Use of lectins to measure mammaglobin A glycoforms in breast cancer.

Abstract

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

Description

[Technical field]

[0001] This application claims the benefit of priority to European Patent Application No. 21196556.1, filed September 14, 2021, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] The present invention relates to a method for diagnosing whether a subject may be at risk for or suffer from breast cancer, in which a (significantly) lower or (significantly) higher binding of a binding agent to a specific glycan structure of the biomarker glycoprotein mammaglobin A compared to a control sample indicates that said subject is at risk for or suffers from breast cancer. The present invention further relates to a kit for carrying out said method for diagnosing whether a subject may be at risk for or suffers from breast cancer, comprising a binding agent capable of binding to the glycan structure of mammaglobin A. [Background technology]

[0003] Breast cancer (BCa) is one of the most common types of cancer along with lung, colon, and prostate cancer (men only), with its peak incidence occurring between 45 and 65 years of age. In 2020, there were 2,261,419 new cases of BCa in women worldwide and 684,996 new deaths from the disease (the fifth most common cause of all cancer deaths) (see Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: Cancer J Clin. 2021; 71: 209-249). However, mortality rates could be much lower if routine screening were performed regularly for women aged 40 years and older. In 2021, the incidence was predicted to further increase to 18 per 100,000 women worldwide (see Akram M, Iqbal M, Daniyal M, et al. Awareness and current knowledge of breast cancer. Biol Res. 2017; 50: 1-23). ​​Today, screening and early diagnosis rely on imaging modalities such as digital mammography, handheld or automated ultrasound, and magnetic resonance imaging (see Schuenemann HJ, Lerda D, Quinn C, et al. Breast cancer screening and diagnosis: a synopsis of the European Breast Guidelines. Ann Intern Med. 2020; 172: 46-56).BCa is strongly correlated with genetic factors (especially mutations in BRCA 1 and 2 genes) and other risk factors (gender, age, or race - African Americans have higher mortality and earlier onset) (see US Breast Cancer Statistics https: / / www.breastcancer.org / symptoms / understand_bc / statistics2021 [August 8, 2021] (Non-Patent Document 4); Yedjou CG, Sims JN, Miele L, et al. Health and racial disparity in breast cancer. Breast Cancer Metast Drug Resist. 2019: 31-49 (Non-Patent Document 5)). Due to the low specificity and sensitivity of using CEA (carcinoembryonic antigen) and CA15-3 (cancer antigen) for BCa, there is a need to identify new, more specific biomarkers.

[0004] WO 02 / 053017 A2 (Patent Document 1) discloses a method and a kit for determining breast cancer. Tian-Hua et al. (2016), Am J Transl Res, 8(10):4250-4264 (Non-Patent Document 6) disclose glycosylation patterns and PHA-E binding glycoprotein profiling associated with early hepatic encephalopathy in Chinese hepatocellular carcinoma patients. Xiong et al. (2002), Journal of Chromatography B, 782(1-2):405-418 (Non-Patent Document 7) disclose the use of lectin affinity selectors in exploring abnormal glycosylation in proteomics. Zehentner et al. (2004), Clinical Biochemistry, 37(4):249-257 (Non-Patent Document 8) disclose mammaglobin as a candidate diagnostic marker for breast cancer. O'Brien et al. (2004), International Journal of Cancer, 114(4):623-627 (Non-Patent Document 9), disclose the existence of multiple molecular forms of mammaglobin.

[0005] Mammaglobin A, also known as mammaglobin 1 or secretoglobin family 2A member 2, is a secreted glycoprotein and is the product of the SCGB2A2 gene (chromosome 11, synonyms: MGB1, UGB2). It is a member of the secretoglobin superfamily, a group of small dimeric, secreted, occasionally glycosylated proteins. Mammaglobin A itself contains the Asn53 residue. a and Asn68 b It is N-glycosylated at 100-fold higher than that of the prostate-specific protein PSA, which is overexpressed in breast cancer (BCa) and is mammary gland specific (similar to the prostate-specific protein PSA), making it a potential biomarker for breast cancer. In particular, the inventors found that investigating the changes in the glycan structure of this protein offers new possibilities for the diagnosis of breast cancer that have not been described before.

[0006] The above-mentioned shortcomings need to be overcome. Therefore, the present invention addresses these needs and technical objectives by providing a solution as described herein and defined in the claims. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO 02 / 053017 A2 [Non-patent literature]

[0008] [Non-Patent Document 1] Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: Cancer J Clin. 2021; 71: 209-249 [Non-Patent Document 2] Akram M, Iqbal M, Daniyal M, et al. Awareness and current knowledge of breast cancer. Biol Res. 2017; 50: 1-23 [Non-Patent Document 3] Schuenemann HJ, Lerda D, Quinn C, et al. Breast cancer screening and diagnosis: a synopsis of the European Breast Guidelines. Ann Intern Med. 2020; 172: 46-56 [Non-Patent Document 4] US Breast Cancer Statistics https: / / www.breastcancer.org / symptoms / understand_bc / statistics2021 [August 8, 2021] [Non-Patent Document 5] Yedjou CG, Sims JN, Miele L, et al. Health and racial disparity in breast cancer. Breast Cancer Metast Drug Resist. 2019: 31-49 [Non-Patent Document 6] Tian-Hua et al. (2016), Am J Transl Res, 8(10):4250-4264 [Non-Patent Document 7] Xiong et al. (2002), Journal of Chromatography B, 782(1-2):405-418 [Non-Patent Document 8] Zehentner et al. (2004), Clinical Biochemistry, 37(4):249-257 [Non-Patent Document 9] O'Brien et al. (2004), International Journal of Cancer, 114(4):623-627 Summary of the Invention

[0009] The present invention relates to a method for diagnosing whether a subject may be at risk for or suffer from breast cancer, said method comprising: (1) contacting a sample obtained from said subject, containing mammaglobin A as a biomarker glycoprotein, with a binding substance capable of (specifically) binding to the glycan structure of mammaglobin A, the presence or overexpression of mammaglobin A (e.g., at least about 1.5-fold, at least about 2-fold, or at least about 3-fold overexpression) or underexpression of mammaglobin A (e.g., at least about 1.5-fold, at least about 2-fold, or at least about 3-fold underexpression) is indicative of a risk of and / or the presence of breast cancer; and The glycan structure deviates from the glycan structure of mammaglobin A expressed in subjects who are not at risk for or have breast cancer. the steps; and (2) determining whether the binding agent binds to the glycan structure of mammaglobin A; Including, Compared to a control sample, (significantly) lower or (significantly) higher (preferably significantly higher) binding of the binding substance to the glycan structure of mammaglobin A indicates that the subject is at risk of breast cancer or is suffering from breast cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] As used herein, and as generally known in the art, "glycoprotein" (or "glycosylated protein") as used herein refers to a protein that contains one or more N-, O-, S-, or C-covalently linked glycans of various types, e.g., from monosaccharides to branched polysaccharides (including modifications such as the attachment of sulfo or phospho groups). N-linked glycans are glycans attached to the -NH2 group of asparagine. O-linked glycans are glycans attached to the -OH group of serine, threonine, or hydroxylated amino acids. S-linked glycans are glycans attached to the -SH group of cysteine. C-linked glycans are glycans attached to tryptophan via a C-C bond.

[0011] The term "glycan" refers to glyco-RNA and / or compounds consisting of glycosidically linked monosaccharides, and can also refer to the glycan portion of glycoconjugates such as glycoproteins, glycolipids, and proteoglycans, even if the glycans consist only of monosaccharides or oligosaccharides.

[0012] In one aspect of the invention, the subject, who may be at risk for or may be afflicted with breast cancer, is a human.

[0013] Surprisingly, as found in the context of the present invention, the biomarker glycoproteins described herein, which can indicate the risk and / or presence of breast cancer, show alterations in glycan structure (statistically relevant alterations in the glycan structure of said biomarker glycoprotein, e.g. the presence or over- or under-expression of said biomarker glycoprotein) if a subject may be at risk for or may be affected by breast cancer. In the context of the present invention, this led to the surprising discovery that certain glycan structures on mammaglobin A that deviate from the "normal" glycan structure of mammaglobin A may indicate the risk and / or presence of breast cancer. According to the present invention, identifying such altered glycan structures on mammaglobin A using a suitable binding agent capable of binding to such glycan structures makes it possible to diagnose whether a subject may be at risk for or affected by breast cancer.

[0014] In this regard, according to the present invention, it is possible to use a binding agent capable of binding to the glycan structure of mammaglobin A in a non-cancerous state, contact the binding agent with a sample according to step (1) of the method provided herein, and compare the binding ability of the binding agent to the glycan structure of mammaglobin A contained in a control sample (healthy sample). The mammaglobin A may have an altered glycan structure compared to the glycan structure of mammaglobin A in a non-cancerous state, as described in the method provided herein, and may, for example, contain more (e.g., at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold more) or less (e.g., at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold less) mammaglobin A as a biomarker glycoprotein in a cancerous state.

[0015] In one embodiment of the method of the present invention, if the binding substance binds to mammaglobin A to a higher extent (preferably a significantly higher extent, e.g., at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold higher) to the glycan structure of mammaglobin A contained in a sample of a subject who may be at risk of breast cancer or who may be suffering from breast cancer compared to that of a control sample, this may indicate that the subject is at risk of breast cancer or is suffering from breast cancer.

[0016] In one embodiment of the method of the present invention, if the binding substance binds to mammaglobin A to a lower extent (preferably a significantly lower extent, for example at least about 1.5-fold, at least about 1 / 2-fold, at least about 1 / 2.5-fold, or at least about 1 / 3-fold lower) to the glycan structure of mammaglobin A contained in a sample from a subject who may be at risk of breast cancer or who may be suffering from breast cancer compared to that of a control sample, this may indicate that the subject is at risk of breast cancer or is suffering from breast cancer.

[0017] Therefore, according to the present invention, it is possible to use a binding substance capable of binding to the glycan structure of mammaglobin A in a cancerous state, by contacting said binding substance with the sample according to step (1) of the method provided herein, and to compare the binding ability of said binding substance to the glycan structure of mammaglobin A contained in a control sample (healthy sample). Preferably, if said binding substance binds to a higher degree (preferably a significantly higher degree, for example at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold higher) of the glycan structure of mammaglobin A contained in a sample of a subject who may be at risk of breast cancer or may be affected by breast cancer compared to that of the control sample, this may indicate that said subject is at risk of breast cancer or is affected by breast cancer.

[0018] In one embodiment of the invention, the glycoprotein mammaglobin A, also called mammaglobin 1 or secretoglobin family 2A member 2, may be mammaglobin A from homo sapiens, for example as displayed in UniProtKB Accession No. Q13296.

[0019] In one aspect of the invention, the breast cancer is characterized as being Her2 negative; estrogen receptor (ER) negative, progesterone receptor (PR) negative, and Her2 negative (triple negative); or estrogen receptor positive, progesterone receptor positive, and Her2 negative.

[0020] In one aspect of the invention, breast cancer comprises invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), lobular carcinoma in situ (ILCIS), non-specialty ductal carcinoma (NST), or invasive lobular carcinoma (ILC).

[0021] According to the present invention, the binding agent capable of (specifically) binding to the glycan structure of mammaglobin A described herein and used in the methods provided herein can be any kind of agent capable of binding to the glycan structure. Preferably, such a binding agent is an agent whose binding to the glycan structure can be measured and quantified, for example, either when the binding itself can be detected and measured, and / or when the glycan structure is recognized by a binding agent that includes a marker molecule (detectable using a suitable method).

[0022] In the context of the present invention, non-limiting examples of suitable binding agents include lectins, anti-glycan antibodies, aptamers (nucleic acid aptamers, such as DNA or RNA aptamers, or peptide aptamers), or boronic acid or its derivatives. In one aspect of the present invention, the binding agent used in the methods described and provided herein is a lectin. In another example in the context of the methods of the invention described and provided herein, the binding agent is capable of (specifically) binding to a glycan structure terminating in an α- or β-linked N-acetylgalactosamine at the 3- or 6-position of galactose or comprises a LacNAc epitope; alternatively, the binding agent is capable of (specifically) binding to an antennary or core fucose, α-2,3-Neu5Ac (α-2,3 linked sialic acid), α-2,6-Neu5Ac (α-2,6 linked sialic acid), α-2,8-Neu5Ac (α-2,8 linked sialic acid), sialic acid (α-2,3-Neu5Ac, α-2,6-Neu5Ac, or α-2,8-Neu5Ac), N-linked tri / tetraantennary, branched β-1,6-GlcNAc, bisected GlcNAc, or branched (LacNAc) nThe binding agent may be capable of (specifically) binding to glycan structures terminating in α- or β-linked N-acetylgalactosamine to the 3- or 6-position of galactose, preferably to glycan structures terminating in N-acetylgalactosamine α- or β-linked to the 3- or 6-position of galactose. The binding agent may be capable of (specifically) binding to glycan structures terminating in antennary or core fucose. The binding agent may be capable of (specifically) binding to α-2,3-Neu5Ac (α-2,3-linked sialic acid). The binding agent may be capable of (specifically) binding to α-2,6-Neu5Ac (α-2,6-linked sialic acid). The binding agent may be capable of (specifically) binding to α-2,8-Neu5Ac (α-2,8-linked sialic acid). The binding agent may be capable of (specifically) binding to sialic acid (α-2,3-Neu5Ac, α-2,6-Neu5Ac, or α-2,8-Neu5Ac). The binding agent may be N-linked tri / tetraantennary, branched β-1,6-GlcNAc, bisected GlcNAc, or branched (LacNAc). n It may be possible to (specifically) bind to

[0023] In general, "binding substances" (or "recognition molecules") as used herein include polypeptides (e.g., lectins or anti-glycan antibodies, or fragments thereof) that contain one or more binding domains capable of binding to a target epitope, as well as other molecules (e.g., aptamers or boronic acids and their derivatives) that can bind to glycan structures. The binding substance, so to speak, provides a scaffold for said one or more binding domains so that said binding domains can bind to / interact with a given target structure / antigen / epitope. The term "binding domain" in the context of the present invention characterizes a domain of a polypeptide that specifically binds / interacts with a given target epitope. An "epitope" is antigenic, and therefore the term epitope may also be referred to herein as an "antigenic structure" or an "antigenic determinant". In the context of the present invention, the glycan structure may serve as an antigen 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 acids or derivatives thereof, preferably one or more lectins and / or anti-glycan antibodies, preferably one or more lectins. Thus, the binding domain is an "antigen interaction site". The term "antigen interaction site" defines, according to the present invention, a motif of a polypeptide 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 is also understood to define "specific recognition".

[0024] The term "epitope" also refers to a site on an antigen to which a binding agent binds. Preferably, an epitope is a site on a molecule to which a binding agent binds, such as a lectin, an anti-glycan antibody, an aptamer (a nucleic acid aptamer, such as a DNA or RNA aptamer, or a peptide aptamer), or a boronic acid or a derivative thereof, preferably one or more lectins and / or an anti-glycan antibody, preferably one or more lectins.

[0025] The term "aptamer" as used herein refers to a nucleic acid, oligonucleotide or peptide molecule that binds to a specific target molecule.As used herein, unless otherwise specified, the term "nucleic acid" or "nucleic acid molecule" is used synonymously with "oligonucleotide", "polynucleotide", "nucleic acid chain" and the like, and refers to a polymer, such as single-stranded or double-stranded, that contains one, two or more nucleotides.

[0026] The term "lectin" as used herein refers to a glycan-binding protein of any type and origin, including, for example, lectins, galectins, selectins, recombinant lectins, or fragments thereof, as well as fragments of the glycan-binding site bound to a scaffold. The term "lectin" as used herein also includes fragments of lectins that are capable of binding to glycan structures. A lectin can be highly specific for one or more glycan moieties (e.g., it specifically reacts with terminal glycosidic residues of other molecules, such as glycans of glycoproteins (e.g., branched sugar molecules of glycoproteins, e.g., target polypeptides within the meaning of the present invention and biomarkers described in Table 1 herein)). Lectins are well known in the art. A person 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 bound to a protein. Preferred lectins to be applied in the context of the present invention are described herein. The term "lectin" also includes siglecs (sialic acid-binding immunoglobulin-like lectins). Of note, the term "lectin" as used herein also refers to glycan-binding antibodies. Thus, the term "lectin" as used herein includes lectins, siglecs, as well as glycan-binding antibodies.

[0027] The lectins described herein and used in the context of 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 sources, they can be purified to homogeneity on a suitable immobilized carbohydrate matrix and eluted with a suitable hapten (see Goldstein & Poretz (1986) In The lectins. Properties, functions and applications in biology and medicine (eds. Liener et al.), pp. 33-247, Academic Press (Orlando, Fla.); Rudiger (1993) In Glycosciences: Status and perspectives (eds. Gabius & Gabius), pp. 415-438, Chapman and Hall (Weinheim, Germany)). Alternatively, lectins can be produced recombinantly according to established methods (Streicher & Sharon (2003) Methods Enzymol. 363: 47-77). As yet another alternative, lectins can be produced based on the amino acid sequences of known lectins or lectins disclosed herein using standard peptide synthesis techniques or chemical cleavage methods well known in the art (e.g., US 9169327 B2). Another alternative can 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).

[0028] In the context of the present invention, when a glycan binds to a lectin (or vice versa), the binding affinity is about 10 -1 ~10 -10 (K D ), preferably about 10 -2 ~10 -8 (K D ), more preferably about 10 -3 ~10 -5 (K D When the binding agent is a lectin, the term "specifically" or "specifically" as used herein in relation to the binding of the binding agent to a glycan structure preferably ranges from about 10 -2 ~10 -8 (K D ), more preferably about 10 -3 ~10 -5 (K D ) binding affinity to the lectin. D Methods for measuring are known in the art and readily available to those of skill in the art.

[0029] In one aspect of the invention, a binding agent used in the context of the present invention may be an antibody. As used herein, an "antibody" is a protein composed of one or more polypeptides (including one or more binding domains, preferably antigen-binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody." Recognized immunoglobulin genes include the kappa (κ), lambda (λ), alpha (α), gamma (γ), delta (δ), epsilon (ε) and mu (μ) constant region genes, as well as the myriad immunoglobulin variable region genes.

[0030] In particular, an "antibody" as used herein is a tetrameric glycosylated protein typically composed of two light chains (L chains) of about 25 kDa each and two heavy chains (H chains) of about 50 kDa each. There can be two types of light chains in an antibody, 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 are composed of five basic heterotetrameric units and an additional polypeptide called the J chain, which contains ten antigen-binding sites; whereas IgA antibodies are composed of two to five basic four-chain units, which can polymerize with the J chain to form multivalent aggregates. In the case of IgG, the four-chain unit is generally about 150,000 daltons.

[0031] 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 domains are not directly involved in binding the antibody to the antigen.

[0032] When VH and VL are paired together, a single antigen-binding site is formed. The CH domain closest to VH is designated CH1. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. The VH and VL domains are composed of four regions of relatively conserved 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 of the antibody with the antigen. The CDRs are termed 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.

[0033] The term "variable" refers to that portion of an immunoglobulin domain (i.e., the "variable domain") that exhibits variability in the immunoglobulin sequence and is responsible for determining the specificity and binding affinity of a particular antibody. The variability is not uniformly distributed throughout the variable domain of an antibody; it is concentrated in subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable" or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called "framework" regions (FRMs). Naturally occurring heavy and light chain variable domains each contain four FRM regions that predominantly adopt a β-sheet configuration, which are connected by three hypervariable regions; the hypervariable regions form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions of each chain are closely connected by the FRM and contribute to the formation of the antigen-binding site together with the hypervariable regions of the other chain (Chothia et al., J MoI Biol (1987), 196: 901; and MacCallum et al., J MoI Biol (1996), 262: 732 et seq.). The constant domains are not directly involved in antigen binding but exhibit various effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement activation.

[0034] The term "CDR" and its plural "CDRs" refer to the complementarity determining regions (CDRs), three of which constitute the binding properties of the light chain variable region (CDRL1, CDRL2, and CDRL3) and three of which constitute the binding properties of the heavy chain variable region (CDRH1, CDRH2, and CDRH3). The CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences that constitute scaffolding or framework regions. The exact definition of the boundaries and lengths of the CDRs varies according to the various classification and numbering systems. Despite the different boundaries, each of these systems has a certain degree of overlap in the parts of the variable sequences that constitute the so-called "hypervariable regions". Thus, the definition of CDRs by these systems may differ in terms of length and border regions with respect to the flanking framework regions (see, e.g., Kabat, Chothia, and / or MacCallum; Chothia et al., J MoI Biol (1987), 196: 901; and MacCallum et al., J MoI Biol (1996), 262: 732).

[0035] As used herein, the term "amino acid" or "amino acid residue" typically refers to an amino acid having a definition generally recognized in the art, e.g., an amino acid selected from the group consisting of 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 (Ile 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, synthetic, or rare amino acids may be used if desired. In general, amino acids can be grouped as having nonpolar side chains (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); negatively charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0036] The term "framework region" refers to the art-recognized portion of an antibody variable region that lies between the more diverse (i.e., hypervariable) CDRs. Such framework regions are usually referred to as frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and provide a scaffold for presenting the six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space to form an antigen-binding surface.

[0037] As used herein, the term "antibody" refers not only to immunoglobulins (i.e., intact antibodies) but also to fragments thereof, including 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 will contain the antigen-binding domain and will have the same properties as the antibodies described herein.

[0038] The term "antibody" as used herein includes antibodies that compete for binding to the same epitope as the antibody of the present invention, preferably such antibodies can be obtained by the methods for producing antibodies described elsewhere herein.

[0039] To determine whether the test antibody can compete for binding to the same epitope, cross-blocking assays, such as competitive ELISA assays, can be carried out.In an exemplary competitive ELISA assay, the wells of a microtiter plate coated with epitopes or the sepharose beads coated with epitopes are pre-incubated in the presence or absence of a candidate competing antibody, and then the biotin-labeled antibody of the present invention is added.The amount of labeled antibody bound to the epitope in the wells or on the beads is measured using avidin-peroxidase conjugate and a suitable substrate.

[0040] Alternatively, the antibody can be labeled, e.g., with a radioactive, enzymatic, fluorescent, or other detectable or measurable label. The amount of labeled antibody that binds to the antigen will be inversely correlated with the ability of a candidate competing antibody (test antibody) to compete for binding to the same epitope on the antigen; i.e., the higher the affinity of the test antibody for the same epitope, the less amount of labeled antibody that will bind to the antigen-coated well.

[0041] A candidate competing antibody is considered to be an antibody that substantially binds to the same epitope as an antibody of the invention, or competes for binding to the same epitope, if the candidate competing antibody is able to block binding of an antibody of the invention by at least 20%, preferably at least 20-50%, more preferably at least 50%, compared to a control run in parallel in the absence of the candidate competing antibody (but which may be in the presence of a known non-competing antibody). It will be understood that variations of this assay may be performed to arrive at the same quantitative value.

[0042] The term "antibody" also includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific such as bispecific, nonspecific, humanized, human, single chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies; polyclonal antibodies are preferred. The term also includes domain antibodies (dAbs) and nanobodies.

[0043] 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, most preferably purified (polyclonal) serum. The antibody / serum can be obtained, and preferably is obtained, for example, by the methods or uses described herein.

[0044] "Polyclonal antibody" or "polyclonal antiserum" refers to an immune serum containing a mixture of antibodies specific for one (monovalent or specific antiserum) or multiple (polyvalent antiserum) antigens, and can be prepared from the blood of animals immunized with one or more antigens.

[0045] Furthermore, the term "antibody" as used herein also relates 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 matured" 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 functions (see, e.g., U.S. Patent No. 5,648,260).

[0046] The terms "antigen-binding domain", "antigen-binding fragment", and "antibody-binding region" as used herein refer to a portion of an antibody molecule that contains the amino acids involved in the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an "epitope" as described herein above. As noted above, an antigen-binding domain typically may contain an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it need not contain 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 antigen-binding fragments of antibodies include: (1) a Fab fragment, which is a monovalent fragment having the VL, VH, CL, and CH1 domains; (2) an F(ab')2 fragment, which is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody; (5) a dAb fragment having a VH domain (see Ward et al., (1989) Nature 341: 544-546); (6) an isolated complementarity determining region (CDR); and (7) a single-chain Fv (scFv). The two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be joined together using recombinant techniques with a synthetic linker that allows the VL and VH regions to be produced as a single protein chain that pairs to form 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 are obtained using conventional techniques known to those skilled in the art and are evaluated for function in the same manner as intact antibodies.

[0047] The term "monoclonal antibody" as used herein includes chemically modified monoclonal antibodies or fragments thereof, as well as antibodies obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible natural mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies against various determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the nature of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described in Kohler et al., Nature (1975), 256: 495, or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature (1991), 352: 624-628; and Marks et al., J Mol Biol (1991), 222: 581-597.

[0048] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins), as well as fragments of such antibodies so long as they exhibit the desired biological activity; portions of the heavy and / or light chains of a chimeric antibody are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass (U.S. Pat. No. 4,816,567; Morrison et al., PNAS USA (1984), 81: 6851-6855). Chimeric antibodies of interest herein include "primitized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0049] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) of mostly human sequence with minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins in which residues from a hypervariable region (or CDR) of a human immunoglobulin (recipient antibody) are replaced by residues from a hypervariable region of a non-human species antibody (donor antibody) such as mouse, rat, rabbit, etc. having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, as used herein, "humanized antibodies" may include residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize antibody performance. A humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a 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.

[0050] The term "human antibody" includes antibodies having variable and constant regions substantially corresponding to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (Kabat et al., supra). The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, particularly CDR3. Human antibodies can have at least one, two, three, four, five, or more positions replaced with an amino acid residue not encoded by a human germline immunoglobulin sequence.

[0051] As used herein, "in vitro generated antibody" refers to an antibody in which all or a portion of the variable region (e.g., at least one CDR) is generated by a non-immune cell selection method (e.g., in vitro phage display, protein chip, or other method that allows testing of candidate sequences for antigen-binding ability). Thus, the term preferably excludes sequences generated by genomic rearrangement in immune cells.

[0052] 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 a variety of ways, such as by fusion of hybridomas or linking of Fab' fragments (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 via an immunoglobulin constant region to a second binding domain polypeptide.

[0053] Numerous methods known to those skilled in the art are available for obtaining antibodies or antigen-binding fragments thereof. For example, antibodies can be produced using recombinant DNA methods (U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be obtained by producing 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 particular antigen can be used as the immunogen, for example, recombinant antigens, naturally occurring forms, variants or fragments thereof, as well as antigenic peptides thereof.

[0054] One exemplary method for producing antibodies includes screening protein expression libraries, such as phage or ribosome display libraries. Phage display is 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.

[0055] In another embodiment, monoclonal antibodies can be obtained from non-human animals, and then modified versions, such as humanized, de-immunized, and chimeric versions, can be produced using recombinant DNA techniques known in the art.Various approaches to make 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, and 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 an exemplary CDR-grafting method that can be used to generate the humanized antibodies described herein (U.S. Patent No. 5,225,539). All CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only a portion of the CDRs may be replaced with a non-human CDR. It is sufficient to replace as many CDRs as are necessary for the humanized antibody to bind to a given antigen.

[0056] Humanized antibodies or fragments thereof can be made by replacing sequences of Fv variable domains that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for making 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 include isolating, manipulating, and expressing nucleic acid sequences that code for all or part of immunoglobulin Fv variable domains from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas that produce antibodies against a given target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0057] In certain embodiments, humanized antibodies are optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions and / or back mutations. Such modified immunoglobulin molecules can be made 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 can be made according to the teachings of WO 92 / 06193 or EP 239400).

[0058] In the case of antibodies, specific binding is believed to be brought about by certain motifs in the amino acid sequence of the binding domain, where antigen and antibody bind to each other as a result of their primary, secondary or tertiary structure, as well as as a result of secondary modifications of said structure. The specific interaction of an antigen interaction site with its specific antigen may also result in a simple binding of said site to said antigen. Moreover, the specific interaction of an antigen interaction site with its specific antigen may instead result in the initiation of a signal, for example due to the induction of a conformational change in the antigen, oligomerization of the antigen, etc. An example of a binding domain in line with the present invention is an anti-glycan antibody. In this context, when the binding substance is an antibody, it is possible to use an antibody with a binding affinity of 10 -1 Binding can be considered "specific" if the binding affinity is greater than about M. Preferably, the binding affinity is about 10 -5 ~10 -12 M(K D ), preferably about 10 -8 ~10 -12 When M (when the binding substance is an antibody), the binding is considered specific. If necessary, the binding conditions can be changed to reduce non-specific binding without substantially affecting specific binding. Whether a recognition molecule reacts specifically as defined herein above can be easily tested, inter alia, by comparing the reaction of the recognition molecule with the epitope with the reaction of said recognition molecule with other proteins.

[0059] According to the present invention, 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) of the biomarker glycoprotein mammaglobin A (also referred to herein as biomarker or biomarker protein) is indicative of risk and / or presence of breast cancer, such biomarker glycoprotein mammaglobin A may be present or overexpressed (e.g., at least 1.5-fold, 2-fold, or 3-fold overexpression) or underexpressed (e.g., at least 1.5-fold, 2-fold, or 3-fold underexpression) in cells of a (human) subject at risk of developing breast cancer or afflicted with breast cancer, compared to cells of a (human) subject not at risk of developing breast cancer or afflicted with breast cancer. Preferably, in the context of the present invention, such mammaglobin A biomarker glycoprotein has a different glycan structure in a cancerous state compared to a non-cancerous state. Thus, in one aspect of the invention, the presence or overexpression (e.g., at least 1.5-fold, 2-fold, or 3-fold overexpression) or underexpression (e.g., at least 1.5-fold, 2-fold, or 3-fold underexpression) of the biomarker glycoprotein mammaglobin A (also referred to herein as biomarker, or biomarker protein) is indicative of risk of and / or the presence of breast cancer.

[0060] As used herein, "overexpression" of a glycoprotein or protein may mean, in any case, resulting in a greater amount of such glycoprotein or protein in cells of a subject at risk of breast cancer or affected by breast cancer as described herein, compared to cells of a subject not at risk of breast cancer or affected by breast cancer. This term also includes a statistically relevant increase in expression of the respective glycoprotein or protein. For example, according to the present invention, "overexpression" may mean an increase in the translation or transcription rate of such glycoprotein or protein, or an increase in the overall synthesis; whereas, "underexpression" may mean a statistically relevant decrease in expression of the respective glycoprotein or protein, for example, a decrease in the translation or transcription rate of such glycoprotein or protein, or a decrease in the overall synthesis.

[0061] As found in the context of the present invention, mammaglobin A exhibits different glycan structures in samples derived from subjects at risk of or suffering from breast cancer compared to mammaglobin A contained in samples derived from subjects neither at risk of breast cancer nor suffering from breast cancer.

[0062] In the context of the present invention, the binding agent used in the methods described and provided herein is capable of binding to the glycan structures of the biomarker glycoprotein mammaglobin A described herein. In one aspect of the present invention, the binding agent (preferably a lectin) is capable of (specifically) binding to any one or more of the following or to glycan structures that contain or terminate with: core fucose, antennary fucose, Fuc-α-1,6-GlcNAc-N-Asn-containing N-linked oligosaccharides, Fuc-α-1,6 / 3-GlcNAc, α-L-Fuc, Fuc-α-1,2-Gal-β-1,4(Fuc-α-1,3)GlcNAc, Fuc-α- 1,2-Gal, Fuc-α-1,6-GlcNAc, Man-β-1,4-GlcNAc-β-1,4-GlcNAc, branched N-linked hexasaccharide, Man-α-1,3-Man, α-D-Man, GlcNAc-β-1,4-Gal, Gal-β-1, 4-GlcNAc, GlcNAc-α-1,4-Gal-β-1,4-GlcNAc, Neu5Ac (sialic acid), Gal-α-1,3-GalNAc, Gal-β-1,6-Gal, Gal-β-1,4-GlcNAc, Gal-β-1,3-Gal NAc, GalNAc-α-1,3-GalNAc, GalNAc-α-1,3-Gal, GalNAc-α / β-1,3 / 4-Gal, α-GalNAc, GalNAc-β-1,4-Gal, GalNAc-α-1,3-(Fuc-α-1,2) Gal, GalNAc-α-1,2-Gal, GalNAc-α-1,3-GalNAc, GalNAc-β-1,3 / 4-Gal, GalNAc-β-1,4-GlcNAc(LacdiNAc), LacNAc, N-glycolylsialic acid, α-2, 3-Neu5Ac (α-2,3-linked sialic acid), α-2,6-Neu5Ac (α-2,6-linked sialic acid), α-2,8-Neu5Ac (α-2,8-linked sialic acid), sialic acid (α-2,3-Neu5Ac, α-2,6-Neu5Ac, or α-2,8-Neu5Ac), N-acetylglucosamine-β-(1,2)-mannopyranosyl, Neu5Ac-α-4 / 9-O-Ac-Neu5Ac, Neu5Ac-α-2,3-Gal-β-1,4-Glc / GlcNAc, Neu5Ac-α-2,6-Gal / GalNAc, N-linked biantennary, N-linked tri / tetraantennary, branched β-1,6-GlcNAc, Gal-α-1,3(Fuc-α-1,2)Gal-β-1,3 / 4-GlcNAc, Gal-β-1,3(Fuc-α-1,4)GlcNAc, NeuAc-α-2,3-Gal-β-1,3(Fuc-α-1,4)Glc NAc, Fuc-α-1,2-Gal-β-1,3(Fuc-α-1,4)GlcNAc, Gal-β-1,4(Fuc-α-1,3)GlcNAc, NeuAc-α-2,3 -Gal-β-1,4(Fuc-α-1,3)GlcNAc, Fuc-α-1,2-Gal-β-1,4(Fuc-α-1,3)GlcNAc, high mannose, sialyl Lewis, a (Sialyl Le a ) antigen, sialyl Lewis x (Sialyl 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, (GlcNAc-β-1,4) n , β-D-GlcNAc, GalNAc, Gal-GlcNAc, GlcNAc, Gal-α-1,3-Gal, Gal-β-1,3-GalNAc, α-Gal, α-GalNAc, (GlcNAc) n , β-1,6-GlcNAc, bisected GlcNAc, or branched (LacNAc) n .

[0063] As described herein, in one aspect of the invention, the binding agent used in the methods described and provided herein may be capable of binding, inter alia, to a glycan structure terminating in an N-acetylgalactosamine α- or β-linked to the 3- or 6-position of galactose, or the binding agent may comprise a LacNAc epitope; alternatively, the binding agent may be capable of binding, inter alia, to an antennary or core fucose, α- 2,3-Neu5Ac (α-2,3-linked sialic acid), α-2,6-Neu5Ac (α-2,6-linked sialic acid), α-2,8-Neu5Ac (α-2,8-linked sialic acid), sialic acid (α-2,3-Neu5Ac, α-2,6-Neu5Ac, or α-2,8-Neu5Ac), N-linked tri- / tetra-antennary, branched β-1,6-GlcNAc, bisected GlcNAc, or branched (LacNAc) n The binding agent may be capable of (specifically) binding to glycan structures terminating in an α- or β-linked N-acetylgalactosamine to the 3- or 6-position of galactose, preferably (specifically) binding to glycan structures terminating in an α- or β-linked N-acetylgalactosamine to the 3- or 6-position of galactose. The binding agent may be capable of (specifically) binding to glycan structures terminating in an antennary or core fucose. The binding agent may be capable of (specifically) binding to α-2,3-Neu5Ac (α-2,3-linked sialic acid). The binding agent may be capable of (specifically) binding to α-2,6-Neu5Ac (α-2,6-linked sialic acid). The binding agent may be capable of (specifically) binding to α-2,8-Neu5Ac (α-2,8-linked sialic acid). The binding agent may be capable of (specifically) binding to sialic acid (α-2,3-Neu5Ac, α-2,6-Neu5Ac, or α-2,8-Neu5Ac). The binding agent may be capable of (specifically) binding to N-linked tri / tetraantennary, branched β-1,6-GlcNAc, bisected GlcNAc, or branched (LacNAc). nIt may be possible to (specifically) bind to

[0064] In one embodiment, the binding agent binds to a glycan structure terminating in an α- or β-linked N-acetylgalactosamine to the 3- or 6-position of galactose or comprises a LacNAc epitope; alternatively, the binding agent binds to a glycan structure terminating in an antennary or core fucose, in α-2,3-Neu5Ac (α-2,3-linked sialic acid), in α-2,6-Neu5Ac (α-2,6-linked sialic acid), in α-2,8-Neu5Ac (α-2,8-linked sialic acid), or in sialic acid (α-2,3-Neu5Ac, α-2,6-Neu5Ac, or α-2,8-Neu5Ac).

[0065] Surprisingly, as found in the context of the present invention, mammaglobin A in samples from subjects at risk of or suffering from breast cancer ("cancerous mammaglobin A") exhibits a different glycan structure compared to mammaglobin A in samples from subjects not at risk of or suffering from breast cancer. According to the present invention, such "cancerous mammaglobin A" can be detected using a binding agent capable of binding to the glycan structure of "cancerous mammaglobin A" described herein. As further found in the context of the present invention, mammaglobin A in samples from subjects at risk of or suffering from breast cancer ("cancerous mammaglobin A") can be bound (and therefore detected) by using a specific lectin, such as, for example, Wisteria floribunda lectin (WFA / WFL). Thus, in one aspect of the invention, the binding agent capable of binding to the glycan structure of the biomarker glycoprotein mammaglobin A described herein and used in the methods provided herein may be capable of (specifically) binding to the same glycan structure as Fuji F. lectin (WFA / WFL) or PHA, preferably PHA-L, or a combination thereof, 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 PHA, preferably PHA-L, or WFL, or a combination thereof, binds to said glycan structure. Methods for measuring the affinity level of a binding agent (e.g., lectin) for a glycan structure are generally known in the art and include, in particular, surface plasmon resonance, isothermal microcalorimetry, or ELISA and ELISA-like formats, preferably surface plasmon resonance.

[0066] In a more specific embodiment of the present invention, the binding agent capable of binding to the glycan structure of the biomarker glycoprotein mammaglobin A described herein and used in the methods provided herein can be WFL, PHA, AAL, UEA-I, LCA, PSL, AAA, LTA, HPA, LBA, PhoSL, AOL, VVA, Siglec 1, Siglec 4, Siglec 8, TJA-I, SCA, WGA, SNA, MAA II, Con A, GNA, MGL, NPA, Jacalin, DBA, Galectin 1, Galectin 3, Galectin 8, RCA I, RCA 120, Bandeiraea simplicifolia lectin I (BS-I), MGL (macrophage galactose-type lectin), P-selectin, H-selectin, and E-selectin, or a combination thereof.

[0067] In another more specific aspect of the present invention, the binding agent capable of binding to the glycan structure of the biomarker glycoprotein mammaglobin A described herein and used in the methods provided herein can be WFL, PHA-L, AAL, UEA-I, LCA, PSL, AAA, LTA, HPA, LBA, PhoSL, AOL, VVA, Siglec-1, Siglec-4, Siglec-8, TJA-I, SCA, WGA, SNA, MAA II, Con A, GNA, MGL, NPA, Jacalin, DBA, PHA-E, Galectin-1, Galectin-3, Galectin-8, RCA I, RCA 120, Bandeiraea simplicifolia lectin I (BS-I), MGL (macrophage galactose-type lectin), P-selectin, H-selectin, and E-selectin, or a combination thereof.

[0068] In a specific embodiment of the present invention, the binding agent is Fuji lectin (WFA / WFL) or PHA, preferably PHA-L, or a combination thereof, preferably Fuji lectin (WFA / WFL).Most preferably, the binding agent is a combination of Fuji lectin (WFA / WFL) and PHA, preferably PHA-L.

[0069] In the context of the present invention, AAA means Anguilla anguilla agglutinin (see, for example, UniProtKB accession number Q7SIC1), AAL means Aleuria aurantia lectin, AOL means Aspergillus oryzae lectin, BS-I means Bandeiraea simplicifolia lectin, also known as Griffonia (Bandeiraea) simplicifolia lectin I, Con A means Concanavalin A, DBA means Dolichos biflorus agglutinin, GNA means Galanthus nivalis agglutinin, HPA means Helix pomatia agglutinin, LBA stands for Phaseolus lunatus (lima bean), LCA stands for Lens culinaris agglutinin, LTA stands for Lotus tetragonolobus lectin, MAA II stands for Maackia amurensis agglutinin II, MGBL 1 stands for macrophage galactose-binding lectin 1, NPA stands for Narcissus pseudonarcissus (Daffodil) lectin, PHA stands for PHA-E and / or PHA-L, PHA-E stands for Phaseolus vulgaris agglutinin E, PHA-L stands for Phaseolus vulgaris agglutinin L, PhoSL stands for Pholiota squarrosa lectin, PSL stands for pea (Pisum sativum) lectin, RCA I stands for castor bean (Ricinus communis) agglutinin I, SCA stands for American elderberry (Sambucus canadensis) agglutinin, and SNA stands for Sambucusnigra agglutinin, TJA-I means Trichosanthes japonica agglutinin I, UEA means Ulex europaeus agglutinin, VVA means Vicia villosa lectin, WFA means Wisteria floribunda lectin, WGA means wheat germ agglutinin, and TVA means Triticum vulgaris agglutinin.

[0070] AAL, UEA-I, LCA, PSL, AAA, LTA, HPA, LBA, PhoSL, AOL, and VVA can recognize fucose. Siglec-1, Siglec-4, Siglec-8, TJA-I, SCA, WGA, SNA, and MAA II can recognize sialic acid. Con A, GNA, MGL, and NPA can recognize mannose. Jacalin, DBA, and PHA-E can recognize branched or bisected glycans. Galectin-1, Galectin-3, Galectin-8, RCA I, and RCA 120 can recognize galactose.

[0071] In the context of the present invention, it is also possible to combine two or more binding substances capable of binding to the glycan structure of the biomarker glycoprotein mammaglobin A described herein, which are used in the methods described and provided herein. In some cases, combining two or more such binding substances may increase the possibility of diagnosis. In this regard, according to the present invention, in step (1) of the method of the present invention, two or more binding substances (e.g., lectins) are used in the same assay, or preferably, such two or more binding substances (e.g., lectins) are used in different assays (using the same sample), and then in step (2), it is possible to separately determine whether each of the binding substances binds to the glycan structure of mammaglobin A, and then combine the information thus obtained to diagnose whether the subject may be at risk of breast cancer or may be affected by breast cancer. In one embodiment of the present invention, when two (or more) such binding substances are used in the method of the present invention, both such binding substances are lectins. In a related specific embodiment, when two (or more) such binding agents are used in the methods of the invention, the lectins are or comprise Fuji lectin (WFA / WFL) and PHA, preferably PHA-L. In one embodiment, the binding agents comprise WFA / WFL and PHA, preferably PHA-L. In a preferred embodiment, the binding agent is a combination of WFA / WFL and PHA, preferably PHA-L.

[0072] In the context of the present invention, for the methods described and provided herein, any suitable assay may be used that allows for detecting and quantifying the binding of the binding agent described herein to the biomarker glycoprotein mammaglobin A described herein. Such suitable assays are generally known in the art and include, inter alia, ELISA or Western blot (particularly when the binding agent is an antibody), or lectin-based assays (see, for example, the assays described in WO 2019 / 185515), or enzyme-linked lectin-binding assay (ELLBA) (on cells, CELLBA; see, for example, Gaverieux et al., J Immunol Methods (1987), 104(1-2): 173-182). In one embodiment of the present invention, a lectin-based assay is used. In a preferred embodiment of the present invention, an enzyme-linked lectin-binding assay (ELLBA) or a magnetic enzyme-linked lectin assay (MELLBA) is used, with MELLBA being preferred.

[0073] The present invention further relates to a kit for carrying out a method for diagnosing whether a subject may be at risk of or suffer from breast cancer, comprising a binding substance capable of binding to the glycan structure of the biomarker protein mammaglobin A described herein.

[0074] In a preferred embodiment of the kit of the present invention, the binding substance may be a lectin.

[0075] In a more preferred embodiment of the kit of the present invention, the binding substance capable of binding to the glycan structure of the biomarker glycoprotein mammaglobin A described herein and used in the methods described and provided herein may be capable of (specifically) binding to the same glycan structure as Fuji Lectin (WFA / WFL) or PHA, preferably PHA-L, 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 Fuji Lectin (WFA / WFL) or PHA, preferably PHA-L, binds to said glycan structure.

[0076] In further specific embodiments of the kit of the present invention, the binding agent may be, for example, WFL, PHA, AAL, UEA-I, LCA, PSL, AAA, LTA, HPA, LBA, PhoSL, AOL, VVA, Siglec-1, Siglec-4, Siglec-8, TJA-I, SCA, WGA, SNA, MAA II, Con A, GNA, MGL, NPA, jacalin, DBA, Galectin-1, Galectin-3, Galectin-8, RCA I, RCA 120, Bandeiraea simplicifolia lectin I (BS-I), MGL (macrophage galactose-type lectin), P-selectin, H-selectin, and E-selectin. In some cases, combining two or more of such binding agents may increase the diagnostic potential. Thus, in one embodiment of the kit of the present invention, the kit described and provided herein comprises two or more of such binding agents. In this context, in a specific embodiment of the kit of the invention, both or at least two of such binding substances comprised in said kit are lectins, and in a more specific embodiment of this context, such two or more lectins comprised in said kit are or comprise WFA / WFL and PHA, preferably PHA-L.

[0077] The kits described and provided in connection with the present invention can also contain further suitable components, such as enzymes and buffers required to carry out the methods using suitable assays described herein (e.g., ELISA, Western blot, lectin-based assays, ELLBA, MELLBA, etc.), as will be readily appreciated by those of skill in the art.

[0078] The kit of the present invention can be used in the method of the present invention.

[0079] The features that characterize the present invention are described herein, illustrated in the examples, and reflected in the claims.

[0080] It should be noted that the singular forms "a", "an" and "the" used herein include plural referents unless the context dictates otherwise.Thus, for example, reference to "a reagent" includes one or more of such various reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that may modify or replace the method described herein.

[0081] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0082] As used herein, the term "and / or" is inclusive of the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0083] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% or 2% of a given value or range, including the given value.

[0084] Throughout this specification and the claims which follow, unless the context dictates otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or groups of integers or steps. As used herein, the term "comprising" can be interchanged with the terms "containing" or "including," and sometimes can also be interchanged with the term "having" as used herein.

[0085] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0086] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms.

[0087] It is to be understood that this invention is not limited to the particular methodology, protocols, reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0088] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether referenced supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention. To the extent that the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification shall supersede such material.

[0089] The present invention is further illustrated by the following examples. However, the examples and specific embodiments described therein should not be construed as limiting the invention to such specific embodiments. EXAMPLES

[0090] The methodology used here is well known and has been published, for example, in Mislovicova et al., Biointerfaces (2012), 94: 163-169. Polyclonal anti-mammaglobin A antibodies were immobilized on the bottom surface of ELISA plate wells. After a washing step, the surface was blocked (with human serum albumin) and washed again using a previously optimized protocol. Subsequently (with an additional washing step after each of the following steps), (i) diluted human serum samples, (ii) biotinylated lectins, and (iii) streptavidin-peroxidase (from horseradish) were added to the plate, completing the sandwich structure. The signal was developed using an OPD / hydrogen peroxide system, the reaction was stopped with sulfuric acid, and the signal was read at 450 nm. The assay format was simplified without the use of magnetic beads because there is no need to pre-enrich mammaglobin A with magnetic beads since mammaglobin A is present in much higher concentrations in blood compared to PSA, even if the use of magnetic beads had been considered and would have at least yielded clear results.

[0091] The response of individual samples to lectin binding (measured at least in duplicate) was evaluated for individual markers (mammaglobin A levels, age, and individual lectins) and their combinations using ROC (Receiver Operating Characteristic) curves and AUC (Area Under the Curve) parameters, respectively, using OriginPro® software and the free version of R in RStudio, as previously reported (see Bertokova et al., Bioorganic & Medicinal Chemistry (2021), 116156; Bertok et al., Glycoconjugate Journal (2020), 37: 703-711). ROC curves were obtained for the two individual lectins PHA-L and WFL, as well as their combinations, in the case of complete early diagnosis (without subtypization) and HER2 subtype. AUC values ​​were below the internal threshold (i.e. 0.8). Proposed N-glycan epitopes recognized by the PHA-L and WFL lectins were used.

[0092] Actual plasma samples were collected from the National Oncology Institute, Bratislava, Slovakia, where serum samples are also available. The total number of plasma samples in this study was n=52. The 52 breast cancer patient samples had the following characteristics: TNM (T1=30, T2=21, T3=1) (no distant metastasis), IDC=47, ILC=3, other=2 (invasive ductal / invasive lobular), HER2(-)=36, triple(-)=19, ER(+), PR(+), HER2(-)=15. 24 controls (anonymized non-BCa patients) were used.

[0093] The results showed that glycoprofiling of mammaglobin A is applicable to the diagnosis of (early) BCa. The optimal lectin for detecting (early) BCa was found to be a combination of WFL and PHA-L, with an AUC of 0.864 (Table 1) (WFL as used herein is Fuji lectin (WFA / WFL)).

[0094] Therefore, it was possible to combine two lectins to further enhance the discrimination power of mammaglobin A glycan profiling. The optimal combination of two lectins was WFL and PHA-L (Table 1).

[0095] Table 1. Parameters (AUC values ​​and left and right confidence intervals), specificity, sensitivity, and analytical accuracy for individual WFL markers, PHA-L markers, and their combinations. TIFF2024531711000001.tif64158

Claims

1. 1. A method for detecting whether a subject, preferably a human, may be at risk for or suffer from breast cancer, comprising: (1) contacting a sample obtained from the subject, containing mammaglobin A as a biomarker glycoprotein, with a binding substance capable of binding to the glycan structure of mammaglobin A, the presence or overexpression of mammaglobin A indicates a risk of and / or the presence of breast cancer, and the glycan structure deviates from the glycan structure of mammaglobin A expressed in subjects who are not at risk for or have breast cancer; the steps; and (2) determining whether the binding substance binds to the glycan structure of mammaglobin A; Including, a lower or higher binding of the binding agent to the glycan structure of mammaglobin A compared to a control sample indicates that the subject is at risk of or has breast cancer; wherein the binding substance is a lectin, the lectin binds to glycan structures terminating in α- or β-linked N-acetylgalactosamine at the 3- or 6-position of galactose or contains a LacNAc epitope; or the lectin binds to glycan structures terminating in antennary or core fucose, α-2,3-Neu5Ac (α-2,3-linked sialic acid), α-2,6-Neu5Ac (α-2,6-linked sialic acid), α-2,8-Neu5Ac (α-2,8-linked sialic acid), sialic acid (α-2,3-Neu5Ac, α-2,6-Neu5Ac, or α-2,8-Neu5Ac), N-linked tri- / tetra-antennary, branched β-1,6-GlcNAc, bisected GlcNAc, or branched (LacNAc)n. The method.

2. the breast cancer is characterized as being Her2-negative; estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and Her2-negative (triple-negative); or estrogen receptor-positive, progesterone receptor-positive, and Her2-negative; and / or the breast cancer comprises invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), non-specialty ductal carcinoma (NST), or invasive lobular carcinoma (ILC); The method of claim 1.

3. The method of claim 1, wherein the lectin binds to the same glycan structure as PHA or WFL or a combination thereof with an affinity that is at least 80% of the affinity with which PHA or WFL or a combination thereof binds to the glycan structure.

4. 2. The method of claim 1, wherein the lectin is WFL, PHA, AAL, UEA-I, LCA, PSL, AAA, LTA, HPA, LBA, PhoSL, AOL, VVA, Siglec-1, Siglec-4, Siglec-8, TJA-I, SCA, WGA, SNA, MAA II, Con A, GNA, MGL, NPA, jacalin, DBA, galectin-1, galectin-3, galectin-8, RCA I, RCA 120, Bandeiraea simplicifolia lectin I (BS-I), P-selectin, H-selectin, and E-selectin, or a combination thereof.

5. The method of claim 4, wherein the lectin is PHA or WFL, or a combination of PHA and WFL.

6. The method of claim 1 , wherein a lectin-based assay is used.

7. 7. The method of claim 6, wherein an Enzyme-Linked Lectin Binding Assay (ELLBA) or a Magnetic Enzyme-Linked Lectin Binding Assay (MELLBA) is used.

8. 10. A kit for carrying out the method of claim 1, comprising a binding substance capable of binding to the glycan structure of mammaglobin A, The kit, wherein the binding substance is one or more lectins and further comprises an anti-mammaglobin A antibody.

9. The lectin WFL and PHA, The kit according to claim 8, wherein

10. Use of the kit according to claim 8 in the method according to claim 1.