sensitivity.

[0176] Prostate cancer Example 7. PSA Glycosylated Variant Lectin-Nanoparticle Assay Reagents and equipment Seminal plasma PSA from healthy donors was obtained with appropriate permission and informed consent from the Department of Clinical Chemistry, Lund University Hospital (Malmo, Sweden). PSA purified from a prostate cancer cell line (LNCaP) was from the Department of Biotechnology, University of Turku. Human MGL (macrophage galactose-type lectin) was from Sino Biological, Inc. (Beijing, China). Unconjugated WGA (wheat germ agglutinin) was from Vector Laboratories (Peterborough, UK). Recombinant human Gal4 (galectin 4) and Dectin-2 were from R&D Systems, Inc. (Minneapolis, Minnesota, USA). Streptavidin-coated microtiter wells, wash buffer concentrate, and RED assay buffer were purchased from Kaivogen Ltd. (Turku, Finland). The DELFIA plate shaker, DELFIA plate washer, and Victor™ fluorometer were manufactured by Wallac Oy (Turku, Finland).

[0177] LNCaP PSA was used as a calibrator, and dilutions (1–200 μg / L LNCaP PSA) were made in Tris-saline-azide buffer (50 mmol / L Tris, 150 mmol / L NaCl, 0.5 g / L NaN3, pH 7.7) containing 5 g / L BSA and run in triplicate.

[0178] Clinical and control samples Prostate tissue samples were obtained immediately after radical prostatectomy (RP) from 36 patients with PCa (12 histologically benign and 24 cancerous, graded Gleason 2–4) at Turku University Hospital (Finland). Sample tissues were stored at −20°C in 1 ml of phosphate-buffered saline (PBS) buffer supplemented with protease inhibitors (cOmplete tablets, Roche Diagnostics, Manheim, Germany) until further processing. Protein pools were recovered by adding 1 / 10 of the sample volume to 10x PBS lysis buffer (pH 7.4, 20 mM EDTA, 10% Triton-X-100 supplemented with protease inhibitors) using Triton-X-100 detergent from Acros Organics, ThermoFischer Scientific, Geel, Belgium. Lysis was performed on ice for 2 h under slow shaking, and the supernatant was stored at −20°C. For analysis from each sample, a 1:10 dilution was made in Tris-saline-azide buffer containing 5 g / L BSA.

[0179] Urine samples were obtained from Turku University Hospital. The study cohort included 143 consecutive, prospectively enrolled men with clinical suspicion of prostate cancer (PCa) and was registered with the IMPROD trial (ClinicalTrials.gov identifier NCT01864135). EDTA-plasma was obtained from 45 men enrolled in the IMPROD trial. Urine samples from 11 male volunteers (<35 years of age) were collected at the Department of Biotechnology, University of Turku.

[0180] method Preparation of biotinylated 5A10 Fab spots Biotinylated 5A10 Fab (100 μg / mL) was printed in an array-in-well format onto a yellow streptavidin-coated 96-well microtiter plate (Kaivogen, 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.

[0181] Lectin nanoparticle assay for tissue lysates Developed PSA MGL The glycovariant lectin-nanoparticle assay was performed in a three-step sandwich format, with all incubations performed at room temperature with slow shaking. Biotinylated free PSA-specific recombinant Fab fragments (150 ng / well), used as capture antibodies, were immobilized on streptavidin-coated wells in 100 μL of RED assay buffer for 1 hour of incubation. After two washes, 50 μL of either tissue lysate dilutions or calibrators and 50 μL of Red assay buffer supplemented with 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. The wells were washed twice, and Eu(III)-coupled MGL-chelating dye nanoparticles (2.0 × 10 7 Nanoparticles (particles / well) were added in 100 μl of RED assay buffer supplemented with 3 mM CaCl. After a 2-hour incubation of the nanoparticles, the wells were washed six times and the time-resolved fluorescence (TRF) of the bound nanoparticle bioconjugates was measured directly from the well surface.

[0182] PSA secreted by LNCaP cells and seminal plasma PSA from healthy donors were analyzed using the developed PSA assay. MGLA glycovariant lectin-nanoparticle assay was used to study the fluorescence from different PSA forms in the concentration range of 1-180 ng / ml is shown in Figure 20. The assay was performed as for tissue lysates, except that 25 μl of different PSA concentrations and 75 μl of RED assay buffer were used in the sample incubation.

[0183] Lectin nanoparticle assay for urine and EDTA plasma samples Biotinylated 5A10 Fab (100 μg / ml) spotted onto a streptavidin-coated microtiter plate was used to detect glycosylation variant fPSA. MGL Used as capture in the assay. デクチン2 fPSA WGA Biotinylated 5A10 Fab (80 ng / well) for assay, and total PSA Gal4 For the assay, H117 mAb (120 ng / well) was added onto streptavidin-coated microtiter plates in RED assay buffer and incubated for 1 hour.

[0184] After two washes, 20 μL of either sample or calibrator and 30 μL of Red assay buffer supplemented with 5 μg / ml native mouse IgG, 5 μg / ml HBR-2, and 5 μg / ml MAK-33 were added to the fPSA plate. MGL For other assays, 10 μl of sample or calibrator and 40 μl of assay buffer were used. Samples and calibrators were added in triplicate and incubated for 1 hour. Lectin nanoparticles were diluted in RED assay buffer containing 6 mM CaCl. After two washes, the added lectin nanoparticle concentrations were 0.01 for the MBL assay, 0.01 for the fPSA assay, 0.01 for the fPSA assay, and 0.01 for the MBL assay. MGL , fPSA デクチン2 , fPSA WGA and all PSA Gal4 7 x 10 per well for the assay 6 , 4×10 7 , 5×10 6 , and 2 × 107 The plates were washed six times, incubated for 2 hours, and TRF was measured.

[0185] result Seminal plasma PSA from healthy donors and PSA derived from a prostate cancer cell line (LNCaP) were used to develop PSA. MGL It is differentially detected by the glycovariant assay because this assay detects only cancerous PSA (Figure 20).

[0186] PSA from 12 benign and 24 cancerous prostate tissue lysates measured using a nanoparticle-based lectin immunoassay (fPSA) in a whole-well format. MGL Tumor tissues were studied using PSA assays compared to benign tissues from PCa patients. MGL There was a statistically significant (p=0.002) increase in glycosylation variants (Figure 21). All benign tissue samples were from PCa patients with normal prostate tissue microscopy, making it highly likely that glycosylation changes may already occur on PSA in benign tissues. MGL The glycovariant assay results in a measurable fluorescent signal.

[0187] Urine samples from 143 men with clinical suspicion of PCa in a prospective controlled trial (IMPROD, NCT01864135) were analyzed for fPSA with captured 5A10 Fab spotted onto streptavidin-coated microtiter plates. MGL Glycovariant assays were used to analyze PSA. Of the 143 men, 74 were diagnosed with clinically significant PCa (Gleason score ≥ 7), 20 men had PCa with a Gleason score of 6, and 49 had negative prostate biopsies (referred to as biopsy-negative). In addition, urine samples from 11 young, healthy men were included. PSA from urine PSA was MGLThe total PSA-corrected fluorescence signal of the glycovariant assay is presented in Figure 22. There was a statistically significant (p=0.006) increase in the median urine PSA glycovariant concentration in men with clinically significant PCa compared to men with negative PCa biopsies or Gleason score 6 PCa. Urinary PSA glycovariant content appeared to be further elevated in the more aggressive Gleason score 9 prostate cancer. Although urinary PSA concentrations were in the same range for all groups tested, urinary PSA from younger men was significantly higher than fPSA. MGL None were detected using the glycovariant assay. Urinary PSA concentrations for patients with clinically significant PCa, negative prostate biopsies or Gleason 6 PCa, and healthy young men are presented in Table 7.

[0188] An additional PSA glycovariant assay with the potential to further improve discrimination of clinically significant prostate cancer is presented in Figure 23. EDTA plasma from men with confirmed PCa or suspected prostate cancer but with negative biopsies was compared to healthy men. PSA WGA , PSA Gal4 , and PSA デクチン2 Our glycovariant assay demonstrated an increased median PSA glycovariant content in men with clinically significant PCa.

[0189] Among the 30 lectins tested, MGL showed excellent ability to discriminate between cancerous and non-cancerous PSA antigens. [Table 7]

[0190] fPSA MGL Improvements through glycosylation variant assays fPSA MGLGlycosylation variant assays can improve the cancer specificity of PSA and aid in the identification of patients with clinically significant PCa. The assay detects glycoforms of PSA present in prostate cancer tissue and urine of PCa patients. Urinary PSA, even at high concentrations, is characterized by the presence of fPSA. MGL It is undetectable in young men by glycosylation variant assays. Using urine samples instead of blood has the advantage that the sample is non-invasive, eliminating the need for personnel for sampling. Because the PSA concentration range is the same in young men, PCa patients, and men with negative PCa biopsies, conventional total or free PSA immunoassays cannot distinguish between them.

[0191] PSA MGL Since the glycovariant assay is suitable for tissue lysates and urine samples, it can be applied to serum samples, as indicated by other tumor markers. The present invention includes the following preferred embodiments. (1) A method for determining prostate cancer disease status in a subject, comprising: assaying a sample obtained from the subject for the level of glycoforms of PSA that bind to macrophage galactose-type lectin (MGL) by determining binding of PSA to MGL; comparing the detected level of the lectin-bound PSA glycoform in the sample with its level in a control sample or with a predetermined threshold value; and determining a prostate cancer disease status in the subject based on said comparison; The above method, comprising: (2) assaying a sample obtained from the subject for PSA protein antigen; and comparing the detected level of the one or more biomarkers in the sample to its level in a control sample or to a predetermined threshold; The method according to (1), further comprising: (3) A method for determining the disease status of colorectal cancer in a subject, comprising: assaying a sample obtained from the subject for the level of CEA glycoforms that bind to one or more lectins selected from the group consisting of mannose-binding lectin (MBL), dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), and MGL by determining binding of CEA to the lectin; comparing the detected level of the lectin-bound CEA glycoform in the sample with that level in a control sample or with a predetermined threshold value; and determining a colorectal cancer disease status in the subject based on said comparison; The method comprising: (4) The method according to (3), wherein the binding of CEA to MBL and DC-SIGN, MBL and MGL, DC-SIGN and MGL, or MBL, DC-SIGN, and MGL is assayed. (5) assaying a sample obtained from the subject for CEA protein antigen; and comparing the detected level of the one or more biomarkers in the sample to its level in a control sample or to a predetermined threshold; The method according to (3) or (4), further comprising: (6) A method for determining the disease status of pancreatic cancer in a subject, comprising: assaying a sample obtained from the subject for the level of glycoforms of CA19-9 that bind to DC-SIGN by determining binding of CA19-9 to DC-SIGN; comparing the detected level of the lectin-binding CA19-9 glycoform in the sample with that level in a control sample or with a predetermined threshold; and determining a prostate cancer disease status in the subject based on said comparison; The method comprising: (7) assaying a sample obtained from the subject for CA19-9 protein antigen; and comparing the detected level of the one or more biomarkers in the sample to its level in a control sample or to a predetermined threshold; The method according to (6), further comprising: (8) A method for determining the disease status of breast cancer in a subject, comprising: assaying a sample obtained from the subject for the level of CA15-3 glycoforms that bind to one or more lectins selected from the group consisting of MGL and wheat germ agglutinin (WGA); comparing the detected level of the lectin-binding CA15-3 glycoform in the sample with that level in a control sample or with a predetermined threshold value; and determining a breast cancer disease status in the subject based on said comparison; The method comprising: (9) assaying a sample obtained from the sample for one or more additional CA15-3 glycoforms that bind to a lectin selected from the group consisting of DSL and Gal4; and comparing the detected level of the one or more additional lectin-binding glycoforms in the sample with its level in a control sample or with a predetermined threshold value; The method according to (8), further comprising: (10) Assaying a sample obtained from the subject for one or more conventional biomarkers selected from the group consisting of CA15-3, CA125, and CEA; and comparing the detected level of the one or more biomarkers in the sample to its level in a control sample or to a predetermined threshold; The method according to (8) or (9), further comprising: (11) The method according to any one of (1) to (10), wherein an increase in the level of the glycoform or the biomarker in the sample compared to the level in a control sample or a predetermined threshold indicates that the subject has or is at risk of having the cancer. (12) The method according to any one of (1) to (11) for screening, diagnosing, predicting the prognosis, or monitoring the cancer. (13) The method according to (12), wherein the monitoring is for monitoring the onset of the cancer, for monitoring any change in the risk of having or developing the cancer, for monitoring response to treatment, for monitoring recurrence of the cancer, or for monitoring recurrence of the cancer. (14) Assaying for the level of the one or more lectin-binding glycoforms of the biomarkers exposing the sample to antibodies specific for the biomarkers to capture the biomarkers contained in the sample; exposing the captured biomarkers to one or more lectins; and measuring the level of binding of said captured biomarkers to said one or more lectins with the aid of a detectable signal; The method according to any one of (1) to (13), wherein the method is carried out by a binding assay comprising: (15) Assaying for the level of the one or more lectin-binding glycoforms of the biomarkers exposing the sample to one or more lectins to capture one or more lectin-binding glycoforms of the biomarkers contained in the sample; exposing the captured biomarker to an antibody specific for the biomarker; and measuring the level of binding of said captured biomarker to an antibody specific for said biomarker with the aid of a detectable signal; The method according to any one of (1) to (14), wherein the method is carried out by a binding assay comprising: (16) The method according to (14) or (15), wherein either the lectin or the antibody is immobilized on a nanoparticle. (17) The method according to any one of (1) to (16), wherein the sample is selected from the group consisting of urine, blood, serum, plasma, peritoneal fluid, and tissue samples. (18) A kit for use in the method according to any one of (1), (2), or (11) to (17), comprising a PSA binding agent and MGL, wherein either the CA15-3 binding agent or the MGL comprises a detectable label. (19) The kit according to (18), further comprising a PSA binding agent. (20) A kit for use in the method according to any one of (3) to (5) or (11) to (17), comprising a CEA-binding agent and one or more lectins selected from the group consisting of MBL, DC-SIGN, and MGL, wherein either the CA15-3-binding agent or the lectins comprises a detectable label. (21) The kit according to (20), comprising MBL and DC-SIGN, MBL and MGL, DC-SIGN and MGL, or MBL, DC-SIGN, and MGL. (22) The kit according to (20) or (21), further comprising a CEA binding agent. (23) A kit for use in the method according to any one of (6), (7), or (11) to (17), comprising a CA19-9 binding agent and DC-SIGN, wherein either the CA19-9 binding agent or the DC-SIGN comprises a detectable label. (24) The kit according to (23), further comprising a CA-19-9 binding agent. (25) A kit for use in the method according to any one of (8) to (17), comprising a CA15-3 binding agent and one or more lectins selected from the group consisting of MGL and WGA, wherein either the CA15-3 binding agent or the one or more lectins comprises a detectable label. (26) The kit according to (25), further comprising a CA-15-3 binding agent. (27) The kit according to any one of (18) to (26), further comprising one or more lectins selected from the group consisting of SBA, SNA, PNA, MAA II, AAL, UEA, PHA-E, RCA, WGA, WFA, PSA, VVL, TJA-II, DSL, HPA, MGL, DC-SIGN, MMR, MBL, Siglec-2, Siglec-3, Siglec-5, Siglec-9, Siglec-10, Siglec-11, galectin-3, galectin-4, E-selectin, DSL, and Gal4. (28) The kit according to any one of (18) to (26), wherein the lectin is immobilized on nanoparticles. (29) The method according to any one of (1) to (17) or the kit according to any one of (18) to (28), wherein all dimensions of the nanoparticles are less than about 1000 nm, about 500 nm or less, about 100 nm or less, or about 50 nm or less. (30) The method or kit according to (29), wherein the nanoparticles are selected from the group consisting of protein nanoparticles, mineral nanoparticles, glass nanoparticles, nanoparticle crystals, metal nanoparticles, plastic nanoparticles, polystyrene nanoparticles, poly(ethylene glycol) nanoparticles, polyethylene nanoparticles, poly(acrylic acid) nanoparticles, poly(methyl methacrylate) nanoparticles, polysaccharide nanoparticles, colloidal gold nanoparticles, silver nanoparticles, quantum dots, carbon nanoparticles, porous silica nanoparticles, and liposomes. (31) The method or kit according to (29) or (30), wherein the nanoparticles are doped with a lanthanide chelate (such as europium(III)). (32) Use of MGL to determine the disease state of prostate cancer in a subject by assaying a sample obtained from the subject for the level of glycoforms of PSA that bind to MGL. (33) Use of MBL, DC-SIGN and / or MGL to determine the disease status of colorectal cancer in a subject by assaying a sample obtained from the subject for the level of CEA glycoforms that bind MBL, DC-SIGN and / or MGL. (34) Use of DC-SIGN to determine the disease status of pancreatic cancer in a subject by assaying a sample obtained from the subject for the level of CA19-9 glycoforms that bind to DC-SIGN. (35) Use of MGL or WGA, either alone or in combination, optionally in combination with DSL and / or Gal4, to determine the disease status of breast cancer in a subject by assaying a sample obtained from the subject for the level of CA15-3 glycoforms that bind to MGL, WGA, DSL, and / or Gal4. (36) The use according to any one of (32) to (35), wherein the lectin is immobilized on a nanoparticle or labeled with a detectable label. (37) The use according to any one of (32) to (36) for screening, diagnosing, predicting the prognosis of, and / or monitoring the cancer in a subject.

Claims

1. 1. A method for aiding in the determination of colorectal cancer disease status in a subject, comprising: assaying a sample obtained from the subject for the level of CEA glycoforms that bind to one or more lectins selected from the group consisting of human mannose-binding lectin (MBL), dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), and macrophage galactose-type lectin (MGL); comparing the detected level of the lectin-bound CEA glycoform in the sample with that level in a control sample or with a predetermined threshold value; and assisting in determining colorectal cancer disease status in the subject based on said comparison; The method comprising:

2. The method of claim 1, wherein the binding of CEA to human MBL, DC-SIGN, or MGL is assayed.

3. The method of claim 1, wherein the binding of CEA to human MBL and DC-SIGN, human MBL and MGL, DC-SIGN and MGL, or human MBL, DC-SIGN and MGL is assayed.

4. Assaying a sample obtained from the subject for a conventional CEA biomarker; and comparing the detected level of said biomarker in said sample to its level in a control sample or to a predetermined threshold; The method of any one of claims 1 to 3, further comprising:

5. 5. The method of any one of claims 1 to 4, wherein an increase in the level of said glycoform or said biomarker in said sample compared to its level in a control sample or a predetermined threshold indicates that said subject has or is at risk of having said cancer.

6. 6. The method of any one of claims 1 to 5 for screening, diagnosing, prognosing or monitoring said cancer.

7. 7. The method of claim 6, wherein the monitoring is for monitoring the onset of the cancer, for monitoring any change in the risk of having or developing the cancer, for monitoring response to treatment, for monitoring the relapse of the cancer, or for monitoring the recurrence of the cancer.

8. Assaying for the level of the one or more lectin-binding CEA glycoforms comprises: exposing the sample to antibodies specific for the biomarkers to capture the biomarkers contained in the sample; exposing the captured biomarkers to one or more lectins; and measuring the level of binding of said captured biomarkers to said one or more lectins with the aid of a detectable signal; The method according to any one of claims 1 to 7, wherein the method is carried out by a binding assay comprising:

9. Assaying for the level of the one or more lectin-binding CEA glycoforms comprises: exposing the sample to one or more lectins to capture one or more lectin-binding glycoforms of the biomarkers contained in the sample; exposing the captured biomarker to an antibody specific for the biomarker; and measuring the level of binding of said captured biomarker to an antibody specific for said biomarker with the aid of a detectable signal; The method according to any one of claims 1 to 8, wherein the method is carried out by a binding assay comprising:

10. The method of claim 8 or 9, wherein either the lectin or the antibody is immobilized on a nanoparticle.

11. The method of any one of claims 1 to 10, wherein the sample is selected from the group consisting of urine, blood, serum, plasma, peritoneal fluid and tissue samples.

12. 11. The method of claim 10, wherein the nanoparticles have a diameter of less than 1000 nm, 500 nm or less, 100 nm or less, or 50 nm or less.

13. 11. The method of claim 10, wherein the nanoparticles are selected from the group consisting of protein nanoparticles, mineral nanoparticles, glass nanoparticles, nanoparticle crystals, metal nanoparticles, plastic nanoparticles, polystyrene nanoparticles, poly(ethylene glycol) nanoparticles, polyethylene nanoparticles, poly(acrylic acid) nanoparticles, poly(methyl methacrylate) nanoparticles, polysaccharide nanoparticles, colloidal gold nanoparticles, silver nanoparticles, quantum dots, carbon nanoparticles, porous silica nanoparticles, and liposomes.

14. 14. The method of claim 10, 12 or 13, wherein the nanoparticles are doped with a lanthanide chelate including europium(III).

15. 15. A kit for use in the method of any one of claims 1 to 14, comprising a CEA-binding agent and one or more lectins selected from the group consisting of human MBL, DC-SIGN and MGL, wherein either the CEA-binding agent or the lectins comprises a detectable label.

16. 16. The kit of claim 15, wherein the CEA binding agent is a monoclonal antibody.

17. 17. The kit of claim 15 or 16, further comprising a CEA-binding agent.

18. 18. The kit of claim 17, wherein the CEA binding agents are two monoclonal antibodies that bind to different epitopes on CEA.

19. The kit according to any one of claims 15 to 18, comprising human MBL and DC-SIGN, human MBL and MGL, DC-SIGN and MGL, or human MBL, DC-SIGN and MGL.

20. 20. The kit of any one of claims 15 to 19, further comprising one or more lectins selected from the group consisting of SBA, SNA, PNA, MAA II, AAL, UEA, PHA-E, RCA, WGA, WFA, PSA, VVL, TJA-II, DSL, HPA, MGL, DC-SIGN, MMR, human MBL, Siglec-2, Siglec-3, Siglec-5, Siglec-9, Siglec-10, Siglec-11, Galectin-3, Galectin-4, E-selectin, DSL, and Gal4.

21. The kit according to any one of claims 15 to 20, wherein the lectin is immobilized on nanoparticles.

22. 22. The kit of claim 21, wherein the nanoparticles have a diameter of less than 1000 nm, 500 nm or less, 100 nm or less, or 50 nm or less.

23. 23. The kit of claim 21 or 22, wherein the nanoparticles are selected from the group consisting of protein nanoparticles, mineral nanoparticles, glass nanoparticles, nanoparticle crystals, metal nanoparticles, plastic nanoparticles, polystyrene nanoparticles, poly(ethylene glycol) nanoparticles, polyethylene nanoparticles, poly(acrylic acid) nanoparticles, poly(methyl methacrylate) nanoparticles, polysaccharide nanoparticles, colloidal gold nanoparticles, silver nanoparticles, quantum dots, carbon nanoparticles, porous silica nanoparticles, and liposomes.

24. The kit according to any one of claims 21 to 23, wherein the nanoparticles are doped with a lanthanide chelate, including europium(III).

25. Use of human MBL, DC-SIGN and / or MGL to determine the disease status of colorectal cancer in a subject by assaying a sample obtained from the subject for the level of CEA glycoforms that bind to human MBL, DC-SIGN and / or MGL.

26. 26. The use according to claim 25, wherein the lectin is immobilized on a nanoparticle or labeled with a detectable label.

27. 27. The use according to claim 25 or 26 for screening, diagnosing, prognosing and / or monitoring said cancer in a subject.

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