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WO2025196374A2 - Biomarqueurs glycovariants pour le diagnostic du cancer - Google Patents

Biomarqueurs glycovariants pour le diagnostic du cancer

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Publication number
WO2025196374A2
WO2025196374A2 PCT/FI2025/050138 FI2025050138W WO2025196374A2 WO 2025196374 A2 WO2025196374 A2 WO 2025196374A2 FI 2025050138 W FI2025050138 W FI 2025050138W WO 2025196374 A2 WO2025196374 A2 WO 2025196374A2
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WIPO (PCT)
Prior art keywords
cancer
ceacam
lectin
ceacam6
monitoring
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WO2025196374A3 (fr
Inventor
Janne LEIVO
Rufus VINOD
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University of Turku
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University of Turku
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Publication of WO2025196374A2 publication Critical patent/WO2025196374A2/fr
Publication of WO2025196374A3 publication Critical patent/WO2025196374A3/fr
Pending legal-status Critical Current
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • 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.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • PET positron emission tomography
  • 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.
  • the assay targets lectin binding glycoforms of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs).
  • CEACAMs carcinoembryonic antigen-related cell adhesion molecules
  • 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.
  • kits for use or, alternatively, use of a kit in the method for determining cancer disease 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.
  • 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.
  • 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.
  • CRC colorectal cancer
  • TLIRP transurethral resection of the prostate
  • Endo Endometriosis serum pool
  • CCSM Cell culture spent media
  • CRC cell lines Cold 205, Colo 320 DM, SW 403, LS17 4T, and SW 1463
  • 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);
  • concentration of CEA in the CEA-ELISA assay was significantly higher in CRC than in healthy and benign controls.
  • 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.
  • S/B signal-to-background
  • Figure 5 illustrates comparison of CEACAM6 ELISA, in-house total CEACAM6 and Glycovariant Immunoassays of CEACAM6.
  • 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
  • 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).
  • Overexpression of proteins is a typical feature of cancer cells.
  • 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.
  • 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.
  • PTM post-translational modification
  • GIcNAc A/-acetylglucosamine
  • sLe a sialyl-Lewis a
  • sLe x sialyl-Lewis x
  • Carcinoembryonic antigen-related cell adhesion molecules 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.
  • 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).
  • 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.
  • 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.
  • Fuc refers to fucose
  • Gal refers to galactose
  • Glc refers to glucose
  • GIcNAc refers to N-acetylglucosamine.
  • 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.
  • AAL 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.
  • WFL Wisteria floribunda lectin
  • GalNAc N-acetylgalactosamine
  • BPL Bauhinia purpurea lectin detects N-terminal acetylgalactosamine, binding specifically to Gal0-1 ,3GalNAc.
  • DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin
  • tissue sample 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.”
  • 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.
  • tissue samples such as biopsy samples taken from a tissue.
  • said tissue sample may be a formalin-fixed or paraffin-embedded tissue sample.
  • 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.
  • the sample is an EDTA plasma sample.
  • 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.
  • 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.
  • 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”.
  • binder molecule refers broadly to any molecule that is able to bind a biomarker or a glycan part thereof.
  • binder molecules include lectins, antibodies, antibody mimetics, and oligonucleotide and peptide aptamers.
  • 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.
  • 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.
  • the terms "human subject”, “patient” and “individual” and the like are interchangeable.
  • 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.
  • the terms “apparently healthy” and “healthy” and the like may in some instances be used interchangeably.
  • benign condition refers to non-cancerous conditions.
  • non-cancerous benign conditions include benign prostatic hyperplasia, Crohn’s disease and endometriosis.
  • 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.
  • the term “cancer” refers broadly to any cancerous condition.
  • 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).
  • the cancer is CRC.
  • “cancer” is broadly referred to herein by using the term "disease”.
  • cancer disease state refers to any distinguishable manifestation of cancer, including non-cancer.
  • 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.
  • 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.
  • 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.
  • 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.
  • level is interchangeable with terms including “amount”, “expression” and “concentration”, unless otherwise indicated.
  • 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.
  • 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.
  • 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.
  • 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.
  • the term “about” refers to a range of values ⁇ 10% of a specified value.
  • 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.
  • 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.
  • a predetermined threshold value i.e. a cut-off value
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • WGA Wheat germ agglutinin
  • DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin
  • HPA Helix pomatia agglutinin
  • ConA Concanavalin A
  • MAA Maackia amurensis lectin
  • 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 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.
  • CEA CEACAM1
  • CEACAM6 CEACAM6
  • the term "antigen” refers broadly to any protein antigen that may be indicative of cancer.
  • 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.
  • the term “antigen” refers broadly to any antigen in soluble form and to an extracellular vesicle which harbors said antigen on its surface.
  • 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 Lewis 3 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.
  • 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”.
  • CEACAM1-UEA-1 and “CEACAM6-UEA-1” refer to UEA-1 lectin binding glycoforms of CEACAM1 and CEACAM6, respectively.
  • CEACAM- UEA-1 refers to a UEA-1 lectin binding glycoform of a protein antigen belonging to the CEACAM family.
  • 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.
  • 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.
  • false positive refers to a test result, which classifies an unaffected subject incorrectly as an affected subject.
  • false negative refers to a test result, which classifies an affected subject incorrectly as an unaffected subject.
  • true positive refers to a test result, which classifies a subject who has a disease correctly as an affected subject.
  • true negative refers to a test result, which classifies an unaffected subject correctly as an unaffected.
  • uccess rate refers to the percentage-expressed proportion of affected individuals with a positive result
  • false positive rate refers to the percentage-expressed proportion of unaffected individuals with a positive result
  • the area under the ROC curve is a measure of the utility of a marker in the correct identification of disease subjects.
  • 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.
  • 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 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.
  • Ulex europaeus agglutinin I UEA-1
  • WFL Wisteria floribunda lectin
  • BPL Bauhinia purpurea lectin
  • 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.
  • 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.
  • lectins may further include WGA, DC-SIGN, jacalin, DSA, HPA, ConA, and MAA.
  • the CEACAM is CEACAM1.
  • 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.
  • the CEACAM is CEACAM 1.
  • the method comprises assaying a sample obtained from said subject for the level of CEACAM1-UEA-1 or CEACAM6-UEA-1 or both.
  • one or more biomarkers selected from the group consisting of CEACAM1-WFL, CEACAM6-WFL, CEACAM1-BPL and CEACAM6-BPL may be assayed.
  • 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.
  • 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.
  • 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.
  • mucins e.g. MLIC1
  • CEACAMs e.g. CA19-9
  • 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.
  • assaying a sample for CEACAM1-UEA-1 glycoform 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.
  • 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.
  • a method for determining cancer disease state in a subject 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.
  • the CEACAM is CEACAM1.
  • 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.
  • the glycan comprises a structure selected from one or more of Fucal ,2Galp1 ,4Glc, Fucal ,2Galp1 ,4GlcNAc and Fucal ,2Galp1 ,4(Fuca1 ,3)GlcNAc.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present method is particularly suitable for early diagnosis of cancer and early detection of cancer relapse, recurrence and progression.
  • 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.
  • 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.
  • a sandwich assay may be conducted in a reversed way.
  • the lectin is used as a capturing agent and a protein antigen-specific antibody, as a directly or indirectly detectably labelled tracer.
  • the reversed sandwich assay may operate better with urine samples than with blood samples.
  • 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.
  • washing solutions and conditions e.g. time and temperature are known to those skilled in the art.
  • Sandwich assays may be performed either on a solid surface, such as a microtiter plate, or in lateral flow format.
  • a solid surface such as a microtiter plate
  • 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.
  • 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.
  • a capturing agent i.e. either an anti-protein antigen antibody or a lectin
  • one or more control antibodies or control lectins may also be attached to the substrate.
  • 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.
  • said anti-protein antigen antibody may detectably labelled indirectly, for example through immobilization to a detectable nanoparticle (NP).
  • lectins or antibodies may be detectably labelled by various ways as is well known in the art.
  • 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.
  • 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).
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • 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.
  • NP nanoparticle
  • the term “nanoparticle” 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.
  • the term “about” refers to a range of values ⁇ 10% of a specified value.
  • 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.
  • 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.
  • 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.
  • 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.
  • the NPs can be made detectable e.g. by fluorescent labels, bioluminescent labels, chemiluminescent labels.
  • 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.
  • 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.
  • 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.
  • 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.
  • said different lectins may have been detectably labelled, either directly or indirectly, with same of different labels.
  • 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.
  • 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.
  • a detectable label such as a lanthanide chelate selected from Eu(lll), Tb(lll), Sm(lll), and Dy(lll) are used as tracers.
  • europium chelate is used as a detectable label.
  • lectin-NP is used as a tracer and is doped with about 30000 Eu-chelates.
  • lectin in question is attached on upconverting phosphorus (UCP) particles, which are particularly suitable for use as tracers in the lateral flow format.
  • UCP upconverting phosphorus
  • 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.
  • a recognition element transducer
  • 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.
  • 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.
  • small target molecules with only one copy of the targeted epitope can be captured with high stability.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a CEACAM-binding agent specific for the CEACAM in question, such as monoclonal anti-CEACAM antibody
  • 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 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.
  • 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.
  • suitable 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.
  • one of the binding agents may be the same as the binding agent provided for assaying a respective CEACAM-lectin.
  • 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.
  • a kit for determining a subject's cancer disease state, or for screening, diagnosing, prognosing, or monitoring cancer in said subject.
  • 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.
  • lectins may further include WGA, DC-SIGN, jacalin, DSA, HPA, ConA, and MAA.
  • the lectin(s) may be immobilized onto same or different NPs.
  • the lectin(s) comprise(s) a detectable label or has (have) been immobilized on a solid surface, such as a microtiter plate.
  • 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.
  • 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.
  • the control is a threshold value for comparing to the measured value.
  • the kit may also comprise a computer readable medium comprising computer-executable instructions for performing any method of the present disclosure.
  • 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.
  • 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.
  • CEACAM carcinoembryonic antigen-related cell adhesion molecule
  • 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.
  • 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.
  • the glycan may be comprised on one or more selected from CEACAM 1 -presenting vesicle, CEACAM6-presenting vesicle, CEACAM1 antigen, and CEACAM6 antigen.
  • the glycan comprises a structure selected from one or more of Fucal ,2Gaipi ,4Glc, Fucal ,2Gaipi ,4GlcNAc and Fucal ,2Gaipi ,4(Fuca1 ,3)GlcNAc.
  • 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.
  • 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).
  • 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.
  • 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.
  • any binding molecule that recognizes said glycan structure may be used.
  • 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 HIDEXTM 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.
  • 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.
  • DMEM Modified Eagle Medium
  • FBS fetal bovine serum
  • CCSM cell culture spent medium
  • 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.
  • 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.
  • NPs Eu-nanoparticles
  • Glycan-binding proteins 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 1e10 12 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.
  • 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.
  • RED assay buffer 25 pL was added per well, containing 1 x 10 7 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.
  • 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).
  • Sandwiched immunoassays were conducted using time-resolved fluorescence (TRF) to detect antibodies against glycoforms of CA19-9, CEACAM1 , CEACAM6 and MUC1.
  • TRF time-resolved fluorescence
  • 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.
  • 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.
  • 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.
  • 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.
  • TLIRP transurethral resection of the prostate
  • 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.
  • 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.
  • CEACAM1 and CEACAM6 have previously shown high expression in CRC in an RNA profiling study.
  • 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.
  • C192 is specific for the sialyl Lewis 3 epitope CA19-9
  • Ma695 recognizes a sialylated carbohydrate antigen on MUC1.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • tCEACAMI time-resolved fluorescence
  • CEACAM6 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.
  • TRF time-resolved fluorescence
  • 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.

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Abstract

La présente divulgation concerne le diagnostic et la surveillance du cancer, et en particulier des procédés consistant à déterminer des glycovariants d'antigènes associés au cancer. La présente invention concerne en outre des kits pour déterminer les glycovariants associés au cancer d'antigènes et des utilisations des glycovariants d'antigènes associés au cancer en tant que biomarqueurs du cancer.
PCT/FI2025/050138 2024-03-22 2025-03-21 Biomarqueurs glycovariants pour le diagnostic du cancer Pending WO2025196374A2 (fr)

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Title
BOJAR ET AL., ACS CHEM. BIOL., vol. 17, no. 11, 2022, pages 2993 - 3012
GIDWANI ET AL., CLIN CHEM., vol. 62, no. 10, 2016, pages 1390 - 1400
TERÄVÄ ET AL., PLOS ONE, vol. 14, no. 7, 2019, pages 0219480

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