Glycoprotein biomarkers for cancer diagnosis

Abstract

The present invention relates to a method for diagnosing whether a subject may be at risk for cancer or may be suffering from cancer, wherein (significantly) lower or (significantly) higher binding of a binding substance to a specific glycan structure of a biomarker glycoprotein compared to a control sample indicates that the subject is at risk for cancer or suffering from cancer. The present invention further relates to a kit for carrying out the method for diagnosing whether a subject may be at risk for cancer or may be suffering from cancer, comprising a binding substance capable of binding to the glycan structure of a biomarker protein.

Description

[Technical Field]

[0001] This application claims priority to European Patent Application No. 21 193 158.9, filed on 26 August 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention relates to a method for diagnosing whether a subject is at risk of cancer or is likely to have cancer, wherein significantly lower or significantly higher binding of a binding agent to a specific glycan structure of a biomarker glycoprotein compared to a control sample indicates that the subject is at risk of cancer or is likely to have cancer. The present invention further relates to a kit for carrying out a method for diagnosing whether a subject is at risk of cancer or is likely to have cancer, the kit comprising a binding agent capable of binding to the glycan structure of a biomarker protein. [Background technology]

[0003] Cancer is currently one of the greatest threats (scarecrows) in civilization. The most common cause of cancer death in men is prostate cancer (PCa). Despite the fact that this diagnosis is serious, the prognosis is good with early detection and appropriate treatment (Tkac et al., Interface Focus (2019), 9: 20180077 (Non-patent Literature 1)). Screening and diagnosis of PCa is usually performed by analysis of prostate-specific antigen (PSA). This protein is formed not only in cancerous prostate tissue but also in healthy prostates and prostates affected by other diseases (Damborska et al., Acta (2017), 184: 3049-3067 (Non-patent Literature 2)). Due to the low specificity of using PSA for PCa, there is a need to identify new biomarkers with higher specificity. The glycoprotein ZAG (zinc α-2-glycoprotein) has been previously identified as a potential biomarker for prostate cancer (Katafigioti et al., Ital. Urol. Androl. (2016), 88: 195-200 (Non-Patent Literature 3)). ZAG is expressed in various tissues, including several types of secretory epithelial cells, such as those found in the breast, prostate, and liver. Several studies have shown that in the early stages of this disease, ZAG levels are elevated in both urine and blood, and may serve as a biomarker for prostate cancer and other genitourinary cancers (Katafigiotis et al., BJU Int. (2012), 110: E688-E693 (Non-Patent Literature 4)). However, since ZAG is also present on the surface of non-cancerous cells, detection of ZAG levels alone is insufficient for diagnosis.

[0004] These and further drawbacks need to be overcome. Therefore, the present invention addresses these needs and technical objectives and provides solutions as described herein and defined in the claims. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Tkac et al., Interface Focus (2019), 9: 20180077 [Non-Patent Document 2] Damborska et al., Acta (2017), 184: 3049-3067 [Non-Patent Document 3] Katafigioti et al., Ital. Urol. Androl. (2016), 88: 195-200 [Non-Patent Document 4] Katafigiotis et al., BJU Int. (2012), 110: E688-E693 [Summary of the Invention]

[0006] The present invention is a method for diagnosing whether a subject may have a risk of cancer or may be suffering from cancer, comprising: (1) contacting a sample containing a biomarker glycoprotein obtained from the subject with a binding substance capable of (specifically) binding to the glycan structure of the biomarker glycoprotein, the presence or overexpression (e.g., at least about 1.5-fold, at least about 2-fold, or at least about 3-fold overexpression) or underexpression (e.g., at least about 1.5-fold, at least about 2-fold, or at least about 3-fold underexpression, e.g., underexpression when the glycan structure is O-glycan or N-glycan, preferably O-glycan) of the biomarker glycoprotein indicates the risk and / or presence of cancer, and the glycan structure deviates from the glycan structure of the biomarker glycoprotein expressed in a subject having no risk of cancer and not suffering from cancer, the step; and (2) A step to determine whether the binding substance has bound to the glycan structure of the biomarker glycoprotein. Includes, Compared to a control sample, a (significantly) lower or (significantly) higher (preferably significantly higher) binding of the binding substance to the glycan structure of the biomarker glycoprotein indicates that the subject is at risk of cancer or has cancer, and Preferably, the biomarker glycoprotein is ZAG and / or PAP, more preferably ZAG. Regarding the aforementioned method. [Invention 1001] A method for diagnosing whether a subject may be at risk of cancer or may have cancer, (1) A step of contacting a sample containing a biomarker glycoprotein obtained from the subject with a binding substance capable of binding to the glycan structure of the biomarker glycoprotein, The presence or overexpression of the biomarker glycoprotein indicates the risk and / or presence of the cancer, and The glycan structure deviates from the glycan structure of the biomarker glycoprotein expressed in subjects who are neither at risk of cancer nor suffering from cancer. The aforementioned process; and (2) A step to determine whether the binding substance has bound to the glycan structure of the biomarker glycoprotein. Includes, Compared to a control sample, lower or higher binding of the binding substance to the glycan structure of the biomarker glycoprotein indicates that the subject is at risk of cancer or has cancer. The biomarker glycoprotein is ZAG and / or PAP. The aforementioned method. [Invention 1002] The method of the present invention 1001, wherein the subject is a human. [Invention 1003] The method of the present invention, wherein the cancer is a genitourinary cancer, preferably prostate cancer (PCa). [Invention 1004] The method of the present invention, wherein the binding substance is a lectin, an antiglycan antibody, an aptamer, or a boronic acid or a derivative thereof. [Invention 1005] Any method of the present invention, wherein one or more further biomarker glycoproteins are selected from the group consisting of PSA, TIMP-1, fPSA, tPSA, osteopontin, and spongin 2. [Invention 1006] The aforementioned binding substances include core-type fucose, antenna-type fucose, Fucα1-6GlcNAc-N-Asn-containing N-linked oligosaccharide, Fucα1-6 / 3GlcNAc, α-L-Fuc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Gal, Fucα1-6GlcNAc, Manβ1-4GlcNAcβ1-4GlcNAc, and branched N-linked hexasaccharide. , Manα1-3Man, α-D-Man, (GlcNAcβ1-4, Galβ1-4GlcNAc, GlcNAcα1-4Galβ1-4GlcNAc, (GlcNAcβ1-4, Ne u5Ac (sialic acid), Galβ1-3GalNAc-serine / threonine, Galα1-3GalNAc, Galβ1-6Gal, Galβ1-4GlcNAc, Galβ1-3Gal NAc, GalNAcα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1 -3(Fucα1-2)Gal, GalNAcα1-2Gal, GalNAcα1-3GalNAc, GalNAcβ1-3 / 4Gal, GalNAc-serine / threonine (Tn antigen) Galβ1-3GalNAc-serine / threonine (T antigen), GalNAcβ1-4GlcNAc (LacdiNAc), α-2,3Neu5Ac (α2-3 linked sialic acid), α-2,6Neu5Ac (α2-6 linked sialic acid), α-2,8Neu5Ac (α2-8 linked sialic acid), sialic acid (α-2,3Neu5Ac, α-2,6Neu5Ac, or α-2,8Neu5Ac), Neu5Acα4 / 9-O-Ac-Neu5Ac, Neu5Acα2-3Galβ1-4Glc / GlcNAc, Neu5Acα2-6Gal / GalNAc, N-coupled bi-antenna type, N-coupled tri / tetra-antenna type, branched β1-6GlcNAc, Galα1-3(Fucα 1-2) Galβ1-3 / 4GlcNAc, Galβ1-3(Fucα1-4)GlcNAc, NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc, Fucα1-2Galβ1-3(Fucα1-4)GlcNAc, Galβ1-4(Fucα1-3)GlcNAc, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, high mannose, sialyl Lewis, a (Sialil Le a ) Antigen, sialyl Lewis x (Sialil Le x ) Antigen, Lewis x (Le x ) Antigen, sialyl Tn antigen, sialyl T antigen, Lewis Y (Le Y ) Antigen, sulfated core 1 glycan, Tn antigen, T antigen, core 2 glycan, Lewis a (Le a ) antigen, (GlcNAcβ1-4) n , β-D-GlcNAc, GalNAc, Gal-GlcNAc, GlcNAc, Galα1-3Gal, Galβ1-3GalNAc, α-Gal, α-GalNAc, (GlcNAc) n , or branched type (LacNAc) n Any method of the present invention, which is combined with one or more of the following: [Invention 1007] The present invention, wherein the binding substance binds to a glycan structure terminated by an N-acetylgalactosamine α or β-bonded to the 3rd or 6th position of galactose, or to a glycan structure containing a LacdiNAc epitope (GalNAc1-4GlcNAc). [Invention 1008] Any method of the present invention, wherein the binding substance binds to the same glycan structure as WFA / WFL with an affinity of at least 80% of the affinity with which WFL binds to the glycan structure. [Invention 1009] The method of the present invention, wherein the binding substance is WFA / WFL, L-selectin, P-selectin, E-selectin, AAL, MAA, GNL, PSL, or PHA-E. [Invention 1010] The method of the present invention, wherein the binding substance is WFL / WFA. [Invention 1011] Any of the methods of the present invention described above, wherein a lectin-based assay is used. [Invention 1012] The method of the present invention 1011, wherein an enzyme-binding lectin-binding assay (ELLBA) is used. [Invention 1013] A kit for carrying out any of the methods of the present invention, comprising a binding substance capable of binding to the glycan structure of a biomarker protein. [Invention 1014] The kit of the present invention 1013, wherein the binding substance is a lectin. [Invention 1015] The lectin mentioned above WFA, or A binding substance that binds to the same glycan structure as WFL / WFA with an affinity of at least 80% of the affinity with which WFL / WFA binds to the glycan structure. A kit according to the present invention 1013 or 1014.

Mode for Carrying Out the Invention

[0007] As used herein and as is commonly known in the art, “glycoprotein” (or “glycosylated protein”) means a protein containing one or more N-, O-, S-, or C-covalent sugar chains of various types, ranging from monosaccharides to branched polysaccharides (including modifications such as the attachment of sulfo or phospho groups). N-linked glycans are sugar chains attached to the -NH2 group of asparagine. O-linked glycans are sugar chains attached to the -OH group of serine, threonine, or hydroxylated amino acids. S-linked glycans are sugar chains attached to the -SH group of cysteine. C-linked glycans are sugar chains attached to tryptophan via C-C bonds.

[0008] The term "glycan" refers to a compound consisting of glycoRNA and / or glycosidically linked monosaccharides, and can also refer to the sugar chain portion of glycoconjugates such as glycoproteins, glycolipids, and proteoglycans, even if the sugar chain consists only of monosaccharides or oligosaccharides.

[0009] In one aspect of the present invention, the subject who may be at risk of cancer or who may have cancer is a human being.

[0010] Surprisingly, as found in connection with the present invention, certain biomarker glycoproteins (e.g., the presence or overexpression of such biomarker glycoproteins) that can indicate the risk and / or presence of cancer (e.g., genitourinary cancers such as prostate cancer, kidney cancer, bladder cancer, and testicular cancer) exhibit altered glycan structures if a subject may be at risk of cancer or may have cancer. In connection with the present invention, this led to the surprising discovery that if a particular glycan structure on such a glycoprotein deviates from the “normal” glycan structure of the same glycoprotein, it may indicate the risk and / or presence of cancer (e.g., genitourinary cancers such as prostate cancer, kidney cancer, bladder cancer, and testicular cancer). According to the present invention, by identifying such altered glycan structures on biomarker glycoproteins using a suitable binding agent capable of binding to such glycan structures, it is possible to diagnose whether a subject may be at risk of cancer (e.g., genitourinary cancers such as prostate cancer, kidney cancer, bladder cancer, and testicular cancer) or may have said cancer.

[0011] In this regard, according to the present invention, a binding substance capable of binding to the glycan structure of a biomarker glycoprotein in a non-cancerous state is used, and the binding substance is brought into contact with a sample according to step (1) of the method provided herein, and the binding ability of the binding substance to the glycan structure of a biomarker glycoprotein in a control sample (a healthy sample, i.e., a sample that does not contain a cancerous biomarker glycoprotein having a modified glycan structure compared to the glycan structure of a non-cancerous biomarker glycoprotein, or a sample that contains less (e.g., at least about 1.5 times, at least about 2 times, at least about 2.5 times, or at least about 3 times less) a cancerous biomarker glycoprotein having a modified glycan structure compared to the glycan structure of a non-cancerous biomarker glycoprotein) is made possible, as described in the method provided herein. If the binding substance binds to the glycan structure of a biomarker glycoprotein in a sample from a subject who may be at risk of cancer or who has cancer to a lower degree (preferably significantly lower, e.g., at least about 1.5 times, at least about 2 times, at least about 2.5 times, or at least about 3 times lower) compared to a control sample, it may indicate that the subject is at risk of cancer or has cancer.

[0012] Similarly, according to the present invention, it is also possible to use a binding substance capable of binding to the glycan structure of a biomarker glycoprotein in a cancerous state, contact the binding substance with a sample according to step (1) of the method provided herein, and compare the binding ability of the binding substance to the glycan structure of a biomarker glycoprotein in a control sample (a healthy sample, i.e., a sample that does not contain a biomarker glycoprotein in a cancerous state and has a modified glycan structure compared to a biomarker glycoprotein in a non-cancerous state, or a sample that contains more (e.g., at least about 1.5 times, at least about 2 times, at least about 2.5 times, or at least about 3 times more) a biomarker glycoprotein in a cancerous state and has a modified glycan structure compared to a biomarker glycoprotein in a non-cancerous state) as described in the method provided herein. If the binding substance binds to the glycan structure of a biomarker glycoprotein in a sample from a subject who may be at risk of cancer or who has cancer to a higher degree (preferably significantly higher, e.g., at least about 1.5 times, at least about 2 times, at least about 2.5 times, or at least about 3 times higher) than a control sample, it may indicate that the subject may be at risk of cancer or has cancer.

[0013] In one aspect of the present invention, the cancer that the subject may be at risk of or may have is a genitourinary cancer. In a specific aspect, such a genitourinary cancer may be prostate cancer, kidney cancer, bladder cancer, or testicular cancer, preferably prostate cancer (PCa).

[0014] According to the present invention, a binding agent capable of (specifically) binding to the glycan structure of a biomarker glycoprotein described herein, as used in the methods described herein and provided herein, can be any type of active agent capable of binding to the glycan structure. Preferably, such a binding agent is an active agent whose binding to the glycan structure can be measured and quantified, for example, if the binding itself can be detected and measured, and / or if the binding agent contains a marker molecule that can be detected in a suitable manner. In relation to the present invention, non-limiting examples of suitable binding agents include lectins, anti-glycan antibodies, aptamers (nucleic acid aptamers, e.g., DNA or RNA aptamers, or peptide aptamers), or boronic acids or derivatives thereof. In one aspect of the present invention, the binding agent used in the methods described herein and provided herein is a lectin. In another example related to the methods of the present invention described and provided herein, the binding substance can (specifically) bind to a glycan structure terminated by an N-acetylgalactosamine α or β-linked to the 3 or 6 position of galactose, or to a glycan structure containing a LacdiNAc epitope (GalNAc1-4GlcNAc), preferably to a glycan structure terminated by an N-acetylgalactosamine α or β-linked to the 3 or 6 position of galactose.

[0015] Generally, as used herein, “binding substances” (or “recognition molecules”) include polypeptides containing one or more binding domains capable of binding to a target epitope (e.g., lectins or anti-glycan antibodies, or fragments thereof), as well as other molecules capable of binding to glycan structures (e.g., aptamers or boronic acids and their derivatives). The binding substance provides, so to speak, a scaffold for the one or more binding domains so that they can bind to / interact with a given target structure / antigen / epitope. In the context of the present invention, the term “binding domain” characterizes a domain of a polypeptide that specifically binds to / interacts with a given target epitope. “Epitope” is antigenic, and therefore the term epitope may also be referred to herein as “antigenic structure” or “antigenic determinant.” In relation to the present invention, the glycan structure can function as an antigenic structure for binding substances, such as lectins, anti-glycan antibodies, aptamers (nucleic acid aptamers, e.g., DNA or RNA aptamers, or peptide aptamers), or boronic acid or its derivatives, preferably one or more types of lectins and / or anti-glycan antibodies, preferably one or more types of lectins. Thus, the binding domain is an "antigen interaction site." The term "antigen interaction site," according to the present invention, defines a polypeptide motif that can specifically interact with a particular antigen or a particular group of antigens (e.g., the same antigen in different species). This binding / interaction can also be understood as defining "specific recognition."

[0016] The term "epitope" also refers to the site on an antigen to which a binding substance binds. Preferably, an epitope is a site on a molecule to which a binding substance, such as a lectin, an antiglycan antibody, an aptamer (nucleic acid aptamer, e.g., a DNA or RNA aptamer, or a peptide aptamer), or a boronic acid or its derivative, preferably one or more types of lectins and / or antiglycan antibodies, preferably one or more types of lectins, binds.

[0017] As used herein, the term “aptamer” refers to a nucleic acid, oligonucleotide, or peptide molecule that binds to a specific target molecule. As used herein, unless otherwise defined, the terms “nucleic acid” or “nucleic acid molecule” are synonymous with “oligonucleotide,” “nucleic acid chain,” etc., and mean a polymer containing one, two, or more nucleotides, e.g., single-stranded or double-stranded.

[0018] As used herein, the term “lectin” refers to any type and origin of glycosylated proteins, including, for example, lectins, galectins, selectins, recombinant lectins, or fragments thereof, as well as fragments of glycan-binding sites attached to a scaffold. As used herein, the term “lectin” also includes fragments of lectins capable of binding to glycan structures. Lectins may be highly specific to one or more glycan moieties (for example, they may react specifically with terminal glycoside residues of other molecules, such as glycans of glycoproteins (e.g., branched sugar molecules of glycoproteins, e.g., target polypeptides within the scope of the present invention and biomarkers listed in Table 1 herein)). Lectins are well known in the art. Those skilled in the art can easily determine which lectins can be used to bind to one or more glycan moieties of interest, e.g., one or more glycan moieties of glycans attached to a protein. Preferred lectins applicable in connection with the present invention are described herein. The term "lectin" also includes Siglec (sialic acid-binding immunoglobulin-like lectin). Notably, as used herein, the term "lectin" also refers to glycan-binding antibodies. Therefore, as used herein, the term "lectin" includes lectins, Siglec, and glycan-binding antibodies.

[0019] Lectins described herein and used in connection with the present invention can be isolated and optionally purified using conventional methods known in the art. For example, when lectins are isolated from their natural source, they can be purified to homogeneity on a suitable immobilized glycan matrix and eluted with a suitable hapten. See Goldstein & Poretz (1986) In The lectins. Properties, functions and applications in biology and medicine (edited by Liener et al.), pp. 33-247. Academic Press (Orlando, Fla.); Rudiger (1993) In Glycosciences: Status and perspectives (edited by Gabius & Gabius), pp. 415-438. See Chapman and Hall (Weinheim, Germany). Alternatively, lectins can be produced by recombinant methods according to established methods. See Streicher & Sharon (2003) Methods Enzymol. 363:47-77. As yet another alternative, lectins can be produced using standard peptide synthesis techniques or chemical cleavage methods well known in the art, based on the amino acid sequences of known lectins or lectins disclosed herein (e.g., US 9169327 B2). Another alternative may be artificial lectins prepared by chemical modification of the lectins identified above (see YW Lu, CW Chien, PC Lin, LD Huang, CY Chen, SW Wu, CL Han, KH Khoo, CC Lin, YJ Chen, BAD-Lectins: Boronic Acid-Decorated Lectins with Enhanced Binding Affinity for the Selective Enrichment of Glycoproteins, Analytical Chemistry, 85 (2013) 8268-8276).

[0020] In relation to the present invention, when a glycan binds to a lectin (or vice versa), the binding affinity is approximately 10 -1 ~10 -10 (K D ), preferably about 10 -2 ~10 -8 (K D ), comfortable for about 10 -3 ~10 -5 (K D ) is within the range. When the binding substance is a lectin, the terms "specifically" or "specific" as used herein in relation to the binding of the binding substance to the glycan structure are preferably about 10 -2 ~10 -8 (K D ), comfortable for about 10 -3 ~10 -5 (K D This can mean the binding affinity of ) to glycans. D Methods for measuring this are well known in the art and are readily available to those skilled in the art.

[0021] In one aspect of the present invention, the binding substance used in connection with the present invention may be an antibody. As used herein, “antibody” is a protein consisting of one or more polypeptides (including one or more binding domains, preferably antigen-binding domains) substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. The term “immunoglobulin” (Ig) is used herein interchangeably with “antibody.” Widely recognized immunoglobulin genes include not only a multitude of immunoglobulin variable region genes, but also the constant region genes of kappa (κ), lambda (λ), alpha (α), gamma (γ), delta (δ), epsilon (ε), and mu (μ).

[0022] In particular, the “antibody” as used herein is typically a tetrameric glycosylated protein composed of two light chains (L chains) of approximately 25 kDa each and two heavy chains (H chains) of approximately 50 kDa each. Antibodies can have two types of light chains, called lambda (λ) and kappa (κ). Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, some of which can be further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called a J chain, containing 10 antigen-binding sites; IgA antibodies, on the other hand, consist of 2 to 5 basic quad units, which can polymerize with the J chain to form a multivalent aggregate. In the case of IgG, a quad unit is generally about 150,000 daltons.

[0023] Each light chain contains an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain contains an N-terminal V domain (VH), three or four C domains (CH), and a hinge region. The constant domain is not directly involved in the binding of the antibody to the antigen.

[0024] When the VH and VL domains pair together, a single antigen-binding site is formed. The CH domain closest to the VH is designated CH1. Each L chain is attached to the H chain by one covalent disulfide bond, while the two H chains are attached to each other by one or more disulfide bonds depending on the H chain isotype. The VH and VL domains consist of four relatively conserved regions of sequence called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence (complementarity-determining regions; CDRs). The CDRs contain most of the residues involved in the specific interaction between the antibody and the antigen. The CDRs are called CDR1, CDR2, and CDR3. Accordingly, the CDR components on the heavy chain are called H1, H2, and H3, while the CDR components on the light chain are called L1, L2, and L3.

[0025] The term "variable" refers to a portion of the immunoglobulin domain (i.e., the "variable domain") that exhibits variability in the immunoglobulin sequence and is involved in determining the specificity and binding affinity of a particular antibody. Variability is not uniformly distributed throughout the antibody's variable domain; it is concentrated in the respective subdomains of the heavy and light chain variable regions. These subdomains are called "hypervariable" regions or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRMs). Naturally occurring heavy and light chain variable domains each contain four FRM regions, primarily in a β-sheet configuration, and these FRM regions are linked to three hypervariable regions; the hypervariable regions form loops connecting the β-sheet structures, and in some cases, form parts of the β-sheet structures themselves. The hypervariable regions of each chain are linked very closely together by the FRM and, together with the hypervariable region of the other chain, contribute to the formation of the antigen-binding site (Chothia et al., J MoI Biol (1987), 196: 901; and MacCallum et al., J MoI Biol (1996), 262: 732 onwards). The constant domain does not directly participate in antigen binding, but exhibits various effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement activation.

[0026] The term "CDR" and its plural form "CDRs" refer to complementarity-determining regions (CDRs), three of which constitute the binding properties of the light chain variable region (CDRL1, CDRL2, and CDRL3), and three which constitute the binding properties of the heavy chain variable region (CDRH1, CDRH2, and CDRH3). CDRs contribute to the functional activity of antibody molecules and are separated by amino acid sequences that constitute the scaffold or framework region. The precise definition of CDR boundaries and lengths varies depending on the various classification and numbering systems. Despite the differing boundaries, each of these systems has some overlap in the portions that constitute the so-called "hypervariable regions" within the variable sequences. Therefore, the definitions of CDRs by these systems may differ in terms of length and boundary regions with respect to adjacent framework regions. See, for example, Kabat, Chothia, and / or MacCallum (Chothia et al., J MoI Biol (1987), 196: 901; and MacCallum et al., J MoI Biol (1996), 262: 732).

[0027] As used herein, the terms “amino acid” or “amino acid residue” typically refer to amino acids with definitions widely recognized in the art, such as alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified amino acids, synthetic amino acids, or rare amino acids may be used as desired. Generally, amino acids can be grouped based on whether they have a nonpolar side chain (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged side chain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0028] The term "framework region" refers to a widely recognized portion of the antibody variable region in the art that exists among more diverse (i.e., hypervariable) CDRs. Such framework regions are typically called frameworks 1 through 4 (FR1, FR2, FR3, and FR4) and provide a scaffold for presenting six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space to form an antigen-binding surface.

[0029] As used herein, the term “antibody” refers not only to immunoglobulins (i.e., intact antibodies) but also to their fragments, encompassing any polypeptide containing an antigen-binding fragment or antigen-binding domain. Preferably, the fragments are, for example, Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Typically, such fragments are considered to contain an antigen-binding domain and to have the same properties as the antibodies described herein.

[0030] As used herein, the term “antibody” includes antibodies that compete for binding to the same epitope as the antibody of the present invention, and preferably such antibodies can be obtained by antibody production methods described elsewhere herein.

[0031] To investigate whether test antibodies can compete for binding to the same epitope, a cross-blocking assay, such as a competitive ELISA assay, can be performed. In an exemplary competitive ELISA assay, wells of a microtiter plate coated with an epitope, or Sepharose beads coated with an epitope, are pre-incubated in or without a candidate competitive antibody, and then the biotin-labeled antibody of the present invention is added. The amount of labeled antibody bound to the epitope in the well or on the bead is measured using an avidin-peroxidase conjugate and a suitable substrate.

[0032] Alternatively, antibodies can be labeled with, for example, radioactive labels, enzyme labels, fluorescent labels, or other detectable and measurable labels. The amount of labeled antibody that binds to an antigen is thought to be inversely correlated with the ability of competing candidate antibodies (test antibodies) to bind to the same epitope on that antigen; that is, the higher the affinity of the test antibody for the same epitope, the less labeled antibody is thought to bind to the well coated with the antigen.

[0033] If a candidate competing antibody can block the binding of the antibody of the present invention by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to a control assay performed in parallel in the absence of the candidate competing antibody (but in the presence of a known non-competitive antibody), then the candidate competing antibody is considered to be an antibody that substantially binds to the same epitope as the antibody of the present invention, or an antibody that competes for binding to the same epitope. It is understood that the same quantitative values ​​can be reached even when this assay is modified.

[0034] Furthermore, the term "antibody" is not limited to polyclonal, monoclonal, monospecific, bispecific, nonspecific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, grafted, and in vitro produced antibodies; polyclonal antibodies are preferred. This term also includes domain antibodies (dAbs) and nanobodies.

[0035] Therefore, the term “antibody” also relates to purified serum, i.e., purified polyclonal serum. Accordingly, the term preferably relates to serum, more preferably polyclonal serum, and most preferably purified (polyclonal) serum. Antibodies / serum are obtainable, and preferably obtained, by, for example, the methods or uses described herein.

[0036] A "polyclonal antibody" or "polyclonal antiserum" refers to an immunoserum containing a mixture of antibodies specific to one (monovalent or specific antiserum) or more (polyvalent antiserum) antigens, which can be prepared from the blood of an animal immunized with one or more antigens.

[0037] Furthermore, the term “antibody” as used in this invention also refers to derivatives or variants of the antibodies described herein that exhibit the same specificity as the antibodies described herein. Examples of “antibody variants” include humanized variants of non-human antibodies, “affinity-mature” antibodies (see, e.g., Hawkins et al., J Mol Biol (1992), 254, 889-896; and Lowman et al., Biochemistry (1991), 30: 10832-10837), and antibody variants with altered effector function (see, e.g., U.S. Patent No. 5,648,260).

[0038] As used herein, the terms “antigen-binding domain,” “antigen-binding fragment,” and “antibody-binding region” refer to a portion of an antibody molecule containing amino acids involved in the specific binding between an antibody and an antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an “epitope,” as described herein. As stated above, an antigen-binding domain may typically include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily have to include both. For example, an Fd fragment has two VH regions and often retains some of the antigen-binding function of the intact antigen-binding domain. Examples of antibody antigen-binding fragments include: (1) Fab fragments, which are monovalent fragments having VL, VH, CL, and CH1 domains; (2) F(ab')2 fragments, which are bivalent fragments having two Fab fragments linked by a disulfide bridge at the hinge region; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains in a single arm of the antibody; (5) dAb fragments having a VH domain (Ward et al., (1989) Nature 341: 544-546); (6) isolated complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv). The two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be joined together using a synthetic linker via recombination; this synthetic linker allows the VL and VH regions to pair up and form a single protein chain that forms a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science (1988), 242: 423-426; and Huston et al., (1988) PNAS USA (1988), 85: 5879-5883). These antibody fragments can be obtained using prior art known to those skilled in the art and evaluated for function in the same manner as intact antibodies.

[0039] As used herein, the term “monoclonal antibody” includes chemically modified monoclonal antibodies or their fragments, as well as antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are directed to a single antigen site and are highly specific. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which generally contain different antibodies against various determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier “monoclonal” refers to the nature of the antibody, that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring antibody production by a specific method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described in Kohler et al., Nature (1975), 256: 495, or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature (1991), 352: 624-628; and Marks et al., J Mol Biol (1991), 222: 581-597.

[0040] Monoclonal antibodies as used herein include, in particular, “chimeric” antibodies (immunoglobulins), and fragments of such antibodies insofar as they exhibit the desired biological activity; a portion of the heavy and / or light chain of a chimeric antibody is identical or homologous to a corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass (U.S. Patent No. 4,816,567; Morrison et al., PNAS USA (1984), 81: 6851-6855). Chimeric antibodies as used herein include “primitized” antibodies that contain a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0041] The “humanized” form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) that is mostly human and contains minimal sequences derived from non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin in which residues from the hypervariable region (or CDR) of a human immunoglobulin (recipient antibody) are replaced with residues from the hypervariable region of a non-human species antibody (donor antibody), such as mouse, rat, or rabbit, that possess the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, as used herein, “humanized antibody” may contain residues not present in either the recipient or donor antibody. These modifications are made to further improve and optimize the performance of the antibody. A humanized antibody is also considered to optimally contain at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulin. For further details, see Jones et al., Nature (1986), 321: 522-525; Reichmann et al., Nature (1988), 332: 323-329; and Presta, Curr. Op. Struct Biol (1992), 2: 593-596.

[0042] The term "human antibody" includes antibodies having variable and constant regions substantially corresponding to human germline immunoglobulin sequences known in the art, such as those described by Kabat et al. (see above). The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-directed mutagenesis, or in vivo by somatic mutation), for example, in the CDR, particularly CDR3. The human antibody may have at least one, two, three, four, five, or more positions replaced by amino acid residues not encoded by human germline immunoglobulin sequences.

[0043] As used herein, “in vitro produced antibody” refers to an antibody in which all or part of the variable region (e.g., at least one CDR) is produced by a non-immune cell selection method (e.g., in vitro phage display, protein chip, or other method that can test candidate sequences for antigen-binding ability). Therefore, this term preferably excludes sequences produced by genomic rearrangement in immune cells.

[0044] A "bispecific antibody" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced in various ways, such as by hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai & Lachmann, Clin Exp Immunol (1990), 79: 315-321; Kostelny et al., J Immunol (1992), 148: 1547-1553. In one embodiment, a bispecific antibody comprises a first binding domain polypeptide, such as a Fab' fragment, linked to a second binding domain polypeptide via an immunoglobulin constant region.

[0045] Numerous methods known to those skilled in the art are available for obtaining antibodies or their antigen-binding fragments. For example, antibodies can be produced using recombinant DNA (U.S. Patent No. 4,816,567). Monoclonal antibodies can also be obtained by the production of hybridomas by known methods (see, for example, Kohler and Milstein, Nature (1975), 256: 495-499). The hybridomas thus formed are then screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (BIACORE®) analysis to identify one or more hybridomas that produce antibodies that specifically bind to a particular antigen. Any form of a specific antigen can be used as an immunogen, for example, recombinant antigens, naturally occurring forms, their variants or fragments, and their antigenic peptides.

[0046] One exemplary antibody production method involves screening a protein expression library, such as a phage or ribosome display library. Phage displays are described, for example, in U.S. Patent No. 5,223,409; Smith, Science (1985), 228: 1315-1317; Clackson et al., Nature (1991), 352: 624-628; Marks et al., J MoI Biol (1991), 222: 581-597; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; and WO 90 / 02809.

[0047] In another embodiment, monoclonal antibodies can be obtained from non-human animals, and then modified forms, such as humanized, deimmunized, and chimeric forms, can be produced using recombinant DNA techniques known in the art. Various approaches for producing chimeric antibodies have been described. See, for example, Morrison et al., PNAS USA (1985), 81: 6851; Takeda et al., Nature (1985), 314: 452; U.S. Patent No. 4,816,567; U.S. Patent No. 4,816,397; EP 171496; EP 173494, GB 2177096. Humanized antibodies can also be produced, for example, using transgenic mice that express human heavy and light chain genes but cannot express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes exemplary CDR-grafting methods that can be used to produce the humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of non-human CDRs, or only a portion of the CDRs may be replaced with non-human CDRs. It is sufficient to replace only the number of CDRs necessary for the humanized antibody to bind to a given antigen.

[0048] Humanized antibodies or fragments thereof can be prepared by replacing the sequence of the Fv variable domain, which is not directly involved in antigen binding, with an equivalent sequence from the human Fv variable domain. Exemplary methods for preparing humanized antibodies or fragments thereof are provided by Morrison, Science (1985), 229: 1202-1207; Oi et al., BioTechniques (1986), 4: 214; US 5,585,089; US 5,693,761; US ​​5,693,762; US 5,859,205; and US 6,407,213. These methods involve isolating, manipulating, and expressing nucleic acid sequences encoding all or part of the immunoglobulin Fv variable domain from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas producing antibodies against a given target, as described above, as well as from other sources. Subsequently, the recombinant DNA encoding the humanized antibody molecule can be cloned into a suitable expression vector.

[0049] In certain embodiments, humanized antibodies are optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or reverse mutations. Such modified immunoglobulin molecules can be produced by any of several techniques known in the art (e.g., Teng et al., PNAS USA (1983), 80: 7308-731; Kozbor et al., Immunology Today (1983), 4: 7279; Olsson et al., Meth Enzymol (1982), 92: 3-16), or according to the teachings of WO 92 / 06193 or EP 239400.

[0050] In the case of antibodies, specific binding is thought to be brought about by the binding of a specific motif in the amino acid sequence of the binding domain to the antigen as a result of their primary, secondary, or tertiary structures, as well as as a result of secondary modifications of said structures. Specific interaction between an antigen interaction site and its specific antigen may also result in simple binding of the site to the antigen. Furthermore, specific interaction between an antigen interaction site and its specific antigen may instead result in signal initiation, for example, induction of conformational changes of the antigen or oligomerization of the antigen. An example of a binding domain in line with the present invention is an anti-glycan antibody. In this regard, if the binding substance is an antibody, the binding affinity is 10 -1 If the binding affinity is higher than M, the binding can be considered "specific". Preferably, the binding affinity is about 10 -5 ~10 -12 M(K D ), preferably about 10 -8 ~10 -12 When the binding is M (i.e., when the binding substance is an antibody), the binding is considered specific. If necessary, nonspecific binding can be reduced without substantially affecting specific binding by changing the binding conditions. Whether a recognition molecule reacts specifically as previously defined herein can be easily tested, in particular, by comparing the reaction between the recognition molecule and the epitope with the reaction between the recognition molecule and other proteins.

[0051] According to the present invention, the presence or overexpression (e.g., at least about 1.5 times, at least about 2 times, or at least about 3 times overexpression) of a biomarker glycoprotein indicates the risk and / or presence of cancer (e.g., genitourinary cancers such as prostate cancer, kidney cancer, bladder cancer, or testicular cancer). Such a biomarker glycoprotein (also referred to herein as a biomarker or biomarker protein) may be any glycoprotein that is present or overexpressed (e.g., at least about 1.5 times, at least about 2 times, or at least about 3 times overexpression) in cells of a (human) subject that are at risk of developing such cancer or are suffering from such cancer, compared to cells of a (human) subject that are not at risk of developing such cancer or are suffering from such cancer. Preferably, in connection with the present invention, such a biomarker glycoprotein has a different glycan structure in the cancerous state compared to the non-cancerous state. For example, in human subjects who are at risk of developing prostate cancer (PCa) or who have prostate cancer (PCa), glycoproteins such as ZAG (zinc α2 glycoprotein), PAP (prostatic acid phosphatase), PSA (prostate-specific antigen), TIMP-1 (tissue inhibitor of metalloproteinase-1), fPSA (free PSA), tPSA (total PSA), osteopontin, PSMA (prostate-specific membrane antigen), and / or spongin 2 may be present or overexpressed in cells, compared to human subjects who are neither at risk of developing prostate cancer nor have prostate cancer, and therefore may function as biomarker glycoproteins according to the present invention.As a result, in one aspect of the present invention, the biomarker glycoprotein (hereinafter also referred to as biomarker or biomarker protein) whose presence or overexpression (e.g., overexpression by at least about 1.5 times, about 2 times, or about 3 times) or underexpression (e.g., underexpression by at least about 1.5 times, about 2 times, or about 3 times) indicates the risk and / or presence of cancer (e.g., genitourinary cancers such as prostate cancer, kidney cancer, bladder cancer, testicular cancer, etc.), particularly when the cancer is prostate cancer, may be ZAG, PAP, PSA, TIMP-1, fPSA, tPSA, osteopontin, PSMA, or spongin 2.

[0052] As used herein, “overexpression” of a glycoprotein or protein may mean, in any case, that a cell of subject that is at risk of or has cancer as described herein brings about a greater amount of glycoprotein or protein compared to a cell of subject that is not at risk of or has cancer as described herein. For example, according to the present invention, “overexpression” may mean an increase in the translation or transcription rate or overall synthesis of such glycoprotein or protein, while “underexpression” may mean a decrease in the translation or transcription rate or overall synthesis of such glycoprotein or protein.

[0053] As found in connection with the present invention, ZAG in samples from subjects at risk of or suffering from prostate cancer exhibits a different glycan structure compared to ZAG in samples from subjects neither at risk of nor suffering from prostate cancer. In particular aspects of the present invention, especially when the cancer of the subject being diagnosed is prostate cancer, the biomarker glycoprotein is ZAG and / or PAP, preferably ZAG or PAP, more preferably ZAG.

[0054] The method of the present invention may also include the additional analysis of further biomarkers. Accordingly, in the method of the present invention, one or more further biomarker glycoproteins may be selected from the group consisting of PSA, TIMP-1, fPSA, tPSA, osteopontin, and spongin 2.

[0055] In relation to the present invention, the binding material used in the method described herein and provided herein, which is capable of binding to the glycan structure of the biomarker glycoprotein described herein, binds to the glycan structure of the biomarker glycoprotein described herein. In one aspect of the present invention, the binding substance (preferably a lectin) can (specifically) bind to one or more of the following: core-type fucose, antenna-type fucose, Fucα1-6GlcNAc-N-Asn-containing N-linked oligosaccharide, Fucα1-6 / 3GlcNAc, α-L-Fuc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Gal, Fucα1-6GlcNAc, Manβ1-4GlcNAcβ1-4GlcNAc, branched N-linked hexasaccharide, Manα1-3Man, α-D-Man, (GlcNAcβ1-4, Galβ1-4GlcNAc, GlcNAcα1-4Galβ1-4GlcNAc, (GlcNAcβ) 1-4Neu5Ac (sialic acid), Galβ1-3GalNAc-serine / threonine, Galα1-3GalNAc, Galβ1-6Gal, Galβ1-4GlcNAc, Galβ1-3GalNAc, GalNAcα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1-3(Fucα1-2)Gal, GalN Acα1-2Gal, GalNAcα1-3GalNAc, GalNAcβ1-3 / 4Gal, GalNAc-serine / threonine (Tn antigen), Galβ1-3GalNAc-serine / threonine (T antigen), GalNAcβ1-4GlcNAc (LacdiNAc), α-2,3Neu5Ac (α2-3 linked sialic acid), α-2,6Neu5Ac (α2-6 linked sialic acid), α-2,8Neu5Ac (α2-8 linked Sialic acid), sialic acid (α-2,3Neu5Ac, α-2,6Neu5Ac, or α-2,8Neu5Ac), Neu5Acα4 / 9-O-Ac-Neu5Ac, Neu5Acα2-3Galβ1-4Glc / GlcNAc, Neu5Acα2-6Gal / GalNAc, N-bonded biantennae type, N-bonded tri / tetraantennae type, branched β1-6GlcNAc, Galα1-3(Fucα1-2)Galβ1-3 / 4 GlcNAc, Galβ1-3(Fucα1-4)GlcNAc, NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc, Fucα1-2Galβ1-3(Fucα1-4)GlcNAc, Galβ1-4(Fucα1-3)GlcNAc, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, high mannose, sialyl Lewis a (Sialil Le a ) Antigen, sialyl Lewis x (Sialil Le x ) Antigen, Lewis x (Le x ) Antigen, sialyl Tn antigen, sialyl T antigen, Lewis Y (Le Y ) Antigen, sulfated core 1 glycan, Tn antigen, T antigen, core 2 glycan, Lewis a (Le a) Antigen, (GlcNAcβ1-4) n , β-D-GlcNAc, GalNAc, Gal-GlcNAc, GlcNAc, Galα1-3Gal, Galβ1-3GalNAc, α-Gal, α-GalNAc, (GlcNAc) n , or branched type (LacNAc) n ).

[0056] As described herein, in one aspect of the present invention, the binding agent used in the method described herein and provided may (specifically) bind, in particular, to a glycan structure terminated by an N-acetylgalactosamine α- or β-linked to the 3- or 6-position of galactose, or to a glycan structure containing a LacdiNAc epitope (GalNAc1-4GlcNAc), preferably to a glycan structure terminated by an N-acetylgalactosamine α- or β-linked to the 3- or 6-position of galactose. Surprisingly, as has been found in connection with the present invention, ZAGs contained in samples from subjects at risk of or suffering from prostate cancer ("cancerous ZAGs") exhibit a different glycan structure compared to ZAGs contained in samples from subjects neither at risk of nor suffering from prostate cancer. According to the present invention, such "cancerous ZAGs" can be detected using a binding agent capable of binding to the glycan structure of such "cancerous ZAGs," as described herein. As further discovered in connection with the present invention, ZAGs ("oncogenic ZAGs") contained in samples from subjects at risk of or suffering from prostate cancer can be conjugated (and therefore detected) by using a specific lectin, such as wisteria floribunda lectin (WFA / WFL). As a result, in one aspect of the present invention, the conjugating substance, which can be used in the methods described herein and provided herein, to bind to the glycan structure of the biomarker glycoprotein described herein may be able to (specifically) bind to the same glycan structure as wisteria lectin (WFA / WFL) with an affinity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the affinity to which wisteria lectin (WFA / WFL) binds to the glycan structure.Methods for measuring the affinity level of a binding substance (e.g., lectin) to a glycan structure are commonly known in the art and include, in particular, surface plasmon resonance, isothermal microcalorimetry, or ELISA and ELISA-like formats, preferably surface plasmon resonance.

[0057] In a more specific embodiment of the present invention, the binding substance capable of binding to the glycan structure of the biomarker glycoprotein described herein, as used in the methods described herein and provided herein, may be Fuji lectin (WFA / WFL), L-selectin, P-selectin, E-selectin, AAL (Aleuria aurantia lectin), MAA (Maackia amurensis aglutinin / lectin), GNL (Galanthus nivalis lectin), PSL (Pisum sativum lectin), or PHA-E (Phaseolus vulgaris erythroaglutinin). In a specific embodiment of the present invention, the binding substance is Fuji lectin (WFA / WFL) or PHA-E, preferably Fuji lectin (WFA / WFL).

[0058] In connection with the present invention, it is also possible to combine two or more binding agents capable of binding to the glycan structure of the biomarker glycoprotein described herein, as used in the methods described herein and provided herein. In some cases, combining two or more such binding agents may increase the likelihood of diagnosis. In this regard, according to the present invention, in step (1) of the method of the present invention, two or more binding agents (e.g., lectins) may be used in the same assay, or preferably, two or more such binding agents (e.g., lectins) may be used in different assays (using the same sample), and then in step (2), it is possible to separately determine whether each of the binding agents has bound to the glycan structure of the biomarker glycoprotein, and then combine the information thus obtained to diagnose whether the subject may be at risk of cancer or may have cancer. In one aspect of the present invention, when two (or more) such binding agents are used in the method of the present invention, all such binding agents are lectins. In a specific embodiment related thereto, when two (or more) such binding substances are used in the method of the present invention, such binding substances are fusi lectin (WFA / WFL) and PHA-E, or include them.

[0059] In the case of the methods described and provided herein in connection with the present invention, any suitable assay may be used to detect and quantify the binding of the binding substance described herein to the biomarker glycoprotein described herein. Such suitable assays are commonly known in the art and include, among others, ELISA or Western blotting (especially when the binding substance is an antibody), or lectin-based assays (see, for example, the assay described in WO2019 / 185515), or enzyme-linked lectin-binding assays (ELLBA) (on cells, CELLBA; see, for example, Gaverieux et al., J Immunol Methods (1987), 104(1-2): 173-182). In one aspect of the present invention, a lectin-based assay is used. In another specific aspect of the present invention, an enzyme-linked lectin-binding assay (ELLBA) or a magnetic enzyme-linked lectin assay (MELLA) is used, with ELLBA being preferred.

[0060] The present invention further relates to a kit comprising a conjugate capable of binding to the glycan structure of the biomarker protein described herein. In one aspect of the present invention, the conjugate may be a lectin. In a more specific aspect of the present invention, the conjugate capable of binding to the glycan structure of the biomarker glycoprotein described herein, used in the method described herein and provided herein, may be capable of (specifically) binding to the same glycan structure as fusi lectin (WFA / WFL) with an affinity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the affinity with which fusi lectin (WFA / WFL) binds to the glycan structure. In a further specific aspect of the present invention, the conjugate may be, for example, WFA / WFL, L-selectin, P-selectin, E-selectin, AAL, MAA, GNL, PSL, or PHA-E, preferably WFA / WFL. In some cases, combining two or more such binding agents can increase the likelihood of diagnosis. Accordingly, in one aspect of the present invention, a kit described and provided herein contains two or more such binding agents. In this regard, in a specific aspect of the present invention, both or at least two of the such binding agents contained in the kit are lectins. In a more specific aspect related thereto, the two or more such lectins contained in the kit are WFA / WFL and PHA-E, or contain them.

[0061] The kit described and provided in connection with the present invention may also include, as will be readily apparent to those skilled in the art, further suitable components, such as enzymes and buffers necessary to carry out the method by using suitable assays described herein (e.g., ELISA, Western blotting, lectin-based assays, ELLBA, MELLA, etc.).

[0062] This invention also relates to the following: 1. A method for diagnosing whether a subject may be at risk of cancer or may have cancer, (1) A step of contacting a sample containing a biomarker glycoprotein obtained from the subject with a binding substance capable of binding to the glycan structure of the biomarker glycoprotein, The presence or overexpression of the biomarker glycoprotein indicates the risk and / or presence of the cancer, and The glycan structure deviates from the glycan structure of the biomarker glycoprotein expressed in subjects who are neither at risk of cancer nor suffering from cancer. The aforementioned process; and (2) A step to determine whether the binding substance has bound to the glycan structure of the biomarker glycoprotein. Includes, Compared to a control sample, lower or higher binding of the binding substance to the glycan structure of the biomarker glycoprotein indicates that the subject is at risk of cancer or has cancer. The aforementioned method. 2. The method of item 1, wherein the subject is a human. 3. Any one of the above methods, wherein the cancer is a genitourinary cancer, preferably prostate cancer (PCa). 4. Any one of the above methods, wherein the binding substance is a lectin, an anti-glycan antibody, an aptamer, or a boronic acid or a derivative thereof. 5. Any one of the above methods, wherein the biomarker glycoprotein is selected from the group consisting of ZAG, PAP, PSA, TIMP-1, fPSA, tPSA, osteopontin, and spongin 2. 6. The binding substance is core-type fucose, antenna-type fucose, Fucα1-6GlcNAc-N-Asn-containing N-linked oligosaccharide, Fucα1-6 / 3GlcNAc, α-L-Fuc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Gal, Fucα1-6GlcNAc, Manβ1-4GlcNAcβ1-4GlcNAc, branched N-linked hexasaccharide. , Manα1-3Man, α-D-Man, (GlcNAcβ1-4, Galβ1-4GlcNAc, GlcNAcα1-4Galβ1-4GlcNAc, (GlcNAcβ1-4, Ne u5Ac (sialic acid), Galβ1-3GalNAc-serine / threonine, Galα1-3GalNAc, Galβ1-6Gal, Galβ1-4GlcNAc, Galβ1-3Gal NAc, GalNAcα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1 -3(Fucα1-2)Gal, GalNAcα1-2Gal, GalNAcα1-3GalNAc, GalNAcβ1-3 / 4Gal, GalNAc-serine / threonine (Tn antigen) Galβ1-3GalNAc-serine / threonine (T antigen), GalNAcβ1-4GlcNAc (LacdiNAc), α-2,3Neu5Ac (α2-3 linked sialic acid), α-2,6Neu5Ac (α2-6 linked sialic acid), α-2,8Neu5Ac (α2-8 linked sialic acid), sialic acid (α-2,3Neu5Ac, α-2,6Neu5Ac, or α-2,8Neu5Ac), Neu5Acα4 / 9-O-Ac-Neu5Ac, Neu5Acα2-3Galβ1-4Glc / GlcNAc, Neu5Acα2-6Gal / GalNAc, N-coupled bi-antenna type, N-coupled tri / tetra-antenna type, branched β1-6GlcNAc, Galα1-3(Fucα 1-2) Galβ1-3 / 4GlcNAc, Galβ1-3(Fucα1-4)GlcNAc, NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc, Fucα1-2Galβ1-3(Fucα1-4)GlcNAc, Galβ1-4(Fucα1-3)GlcNAc, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, high mannose, sialyl Lewis, a (Sialil Le a ) Antigen, sialyl Lewis x (Sialil Le x ) Antigen, Lewis x (Le x ) Antigen, sialyl Tn antigen, sialyl T antigen, Lewis Y (Le Y ) Antigen, sulfated core 1 glycan, Tn antigen, T antigen, core 2 glycan, Lewis a (Le a ) antigen, (GlcNAcβ1-4) n , β-D-GlcNAc, GalNAc, Gal-GlcNAc, GlcNAc, Galα1-3Gal, Galβ1-3GalNAc, α-Gal, α-GalNAc, (GlcNAc) n , or branched type (LacNAc) n One way of combining with one or more of the above items. 7. Any one of the above methods, wherein the binding substance binds to a glycan structure terminated by an N-acetylgalactosamine α or β-linked to the 3rd or 6th position of galactose, or binds to a glycan structure containing a LacdiNAc epitope (GalNAc1-4GlcNAc). 8. Any one of the above methods, wherein the binding substance binds to the same glycan structure as WFA / WFL with an affinity of at least 80% of the affinity with which WFL binds to the glycan structure. 9. Any one of the above methods, wherein the binding substance is WFA / WFL, L-selectin, P-selectin, E-selectin, AAL, MAA, GNL, PSL, or PHA-E. 10. Any one of the above methods, wherein the binding substance is WFL / WFA. 11. Any one of the above methods, in which a lectin-based assay is used. 12. The method of item 11, using an enzyme-binding lectin-binding assay (ELLBA). 13. A kit for performing any one of the methods described above, comprising a binding substance capable of binding to the glycan structure of a biomarker protein. 14. The kit of item 13, wherein the binding substance is a lectin. 15. The lectin is WFA, or A binding substance that binds to the same glycan structure as WFL / WFA with an affinity of at least 80% of the affinity with which WFL / WFA binds to the glycan structure. This is a kit containing item 13 or 14.

[0063] The embodiments characterizing the present invention are described herein, illustrated in the examples, and reflected in the claims.

[0064] It should be noted that the singular forms “a,” “an,” and “the” used herein include plural referents unless otherwise indicated by the context. Therefore, for example, a reference to “a reagent” includes one or more of such reagents, and a reference to “the method” includes equivalent steps and methods known to those skilled in the art that may be used to modify or substitute the method described herein.

[0065] Unless otherwise indicated, the term “at least” preceding a set of elements should be understood to refer to all elements of that set. Those skilled in the art will recognize, or can verify by routine experimentation, many equivalents corresponding to specific embodiments of the invention described herein. Such equivalents shall be incorporated herein.

[0066] As used herein, the terms “and / or” include the meanings of “and,” “or,” and “all or any other combination of the elements linked by the terms above.”

[0067] As used herein, the terms “about” or “approximately” mean within 20%, preferably within 10%, more preferably within 5% or 2% of a given value or range.

[0068] Throughout this specification and the subsequent claims, unless otherwise indicated by context, the word “comprise,” and variations such as “comprises” and “comprising,” are understood to mean encompassing the integer or process or group of integers or processes described, but not to mean excluding other integers or processes or groups of integers or processes. The term “comprising” as used herein may be replaced with the terms “containing” or “including,” and sometimes with the term “having” as used herein.

[0069] As used herein, "consisting of" excludes elements, processes, or components not explicitly stated in the claims. As used herein, "consisting essentially of" does not exclude materials or processes that do not significantly affect the basic and novel properties of the claims.

[0070] In each of the cases herein, the terms “include,” “essentially consist of,” and “consist of” may be replaced with any of the other two terms.

[0071] The present invention is not limited to the specific methodologies, protocols, reagents, etc., described herein, and should therefore be understood to be subject to change. The terms used herein are for the sole purpose of describing specific aspects and are not intended to limit the scope of the present invention as defined solely by the claims.

[0072] All publications and patents cited throughout this Specified (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) are incorporated herein by reference in their entirety, regardless of whether they are referenced above or below. Nothing in this Specified should be construed as admitting that the present invention has no prior rights to such disclosures for the sake of prior art. This Specified shall prevail only in the event that any material incorporated by reference is inconsistent or contradictory to this Specified.

[0073] The present invention is further illustrated by the following embodiments. However, the embodiments and specific aspects described herein should not be construed as limiting the present invention to such specific aspects. [Examples]

[0074] The methodology used herein is well known and has been published, for example, in Mislovicova et al., Biointerfaces (2012), 94: 163-169. Polyclonal anti-ZAG antibody was immobilized on the bottom surface of ELISA plate wells. After the washing step, the surface was blocked (with human serum albumin) and washed again using a pre-optimized protocol. Subsequently (with additional washing steps after each of the following steps), (i) diluted human serum sample, (ii) biotinylated lectin, and (iii) streptavidin-peroxidase (derived from horseradish) were added to the plate to complete the sandwich structure. A signal was generated using an OPD / hydrogen peroxide system, the reaction was stopped using sulfuric acid, and the signal was read at 450 nm. The assay format was simplified without the use of magnetic beads because, even if the use of magnetic beads were considered and would at least yield clear results, ZAG is present in blood at considerably higher concentrations than PSA, making it unnecessary to pre-concentrate ZAG using magnetic beads.

[0075] The response of individual samples to lectin binding (measured in pairs) was evaluated for individual markers (PSA level, ZAG level, age, and individual lectins) and their combinations using ROC curves and AUC parameters, respectively, with OriginPro® software and the free version of RStudio R, as previously reported (see Bertokova et al., Bioorganic & Medicinal Chemistry (2021), 116156; Bertok et al., Glycoconjugate Journal (2020), 37: 703-711).

[0076] Actual serum samples were collected from a university hospital in Slovakia. The total number of serum samples in this study was n=69. Two separate experiments were conducted: CASE 1: a comparison between a benign cohort (n=18) and a PCa cohort already receiving treatment (n=15); and CASE 2: a comparison between a BPH cohort (n=21) and early-detection PCa patients (n=15).

[0077] The results showed that ZAG glycan profiling is applicable to identifying early PCa from BPH (CASE 2). The lectin best suited for identifying early PCa from BPH (CASE 2) was shown to be WFL with an AUC of 0.892 (Table 1) (WFL as used herein is Fuji lectin (WFA / WFL)).

[0078] To further enhance the discriminative ability of ZAG glycan profiling, it was possible to combine two types of lectins. In some cases, the use of two lectins was more suitable for identifying PCa from BPH (CASE 2), and the optimal combination of two lectins was WFL+PHA-E with an AUC of 0.917 (Table 1).

[0079] (Table 1) Parameters (AUC value and left and right confidence intervals (CI)), specificity, sensitivity, and assay precision for individual WFL markers (1st column) and combinations with other markers. TIFF0007876222000001.tif108150

Claims

1. A method for detecting whether a subject may be at risk of cancer or may have cancer, (1) A step of contacting a serum sample containing a biomarker glycoprotein obtained from the subject with a binding substance capable of binding to the glycan structure of the biomarker glycoprotein, The glycan structure is a modified glycan structure compared to the glycan structure of the biomarker glycoprotein expressed in subjects who are neither at risk of cancer nor suffering from cancer. The aforementioned process; and (2) A step to determine whether the binding substance has bound to the glycan structure of the biomarker glycoprotein. Includes, Compared to a control serum sample, lower or higher binding of the binding substance to the glycan structure of the biomarker glycoprotein indicates that the subject is at risk of cancer or has cancer. The aforementioned biomarker glycoprotein is ZAG. The aforementioned cancer is prostate cancer (PCa), and The binding substance is a lectin, and The lectin binds to a glycan structure terminated by an N-acetylgalactosamine α or β-linked to the 3rd or 6th position of galactose, or to a glycan structure containing a LacdiNAc epitope (GalNAc1-4GlcNAc). The aforementioned method.

2. The method according to claim 1, wherein the subject is a human.

3. The method according to claim 1 or 2, wherein one or more further biomarker glycoproteins are selected from the group consisting of PSA, TIMP-1, fPSA, tPSA, osteopontin, and spongin 2.

4. The method according to claim 1 or 2, wherein the lectin binds to the same glycan structure as WFA / WFL with an affinity of at least 80% of the affinity with which WFL binds to the glycan structure.

5. The method according to claim 1 or 2, wherein the lectin is WFL / WFA.

6. The method according to claim 1 or 2, wherein a lectin-based assay is used.

7. The method according to claim 6, wherein an enzyme-binding lectin-binding assay (ELLBA) is used.

8. A kit for carrying out the method of claim 1 or 2, comprising a binding substance capable of binding to the glycan structure of the biomarker glycoprotein, wherein the binding substance is a lectin, and the lectin is WFL and PHA-E, and the kit further comprises an anti-ZAG binding antibody.

9. Use of the kit according to claim 8 in the method according to claim 1 or 2.

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