WO2025196374A2 - Glycovariant biomarkers for diagnosis of cancer - Google Patents

Abstract

The present disclosure relates to cancer diagnostics and monitoring, and particularly to methods comprising determining cancer-associated glycovariants of antigens. The present disclosure further concerns kits for determining the cancer-associated glycovariants of antigens and uses of the cancer-associated glycovariants of antigens as cancer biomarkers.

Description

GLYCOVARIANT BIOMARKERS FOR DIAGNOSIS OF CANCER

FIELD OF THE DISCLOSURE

The present disclosure relates to cancer diagnostics and monitoring, and particularly to methods comprising determining cancer-associated glycovariants of antigens. The present disclosure further concerns kits for determining the cancer-associated glycovariants of antigens and uses of the cancer-associated glycovariants of antigens as cancer biomarkers.

BACKGROUND OF THE DISCLOSURE

Cancer is the second leading cause of death worldwide, and 10 million deaths in 2020 were attributed to cancer. The rise in prevalence of cancer in developed countries is attributed to aging population, unhealthy dietary habits, and risk factors such as obesity, diabetes, smoking and insufficient exercise. Given the slow progression of many cancers from precancerous lesions, early detection is paramount in curbing the incidence and mortality rates associated with this disease. Several non-invasive modalities for screening and diagnosing are available, ranging from blood and stool tests to imaging tests including magnetic resonance imaging (MRI), computed tomography (CT) scan, ultrasound, positron emission tomography (PET) scan and X-ray. While non-invasive tests may at times produce negative results even when cancer is present, more invasive modalities like surgery, radiotherapy, endoscopy and colonoscopy commonly employed for both screening and treatment may also occasionally yield negative results despite the presence of cancer. This accentuates the necessity for more sensitive diagnostic approaches in screening to address the limitations of current methods and improve accuracy of cancer detection.

Early diagnosis and long-term monitoring are crucial for improved survival in cancers. Current screening programs and existing markers often have insufficient diagnostic performance. Also, there is a need for biomarkers that are able to distinguish cancer patients from healthy subjects and subjects with a benign condition.

BRIEF DESCRIPTION OF THE DISCLOSURE

An object of the present disclosure is to provide a method for determining cancer disease state in a subject, so as to alleviate the above disadvantages. The method comprises assaying a sample obtained from a subject for the level of cancer-associated glycovariant(s) of antigens. Particularly, the assay targets lectin binding glycoforms of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). As an example, the target of the assay may be a glycan comprising Fuca1 ,2Gal structure, said glycan being comprised on a CEACAM-presenting vesicle or a CEACAM antigen.

It is also an object of the present disclosure to provide a kit for use or, alternatively, use of a kit in the method for determining cancer disease. The kit comprises binding agent(s) and lectin(s) for detection of cancer-associated glycovariant(s) of antigens.

A further object of the present disclosure is to provide a use of lectin binding glycovariants of CEACAMs as cancer biomarkers. As an example, it is provided a use of a glycan comprising Fuca1 ,2Gal structure, said glycan being comprised on a CEACAM-presenting vesicle or a CEACAM antigen.

The object of the disclosure is achieved by the method, kit and use which are characterized by what is stated in the independent claims. The preferred embodiments of the disclosure are disclosed in the dependent claims.

The disclosure is based on the idea of employing cancer-associated glycovariant(s) of antigen(s) in determining cancer disease state by way of screening, diagnosing, prognosing and/or monitoring cancer, or for selecting or assigning a treatment, or for patient stratification.

Advantages of the disclosure include that sensitive and specific cancer disease state determination is achieved with non-invasive or minimally invasive methods.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the disclosure will be described in greater detail by means of preferred embodiments with reference to the accompanying drawings, in which:

Figure 1 illustrates detection and characterization of glycoforms and conventional cancer biomarkers by a passive coating assay. The binding of lectins to glycoforms was evaluated in serum pool samples from colorectal cancer (CRC) patients and benign sources, including transurethral resection of the prostate (TLIRP) and endometriosis serum pool (Endo) samples. Cell culture spent media (CCSM) from CRC cell lines (Colo 205, Colo 320 DM, SW 403, LS17 4T, and SW 1463) and negative control cell lines HEK 293 and MCF-10 were also assayed. The identification of targeted glycoforms was conducted using a panel of europium-labelled lectins. The conventional biomarkers CEA, CA19-9, MLIC1 , and CA125 were assayed using europium-labelled antibodies;

Figure 2 shows characterization of glycoforms in a sandwiched assay format. The capture antibody used targeted a) CA19-9, b) MLIC1 , c) CEACAM1 , or d) CEACAM6. Results represent signal to background (S/B) ratios related to amount of glycoforms present in serum pool samples of CRC and TLIRP (negative control) patients, as well as CRC, HEK 293 (negative control) and MCF-10 (negative control) CCSMs. The signal was obtained using as europium-labelled tracer a glycan-targeting lectin (UEA, WFL, BPL or DSL) or antibody (Ma695 or C192);

Figure 3 illustrates discrimination of healthy (n=18), benign (n=22) and CRC (n=30) samples using conventional CEA-ELISA and glycovariant CEA-UEA-1 immunoassay assay. The concentration of CEA in the CEA-ELISA assay was significantly higher in CRC than in healthy and benign controls. However, most of the samples fell below the cut-off value of CEA-ELISA (5 ng/mL, marked with a dashed line), so the assay result had no significant prognostic value. The glycovariant CEA-UEA-1 assay did not discriminate between CRC and healthy and benign controls in terms of signal-to-background (S/B) ratio. The uncorrected p-values displayed above each plot were computed using the Wilcoxon rank-sum test;

Figure 4 shows comparison of CEACAM1 ELISA, total CEACAM1 and CEACAM1-UEA- 1 glycovariant assay. Discrimination of healthy (n=18), benign (n=22) and CRC (n=30) singlet serum samples was performed using CEACAM1 ELISA, total CEACAM1 and CEACAM1-UEA-1 assays. The expression of the CEACAM1-UEA-1 glycoform in CRC was significantly higher than in healthy and benign controls. The control assays CEACAM1 ELISA and total CEACAM1 showed no discrimination between CRC, healthy and benign samples. The uncorrected p-values displayed above each plot were computed using the Wilcoxon rank-sum test;

Figure 5 illustrates comparison of CEACAM6 ELISA, in-house total CEACAM6 and Glycovariant Immunoassays of CEACAM6. Assays of healthy (n=18), benign (n=22) and CRC (n=30) singlet serum samples was performed with CEACAM6 ELISA, tCEACAM6 assay and the glycovariant immunoassays CEACAM6-UEA-1 and CEACAM6-WFL. Based on the signal-to-background ratio, the expression of CEACAM6-UEA-1 and CEACAM6-WFL in CRC was significantly higher than in healthy and benign controls. Conversely, conventional CEACAM6 ELISA and tCEACAM6 showed no discrimination between any group. The uncorrected p-values displayed above each plot were computed using the Wilcoxon rank-sum test; and

Figure 6 shows a Receiver Operating Characteristics (ROC) plot displaying the AUC of conventional CEA ELISA, CEACAM1 glycovariant and CEACAM6 glycovariant assays. Discrimination between healthy (n=18), benign (n=22) and CRC (n=30) samples in the ROC plot is shown in different colours for different assays. The 95% confidence intervals (Cl) are displayed numerically in brackets after each area under curve (AUC) value. The conventional CEA ELISA (grey) has the lowest AUC of 0.787. The highest AUC was obtained by the CEACAM1-UEA-1 (blue) assay followed by CEACAM6-UEA-1 (green) CEACAM6-BPL (yellow) and CEACAM6-WFL (orange).

DETAILED DESCRIPTION OF THE DISCLOSURE

Overexpression of proteins is a typical feature of cancer cells. As such, tumor markers, each indicative of a particular disease process, may be utilized in oncology to help detect the presence of cancer or even a specific type of cancer. In addition to overexpression, aberrant glycosylation of proteins is a frequently observed phenomenon in cancers. Glycosylation is involved in several physiological processes regulating the development and progression of cancer. For example, glycans play a role in cell signaling, cell-matrix interactions, tumor cell dissociation and invasion, metastasis formation, angiogenesis and immune modulation.

Glycosylation of proteins is a structurally diverse and complex post-translational modification (PTM) determining protein structure, function, and stability. Changes in protein glycosylation are universally observed in different types of cancer which have been reported to display altered N- and/or O-glycosylation. The most common cancer- associated changes in protein glycosylation are increased sialylation, increased branched- glycan structures and overexpression of core fucosylation. For example, overexpression of A/-acetylglucosamine (GIcNAc) and oversialylation and/or overexpression of sialylated Lewis antigens such as sialyl-Lewis^a (sLe^a) and sialyl-Lewis^x (sLe^x) have been reported. Altered O-glycosylation is a characteristic alteration in mucins, preventing normal maturation of glycans and causing overexpression of Thomsen-Friedenreich-related T, Tn, and STn antigens. For example, in colon cancer, increased 1 ,6-branching and a corresponding higher abundance of (poly-)N-acetyl-lactosamine extensions of N-glycans as well as an increase in (truncated) high-mannose type glycans are seen, while the amount of bisected structures decreases. According to some studies, reduced expression of core 3 and 4 glycans predominates in ORC associated O-glycan alterations, although increased levels of core 1 glycans, (sialyl) T-antigen, (sialyl) Tn-antigen, and an overall higher density of O-glycans are also seen.

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are highly glycosylated protein members of a larger family in the immunoglobulin superfamily. They consist of 12 transmembrane proteins capable of hetero- or homodimerization with other CEACAM members or other transmembrane proteins such as integrins. They are involved in many cellular processes, such as neovascularization, angiogenesis, cellular adhesion, and tumorigenesis. CEACAM5 (CEA) is currently used in clinical practice as a prognostic biomarker for cancer recurrence such as CRC recurrence. However, it exhibits low sensitivity and specificity, ranging from 40 to 70%, rendering it unreliable as a diagnostic biomarker for confirming the presence of CRC. Additionally, its limitations make it unsuitable as a screening biomarker for detecting the disease at an early stage. While CEA is valuable for predicting likelihood of CRC recurrence after treatment (prognostic), it is not recommended for use in the diagnostic or screening contexts due to its suboptimal sensitivity and specificity.

CEACAM1 (also denoted as CD66a, BGP, BGP1 or BGPI) is a transmembrane protein containing an extracellular N-terminal variable domain followed by up to three constant C2-like immunoglobulin domains. The extracellular domains of CEACAM1 are essential in its function, as they are required for homophilic (CEACAM1-CEACAM1 ) and heterophilic intercellular adhesion with CEA and with T cell-immunoglobulin and mucin-domain containing 3 (TIM-3) protein. CEACAM1 is a candidate biomarker in e.g. respiratory, genitourinary, breast, skin and gastrointestinal cancer. CEACAM1 has been implicated with the progression of colon cancer and is recognized as a biomarker for CRC.

CEACAM6 (also denoted as CD66c, CEAL, NCA, NCA90 or NCA50/90), is a multifunctional glycoprotein that mediates homotypic binding among other CEA family members and heterotypic binding with integrin receptors. CEACAM6 modulates the development of cancer through many processes such as aberrant cell differentiation, antiapoptosis, cellular growth, and resistance to therapeutic agents. CEACAM6 is a potential tumor marker for a number of aggressive cancers such as CRC, pancreatic cancer and non-small-cell lung carcinoma (NSCLC).

Aberrant glycosylation of cell surface proteins is a distinct marker of cancer cells that influences progression and metastasis of cancer. Since CEACAM antigens are heavily glycosylated, this study was conducted to investigate the glycovariant profiles of CEACAM1 , CEACAM6 and CEA compared to other cancer biomarkers. Here, we report assays that employ lectins in detecting altered glycosylation of CEACAMs such as CEACAM1 and CEACAM6 for diagnosing cancer with high sensitivity and specificity. As an example, assays detecting a glycan comprising the Fucal ,2Gal structure comprised on a CEACAM-presenting vesicle or a CEACAM antigen for determining cancer disease state are reported.

Lectins are a diverse class of proteins that bind to specific carbohydrates (glycans) through their carbohydrate recognition domain (CRD). They may be used as a valuable diagnostic tool for their ability to recognize specific glycans on the surface of cells or on glycosylated molecules present in body fluids. Ulex europaeus agglutinin I (also denoted as UEA, UEA 1 , UEA-1 , UEA I or LIEA-I) is a plant lectin that binds to many glycoproteins and glycolipids containing a-linked fucose residues, such as those in the ABO blood group glycoform precursors. It is also known that UEA-1 binds poorly or not at all to alpha(1 ,3) or alpha(1 ,6 )-lin ked fucose, and it is also believed to be unable to bind internal fucose structures. UEA-1 is well-known to recognize Fucal ,2Gal. A recent analysis (Bojar et al., ACS Chem. Biol. 2022, 17, 11 , 2993-3012) shows type 2 blood group H (Fucal ,2Gal01 ,4GlcNAc) as the predominant binding epitope. No binding is observed to type 1 H epitopes (Fucal ,2Gal01 ,3GlcNAc), indicating that the nature of the Gal linkage is important. Epitopes containing Glc (e.g. Fucal ,2Gal01 ,4Glc) or substituted on the GIcNAc (e.g. Le^y i.e. Fucal ,2Gal01 ,4(Fuca1 ,3)GlcNAc) are also tolerated. In addition, UEA-1 was found to tolerate sulfation on the 6-position of the terminal Gal. Generally recognized nomenclature related to glycans and glycosidic bonds is used for the glycans discussed herein. For instance, “Fuc” refers to fucose, "Gal" refers to galactose, “Glc” refers to glucose, and "GIcNAc" refers to N-acetylglucosamine. Also, for instance, a1 ,3-linked fucose means that the fucose is attached via its anomeric carbon 01 , which has a (alpha) configuration, to the 03 position of another sugar, whereas in a Gal01 ,4 bond the anomeric carbon of the galactose has 0 (beta) configuration and is attached with a glycosidic bond to the C4 position of another sugar.

Other fucose-binding lectins, such as Aleuria aurantia lectin (AAL), may detect fucosylation differently. AAL recognizes fucose linked a-1 ,6 to N-acetylglucosamine. Lectins that target other than fucose residues include Wisteria floribunda lectin (WFL) which recognizes carbohydrate structures terminating in N-acetylgalactosamine (GalNAc) linked a or 0 to the 3 or 6 position of Gal. Bauhinia purpurea lectin (BPL) detects N-terminal acetylgalactosamine, binding specifically to Gal0-1 ,3GalNAc. Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) detects nonsialylated Lewis antigens and high mannose-type structures.

Unless otherwise defined, the terms and expressions used in this specification and claims have the meanings generally applicable in the field of cancer diagnostics. Some of the terms and expressions used herein have the meanings defined below.

As used herein, the meaning of a singular noun includes that of a plural noun and thus a singular term, unless otherwise specified, may also carry the meaning of its plural form. In other words, the term "a" or "an" may mean one or more.

As used herein, the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or when the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein, the terms "biological sample" and "sample" are interchangeable and refer in particularto a sample of a bodily fluid, such as ascites fluid, urine, blood, plasma, semen, serum, and peritoneal cavity fluid, obtained from a subject. However, the terms encompass also tissue samples, such as biopsy samples taken from a tissue. In some embodiments, said tissue sample may be a formalin-fixed or paraffin-embedded tissue sample. Generally, obtaining the sample to be analysed from a subject is not part of the present method for determining a subject’s cancer disease state. A urine, blood, serum or plasma sample is the most preferred sample type to be used in the present method and all its embodiments. In some embodiments, the sample is an EDTA plasma sample.

In embodiments which concern assessment of the level of more than one biomarker, same or different samples obtained from a subject whose cancer disease state is to be determined may be used for each assessment. Said different samples may be of the same or different type.

As used herein, the terms "biomarker" and "marker" are interchangeable and refer broadly to a molecule which is differentially present in a sample taken from a subject with cancer as compared to a comparable sample take from a control subject, such as an apparently healthy subject. More specifically, the terms refer to certain glycovariants of antigens indicative of a given cancer. Particularly, the terms may refer to glycovariants of CEACAMs, having certain glycan structures present on them.

As used herein, the terms "glycoform" and "glycovariant" and the like are interchangeable and refer to a particular form of a glycosylated biomarker. That is, when the same protein backbone that is part of a biomarker has the potential to be linked to different glycans or sets of glycans, then each different version of the biomarker is referred to as a "glycoform".

As used herein, the term "binder molecule" refers broadly to any molecule that is able to bind a biomarker or a glycan part thereof. Non-limiting examples of binder molecules include lectins, antibodies, antibody mimetics, and oligonucleotide and peptide aptamers.

As used herein, the term "antibody", in short, “Ab”, refers to an immunoglobulin structure comprising two heavy (H) chains and two light (L) chains interconnected by disulphide 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 herein encompassed by the term "antibody". Non-limiting examples of said antigen-binding fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments. Said fragments and variants may be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins as is well known in the art. As used herein, the term "subject" refers to an animal, preferably to a mammal, more preferably to a human, and in some embodiments most preferably to a female, while in some other embodiments most preferably to a male. Depending on an embodiment in question, said subject may suffer from cancer with or without diagnosis, be suspected to suffer from cancer, be at risk of cancer, or may have already been treated for cancer. Herein, the terms "human subject", "patient" and "individual" and the like are interchangeable.

As used herein, the term "apparently healthy" refers to an individual or a pool of individuals who show no signs or symptoms of cancer or a benign condition and thus are believed not to be affected by cancer or a benign condition or who are predicted not to develop cancer or a benign condition. Herein, the terms "apparently healthy" and "healthy" and the like may in some instances be used interchangeably.

As used herein, terms such as "benign condition" and the like refer to non-cancerous conditions. Non-limiting examples of non-cancerous benign conditions include benign prostatic hyperplasia, Crohn’s disease and endometriosis. Similarly, as used herein, terms such as "benign control" and "benign cohort" and the like refer to subjects or patients having a benign condition and/or to samples from subjects or patients having a benign condition. Those skilled in the art readily understand which benign conditions are appropriate to consider which respect to a given cancer.

As used herein, the term "cancer" refers broadly to any cancerous condition. In some embodiments, the cancerous condition is urological cancer such as bladder cancer (BICa), prostate cancer (PrCa) or renal cell carcinoma (RCC), gastrointestinal cancer such as colorectal cancer (CRC), colon cancer (ColCa) or pancreatic cancer (PanCa), gynaecological cancer such as ovarian cancer (OvCa), or breast cancer (BrCa), lung cancer (LunCa) or head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is CRC. In some instances, "cancer" is broadly referred to herein by using the term "disease".

As used herein, the term "cancer disease state" refers to any distinguishable manifestation of cancer, including non-cancer. For example, the term includes, without limitation, information regarding the presence or absence of cancer, the presence or absence of a preclinical phase of cancer, the risk of having or developing cancer, the stage of cancer, and progression of cancer.

As used herein, the term "indicative of cancer", when applied to a biomarker, refers to a level which, using routine statistical methods setting confidence levels at a minimum of 95%, is diagnostic of said cancer or a stage of said cancer such that the detected level is found significantly more often in subjects with said cancer or a stage of said cancer than in subjects without said cancer or another stage of said cancer. Preferably, the level which is indicative of cancer is found in at least 80% of subjects who have the cancer and is found in less than 10% of subjects who do not have the cancer. More preferably, the level which is indicative of said cancer is found in at least 90%, at least 95%, at least 98% or more in subjects who have the cancer and is found in less than 10%, less than 8%, less than 5%, less than 2% or less than 1% of subjects who do not have the cancer.

As used herein, the term "level" is interchangeable with terms including "amount", “expression” and "concentration", unless otherwise indicated.

To determine whether a detected level of a biomarker is indicative of the presence or risk of the presence of a cancer associated with said biomarker, its level in a relevant control has to be determined. Once the control levels are known, the determined marker levels can be compared therewith and the significance of the difference can be assessed using standard statistical methods. In some embodiments, a statistically significant difference between the determined biomarker level and the control level is indicative of the cancer in question. In some further embodiments, before comparing with the control, the biomarker levels are normalized using standard methods.

Additionally or alternatively, the detected level of a biomarker may be compared with a predetermined threshold value. Comparison of the assayed level of a biomarker in a sample to be analysed with that of a relevant control or a predetermined threshold value may in some embodiments be performed by a processor of a computing device.

Regardless of whether or not the processor of the computing device is used for said comparison, the level of the assayed level of a biomarker is, at least in some embodiments, determined as "increased" or "higher" if the level of the biomarker in the sample is, for instance, at least about 1 .5 times, at least about 1 .75 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times or at least about 30 times the predetermined threshold level or the level of the biomarker in a control sample. In some embodiments, the difference between the level of the biomarker in the sample to be analyzed and the predetermined threshold level or the level of the biomarker in a control sample has to be statistically significant in order to provide a proper diagnostic, prognostic or predictive result. As used herein, the term "about" refers to a range of values ± 10% of a specified value. For example, the phrase "about 10 times the predetermined threshold level or the level of the biomarker in a control sample " includes ± 10% of 10 times, or from 9 to 1 1 times the predetermined threshold level or the level of the biomarker in a control sample.

Concentration of a biomarker in a sample obtained from a subject whose cancer disease state is to be determined or who is to be screened, diagnosed, prognosed, or monitored for cancer is considered "non-increased" or "normal" if the detected concentration thereof is lower, essentially the same or essentially non-altered as compared with that of a relevant control sample or a predetermined threshold value.

As used herein, the term "control" may refer to a control sample obtained from an apparently healthy individual or pool of apparently healthy individuals, or it may refer to a control sample obtained from an individual or a pool of individuals with a benign condition such as a benign urological condition such as benign prostatic hyperplasia, or it may refer to a predetermined threshold value, i.e. a cut-off value, which is indicative of the presence or absence of the cancer in question. Statistical methods for determining appropriate threshold values will be readily apparent to those of ordinary skill in the art. The threshold values may have been determined, if necessary, from samples of subjects of the same age, demographic features, and/or disease status, etc. The threshold value may originate from a single individual not affected by the cancer in question or be a value pooled from more than one such individual.

In some embodiments, the term "control sample" refers to a sample obtained from the same subject whose cancer disease state is to be determined but obtained at a time point different from the time point of the disease state 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 control samples obtained from the same subject are used when the purpose of cancer disease state determination is to monitor said disease, especially to monitor the onset of the disease, or risk development of the disease, response to treatment, relapse of the disease, or recurrence of the disease.

The present disclosure is, at least partly, based on studies aiming to identify cancer-related glycovariants of antigens with improved sensitivity over other variants of the same antigens as cancer biomarkers. In accordance with this aim, the present disclosure provides means and methods for determining cancer disease state in a subject who is suspected to suffer from or be at risk of suffering from cancer. Said means and methods are provided especially for screening, diagnosing, prognosing or monitoring cancer.

Here, a glycoprofiling analysis was carried out by a passive coating assay of pooled serum samples from subjects with benign condition, CRC patients and CRC cell lines. Benign cell lines were employed as negative controls to investigate the glycosylation status of the samples. Glycovariant CEACAM expression such as CEACAM1 and CEACAM6 expression as detected with lectins including Ulex europaeus I lectin (UEA-1 ), Wisteria floribunda lectin (WFL) and Bauhinia purpurea lectin (BPL) showed a higher significance in differentiating between CRC samples and healthy or benign samples than conventional immunoassays. Other possible lectins that may be employed in glycovariant CEACAM assays include Wheat germ agglutinin (WGA), Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), Artocarpus integrifolia lectin (jacalin), Datura stramonium lectin (DSL), Helix pomatia agglutinin (HPA), Concanavalin A (ConA), and Maackia amurensis lectin (MAA). The results indicate that the aberrant glycosylation of CEACAMs is useful as a predictive biomarker for the diagnosis of cancer such as CRC.

It is thus shown herein that detection of certain glycovariants of protein antigens instead of using conventional immunoassays improves the sensitivity by which patients with cancer can be distinguished from subjects without cancer. Also, as reported herein, the cutoff value of the conventional CEA ELISA (5 ng/mL) often has no significant prognostic or diagnostic value as biomarker levels in many cancer patient samples fall below the cutoff value. The term “ELISA” refers to enzyme-linked immunosorbent assay.

Conventional immunoassays including ELISA assays are based on the determination of antigen levels in a sample such as serum or plasma by two monoclonal antibodies. Conventional CEA, CEACAM 1 or CEACAM6 immunoassays, for example, are based on the determination of CEA, CEACAM1 or CEACAM6 antigen levels by two monoclonal antibodies which recognize different or repeating epitopes of CEA, CEACAM 1 or CEACAM6, respectively. Such conventional immunoassays may herein be referred to as "total CEA", “CEA ELISA”, "total CEACAM1", “CEACAM1 ELISA”, "total CEACAM6" or “CEACAM6 ELISA” assays, respectively. Additionally, the term "CEA", “CEACAM1” or “CEACAM6” may refer to the CEA, CEACAM1 or CEACAM6, respectively, species recognizable by conventional immunoassays. Although not repeated here, corresponding terminology may be used herein also for conventional immunoassays for other antigens.

As used herein, the term "antigen” refers broadly to any protein antigen that may be indicative of cancer. In some embodiments, the antigen is a member of the CEACAM family such as CEACAM1 , CEA or CEACAM6. The antigen may also be for example CA125, CA19-9 or MLIC1. Also, as used herein, the term "antigen" refers broadly to any antigen in soluble form and to an extracellular vesicle which harbors said antigen on its surface. Thus, the term "CEACAM1", for example, may encompass soluble CEACAM1 as well as CEACAM1 present on extracellular vesicles.

Mucin 1 (MLIC1 , MLIC-1 ), also known as mucin I, cell surface associated mucin 1 or cancer antigen 15-3 (CA15-3) is a large transmembrane glycoprotein with molecular weight ranging from 500 to 1000 kDa. MLIC1 is secreted from tumor cells and is a well-established marker for e.g. breast cancer.

CA19-9 i.e. carbohydrate antigen 19-9, also called cancer antigen 19-9 or sialyl Lewis³ antigen, is a commonly used serum tumor marker for e.g. pancreatic cancer diagnosis and for monitoring therapy in cancer patients. CA19-9 is normally present in small amounts in serum and can be overexpressed in several benign gastrointestinal disorders.

CA125 i.e. cancer antigen 125 or ovarian cancer-related tumor marker CA125, also called Mucin-16 (MllC-16, MLIC16) is an antigenic tumor marker that is commonly expressed by the epithelial ovarian neoplasms and other tissues, such as cells lining the endometrium, fallopian tubes, pleura, peritoneum, and pericardium. CA125 is composed of three domains: an N-terminal domain, a tandem repeat domain and a C-terminal domain, of which the first two domains are extracellular and heavily glycosylated.

CEA, CEACAM1 , CEACAM6, CA19-9, CA125 and MUC1 can all be referred to as conventional cancer biomarkers.

Herein, preferred cancer biomarkers include CEACAM1 and CEACAM6, and especially certain lectin binding glycoforms thereof. Lectin binding glycoforms of CEACAMs may be indicated by the general term “CEACAM-lectin”. Also, as used herein, for example the terms “CEACAM1-UEA-1 ” and “CEACAM6-UEA-1” refer to UEA-1 lectin binding glycoforms of CEACAM1 and CEACAM6, respectively.

In the context of this application, similar terminology may be used for lectin binding glycoforms of any biomarker binding to any lectin. By way of example, the term “CEACAM- UEA-1” refers to a UEA-1 lectin binding glycoform of a protein antigen belonging to the CEACAM family. Similarly, the term “CEACAM-WFL” refers to a WFL lectin binding glycoform of a protein antigen belonging to the CEACAM family. The lectin binding glycoform may be in a soluble form and/or present on an extracellular vesicle which harbors said lectin binding glycoform on its surface. 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. Receiver Operating Characteristic (ROC) curves may be utilized to demonstrate the tradeoff between the sensitivity and specificity of a marker, as is well known to skilled persons. Sensitivity is a measure of the ability of the marker to detect the disease, and the specificity is a measure of the ability of the marker to detect the absence of the 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 curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cut-off selected, the values of specificity and sensitivity may be determined. In other words, data points on the ROC curves represent the proportion of true-positive and false-positive classifications at various decision boundaries. Optimum results are obtained as the true-positive proportion approaches 1.0 and the false-positive proportion approaches 0.0. However, as the cut-off is changed to increase specificity, sensitivity usually is reduced and vice versa.

As used herein, the term "false positive" refers to a test result, which classifies an unaffected subject incorrectly as an affected subject. Likewise, "false negative" refers to a test result, which classifies an affected subject incorrectly as an unaffected subject.

As used herein, the term "true positive" refers to a test result, which classifies a subject who has a disease correctly as an affected subject. Likewise, "true negative" refers to a test result, which classifies an unaffected subject correctly as an unaffected.

In accordance with the above, the term "success rate" refers to the percentage-expressed proportion of affected individuals with a positive result, while the term "false positive rate" refers to the percentage-expressed proportion of unaffected individuals with a positive result.

The area under the ROC curve, often referred to as the AUC, is a measure of the utility of a marker in the correct identification of disease subjects. Thus, the AUC can be used to determine the effectiveness of the test. An area of 1 represents a perfect test; an area of 0.5 represents a worthless test.

As demonstrated in the experimental part, the AUC for the conventional CEA ELISA immunoassay was 0.787, while the highest AUC was obtained in the assay for CEACAM1- UEA-1 (0.883) followed by CEACAM6-UEA-1 (0.871 ), CAECAM-BPL (0.831 ) and CEACAM-WFL (0.830). It is thus envisaged that, in some embodiments, it would be advantageous to use the glycovariant assays in combination to complement their performance. According to one aspect of the disclosure, it is provided a method for determining cancer disease state in a subject. The method comprises assaying a sample obtained from a subject for the level of lectin binding glycoforms of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). The lectin binding glycoform of CEACAM (CEACAM- lectin) may be one or more selected from Ulex europaeus agglutinin I (UEA-1 ) binding glycoform of CEACAM1 , the UEA-1 binding glycoform of CEACAM6, the Wisteria floribunda lectin (WFL) binding glycoform of CEACAM1 , the WFL binding glycoform of CEACAM6, the Bauhinia purpurea lectin (BPL) binding glycoform of CEACAM1 , and the BPL binding glycoform of CEACAM6.

The method further comprises comparing the detected level(s) of CEACAM-lectin in said sample with that of a control sample or a predetermined threshold value. Cancer disease state is determined on the basis of said comparison.

In other words, the CEACAM may be one or more selected from CEACAM1 and CEACAM6 and/or the lectin is one or more selected from UEA-1 , WFL and BPL. In some embodiments, lectins may further include WGA, DC-SIGN, jacalin, DSA, HPA, ConA, and MAA. In some embodiments, the CEACAM is CEACAM1.

In an embodiment, the lectin is UEA-1 , and one or more lectins selected from WFL, BPL, WGA, DC-SIGN, jacalin, DSA, HPA, ConA, and MAA is (are) optionally assayed. Optionally, the CEACAM is CEACAM 1.

In an embodiment, the method comprises assaying a sample obtained from said subject for the level of CEACAM1-UEA-1 or CEACAM6-UEA-1 or both. Optionally, one or more biomarkers selected from the group consisting of CEACAM1-WFL, CEACAM6-WFL, CEACAM1-BPL and CEACAM6-BPL may be assayed.

In a further embodiment, an increased level of CEACAM-lectin as compared with that of the control sample or predetermined threshold value indicates that said subject has or is at risk of having cancer. Conversely, a non-increased or decreased level of CEACAM- lectin as compared with that of the control sample or predetermined threshold value indicates that said subject does not have or is not at risk of having cancer.

Said assaying for the level of CEACAM-lectin can be based on a binding reaction between CEACAM and lectin. That is, the CEACAM-lectin biomarker which is a lectin binding glycoform of CEACAM may be present in the sample in soluble form and/or on an extracellular vesicle (EV) harboring said lectin binding glycoform on its surface. The method may detect the soluble form of the lectin binding glycoform and/or the lectin binding glycoform present on EVs. Alternatively or additionally, as explained in more detail below, the glycan structure(s) of the lectin binding glycoform may not be directly associated with the CEACAM on the EVs but are located elsewhere on the EVs.

EVs are lipid bilayer-covered particles that are naturally secreted from cells, and found in biological fluids including, but not limited to, blood, urine and cerebrospinal fluid. EVs carry cargos of proteins, nucleic acids, lipids and metabolites from the parent cell, and may present various antigens such as mucins (e.g. MLIC1 ), CEACAMs and CA19-9 on their surfaces. However, these antigens may also be found in biological fluids as soluble proteins, without being comprised on antigen-presenting vesicles.

The present methods encompass determining levels of certain biomarkers in biological samples regardless of whether they exist as soluble glycovariant antigens or are comprised on antigen-presenting EVs. Thus, assaying a sample for CEACAM1-UEA-1 glycoform, as an example, refers not only to instances wherein the sample is assayed for the presence of CEACAM1 species comprising glycan structure(s) binding to UEA-1 (either in soluble form or on EVs) but also to instances where the sample is assayed for EVs presenting both CEACAM1 and glycan structure(s) binding to UEA-1 on their surface, where the glycan structure(s) may not be associated with CEACAM1 but is (are) located elsewhere on the EV. The latter may be achieved by assaying the sample for EVs that can be captured by an anti-CEACAM1 antibody and comprise glycan structure(s) binding to UEA-1 on their surface. Some embodiments may specifically concern soluble lectin binding glycoforms of antigens, while some other embodiments may specifically concern lectin binding glycan structures presented by EVs.

In an aspect, the preferred cancer biomarkers include glycans comprising the Fuccd ,2Gal structure. Fuccd ,2Gal corresponds to glycan binding specificity of UEA-1 and is the glycan structure recognized by UEA-1 in the lectin binding assay according to the present disclosure. Said glycan is preferably comprised on a CEACAM-presenting vesicle or on a CEACAM antigen.

Thus, according to one aspect of the disclosure, it is provided a method for determining cancer disease state in a subject. The method comprises assaying a sample obtained from said subject for the level of a glycan comprising Fuccd ,2Gal structure. Said glycan is preferably comprised on a CEACAM-presenting vesicle or on a CEACAM antigen. The CEACAM may be one or more selected from CEACAM 1 and CEACAM6. In some preferred embodiments, the CEACAM is CEACAM1. Particularly, the glycan may be comprised on one or more selected from CEACAM 1- presenting vesicle, CEACAM6-presenting vesicle, CEACAM1 antigen, and CEACAM6 antigen.

The method further comprises comparing the detected level of the glycan in said sample with that of a control sample or a predetermined threshold value. Cancer disease state is determined on the basis of said comparison.

In some instances, the glycan comprises a structure selected from one or more of Fucal ,2Galp1 ,4Glc, Fucal ,2Galp1 ,4GlcNAc and Fucal ,2Galp1 ,4(Fuca1 ,3)GlcNAc. Particularly, the glycan comprises the Fucal ,2Gaipi ,4GlcNAc structure.

The CEACAM of the CEACAM-presenting vesicle or a CEACAM antigen may be one or more selected from CEACAM1 and CEACAM6. In some embodiments, the CEACAM is CEACAM 1.

In a further embodiment, an increased level of the glycan comprising Fucal ,2Gal structure as compared with that of the control sample or predetermined threshold value indicates that said subject has or is at risk of having cancer. Conversely, a non-increased or decreased level of the glycan comprising Fucal , 2Gal structure as compared with that of the control sample or predetermined threshold value indicates that said subject does not have or is not at risk of having cancer.

In any of the embodiments set forth above, the methods of determining a cancer disease state in a subject may be more specifically formulated as a method of screening, diagnosing, prognosing and/or monitoring cancer, be it de novo or recurrent appearance or suspicion of cancer.

In accordance with the above, the present methods are in some embodiments directed to diagnosing of cancer, i.e. determining whether or not a subject has or is at risk of having or developing cancer. This is also meant to include instances where the presence or the risk of cancer is not finally determined but that further diagnostic testing is warranted. In such embodiments, the method is not by itself determinative of the presence or absence, or of the risk of cancer in the subject but can indicate that further diagnostic testing is needed or would be beneficial. Therefore, the present method may be combined with one or more other diagnostic methods for the final determination of the presence or absence, or of the risk of cancer in the subject. Such other diagnostic methods are well known to a person skilled in the art.

Being non- or minimally invasive and suitable for analysing for example urine and serum samples, the present method and its various embodiments may be easily incorporated into a population screening protocol to identify subjects having or being at risk of having or developing cancer. This would enable not only early diagnosis of cancer, but also active surveillance for the onset of cancer in subjects with identified increased risk of developing cancer later in life. Moreover, early detection of cancer would allow treating the disease early when chances of cure are at their highest.

The present methods and their various embodiments may be used for screening or diagnosing cancer, or for selecting or assigning a treatment, or for patient stratification. Further uses of the present methods and their various embodiments include prognosing or predicting the outcome of cancer, or monitoring onset of cancer, any development in risk of cancer, the subject's recovery or survival from cancer, any possible relapse or recurrence of the disease, or response to treatment.

In some embodiments, the present methods comprise monitoring cancer in said subject by comparing the level of one or more CEACAM-lectin species or the glycan comprising Fuca1 ,2Gal structure set forth above, with the respective level in one or more other samples obtained from the same subject at a different time point. Samples that may be employed in the monitoring include, but are not limited to, samples collected at different time points after diagnosis of cancer and/or before, during, and after therapeutic intervention, e.g. by surgery, radiation therapy, chemotherapy, any other suitable therapeutic treatment, or any combination thereof, to relieve or cure cancer. In some embodiments, said monitoring is carried out by repeating the assaying step at least twice at different time points, wherein said time points are selected, independently from each other, from the time points set forth above.

In some embodiments, the monitoring is carried out during or after treatment of cancer, and/or the method comprises determining said subject as having relapse or recurrence of cancer or as being at risk of relapse or recurrence of cancer, if the level of at least one of the CEACAM-lectin biomarkers or the glycan comprising Fuca1 ,2Gal structure is higher than in one or more earlier samples obtained from the same subject, or higher than in a relevant control or above a predetermined threshold value.

In some embodiments, the present method is particularly suitable for early diagnosis of cancer and early detection of cancer relapse, recurrence and progression. Thus, the present method and any lectin binding glycoform biomarker combinations disclosed herein may be used not only for diagnostic, prognostic and monitoring purposes but also for screening of asymptomatic subjects for cancer or a risk of developing cancer. In some embodiments, in case the level of CEACAM-lectin or the glycan comprising Fuca1 ,2Gal structure assayed in the present method is higher than in a control sample or above a predetermined threshold value, a treatment is selected for or assigned to said patient. The treatment may be selected from the group consisting of surgery, radiation therapy, immunotherapy, targeted therapy and chemotherapy.

Assaying a sample for the level any one or more of the specific biomarker(s) set forth above may further comprise assaying also for the level of one or more conventional biomarkers selected from for example MLIC1 , CA125, CEA, CEACAM1 , CEACAM6, and CA19-9.

Assaying a sample for the level of a biomarker may be performed by sandwich immunoassays where a protein antigen-specific (monoclonal) antibody is employed as a capturing agent to bind the biomarker which is then detected with a tracer, a lectin that is labelled. The label may be detectable directly or indirectly. In some cases, a sandwich assay may be conducted in a reversed way. In such cases, the lectin is used as a capturing agent and a protein antigen-specific antibody, as a directly or indirectly detectably labelled tracer. For example, since urine contains less interfering glycosylated molecules than blood, the reversed sandwich assay may operate better with urine samples than with blood samples.

In some embodiments, a sandwich assay may comprise one or more washing steps after a capturing step in order to remove any molecular species not specific for the capturing agent. Appropriate washing solutions and conditions (e.g. time and temperature) are known to those skilled in the art.

Sandwich assays according to various embodiments of the present invention may be performed either on a solid surface, such as a microtiter plate, or in lateral flow format. Means and methods for binding a capturing agent to a solid surface, e.g. via a streptavidinbiotin complex, or incorporating a capturing agent to a lateral flow assay are known in the art and readily apparent to a skilled person.

Suitable substrates for use in the 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 indicated above, the substrate may in some embodiments be coated with an appropriate compound to enhance binding of a capturing agent (i.e. either an anti-protein antigen antibody or a lectin) to the substrate. In some further embodiments, one or more control antibodies or control lectins may also be attached to the substrate. For use as a tracer, an anti-protein antigen antibody or lectin may be labelled with any appropriate label known in the art including, but not limited, to fluorescent labels, bioluminescent labels, chemiluminescent labels. In some embodiments, said anti-protein antigen antibody may detectably labelled indirectly, for example through immobilization to a detectable nanoparticle (NP).

Generally, for use as a tracer, lectins or antibodies may be detectably labelled by various ways as is well known in the art. In some embodiments, one or more lectins or antibodies to be employed for assaying a sample for the level of lectin binding glycoform of a protein antigen may be directly labelled with any available detectable label using standard techniques. For example, a lectin or an antibody may be directly labelled with a lanthanide chelate, such as a europium (III), terbium (III), samarium (III), dysprosium (III), ytterbium (III), erbium (III) or neodymium (III) chelate, or made detectable through colorimetric detection, e.g. by conjugating with horseradish peroxidase (HRP) or alkaline phosphatase (AP). In some other embodiments, one or more lectins or antibodies to be employed may be detectably labelled indirectly, for example by immobilizing said one or more lectins or antibodies on a detectable NP. NP-immobilized lectins are called lectin-NPs for short.

As used herein, the term "nanoparticle" (NP) refers to a particle, synthetic or natural, having one or more dimensions, e.g. a diameter, of less than about 1000 nm, e.g. about 500 nm or less, about 100 nm or less, or about 50 nm or less. As used herein, the term "about" refers to a range of values ± 10% of a specified value. For example, the phrase "about 100 nm" includes ± 10% of 100 nm, or from 90 nm to 110 nm. The NPs may generally have a spherical shape but also non-spherical shapes such as ellipsoidal shapes can be used. In some embodiments, all the dimensions of said nanoparticle are less than about 1000 nm, about 500 nm or less, about 100 nm or less, or about 50 nm or less. A variety of different materials may be utilized in the present NPs. Non-limiting examples of suitable polymers include polyethylene glycol) (PEG), polystyrene, polyethylene, poly(acrylic acid), poly(methyl methacrylate) (PMMA), polysaccharides, and copolymers or combinations thereof. Other suitable NP materials include, but are not limited to, colloidal gold, silver, quantum dots, carbon, porous silicon, and liposomes. Further suitable NP materials include protein NPs, mineral NPs, glass NPs, NP crystals, metal NPs, and plastic NPs.

NPs suitable for use in various embodiments of the present invention or disclosure, may be directly or indirectly qualitatively or quantitatively detectable by any known means. For instance, the NPs may be detectable owing to an inherent quality as in the case of e.g. upconverting nanoparticles (LICNP), resonance particles, quantum dots, and gold particles. In some other embodiments, the NPs can be made detectable e.g. by fluorescent labels, bioluminescent labels, chemiluminescent labels. In some further embodiments, labelling or doping with lanthanides, i.e. luminescent lanthanide ions with luminescence emission in visible or near-infrared or infrared wavelengths and long fluorescence decay, such as europium (III), terbium (III), samarium (III), dysprosium (III), ytterbium (III), erbium (III) and neodymium (III), are preferred means for making the present NPs detectable.

Lectins or antibodies may be immobilized on NPs by any suitable method known in the art. In embodiments which involve more than one different lectin or antibody, said different lectins may be immobilized either on the same or different NPs in any desired ratios.

In some non-limiting embodiments, the most preferred NPs are polystyrene NPs having a diameter of either about 97 nm or about 107 nm. Such NPs are commercially available at least from Thermo Scientific Seradyn Inc. Further preferred NPs include europium chelate- doped NPs. Advantages of such NPs include i) signal amplification provided by a great number of chelates per particle, ii) strengthened functional affinity (avidity) of the lectins to their target glycostructure epitopes enabled by the high density of immobilized lectins on the particle, and iii) the glycostructure specificity of the lectins used as enabled by creation of the multivalent NPs. However, NPs are only one preferred way of providing adequate avidity effect and signal amplification for carrying out the present invention or disclosure and their various embodiments.

Furthermore, in those embodiments which involve more than one different lectin, a sample may be assayed for different CEACAM-lectin species either in a same assay (i.e. concomitantly) or in different assays (i.e. in parallel), either simultaneously or sequentially. In any such assays, said different lectins may have been detectably labelled, either directly or indirectly, with same of different labels. In some embodiments, multiplexing, for example, by using differently labelled NPs bearing different lectin species in a single assay may be a preferred format for carrying out any of the methods or embodiments thereof disclosed herein.

In some specific embodiments, one or more lectins immobilized on a same or different NPs labelled with a detectable label such as a lanthanide chelate selected from Eu(lll), Tb(lll), Sm(lll), and Dy(lll) are used as tracers. In some more specific embodiments, europium chelate is used as a detectable label. In a non-limiting preferred embodiment, lectin-NP is used as a tracer and is doped with about 30000 Eu-chelates. In some other specific embodiments, lectin in question is attached on upconverting phosphorus (UCP) particles, which are particularly suitable for use as tracers in the lateral flow format. It is also possible to create a detectable signal by using any available sensor technology. For example, a solid surface may incorporate a recognition element (transducer) capable of converting the binding reaction into a detectable signal with or without the use of label moieties. Different types of transducers can be employed, including those based on electrochemical or optical detection. Detection may also be based on either homogeneous or heterogeneous detection techniques, as is apparent to skilled persons.

Coating the lectins on the surface of NPs instead of coating them onto solid surfaces such as microtiter wells, arrays or sensors, brings about significant benefits in terms of assay performance especially in non-competitive assay formats where the lectins are used in combination with specific antibodies. When such antibodies are bound to the solid phase, the typically significantly higher affinity of antibodies as compared to that of lectins can be exploited in full to capture the target biomarker molecules onto the solid phase with high efficiency and stability as the first step. Given the high affinity of antibodies, also small target molecules with only one copy of the targeted epitope can be captured with high stability. Once the target molecules are captured, the avidity effect of the lectin NPs, i.e. the effect where several adjoining lectins can bind to adjoining captured target molecules thereby significantly increasing the binding force compared to singular lectin molecules, can also be utilized in full.

Further combined with the fact that the NPs typically allow significant intensification of the measurable signal, the lectin-NP/solid-phase -antibody approach optimally combines the high specificity of both binding partners, the high binding force of singular antibodies, the high binding force of multiple adjoining lectins on multiple adjoining glycostructures (the avidity effect), and the high level of signal detectable for each bound NP, resulting in the optimal combination of high sensitivity and high specificity, both in terms of both analytical and clinical attributes.

Furthermore, while lectins typically have a high specificity against the target glycoforms, such forms may also exist in other molecules than the targeted one. Therefore, in some preferred embodiments, a wash step is employed between the target molecule antibody capture phase and the lectin-NP binding phase, to wash out all non-targeted and unbound molecules that may in some cases comprise the same glycostructures as the targeted molecules and hence pose a risk of unspecific detection based on cross-reactivity between species. Once the specific target molecules are captured by the use of antibodies, and other molecules washed away before the lectin binding step, the risk of unspecific binding of unwanted targets by lectins becomes eliminated. Such a wash step is also preferred to prevent competition of the target molecule for binding to distant sites around the lectin-NP, although this would occur with a low affinity in many cases. However, both the risk of crossreactivity and distant binding increase when the target molecule has several reactive glycostructures where adjoining lectins can bind to adjoining glycostructures in the same target molecule. In such case the use of the non-competitive lectin-NP/solid-phase - antibody approach with a wash step before the lectin-NP binding reaction results in a significantly more sensitive and target specific assay compared to platforms where the lectins are bound to the solid phase, or where the lectin-NPs are employed without an intermediate wash step. Similarly, because of an almost complete lack of the avidity effect, assay platforms where the lectins (instead of the antibodies) are bound to the solid phase have poor performance with small target molecules which only have one glycostructure moiety per molecule.

Accordingly, in the method according to the present disclosure, assaying for the level of the glycan comprising Fuca1 ,2Gal structure may be based on a binding reaction between the glycan and UEA-1 lectin. Particularly, the level of the glycan comprising Fuca1 ,2Gal structure may be determined by assaying the level of CEACAM-presenting vesicle or a CEACAM antigen which binds to nanoparticle-immobilized UEA-1 (UEA-1 -NP). However, it can be envisaged that any binding molecule that recognizes said glycan structure, such as a lectin, an antibody, an aptamer, a glycan-binding protein or a glycan-binding peptide, or any mixture thereof, may be employed. In some instances, the glycan to be assayed comprises a structure selected from one or more of Fuccd ,2Gaipi ,4Glc, Fuca1 ,2Gaipi ,4GlcNAc and Fucal ,2Gaipi ,4(Fuca1 ,3)GlcNAc, particularly Fuca1 ,2Gaipi ,4GlcNAc. In such cases, besides UEA-1 , any binding molecule that recognizes said glycan structure may be used.

The present disclosure also provides a kit for use or use of a kit in the present methods and various embodiments thereof. In its broadest form, the kit comprises reagents for assaying one or more lectin binding species of CEACAM cancer-associated glycoprotein biomarkers i.e. a CEACAM-binding agent and lectin. In an embodiment, the kit comprises reagents for assaying a sample for one or more CEACAM-lectin species, optionally selected from the group consisting of CEACAM6-UEA, CEACAM1-UEA, CEACAM1-WFL, CEACAM6-WFL, CEACAM1-BPL and CEACAM6-BPL. For each CEACAM-lectin to be assayed, at least one reagent is a CEACAM-binding agent specific for the CEACAM in question, such as monoclonal anti-CEACAM antibody, and at least one of the reagents is the lectin in question, preferably immobilized on a NP. Either said CEACAM-binding agent or said lectin is detectably labelled. The lectin may be indirectly labelled through a detectable NP on which it is immobilized. CEACAM-lectin species to be assayed for, and thus the reagents to be included in the kit, depend on the intended purpose of using the kit, especially on cancer that is to be screened, diagnosed, prognosed, or monitored, and preferred combinations are apparent from the disclosure.

Optionally, the kit may also comprise reagents for assaying one or more conventional antigens, preferably selected from the group consisting of MLIC1 , CEACAM1 , CEACAM6, CA125, CA19-9, and CEA. Non-limiting examples of typical reagents for assaying said conventional antigens include two binding agents for each conventional antigen to be assayed, such as two monoclonal antibodies, which bind to different epitopes in said conventional antigen. For CEACAM1 and CEACAM6 conventional antigens, one of the binding agents may be the same as the binding agent provided for assaying a respective CEACAM-lectin. Nevertheless, one of the conventional antigen-binding agents may be immobilized on a solid surface or provided for use as a capturing agent in lateral flow format, while the other conventional antigen-binding agent may comprise a detectable label. Conventional antigens to be assayed for, and thus the reagents to be included in the kit, depend on the intended purpose of using the kit, especially on cancer that is to be screened, diagnosed, prognosed, or monitored, and preferred combinations become apparent from the disclosure above.

In some embodiments, a kit is provided for determining a subject's cancer disease state, or for screening, diagnosing, prognosing, or monitoring cancer in said subject. In such cases, the kit comprises a CEACAM-binding agent, such as a monoclonal anti-CEACAM antibody, optionally wherein the CEACAM is selected from CEACAM1 and CEACAM6, and at least one lectin selected from the group consisting of UEA-1 , WFL and BPL, optionally immobilized onto a NP. In some embodiments, lectins may further include WGA, DC-SIGN, jacalin, DSA, HPA, ConA, and MAA. If more than one lectin is to be used, they may be immobilized onto same or different NPs. In some embodiments, the lectin(s) comprise(s) a detectable label or has (have) been immobilized on a solid surface, such as a microtiter plate. In some further embodiments, streptavidin coating of the plates and biotinylation of the antibody are used for said attaching. Alternative ways of achieving the same are readily available for a skilled person.

In some embodiments, the kit may also comprise a control for comparing to a measured value of CEACAM-lectin, optionally wherein the CEACAM is selected from CEACAM 1 and CEACAM6 and/or the lectin is at least one selected from the group consisting of UEA-1 , WFL and BPL. In some embodiments, the control is a threshold value for comparing to the measured value. In some further embodiments, the kit may also comprise a computer readable medium comprising computer-executable instructions for performing any method of the present disclosure.

In addition to the reagents for assaying a sample for the biomarker combinations set forth above, the kit may also comprise reagents for assaying said samples for any other biomarker, especially 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 for screening, diagnosing, prognosing, or monitoring cancer but also for screening, diagnosing, prognosing, or monitoring, for example, other cancers, depending on the specificity and sensitivity of the one or more other biomarkers whose concentrations are to be assayed.

Various details and embodiments of the present method apply also to the present kit, as is readily understood by a skilled person. Thus, properties and features of suitable NPs, for instance, are not repeated herein with respect to the kit.

Also provided is the use of a lectin binding glycoform of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) as a cancer biomarker for screening, diagnosing, prognosing and/or monitoring cancer, or for selecting or assigning a treatment, or for patient stratification. Optionally, the lectin binding glycoform of CEACAM is at least one selected from CEACAM 1-UEA-1 , CEACAM6-UEA-1 , CEACAM1-WFL, CEACAM6-WFL, CEACAM1-BPL and CEACAM6-BPL.

Further is provided the use of a glycan comprising Fuca1 ,2Gal structure as a cancer biomarker for screening, diagnosing, prognosing and/or monitoring cancer, or for selecting or assigning a treatment, or for patient stratification. Said glycan is optionally comprised on a CEACAM-presenting vesicle or a CEACAM antigen. The CEACAM may be one or more selected from CEACAM 1 and CEACAM6. Particularly, the glycan may be comprised on one or more selected from CEACAM 1 -presenting vesicle, CEACAM6-presenting vesicle, CEACAM1 antigen, and CEACAM6 antigen. In some instances, the glycan comprises a structure selected from one or more of Fucal ,2Gaipi ,4Glc, Fucal ,2Gaipi ,4GlcNAc and Fucal ,2Gaipi ,4(Fuca1 ,3)GlcNAc. Particularly, the glycan is Fucal ,2Galp1 ,4GlcNAc.

Said use of a glycan comprising Fucal ,2Gal structure as a cancer biomarker may be based on a binding reaction between the glycan and UEA-1 lectin. Particularly, the use may comprise determining the glycan comprising Fucal , 2Gal structure on a CEACAM- presenting vesicle or a CEACAM antigen by binding to nanoparticle-immobilized UEA-1 (UEA-1-NP). However, it can be envisaged that any binding molecule that recognizes said glycan structure, such as a lectin, an antibody, an aptamer, a glycan-binding protein or a glycan-binding peptide, or any mixture thereof, may be employed. In some instances, the glycan to be assayed comprises a structure selected from one or more of Fucal ,2Galp1 ,4Glc, Fucal ,2Galp1 ,4GlcNAc and Fucal ,2Galp1 ,4(Fuca1 ,3)GlcNAc, particularly Fucal ,2Gaipi ,4GlcNAc. In such cases, besides UEA-1 , any binding molecule that recognizes said glycan structure may be used.

In addition, any details disclosed with respect to the present method and its embodiments apply to the various uses of these biomarkers even though the details are not repeated herein. Uses of various other lectin binding biomarker species and any combinations thereof are also provided. Any details and characteristics of such uses become apparent from the disclosure.

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

EXAMPLES

Example 1

1 Materials and Methods

1.1 Clinical Samples

Serum samples of healthy volunteers (n = 18) were obtained from University of Turku, Department of Biotechnology (Turku, Finland). Benign serum samples (n = 22) were obtained from patients having benign prostatic hyperplasia (BPH) or Crohn’s disease. Serum samples from CRC patients (n = 30) having early stage l-ll (n = 8) or late stage III- IV (n = 19) cancer or undefined (NA) CRC status (n = 3) were provided by Stavanger University Hospital (Stavanger, Norway). Study subjects’ demographic characteristics are presented in Table 1 , and the clinicopathological characteristics of CRC patients are presented in Table 2. The serum samples from patients with CRC were collected prior to surgical resection of the tumour. The undefined samples were from subjects diagnosed with CRC that did not undergo surgery due to their weak immune system or low recovery rate due to elderly age. Therefore, their clinical stage is unknown. Table 1. Study subjects’ demographic characteristics.

Table 2. CRC patients’ clinicopathological characteristics. Total number of CRC patients:

N=30. Location of CRC: colon N=19 (63%); rectum N=11 (37%).

1.2 Reagents

The 96-well microtiter plates coated with streptavidin (SA plates, product #: 41-07TY), wash buffer (product #: 42-01 TY), and RED assay buffer (product #: 42-02TY) were purchased from Kaivogen Oy (Turku, Finland). Antibodies used in the study are listed in Table 3. The plate washer (Delfia PlateWash 1296-026), plate shaker (Delfia PlateWash 1296-026) were from Wallac Oy (Turku, Finland), and HIDEX™ fluorimeter from HIDEX Oy (Turku, Finland). The reagents for cell line cultures were Gibco brand of Thermo Fisher Scientific (Waltham, Massachusetts, US) except for glutamine, which was Ultraglutamine from Lonza (Basel, Switzerland), and phosphate buffered saline which was from GE Healthcare (Chicago, Illinois, US). Fujirebio Diagnostics (Gothenburg, Sweden) provided the conventional CEA kit used as reference assays.

Table 3. Monoclonal antibodies used in the study. 1.3 Cell culture

The human CRC cell lines Colo 205, Colo 320 DM, SW 403, LS17 4T, and SW 1463 were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% inactivated fetal bovine serum (FBS), 1% glutamine, and 1% penicillin-streptomycin. The cell culture spent medium (CCSM) was from Fujirebio Diagnostics. The human embryonic kidney 293 cell line HEK293 was cultured in Expi293 medium, commercially purchased from Thermo Fischer Scientifics (Waltham, MA, USA). The cells were cultured at 37 °C under 5% CO2. When the cells reached the confluence of approximately 70%, the medium was collected and centrifuged for 3 min at 161 x g. The CCSM was collected and stored at -80 °C. The spent medium was then concentrated 5 times with a Vivaspin Turbo 15 filter (Sartorius Stedim Lab Ltd., Stonehouse, UK) and stored at -20 °C.

1.4 Conjugation of lectins and glycan binding antibodies on Eu-nanoparticles (NPs)

Glycan-binding proteins (lectins) and monoclonal antibodies (mAbs) were linked to the activated carboxyl group of the Eu-NPs through covalent bonding of amino groups following procedures previously outlined in Gidwani et al. Clin Chem. 2016;62(10):1390- 1400 and Terava et al. PLoS One 2019;14(7):e0219480. A coupling reaction was carried out by mixing N-hydroxysulfosuccinimide and N-(3-dimethylaminopropyl)-N'- ethylcarbodimide with 1e10¹² NPs in 50 mM MES buffer (pH 6.1 ) at room temperature for 15 minutes. The final concentrations of N-hydroxysulfosuccinimide and N-(3- dimethylaminopropyl)-N'-ethylcarbodimide were 10 and 0.75 mmol/L, respectively. The lectins or mAbs were present in the reaction at a concentration of 0.625 g/L. The reaction was carried out for 2 hours with continuous mixing at room temperature. Afterwards, the remaining active groups were blocked by washing the NP-protein conjugates in Tris buffer (10 mmol/L Tris, 0.5 g/L NaNs, pH 8.5) and storing the conjugates in the same buffer with 2 g/L BSA at 4°C. Before each use, the particles were thoroughly mixed by vortexing and sonicated to prevent large aggregates from forming.

1.5 Biotinylation of antibodies (solid phase surface) for capturing the antigen

MAbs to be used as capture antibodies were biotinylated for 4 hours at room temperature using a procedure described in Gidwani et al. Clin Chem. 2016;62(10):1390-1400. The biotinylated antibodies were purified using NAPTM-5 and NAPTM-10 gel-filtration columns in a solution of 50 mmol/L Tris-HCI (pH 7.75) containing 150 mmol/L NaCI and 0.5 g/L NaNs. The biotinylated antibodies were stored in 1 g/L BSA at 4°C.

1.6 Passive coating time-resolved fluorometry assay for cancer-related glycosylation An immunoassay was employed in profiling glycoforms in CRC cell lines and pooled serum samples from healthy, benign and CRC sources. The assay utilized lectins and glycan- binding antibodies coated on europium NPs to target glycoforms and known cancer biomarkers present in the samples. Benign sources included a pooled serum sample from patients that have undergone transurethral resection of the prostate (TLIRP) (Turku Prostate Cancer Consortium) and a pooled serum sample obtained from endometriosis patients (Gothenburg, Sweden). In addition to CRC cell lines (Colo 205, Colo 320 DM, SW 403, LS17 4T, and SW 1463), the human cell lines HEK 293 and MCF-10 were utilized as negative control CCSMs.

Antigens with the concentration of 1000ng/25ul and 4000ng/25ul prepared in PBS from CRC cell lines and pooled serum samples, respectively, were added on pre-washed 96- welled (streptavidin microtitration “normal” yellow) plates and incubated at 37°C for two hours. After incubation, 4 x wash steps were done by using Kaivogen wash buffer diluted to 1 x concentration. After washing, BSA with the 1 x concentration was added into the wells to block the remaining sites and refrigerated at 4°C for two hours. All wells were sealed with plastic tape. After incubation the wells were again washed 4 times to remove the un-conjugated BSA from the wells. Following washing, 25 pL of RED assay buffer was added per well, containing 1 x 10⁷ europium NPs coated with lectin or monoclonal antibody, for a 90-minute incubation with shaking. Table 4 lists lectins employed in the assays herein as well as their major carbohydrate binding specificities. Of the lectins used in the passive coating assay, AAL, BPL, Con-A, DBA, HPA, Jacalin, LCA, MAA, UEA, WFL, WGA and WL were from Vector Laboratories (Burlingame, CA, USA), and DC-Sign was from R&D Systems (Minneapolis, Ml, USA). Conventional biomarkers CEA, CA19-9, MUC1 , and CA125 were also tested in a passive coating assay employing as tracer Eu- NPs coated with monoclonal antibodies specific for the conventional biomarker to be assayed. The wells were washed six times, and time-resolved fluorescence of Eu³⁺ was recorded using a HI DEX fluorometer (A_ex at 340 nm, A_em at 615 nm).

Table 4. Lectins employed in the studies reported herein, and their major carbohydrate binding specificities.

1.7 Sandwiched time-resolved fluorometry immunoassay for cancer-related glycoforms

Sandwiched immunoassays were conducted using time-resolved fluorescence (TRF) to detect antibodies against glycoforms of CA19-9, CEACAM1 , CEACAM6 and MUC1. The assay was performed at room temperature using as tracer lectins or mAbs coated on Eu- NPs. Biotinylated monoclonal capture antibodies for CA19-9 (C192), CEA (12-140-10), CEACAM1 (MAB22441 ), CEACAM6 (MAB3934) and MUC1 (Ma552) were immobilized at a concentration of 50ng I 25ul onto streptavidin-coated low-fluorescence wells for 45 minutes without shaking. The wells were washed twice and then incubated with RED assay buffer and CCSM sample or diluted serum sample for 45 minutes. After washing, Eu-NP coated with a lectin (UEA-1 , WFL, BPL, or DSL) or a glycan-binding mAb (C192 for CA19- 9, 12-140-1 for CEA or Ma695 for Muc1 ) was added to the wells and incubated for 60 minutes. DSL was obtained from Vector Laboratories (Burlingame, CA, USA). The wells were washed six times and TRF was measured using HI DEX fluorometer with an excitation wavelength of 340 nm and an emission wavelength of 615 nm.

1.8 Statistical analysis

The results were visualized using RStudio and ggplot2 R packages. The heatmap analysis was performed using ggplot2. The marker concentrations in disease groups were compared using Kruskal-Wallis one-way ANOVA with a post hoc Dunn test on rank- transformed data. Statistical analyses were conducted using R studio, and a significant difference was considered if the p-value was 0.05. The normality of the values was evaluated using the Shapiro-Wilk test and visually. Medians and quartiles for conventionally measured markers and CEACAM1 and CEACAM6 assays were calculated for different diagnostic groups and tested for equal variances using Leven's test.

2 Results and Discussion

2.1 Characterization of different glycoforms in CRC cell lines and pooled serum samples by a passive coating assay

Pooled CRC and benign serum samples, and cell culture spent medium (CCSM) collected from CRC cell line cultures were bound to a solid surface in a 96-well plate in a passive coating assay as described in paragraph 1.6. The remaining sites of the bound antigen were blocked by BSA to minimize noise from the samples. Different conventional biomarkers (CEA, CA19-9, MUC1 , and CA125) were tested from the serum samples and CRC cell lines along with different lectins and glycan-binding antibodies. The results are shown in Figure 1.

The signal-to-background (S/B) ratio varied among different lectins and antibodies, representing the amount or expression level of the molecules they bind to in the samples. Notably, in these initial results, UEA-1 , WFL and BPL lectins exhibited relatively strong binding in early stage CRC samples T1 Pool and T2 Pool. UEA-1 for instance showed strong binding to T1 rectum samples but low binding to transurethral resection of the prostate (TLIRP) benign pool samples. A similar trend was observed in CCSM samples, where UEA-1 relatively strongly bound to 3 out 5 CRC cell lines but showed minimal binding to COLO 205 and COLO 320 DM. COLO 320 DM is a cell line representing the less common neuroendocrine type of CRC. UEA-1 showed little or no binding to pooled samples from the benign cell lines HEK 293 and MCF-10.

WFL is believed to prefer binding carbohydrate structures terminating in GalNAc linked or or /? to the 3 or 6 positions of Gal. As seen from Figure 1 , WFL bound to almost all of the samples as indicated by the high S/B ratio but showed exceptionally strong binding with early stage T 1 pool colon samples and LS 147T CRC cell line.

Among the conventional biomarkers, level of CEA was low in early stage (T1 and T2) colon and rectum samples but a higher level of CEA was detected in late stage T3 colon samples and NA pool rectum samples, which can be considered late stage due to the severity of the CRC disease state in the elderly patients. CA125 showed high expression in late stage T4-colon sample but also in the benign endometrium pool and TURP samples. CA19-9 and MUC1 expression was observed in almost all the colon pool samples except in T3 Pool of the colon for CA19-9 and in T3 Pool Rectum for both. CA19-9 expression was low in benign endometrium and TURP samples, while all the CRC cell lines exhibited very high CA19-9 expression and mild signals in benign HEK293 and MCF10 cell lines.

2.2 Characterization of glycoforms in CRC cell lines and pooled serum samples by sandwiched immunoassays

CEACAM1 and CEACAM6 have previously shown high expression in CRC in an RNA profiling study. To assess glycoforms of these biomarkers, we conducted sandwiched immunoassays using as capture antibodies mAbs specific to CEACAM1 and CEACAM6 and as tracers Eu-NPs coated with lectins (UEA-1 , WFL, BPL, DSL) or glycan-binding antibodies (C192, Ma695). The conventional biomarkers CA19-9 and MUC1 were also assayed in the sandwich format. Of the antibodies used, C192 is specific for the sialyl Lewis³ epitope CA19-9, and Ma695 recognizes a sialylated carbohydrate antigen on MUC1.

Altogether 13 lectins with different carbohydrate-binding specificities and 4 antibodies were tested in the passive coating assay described in paragraphs 1.6 and 2.1. Some lectins considered promising as well as certain antibodies were then tested with CRC and benign pooled samples in a sandwich-immunoassay as described in paragraph 1.7. An assay with CA19-9 capture antibody and DSL lectin was also included in the tests at this stage. Wells were coated with CA19-9, MLIC1 , CEACAM1 or CEACAM6 capture antibodies. Thereafter, first the sample and then an Eu-NP coated with a lectin or glycan- binding antibody was added to the well. Only the pooled samples which had a large enough volume left after the passive coating assay were tested. The detailed results of the sandwich immunoassays are presented in Figure 2 a)-d).

UEA-1 performed well in the sense that it showed strong binding in all of the serum pool samples in CEACAM1 (Fig. 2c) and CEACAM6 (Fig. 2d) assays but showed weaker binding in MLIC1 (Fig. 2b) and CA19-9 (Fig. 2a) assays. WFL and BPL exhibited somewhat weaker binding to CEACAM1 and CEACAM6 than UEA-1 but represent usable lectins in CRC diagnostics.

Of the conventional biomarkers, CA19-9 was strongly expressed in all CRC serum samples and most of the CRC cell lines but was not detected in the benign cell lines HEK293 and MCF10. On the other hand, MUC1 is strongly expressed in the CRC serum samples but is not detected in any of the CRC cell lines. Assays using UEA-1-NPs combined and CA19-9 or MUC1 capture antibody showed promising results with pooled samples in these initial tests (Figure 2a-b), but when the same assays were performed using individual samples instead of sample pools (results not shown), the results were not up to the mark of CEACAM1 and CEACAM6.

2.3 Comparison of conventional CEA-ELISA with CEA-UEA-1 glycovariant assay

The concentration of CEA, the conventional prognostic biomarker for CRC, was analyzed in serum samples from healthy subjects (n = 18), subjects with benign condition (n = 22), and CRC (n = 30) patients using a CEA ELISA kit by Fujirebio Diagnostics (Gothenburg, Sweden). The samples underwent testing in the sandwiched time-resolved format as singlet samples, not as pooled samples. Discrimination between healthy, benign, and CRC serum samples in this conventional ELISA assay was significant as shown in Figure 3. The results showed significant discrimination of CRC compared to healthy and benign, with p = 0.000020 and p = 0.00098, respectively. However, the cutoff for the conventional CEA-ELISA kit is 5 ng/ml. Most of the CRC samples tested here fall below the cut-off value which makes the conventional assay unreliable.

We also performed a sandwiched TRF immunoassay for the CEA-UEA-1 glycovariant using an anti-CEA antibody 12-140-10 as capture Ab in combination with the lectin UEA- 1 conjugated on an Eu³⁺ NP (Figure 3). The specificity of this CEA-UEA-1 assay in the samples was poor and it did not give significant discrimination among healthy, benign, and CRC samples (p = 0.1 and p = 1 , respectively). The results indicate that the glycovariant CEA-UEA-1 exhibits low sensitivity and specificity and that there may not be high enough expression of CEA in the samples for diagnosis.

2.4 Comparison of CEACAM1 ELISA, total CEACAM1 and glycovariant CEACAM1-UEA- 1 assays

Expression of CEACAM1 was studied in singlet serum samples of healthy subjects (n = 18), subjects with benign condition (n = 22), and CRC (n = 30) patients. For this purpose, we ran a total CEACAM1 (tCEACAMI ) time-resolved fluorescence (TRF) sandwich immunoassay utilizing MAB22441 as both the capture and tracer antibody. Additionally, we employed a commercial CEACAM1 ELISA kit from Abeam (USA). These CEACAM1 assays were compared with the new CEACAM1-UEA-1 glycovariant assay performed as described in paragraph 1.7 using capture Ab MAB22441 and UEA-1-coated Eu-NPs.

The results of the tCEACAMI immunoassay and CEACAM1 ELISA are shown in Figure 4. There was no significant discrimination between benign and CRC samples in the tCEACAMI assay. Signal-to-background ratio of the tCEACAMI assay indicated higher CEACAM1 expression in healthy samples compared to benign and CRC. Furthermore, CEACAM1 ELISA demonstrated no discrimination across any group. The new CEACAM1- UEA-1 glycovariant assay emerged as the most effective, exhibiting significant discrimination among healthy, benign, and CRC samples. The discrimination was significant between healthy and CRC samples (p = 0.0000014) and between benign and CRC samples (p = 0.0000063). The glycovariant assay CEACAM1-UEA-1 also displayed a remarkably high signal-to-background ratio, indicating high assay sensitivity and specificity.

2.5 Comparison of CEACAM6 ELISA, total CEACAM6 and glycovariant CEACAM6-UEA- 1 and CEACAM6-WFL assays

Expression of CEACAM6 was studied in serum samples of healthy subjects, subjects with benign condition and CRC patients. For this purpose, we ran a total CEACAM6 (tCEACAM6) time-resolved fluorescence (TRF) sandwich immunoassay utilizing an anti- CEACAM6 antibody MAB3934 as both the capture and tracer antibody. Additionally, we employed a commercial CEACAM6 ELISA kit from Abeam (USA). These CEACAM6 assays were compared with the new CEACAM6-UEA-1 and CEACAM6-WFL glycovariant assays performed as described in paragraph 1.7 using a CEACAM6 capture Ab and a UEA-1 or WFL coated Eu-NP. The discriminative ability of total CEACAM6 (tCEACAM6) in the healthy vs. CRC sample (p = 0.36) and benign vs. CRC (p = 0.50) was not significant as shown in Figure 5. With CEACAM6 ELISA the discrimination was not significant (p = 0.85) between benign and CRC samples but between healthy and CRC samples discrimination was slightly significant (p = 0.026).

The new CEACAM6-UEA-1 glycovariant assay demonstrated significant discrimination between benign and CRC samples (p = 0.0000018) as well as between healthy and CRC samples (p = 0.0000028). A similar trend was observed in the CEACAM6-WFL glycovariant assay, where the CRC subject group exhibits a higher signal-to-background ratio than the benign (p = 0.000013) and healthy (p = 0.000010) samples.

2.6 ROC curve analysis of sandwich immunoassays and glycovariant assays

As discussed above, conventional ELISA assays were conducted for CEA, CEACAM1 , and CEACAM6, alongside tCEACAMI and tCEACAM6 immunoassays. Other than CEA ELISA, these assays had very low area under the curve (AUC) and so are not included in the Receiver Operating Characteristics (ROC) curve presented in Figure 6., An immunoassay for CEACAM6-BPL glycoform using Bauhinia purpurea lectin (BPL) conjugated on Eu-NPs was performed. The results of this assay are presented in the ROC curve along with glycovariant assays CEACAM1-UEA-1 , CEACAM6-UEA-1 , and CEACAM6-WFL. The AUC was calculated to determine the overall clinical performance as displayed by the 95% confidence interval of each assay. The glycovariant assays CEACAM1-UEA-1 , CEACAM6-BPL, CEACAM6-UEA-1 and CEACAM6-WFL performed similarly, showing improved clinical performance in comparison to CEA ELISA, which is currently the “gold standard” for CRC diagnosis.

Claims

1 . A method for determining cancer disease state in a subject, the method comprising: a) Assaying a sample obtained from said subject for the level of lectin binding glycoform of carcinoembryonic antigen-related cell adhesion molecule (CEACAM), wherein preferably the lectin binding glycoform of CEACAM (CEACAM-lectin) is one or more selected from Ulex europaeus agglutinin I (UEA- 1 ) binding glycoform of CEACAM1 (CEACAM1-UEA-1 ), the UEA-1 binding glycoform of CEACAM6 (CEACAM6-UEA-1 ), the Wisteria floribunda lectin (WFL) binding glycoform of CEACAM1 (CEACAM1-WFL), the WFL binding glycoform of CEACAM6 (CEACAM6-WFL), the Bauhinia purpurea lectin (BPL) binding glycoform of CEACAM1 (CEACAM 1 -BPL), and the BPL binding glycoform of CEACAM6 (CEACAM6-BPL), b) Comparing the detected level(s) of CEACAM-lectin in said sample with that of a control sample or a predetermined threshold value, and c) Determining the cancer disease state on the basis of said comparison.

2. The method according to claim 1 , wherein increased level of CEACAM-lectin as compared with that of the control sample or predetermined threshold value indicates that said subject has or is at risk of having cancer.

3. The method according to claim 1 or 2, wherein non-increased or decreased level of CEACAM-lectin as compared with that of the control sample or predetermined threshold value indicates that said subject does not have or is not at risk of having cancer.

4. The method according to any one of the preceding claims, wherein said assaying for the level of CEACAM-lectin is based on a binding reaction between CEACAM and lectin.

5. The method according to any one of the preceding claims, wherein CEACAM is one or more selected from CEACAM 1 and CEACAM6, and/or the lectin is one or more selected from UEA-1 , WFL and BPL.

6. The method according to any one of the preceding claims, wherein the method comprises assaying a sample obtained from said subject for the level of CEACAM1- UEA-1 and/or CEACAM6-UEA-1 , and optionally one or more biomarkers selected from the group consisting of CEACAM1-WFL, CEACAM6-WFL, CEACAM1-BPL and CEACAM6-BPL.

7. The method according to any one of the preceding claims for screening, diagnosing, prognosing and/or monitoring cancer, or for selecting or assigning a treatment, or for patient stratification.

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

9. The method according to claim 7 or 8, wherein said monitoring is carried out by repeating the assaying step at least twice at different time points, optionally wherein said time points are selected, independently from each other, from the group consisting of time points before diagnosis, time points after diagnosis, time points before treatment of said cancer, time points during treatment of said cancer, and time points after treatment of said cancer.

10. The method according to any one of claims 7 - 9, wherein the monitoring is carried out during or after treatment of cancer, and wherein said subject is determined as having relapse or recurrence of cancer or as being at risk of relapse or recurrence of cancer, in case the level of CEACAM-lectin is higher than in a control sample or above a predetermined threshold value.

11. The method according to claim 10, wherein said control sample is a sample obtained from the same subject before the treatment of cancer.

12. The method according to any one of the preceding claims, wherein in case the level of CEACAM-lectin is higher than in a control sample or above a predetermined threshold value, a treatment is selected for or assigned to said patient, optionally wherein the treatment is selected from the group consisting of surgery, radiation therapy, immunotherapy, targeted therapy and chemotherapy.

13. The method according to any one of the preceding claims, wherein the level of CEACAM-lectin is determined by assaying the level of CEACAM which binds to nanoparticle-immobilized lectin (lectin-NP).

14. The method according to any one of the preceding claims, wherein said sample is selected from the group consisting of urine, blood, serum, plasma, semen, peritoneal cavity fluid, ascites fluid and tissue samples.

15. The method according to any one of the preceding claims, wherein the cancer is colorectal cancer (CRC).

16. A kit for use or use of a kit in the method according to any one of claims 1 to 15, wherein the kit comprises a CEACAM-binding agent and lectin, wherein either said CEACAM-binding agent or said lectin comprises a detectable label, optionally wherein the CEACAM-binding agent is selected from agents that bind to CEACAM1 and/or CEACAM6 and/or the lectin is one or more selected from UEA-1 , WFL and BPL.

17. Use of a lectin binding glycoform of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) as a cancer biomarker for screening, diagnosing, prognosing and/or monitoring cancer, or for selecting or assigning a treatment, or for patient stratification, wherein preferably the lectin binding glycoform of CEACAM (CEACAM- lectin) is one or more selected from CEACAM 1 -UEA-1 , CEACAM 6- UEA-1 , CEACAM1-WFL, CEACAM6-WFL, CEACAM1-BPL and CEACAM6-BPL.

18. A method for determining cancer disease state in a subject, the method comprising: a) Assaying a sample obtained from said subject for the level of a glycan comprising

Fucal ,2Gal structure, wherein said glycan is comprised on a CEACAM-presenting vesicle or a CEACAM antigen; b) Comparing the detected level of the glycan in said sample with that of a control sample or a predetermined threshold value, and c) Determining the cancer disease state on the basis of said comparison.

19. The method according to claim 18, wherein the glycan comprises a structure selected from one or more of Fucal ,2Gal01 ,4Glc, Fucal ,2Gal01 ,4GlcNAc and Fucal ,2Galp1 ,4(Fuca1 ,3)GlcNAc.

20. The method according to claim 18 or 19, wherein increased level of the glycan comprising Fucal ,2Gal structure as compared with that of the control sample or predetermined threshold value indicates that said subject has or is at risk of having cancer.

21. The method according to any one of claims 18 to 20, wherein non-increased or decreased level of the glycan comprising Fucal ,2Gal structure as compared with that of the control sample or predetermined threshold value indicates that said subject does not have or is not at risk of having cancer.

22. The method according to any one of claims 18 to 21 , wherein said assaying for the level of the glycan comprising Fucal , 2Gal structure is based on a binding reaction between the glycan and UEA-1 lectin.

23. The method according to any one of claims 18 to 22, wherein the CEACAM is one or more selected from CEACAM 1 and CEACAM6.

24. The method according to any one of claims 18 to 23, wherein the method comprises assaying a sample obtained from said subject for the level of a glycan comprising Fucal ,2Gal structure, wherein said glycan is comprised on one or more selected from CEACAM 1 -presenting vesicle, CEACAM6-presenting vesicle, CEACAM1 antigen, and CEACAM6 antigen.

25. The method according to any one of claims 18 to 24 for screening, diagnosing, prognosing and/or monitoring cancer, or for selecting or assigning a treatment, or for patient stratification.

26. The method according to claim 25, wherein said monitoring is for monitoring onset of cancer, for monitoring any change in risk of having or developing cancer, for monitoring response to treatment, for monitoring relapse of cancer, or for monitoring recurrence of cancer.

27. The method according to claim 25 or 26, wherein said monitoring is carried out by repeating the assaying step at least twice at different time points, optionally wherein said time points are selected, independently from each other, from the group consisting of time points before diagnosis, time points after diagnosis, time points before treatment of said cancer, time points during treatment of said cancer, and time points after treatment of said cancer.

28. The method according to any one of claims 25 to 27, wherein the monitoring is carried out during or after treatment of cancer, and wherein said subject is determined as having relapse or recurrence of cancer or as being at risk of relapse or recurrence of cancer, in case the level of the glycan comprising Fucal ,2Gal structure is higher than in a control sample or above a predetermined threshold value.

29. The method according to claim 28, wherein said control sample is a sample obtained from the same subject before the treatment of cancer.

30. The method according to any one of claims 18 to 29, wherein in case the level of the glycan comprising Fuca1 ,2Gal structure is higher than in a control sample or above a predetermined threshold value, a treatment is selected for or assigned to said patient, optionally wherein the treatment is selected from the group consisting of surgery, radiation therapy, immunotherapy, targeted therapy and chemotherapy.

31 . The method according to any one of claims 18 to 30, wherein the level of the glycan comprising Fuca1 ,2Gal structure is determined by assaying the level of CEACAM- presenting vesicle or a CEACAM antigen which binds to nanoparticle-immobilized UEA-1 (UEA-1-NP).

32. The method according to any one of claims 18 to 31 , wherein said sample is selected from the group consisting of urine, blood, serum, plasma, semen, peritoneal cavity fluid, ascites fluid and tissue samples.

33. The method according to any one of claims 18 to 32, wherein the cancer is colorectal cancer (ORC).

34. Use of a glycan comprising Fucal ,2Gal structure as a cancer biomarker for screening, diagnosing, prognosing and/or monitoring cancer, or for selecting or assigning a treatment, or for patient stratification, wherein said glycan is comprised on a CEACAM-presenting vesicle or a CEACAM antigen.

35. The use according to claim 34, wherein the glycan comprises a structure selected from one or more of Fucal ,2Gal01 ,4Glc, Fucal ,2Gal01 ,4GlcNAc and Fucal ,2Galp1 ,4(Fuca1 ,3)GlcNAc.

36. The use according to claim 34 or 35, wherein said use is based on a binding reaction between the glycan and UEA-1 lectin.

37. The use according to any one of claims 34 to 36, wherein the CEACAM is one or more selected from CEACAM1 and CEACAM6.

38. The use according to any one of claims 34 to 37, wherein said glycan is comprised on one or more selected from CEACAM 1 -presenting vesicle, CEACAM6-presenting vesicle, CEACAM1 antigen, and CEACAM6 antigen.

39. The use according to any one of claims 34 to 38, wherein said monitoring is for monitoring onset of cancer, for monitoring any change in risk of having or developing cancer, for monitoring response to treatment, for monitoring relapse of cancer, or for monitoring recurrence of cancer.

40. The use according to any one of claims 34 to 39, comprising determining the glycan comprising Fuca1 ,2Gal structure on a CEACAM-presenting vesicle or a CEACAM antigen by binding to nanoparticle-immobilized UEA-1 (UEA-1-NP).

41. The use according to any one of claims 34 to 40, wherein the cancer is colorectal cancer (ORC).