Glycopeptide, method for analyzing glycopeptide, and method for analyzing cancer

Abstract

The present invention addresses the problem of providing a material for systematically searching for and identifying epitopes specific to various diseases including cancer. The present invention also addresses the problem of establishing a method for disease analysis using epitopes specific to various diseases including cancer. The present invention relates to a glycopeptide in which one sugar chain comprising not more than 6 sugars is bonded to each of one or two or more of the threonines in a peptide comprising the amino acid sequence : TTPPTTATPIR, wherein the sugar chain is an O-linked sugar chain. The present invention further relates to a method for analyzing glycopeptides, comprising analyzing whether an epitope glycopeptide is present in a sample containing glycopeptide obtained by protease digestion of serum or plasma, wherein the glycopeptide is glycopeptide in which one sugar chain comprising not more than 6 sugars is bonded to each of one or two or more of the threonines in a peptide comprising the amino acid sequence : TTPPTTATPIR, wherein the sugar chain is an O-linked sugar chain.

Description

Glycopeptides, glycopeptide analysis method, and cancer analysis method

[0001] The present invention relates to glycopeptides, methods for analyzing glycopeptides, and methods for analyzing cancer.

[0002] Fibronectin is a glycoprotein essential for cancer proliferation, growth, invasion, and the formation of the cancer microenvironment, and is known as the most important extracellular matrix component. Fibronectin is also widely known as one of the most important ligand molecules for various integrin heterodimers that are highly expressed on the surface of many cancer cells (e.g., Non-Patent Document 1). For this reason, cancer therapeutic drugs based on the molecular mechanism that inhibits disease-specific binding between fibronectin (a glycoprotein molecule) and integrins highly expressed on the surface of cancer cells are being developed (e.g., Non-Patent Document 2).

[0003] It is known that fibronectin from cancer patients and fetuses contains three domains, IIICS (type III homology connective segment domain), ED-A (extra domain A), and ED-B (extra domain B), which are not found in adult fibronectin (expression levels are low in adults), and are generated by alternative splicing (e.g., Non-Patent Document 3). Active research and development efforts are being conducted on drug discovery, particularly drug delivery systems (DDS) for cancer therapy, targeting fibronectin isomers containing domains consisting of regions significantly expressed in cancer (e.g., Non-Patent Document 4). Furthermore, Non-Patent Documents 5 and 6 investigate cancer diagnostic agents using antibodies targeting ED-A and ED-B, respectively, as biomarkers (targets). Currently, clinical trials are ongoing on antibody complexes bound to cytokines such as IL-2, but there are no examples of these being put to practical use.

[0004] Non-Patent Document 7 shows that mouse monoclonal antibody FDC-6, which was established by immunization with isolated fibronectin produced by human hepatocellular carcinoma cells (HUH-7), reacts with fibronectin derived from cancer patients and fetuses but does not react with adult fibronectin, and this fibronectin is defined as oncofetal fibronectin (onfFN). FDC-6 binds to a glycopeptide epitope in the IIICS domain of onfFN, ２１１４ Val-Thr(GalNAcα)-His-Pro-Gly-Tyr ２１１９ and ２１１４ Val-Thr(Galβ1,3GalNAcα)-His-Pro-Gly-Tyr ２１１９ It is a mouse antibody that binds to FDC-6 (Non-Patent Documents 8 and 9), and it has been reported that onfFN containing these glycosylation regions is deeply involved in the process of epithelial-mesenchymal transition (EMT) induced by TGF-β in cancer progression and metastasis (Non-Patent Document 10). However, the significance and specific function of this glycosylation region in cancer biology, as well as the possibility of developing new cancer diagnostic and therapeutic methods based on FDC-6 or this epitope, remain unknown.

[0005] R. O. Hynes, Fibronectin, Springer, New York, 1990H. Kessler, et al., A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins. Sci. Rep.2017, 7, 39805J. E. Schwarzbauer, Alternative splicing of fibronectin: Three variants, three functions. BioEssays 1991, 13, 527-533D. P. Reinhardt, et al., Fibronectin-targeted drug delivery in cancer. Adv. Drug Deliv. Rev. 2016, 97, 101-110D. Neri, et al., The extra-domain A of fibronectin is a vascular marker of solid tumors and metastasis. Cancer Res. 2007, 67, 10948-10957L. J. Dubois, et al., Human fibronectin extra domain B as a biomarker for targeted therapy in cancer. Mol. Oncol. 2020, 14, 1555-1568Hakomori, S.-i. et al., The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6: Its presence in fibronectins from fetal and tumor tissues and its absence in those from normal adult tissues and plasma. Pro. Natl. Acad. Sci. USA 1985, 82, 6517-6521Hakomori, S.-i. et al.J. Biol. Chem1988, 263, 3314-3322Hakomori, S.-i. et al., An alpha N-acetylgalactosaminylation at the threonine residue of a defined peptide sequence creates the oncofetal peptide epitope in human fibronectin. ibid, 1989, 264, 10472-10476Hakomori, S.-i. et al., Involvement of O-glycosylation oncofetal fibronectin in epithelial-mesenchymal-transition process. Pro. Natl. Acad. Sci. USA 2011, 108, 17690-17695.

[0006] As mentioned above, various research efforts have been actively conducted for over 40 years targeting fibronectin, a multifunctional, high-molecular-weight glycoprotein that is the most important component of the extracellular matrix, as a target for cancer diagnostic and therapeutic agents. However, no pharmaceuticals or diagnostic technologies targeting this molecule have yet been put into practical use.

[0007] In order to solve these problems of the conventional technology, the present inventors have conducted extensive research into epitopes of fibronectin that are specific to various diseases, including cancer (cancer-specific structural changes caused by post-translational glycosylation: dynamic epitopes).

[0008] An object of the present invention is to provide materials for systematically searching for and identifying epitopes specific to various diseases, including cancer. Another object of the present invention is to identify dynamic epitopes specific to various diseases, including cancer, and to establish a method for analyzing diseases using these dynamic epitopes.

[0009] Specific embodiments of the present invention are described below. [1] A glycopeptide comprising the amino acid sequence TTPPTTATPIR, wherein one or more threonines are each bound to one sugar chain consisting of six or fewer sugars, and the sugar chain is an O-linked sugar chain. [2] The glycopeptide according to [1], wherein the sugar chain is selected from the group consisting of the following (1) to (4): (1) GalNAcα1 (2) Galβ1,3GalNAcα1 (3) Neu5Acα2,6GalNAcα1 (4) Neu5Acα2,3Galβ1,3GalNAcα1 [3] The glycopeptide according to [1] or [2], wherein the sugar chain is bound to one, two, or three positions selected from the group consisting of the second threonine, the fifth threonine, and the sixth threonine from the N-terminus. [4] The glycopeptide according to any one of [1] to [3], wherein the sugar chains are attached to the second threonine, the fifth threonine, the sixth threonine, the second and fifth threonines, the second and sixth threonines, the fifth and sixth threonines, or the second, fifth, and sixth threonines from the N-terminus. [5] The glycopeptide according to any one of [1] to [4], wherein the sugar chains attached to two or more threonines are of the same or different species. [6] The glycopeptide according to any one of [2] to [5], wherein the glycopeptide having the sugar chain of (2) is a glycopeptide represented by the following structural formula: [7] The glycopeptide according to any one of [2] to [6], wherein the glycopeptide having the sugar chain of (4) is a glycopeptide represented by the following structural formula: [8] The glycopeptide according to any one of [2] to [7], wherein the glycopeptide having the sugar chain of (1) is a glycopeptide represented by the following structural formula: [9] The glycopeptide according to any one of [2] to [8], wherein the glycopeptide having the sugar chain of (3) is a glycopeptide represented by the following structural formula:

[10] The glycopeptide according to any one of [1] to [9], which is used as a tumor marker.

[11] The glycopeptide according to any one of [1] to [9], which is used as an immunogen for producing an antibody.

[12] A method for analyzing a glycopeptide, comprising analyzing whether a glycopeptide is contained in a sample containing glycopeptides obtained by protease digestion of serum or plasma, wherein the glycopeptide is a peptide consisting of the amino acid sequence: TTPPTTATPIR, in which one or more threonines are each bound to one sugar chain consisting of six or fewer sugars, and the sugar chain is an O-linked sugar chain.

[13] The method for analyzing a glycopeptide according to

[12] , which comprises analyzing whether the sample contains glycopeptide 15 and / or glycopeptide 20 represented by the following structural formula:

[14] The method for analyzing glycopeptides according to

[12] or

[13] , further comprising analyzing whether the sample contains one or more selected from the group consisting of glycopeptide 5, glycopeptide 9, glycopeptide 10, and glycopeptide 16, which are represented by the following structural formulas:

[15] A method for analyzing glycopeptides, comprising analyzing whether a sample containing glycopeptides obtained by protease digestion of serum or plasma contains one or more types selected from the group consisting of glycopeptide 5, glycopeptide 9, glycopeptide 10, and glycopeptide 16, each represented by the following structural formulas:

[16] The method for analyzing glycopeptides according to any one of

[12] to

[15] , wherein the analysis comprises performing mass spectrometry of glycopeptides.

[17] The method for analyzing glycopeptides according to any one of

[12] to

[16] , wherein the analysis comprises performing mass spectrometry of glycopeptides by tandem mass spectrometry.

[18] The method for analyzing glycopeptides according to any one of

[12] to

[17] , wherein the method is used for detecting digestive system cancer and / or detecting the stage of digestive system cancer.

[19] The method for analyzing glycopeptides according to

[18] , wherein the digestive system cancer is liver cancer or pancreatic cancer.

[20] The method for analyzing glycopeptides according to any one of

[12] to

[19] , wherein the sample is obtained by removing a portion of serum proteins or plasma proteins.

[21] A method for analyzing cancer, comprising the method for analyzing glycopeptides according to any one of

[12] to

[20] .

[0010] According to the present invention, novel glycopeptides can be provided for systematically searching for and identifying epitopes specific to various diseases, including cancer. Furthermore, by using these glycopeptides, disease-specific dynamic epitopes can be identified, and methods for analyzing diseases using these dynamic epitopes can be established. Identifying disease-specific dynamic epitopes is also expected to lead to the development of new therapeutic antibody drugs.

[0011] Figure 1 shows the results of mass analysis by MALDI-TOF / MS for peptide 1. Figure 2 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 2. Figure 3 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 3. Figure 4 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 4. Figure 5 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 5. Figure 6 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 6. Figure 7 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 7. Figure 8 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 8. Figure 9 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 9. Figure 10 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 11. Figure 12 shows the results of mass analysis by MALDI-TOF / MS for glycopeptide 12. Figure 1 shows the results of mass analysis of glycopeptide 13 by MALDI-TOF / MS. Figure 2 shows the results of mass analysis of glycopeptide 14 by MALDI-TOF / MS. Figure 3 shows the results of mass analysis of glycopeptide 15 by MALDI-TOF / MS. Figure 4 shows the results of mass analysis of glycopeptide 16 by MALDI-TOF / MS. Figure 5 shows the results of mass analysis of glycopeptide 17 by MALDI-TOF / MS. Figure 6 shows the results of mass analysis of glycopeptide 18 by MALDI-TOF / MS. Figure 7 shows the results of mass analysis of glycopeptide 19 by MALDI-TOF / MS. Figure 8 shows the results of mass analysis of glycopeptide 20 by MALDI-TOF / MS. Figure 9 shows the results of mass analysis of glycopeptide 21 by MALDI-TOF / MS. (a) Spectrum of glycopeptide 16 in ER mode. (b) Observation of fragment ions generated by CID for optimization of Q3. Schematic diagram explaining the quadrupole mass spectrometry method. Calibration curve for peptide 1. This is a calibration curve for glycopeptide 2. This is a calibration curve for glycopeptide 3. This is a calibration curve for glycopeptide 4. This is a calibration curve for glycopeptide 5. This is a calibration curve for glycopeptide 6. This is a calibration curve for glycopeptide 7. This is a calibration curve for glycopeptide 8. This is a calibration curve for glycopeptide 9. This is a calibration curve for glycopeptide 10. This is a calibration curve for glycopeptide 11. This is a calibration curve for glycopeptide 12. This is a calibration curve for glycopeptide 13. This is a calibration curve for glycopeptide 14.1 shows a calibration curve for glycopeptide 15. 2 shows a calibration curve for glycopeptide 16. 3 shows a calibration curve for glycopeptide 17. 4 shows a calibration curve for glycopeptide 18. 5 shows a calibration curve for glycopeptide 19. 6 shows a calibration curve for glycopeptide 20. 7 shows a calibration curve for glycopeptide 21. 8 shows a box plot of the blood concentrations (nM) of peptide 1 in healthy subjects and liver cancer specimens. 9 shows a box plot of the blood concentrations (nM) of glycopeptide 2 in healthy subjects and liver cancer specimens. 10 shows a box plot of the blood concentrations (nM) of glycopeptide 3 in healthy subjects and liver cancer specimens. 11 shows a box plot of the blood concentrations (nM) of glycopeptide 4 in healthy subjects and liver cancer specimens. 12 shows a box plot of the blood concentrations (nM) of glycopeptide 5 in healthy subjects and liver cancer specimens. 1 shows a box plot of the blood concentration (nM) of glycopeptide 6 in healthy subjects and each liver cancer specimen. 2 shows a box plot of the blood concentration (nM) of glycopeptide 7 in healthy subjects and each liver cancer specimen. 3 shows a box plot of the blood concentration (nM) of glycopeptide 8 in healthy subjects and each liver cancer specimen. 4 shows a box plot of the blood concentration (nM) of glycopeptide 9 in healthy subjects and each liver cancer specimen. 5 shows a box plot of the blood concentration (nM) of glycopeptide 10 in healthy subjects and each liver cancer specimen. 6 shows a box plot of the blood concentration (nM) of glycopeptide 11 in healthy subjects and each liver cancer specimen. 7 shows a box plot of the blood concentration (nM) of glycopeptide 12 in healthy subjects and each liver cancer specimen. 8 shows a box plot of the blood concentration (nM) of glycopeptide 13 in healthy subjects and each liver cancer specimen. 1 shows the results of a box plot showing the blood concentration (nM) of glycopeptide 14 in healthy subjects and each liver cancer specimen. 2 shows the results of a box plot showing the blood concentration (nM) of glycopeptide 15 in healthy subjects and each liver cancer specimen. 3 shows the results of a box plot showing the blood concentration (nM) of glycopeptide 16 in healthy subjects and each liver cancer specimen. 4 shows the results of a box plot showing the blood concentration (nM) of glycopeptide 17 in healthy subjects and each liver cancer specimen.Figure 1 shows a box plot of the blood concentrations (nM) of glycopeptide 18 in healthy subjects and liver cancer specimens. Figure 2 shows a box plot of the blood concentrations (nM) of glycopeptide 19 in healthy subjects and liver cancer specimens. Figure 3 shows a box plot of the blood concentrations (nM) of glycopeptide 20 in healthy subjects and liver cancer specimens. Figure 4 shows a box plot of the blood concentrations (nM) of glycopeptide 21 in healthy subjects and liver cancer specimens. Figure 5 shows the results of a statistical significance test for glycopeptides 5, 9, 15, 16, and 20, which show a significant correlation with the progression (stage) of liver cancer. Figure 6 illustrates structural changes caused by post-translational modifications in the peptide region. Figure 7 shows a box plot of the blood concentrations (nM) of glycopeptide 5 in healthy subjects and four liver cancer and pancreatic cancer specimens at each stage. Figure 8 shows a box plot of the blood concentrations (nM) of glycopeptide 7 in healthy subjects and four liver cancer and pancreatic cancer specimens at each stage. 1 shows a box plot of the blood concentrations (nM) of glycopeptide 8 in healthy subjects and four specimens at each stage of liver cancer and pancreatic cancer. 2 shows a box plot of the blood concentrations (nM) of glycopeptide 9 in healthy subjects and four specimens at each stage of liver cancer and pancreatic cancer. 3 shows a box plot of the blood concentrations (nM) of glycopeptide 10 in healthy subjects and four specimens at each stage of liver cancer and pancreatic cancer. 4 shows a box plot of the blood concentrations (nM) of glycopeptide 11 in healthy subjects and four specimens at each stage of liver cancer and pancreatic cancer. 5 shows a box plot of the blood concentrations (nM) of glycopeptide 15 in healthy subjects and four specimens at each stage of liver cancer and pancreatic cancer. 6 shows a box plot of the blood concentrations (nM) of glycopeptide 16 in healthy subjects and four specimens at each stage of liver cancer and pancreatic cancer. The blood concentration (nM) of glycopeptide 20 in healthy individuals and four specimens of liver cancer and pancreatic cancer at each stage is displayed as a box plot.

[0012] The present invention will be described in detail below. The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0013] (Glycopeptide) This embodiment relates to a glycopeptide comprising the amino acid sequence TTPPPTTATPIR (SEQ ID NO: 1: Thr-Thr-Pro-Pro-Thr-Thr-Ala-Thr-Pro-Ile-Arg), in which one or more threonines are each bound to one sugar chain consisting of six or fewer sugars, and the sugar chain is an O-linked sugar chain. An O-linked sugar chain is a sugar chain that is bound to the hydroxyl group of a threonine via an O-glycosidic bond. In the amino acid sequence, T represents threonine (Thr), P represents proline (Pro), A represents alanine (Ala), I represents isoleucine (Ile), and R represents arginine (Arg). In addition, in the amino acid sequences shown herein, the left end is the N-terminus and the right end is the C-terminus.

[0014] The glycopeptide in this embodiment also includes glycopeptide derivatives, such as those in which cysteine ​​has been introduced at the C-terminus of the above-mentioned glycopeptide or those in which levulinic acid has been introduced at the N-terminus.

[0015] The glycopeptide in this embodiment is, for example, a synthetic glycopeptide that can be produced by in vitro synthesis, and is a novel substance (novel compound).

[0016] As used herein, the term "glycan" refers to a compound formed by a chain of one or more sugar units (monosaccharides and / or derivatives thereof). When two or more sugar units are linked, the sugar units are bonded to each other by dehydration condensation via a glycosidic bond. The sugar constituting the O-linked sugar chain may be a monosaccharide, and the O-linked sugar chain may be composed of one, two, three, four, five, or six sugars, preferably one, two, or three sugars, and more preferably one or two sugars.

[0017] The sugar chain preferably has N-acetylgalactosamine (GalNAc) as a constituent sugar. When the sugar chain is composed of one sugar, the sugar chain is preferably composed of N-acetylgalactosamine (GalNAc), and is particularly preferably a sugar chain represented by the following (1): (1) GalNAcα1

[0018] When the sugar chain is composed of two or more sugars, it preferably has N-acetylgalactosamine (GalNAc), galactose (Gal), or N-acetylneuraminic acid (Neu5Ac) as a constituent sugar, and the sugar chain is preferably selected from the group consisting of the following (2) to (4). In the sugar chains (2) to (4) below, GalNAcα1 is linked to threonine: (2) Galβ1,3GalNAcα1 (3) Neu5Acα2,6GalNAcα1 (4) Neu5Acα2,3Galβ1,3GalNAcα1

[0019] The sugar chains represented by (1) to (4) above are four O-glycan structures known to be prominently observed in cancer cells. Among these sugar chains, GalNAcα1 is sometimes called the Tn antigen, Galβ1,3GalNAcα1 the T antigen, Neu5Acα2,6GalNAcα1 the sialylated T antigen (STn antigen), and Neu5Acα2,3Galβ1,3GalNAcα1 the sialylated T antigen (ST antigen).

[0020] When the sugar chain is composed of three or more sugars, it preferably has one or more sugars selected from the group consisting of N-acetylgalactosamine (GalNAc), galactose (Gal), N-acetylneuraminic acid (Neu5Ac), fucose (Fuc), and N-acetylglucosamine (GlcNAc) as constituent sugars. Examples of the sugar chain include disialyl T antigen (Neu5Acα2,3Galβ1,3[Neu5Acα2,6]GalNAcα1) and sialyl Lewis x / a sugar chain (Neu5Acα2,3Galβ1,4[Fucα1,3]GlcNAc or Neu5Acα2,3Galβ1,3[Fucα1,4]GlcNAc).

[0021] In the glycopeptide of this embodiment, a sugar chain is bound to each of one or more threonines in the peptide having the predetermined sequence. In this case, a sugar chain is bound to each of the following threonines. In this specification, for example, a sugar chain bound to the threonine located at the first position from the N-terminus is represented as T1, a sugar chain bound to the threonine located at the second position from the N-terminus is represented as T2, a sugar chain bound to the threonine located at the fifth position from the N-terminus is represented as T5, a sugar chain bound to the threonine located at the sixth position from the N-terminus is represented as T6, and a sugar chain bound to the threonine located at the eighth position from the N-terminus is represented as T8. (1) threonine located at the first position from the N-terminus (T1) (2) threonine located at the second position from the N-terminus (T2) (3) threonine located at the fifth position from the N-terminus (T5) (4) threonine located at the sixth position from the N-terminus (T6) (5) threonine located at the eighth position from the N-terminus (T8)

[0022] When sugar chains are bound to two threonines in a peptide having the above-mentioned predetermined sequence, the combinations of threonines to which sugar chains are bound are, for example, as follows: In this specification, for example, a combination in which sugar chains are bound to both the threonine located at the first position from the N-terminus and the threonine located at the second position from the N-terminus is represented as T1 / T2. (6) T1 / T2, T1 / T5, T1 / T6, T1 / T8 (7) T2 / T5, T2 / T6, T2 / T8 (8) T5 / T6, T5 / T8 (9) T6 / T8

[0023] When sugar chains are bound to three threonines in a peptide having the above-mentioned predetermined sequence, the combinations of sugar chain-bound threonines are, for example, as follows: In this specification, for example, the combinations in which sugar chains are bound to all of the threonines located at the first position from the N-terminus, the second position from the N-terminus, and the fifth position from the N-terminus are represented as T1 / T2 / T5: (10) T1 / T2 / T5, T1 / T2 / T6, T1 / T2 / T8, T1 / T5 / T6, T1 / T5 / T8, T1 / T6 / T8, (11) T2 / T5 / T6, T2 / T5 / T8, T2 / T6 / T8, T5 / T6 / T8

[0024] When sugar chains are bound to four threonines in the peptide having the above-mentioned predetermined sequence, the combinations of threonines to which sugar chains are bound are, for example, as follows: (12) T1 / T2 / T5 / T6, T1 / T2 / T5 / T8, T2 / T5 / T6 / T8

[0025] When sugar chains are bound to five threonines in the peptide having the above-mentioned predetermined sequence, the combination of threonines to which sugar chains are bound is, for example, as follows: (13) T1 / T2 / T5 / T6 / T8

[0026] Among these, it is preferable that the sugar chain binding positions are one, two, or three positions selected from the group consisting of the second threonine (T2), the fifth threonine (T5), and the sixth threonine (T6) from the N-terminus. More specifically, the sugar chain binding sites are preferably the second threonine (T2), the fifth threonine (T5), the sixth threonine (T6), the second and fifth threonines (T2 / T5), the second and sixth threonines (T2 / T6), the fifth and sixth threonines (T5 / T6), or the second, fifth, and sixth threonines (T2 / T5 / T6), and more preferably the second threonine (T2), the fifth threonine (T5), the sixth threonine (T6), the fifth and sixth threonines (T5 / T6), or the second, fifth, and sixth threonines (T2 / T5 / T6).

[0027] When two or more sugar chains are bound to two or more threonines, the sugar chains bound to each threonine may be of the same type or different types, but are preferably of the same type. In particular, it is preferable that the same sugar chains are bound to the threonines at the fifth and sixth positions (T5 / T6).

[0028] The glycopeptide of this embodiment is preferably represented by, for example, the following structural formula: For example, the glycopeptide having the above-mentioned (1) sugar chain (GalNAcα1) is preferably represented by the following structural formula:

[0029] The glycopeptide having the sugar chain (Galβ1,3GalNAcα1) of (2) above is preferably a glycopeptide represented by the following structural formula:

[0030] The glycopeptide having the sugar chain (Neu5Acα2,6GalNAcα1) of (3) above is preferably a glycopeptide represented by the following structural formula:

[0031] The glycopeptide having the sugar chain (Neu5Acα2,3Galβ1,3GalNAcα1) of (4) above is preferably a glycopeptide represented by the following structural formula:

[0032] In this specification, the above-mentioned glycopeptides 2 to 21 are sometimes referred to as Compounds 2 to 21 or Compounds 2 to 21. Furthermore, a peptide having no sugar chain is referred to as Peptide 1 (Compound 1 or Compound 1).

[0033] In the glycopeptide of this embodiment, a sugar chain may be bound to one or more threonines in the peptide, and a sugar chain may be bound to an amino acid other than threonine. On the other hand, in the glycopeptide of this embodiment, amino acids other than threonine may be blocked so that a sugar chain is not introduced thereto.

[0034] The amino acid sequence of the above-mentioned synthetic glycopeptide contains consecutive threonines present in the IIICS domain (type III homology connective segment domain) of onfFN (oncofetal fibronectin). ２１５１ Thr-Thr-Pro-Pro-Thr-Thr-Ala-Thr-Pro-Ile-Arg ２１６１ The synthetic glycopeptide is identical to the amino acid sequence of the peptide region in the IIICS domain of onfFN. ２１５１ Thr-Thr-Pro-Pro-Thr-Thr-Ala-Thr-Pro-Ile-Arg ２１６１By attaching the above-mentioned O-linked sugar chains that may be possessed by the sugar chains, it becomes possible to track the structural changes of the sugar chains in vivo.

[0035] In this embodiment, by using such synthetic glycopeptides, disease-specific "dynamic epitopes" can be systematically searched for and identified. For example, when the structure of a glycan in a glycopeptide changes due to the canceration of cells, the glycopeptide having the glycan before and after the structural change can be identified and quantified to detect and diagnose cancer. Furthermore, when the structure of a glycan in a glycopeptide changes depending on the progression of cancer, the stage of cancer can be determined by identifying and quantitating the glycopeptide having the glycan before and after the structural change. Therefore, the glycopeptide of this embodiment can be used as a tumor marker (e.g., a biomarker for cancer diagnosis).

[0036] The glycopeptide of this embodiment is useful not only for cancer diagnosis but also for the detection of the nucleotide sequence in the IIICS domain of onfFN. ２１５１ Thr-Thr-Pro-Pro-Thr-Thr-Ala-Thr-Pro-Ile-Arg ２１６１ This method can also be used to detect and diagnose diseases in which the structure of glycans changes due to post-translational modifications in the peptide region.

[0037] The glycopeptide of the present embodiment can also be used as an immunogen for antibody production or a vaccine. The glycopeptide of the present embodiment can also be used to develop new therapeutic antibody drugs. In this embodiment, the immunogen for antibody production can be synthesized, significantly reducing the cost and time required for immunogen production and enabling the efficient development of antibody drugs against antigen epitopes with defined structures.

[0038] Thus, we have successfully synthesized novel glycopeptides for systematically exploring and identifying epitopes specific to various diseases, including cancer. Using these synthetic glycopeptides, we have identified disease-specific dynamic epitopes and established a method for analyzing these diseases, as described below. In particular, these "dynamic epitopes," which emerge as a result of specific post-translational glycosylation observed at the spatiotemporal level during cancer progression (the difference in the site and combination of glycosylation and the glycan structure are defined by the stage of disease progression and / or type of cancer), serve as novel diagnostic biomarkers and potential targets for novel therapeutic antibodies. Identifying new cancer-specific epitopes present in molecules produced by cancer cells will accelerate the development of early cancer detection and diagnosis methods using various advanced instrumental analyses, as well as the development of novel therapeutic antibody drugs.

[0039] (Glycopeptide-containing substance) This embodiment may relate to a solution containing the above-described glycopeptide. For example, this embodiment may be a solution containing the above-described glycopeptide at a predetermined concentration, which is a glycopeptide solution for preparing a calibration curve used for cancer analysis as described below. The glycopeptide solution for preparing a calibration curve may contain optional components such as antioxidants, preservatives, and coloring agents, as long as they do not interfere with the preparation of the calibration curve.

[0040] Furthermore, this embodiment may also relate to a solid (e.g., granules or powder) containing the above-described glycopeptide. The solid may be obtained, for example, by freeze-drying a solution containing the glycopeptide.

[0041] (Method for Producing Glycopeptides) This embodiment relates to a method for producing the above-described glycopeptide. The method for producing a glycopeptide includes synthesizing a glycopeptide by solid-phase peptide synthesis. In solid-phase peptide synthesis, for example, when synthesizing TTPPPTTATPIR (Thr-Thr-Pro-Pro-Thr-Thr-Thr-Ala-Thr-Pro-Ile-Arg) as peptide 1, solid-phase synthesis is performed using Fmoc amino acid derivatives as the amino acids constituting peptide 1, followed by deprotection of the Fmoc group. The target peptide can be synthesized by repeating the elongation of the Fmoc amino acid and deprotection.

[0042] When synthesizing glycopeptides, solid-phase synthesis is performed using Fmoc-threonine having a predetermined sugar chain. As described above, the target glycopeptide can be synthesized by introducing Fmoc-threonine having a predetermined sugar chain into a predetermined site during solid-phase synthesis using an Fmoc amino acid derivative.

[0043] Examples of Fmoc-threonine having a predetermined sugar chain include the following:

[0044] Glycopeptides containing the ST antigen (Neu5Acα2,3Galβ1,3GalNAcα1) can be derived from glycopeptides containing the T antigen (Galβ1,3GalNAcα1) (T antigen) by enzymatic transglycosylation. In the transglycosylation reaction, CMP-sialic acid is used as a glycosyl donor, and sialic acid is attached to Gal at the non-reducing end of the glycan, thereby elongating the glycan. Examples of sialyltransferases include α2,8-sialyltransferase and α-2,3-sialyltransferase, with α-2,3-sialyltransferase being preferred. Commercially available sialyltransferases can also be used, such as Pasteurella multocida-derived recombinant α-2,3-sialyltransferase (Sigma-Aldrich). The reaction temperature in the transglycosylation reaction is preferably, for example, 15 to 60° C., and the reaction time is preferably about 10 minutes to 100 hours.

[0045] For glycopeptides containing the ST antigen (Neu5Acα2,3Galβ1,3GalNAcα1), it is preferable to deprotect the acetyl groups in the glycan region after solid-phase synthesis. For example, the acetyl groups may be deprotected by dissolving the lyophilized glycopeptide in methanol, adding 1N aqueous sodium hydroxide, and shaking at room temperature.

[0046] Glycopeptides containing the ST antigen (Neu5Acα2,3Galβ1,3GalNAcα1) can also be obtained by solid-phase synthesis using Fmoc-threonine. For example, Fmoc-Thr(Neu5Aca2,3Galβ1,3GalNAcα1)-OH can be used as Fmoc-threonine. In this case, the glycopeptide can be synthesized by following the procedure described in S. Dziadek, et al., Chem. Eur. J. 2004, 10, 4150-4162.

[0047] The glycopeptide synthesized by the solid phase synthesis method described above may be purified as needed. For example, purified glycopeptides can be obtained by reversed-phase high performance liquid chromatography.

[0048] (Method for Analyzing Glycopeptides) This embodiment relates to an analytical method for determining whether or not the above-described glycopeptides are contained in serum or plasma. Specifically, the method for analyzing glycopeptides of this embodiment involves analyzing whether or not a glycopeptide is contained in a sample containing glycopeptides obtained by protease digestion of serum or plasma. The glycopeptide is the glycopeptide described above, and is a peptide consisting of the amino acid sequence TTPPTTATPIR, in which one or more threonines are bound to each other with one sugar chain consisting of six or fewer sugars, and the sugar chain is an O-linked sugar chain. Furthermore, examples of proteases used in the method for analyzing glycopeptides of this embodiment include chymotrypsin and trypsin, with trypsin being preferred.

[0049] By digesting serum or plasma with a protease, a peptide consisting of the amino acid sequence TTPPTTATPIR from the IIICS domain of onfFN contained in the serum or plasma can be obtained. In the peptide consisting of the amino acid sequence, one or more threonines are bound to each of which is a single sugar chain (O-linked sugar chain) consisting of six or fewer sugars. This embodiment relates to a method for analyzing the type and amount of glycopeptides contained in a protease digest of serum or plasma. In particular, this embodiment preferably relates to a method for analyzing the type and amount of glycopeptides contained in a tryptic digest of serum.

[0050] The analytical method of this embodiment preferably includes analyzing whether a sample containing glycopeptides obtained by protease digestion of serum or plasma (a protease digest of serum or plasma) contains one or more glycopeptides selected from the group consisting of glycopeptide 5, glycopeptide 8, glycopeptide 9, glycopeptide 10, glycopeptide 15, glycopeptide 16, and glycopeptide 20. The analytical method of this embodiment particularly preferably includes performing mass spectrometry on each of glycopeptides 8, glycopeptide 9, glycopeptide 10, glycopeptide 15, and glycopeptide 20 in a sample containing glycopeptides obtained by protease digestion of serum or plasma (a protease digest of serum or plasma). The structures of glycopeptides 8, glycopeptide 9, glycopeptide 10, glycopeptide 15, and glycopeptide 20 are as follows:

[0051]

[0052]

[0053]

[0054]

[0055] Furthermore, in the case of a liver cancer patient, the analytical method of this embodiment preferably further comprises analyzing whether a glycopeptide-containing sample obtained by protease digestion of serum or plasma (a protease digest of serum or plasma) contains one or more types selected from the group consisting of glycopeptide 5, glycopeptide 9, glycopeptide 10, glycopeptide 15, glycopeptide 16, and glycopeptide 20, and more preferably further comprising analyzing whether one or two types selected from glycopeptide 15 and glycopeptide 20 are contained. The analytical method of this embodiment preferably comprises performing mass spectrometry on glycopeptides 5, 9, glycopeptide 10, glycopeptide 15, glycopeptide 16, and glycopeptide 20 in a glycopeptide-containing sample obtained by protease digestion of serum or plasma (a protease digest of serum or plasma), and particularly preferably comprises performing mass spectrometry on glycopeptides 15 and glycopeptide 20, respectively.

[0056] Furthermore, in the case of a pancreatic cancer patient, the analytical method of this embodiment preferably includes analyzing whether a glycopeptide-containing sample obtained by trypsin digestion of serum or plasma (a tryptic digest of serum or plasma) contains one or more types selected from the group consisting of glycopeptide 5, glycopeptide 8, glycopeptide 9, glycopeptide 10, glycopeptide 11, and glycopeptide 16, and more preferably includes analyzing whether one or more types selected from the group consisting of glycopeptide 8, glycopeptide 9, and glycopeptide 10 are contained. The analytical method of this embodiment preferably includes performing mass spectrometry on glycopeptides 5, 8, 9, 10, 11, and 16 in a glycopeptide-containing sample obtained by protease digestion of serum or plasma (a protease digest of serum or plasma), and particularly preferably includes performing mass spectrometry on glycopeptides 8, 9, and 10, respectively.

[0057] In this embodiment, it is preferable to remove a portion of serum proteins or plasma proteins from serum or plasma before subjecting the serum or plasma to protease digestion. Specifically, it is preferable to remove the top 14 major serum proteins or plasma proteins with the highest blood concentrations. Removal of serum proteins or plasma proteins can be performed by known methods, for example, using a multiple-affinity removal system ("MARS", Agilent Technologies spin columns). Removing a portion of serum proteins or plasma proteins allows for efficient protease digestion and improves the accuracy of mass spectrometry of the target glycopeptide.

[0058] When performing mass spectrometry of glycopeptides, it is preferable to use tandem mass spectrometry (selective reaction monitoring method: SRM) as the analytical method. By using tandem mass spectrometry (selective reaction monitoring method: SRM), quantitative analysis of glycopeptides can be determined quickly and accurately. Here, the SRM (selective reaction monitoring) parameters of glycopeptides are determined by LC-MS / MS (4000 QTRAP triple quadrupole mass spectrometer (AB Sciex Pte. Ltd.)) and Exigent NanoLC Expert. TMUsing a system consisting of a 400 (AB Sciex Pte. Ltd.), the SRM parameters for each glycopeptide can be optimized by the following steps: (a) parent ion selection in the first quadrupole (Q1), (b) optimization of collision-induced dissociation (CID) in the second quadrupole (Q2), and (c) evaluation and selection of fragment ions specific to each glycopeptide in the third quadrupole (Q3) (e.g., S.-I. Nishimura, et al., Selective reaction monitoring approach using structure-defined synthetic glycopeptides for validating glycopeptide biomarkers pre-determined by bottom-up glycoproteomics. RSC Adv 2022, 12, 21385-21393).

[0059] In a quadrupole mass analyzer, four parallel rod-shaped electrodes function as mass filters, selectively passing only ions within a specific mass range by adjusting the radio frequency (RF) and direct current (DC) voltages. Therefore, once the target ion Q1 and fragment ion Q3 are determined, the voltages must be set to allow only these ions to pass through each filter. Four parameters must be optimized: Declustering Potential (DP), Entrance Potential (EP), Collision Energy (CE), and Collision Cell Exit Potential (CXP). DP is the voltage used to guide all ions generated by the ion generator to Q0; EP is the voltage used to guide ions from Q0 to Q1; CE is the voltage used to fragment target ions; and CXP is the voltage used to guide the generated fragment ions into Q3. These voltages are adjusted to maximize detection sensitivity. Of the four parameters, CE has the greatest impact on the sensitivity of parent ions and fragment ions.

[0060] As mentioned above, LC-coupled triple quadrupole tandem mass spectrometers are used in quantitative proteomics research using the multiple reaction monitoring (MRM) method, but there are no known cases of successful quantitative analysis of glycopeptides using the MRM / SRM method. The glycopeptide analysis method of this embodiment is the world's first rapid and precise diagnostic technology using the MRM / SRM method, which can determine the stage of cancer by quantitative analysis of specific glycopeptide fragments in protease digests of blood proteins.

[0061] In the analytical method of this embodiment, first, the above-described parameter set is determined for the synthetic glycopeptides 2 to 21, followed by mass spectrometry to generate a calibration curve for each glycopeptide. Then, mass spectrometry is performed on glycopeptides 2 to 21 contained in samples obtained by protease digestion of serum or plasma from liver cancer patients and healthy individuals, and the content (concentration) of each glycopeptide contained in the serum or plasma is calculated from the calibration curve obtained above. Through this analysis, the inventors have found that the concentrations of glycopeptides 15 and 20 significantly increase in liver cancer patients at stage 2 or later. On the other hand, they have found that the concentrations of glycopeptides 5, glycopeptide 9, glycopeptide 10, and glycopeptide 16 are high in healthy individuals or liver cancer patients at stage 1, and significantly decrease as the stage progresses. More specifically, glycopeptide 9 was found to be highly concentrated in healthy individuals, but its concentration significantly decreases as cancer progresses beyond stage 1. Furthermore, it was found that concentrations of glycopeptide 5, glycopeptide 10, and glycopeptide 16 are high in stage 1 liver cancer patients, and that these concentrations decrease significantly as the cancer progresses to stage 2 or beyond. Based on this, a glycopeptide-containing sample (a protease digest of serum or plasma) obtained by protease digestion of serum or plasma can be analyzed for the presence of glycopeptide 15 and / or glycopeptide 20, and mass spectrometry of glycopeptide 15 and / or glycopeptide 20 can be performed. If high concentrations of glycopeptides 15 and 20 are detected, it can be determined that liver cancer is progressing. On the other hand, if low concentrations of glycopeptide 5, glycopeptide 9, glycopeptide 10, and / or glycopeptide 16 are detected in a glycopeptide-containing sample (a protease digest of serum or plasma) obtained by protease digestion of serum or plasma, or if these glycopeptides are not detected, it can also be determined that liver cancer is progressing or that the patient may be suffering from a disease including liver cancer.Furthermore, when the concentrations of glycopeptides 15 and 20 are significantly increased and the concentrations of glycopeptides 5, glycopeptide 9, glycopeptide 10, and glycopeptide 16 are significantly decreased, it can be more accurately determined that liver cancer is progressing, and it can also be determined that the liver cancer is at least at stage 1 or 2 or later. Thus, the analysis method of this embodiment can identify glycopeptides whose content changes depending on the progression of a specific disease, and after identifying glycopeptides involved in the disease, the presence or absence and progression of the disease can be determined by detecting changes in their content.

[0062] Furthermore, when mass spectrometry was performed on glycopeptides 2 to 21 contained in samples obtained by protease digestion of serum or plasma from pancreatic cancer patients and healthy individuals, and the content (concentration) of each glycopeptide contained in the serum or plasma was calculated from the calibration curve obtained above, it was found that the concentrations of glycopeptides 8, 9, and 10 were significantly increased in the serum of stage 4 pancreatic cancer patients. On the other hand, it was found that while the concentrations of glycopeptides 5, 11, and 16 were high in healthy individuals, their concentrations were significantly decreased in stage 4 pancreatic cancer patients.

[0063] In particular, the analytical method of this embodiment is useful for a method for analyzing cancer, and this embodiment also relates to a method for analyzing cancer including the above-mentioned method for analyzing glycopeptides. The method for analyzing cancer in this specification includes both detection of the presence or absence of cancer and determination of the stage of cancer.

[0064] The cancer analysis method of this embodiment preferably includes the following series of steps: (1) Mass spectrometry is performed on synthetic glycopeptides, and a calibration curve for each glycopeptide is created. (2) Serum or plasma is obtained from a subject, and a portion of the serum proteins or plasma proteins is removed from the serum or plasma. (3) The serum or plasma from which the serum proteins or plasma proteins have been removed is subjected to protease digestion to prepare a measurement sample. (4) Mass spectrometry is performed on the glycopeptides contained in the sample, and the concentration of the glycopeptide contained in the serum or plasma is calculated from the calibration curve. (5) The onset and progression of cancer are determined from the concentration of glycopeptides, the content of which changes depending on the onset and progression of cancer. This embodiment may also relate to a system for determining the onset and progression of cancer that performs the above steps (1) to (5).

[0065] The cancer to be analyzed is not particularly limited, and examples thereof include liver cancer, stomach cancer, colon cancer, gallbladder cancer, esophageal cancer, pharyngeal cancer, pancreatic cancer, breast cancer, lung cancer, melanoma, uterine cancer, osteosarcoma, skin cancer, ovarian cancer, and kidney cancer. Of these, the cancer to be analyzed is preferably a cancer of the digestive system, and the analysis method of this embodiment is suitable for detecting digestive system cancer and / or detecting the stage of digestive system cancer. In particular, the cancer to be analyzed is preferably liver cancer and / or pancreatic cancer, and liver cancer is particularly preferred.

[0066] The present inventors have identified a threonine residue sequence containing consecutive threonines present in the type IIIC domain (type III homology connective segment domain) of onfFN (oncofetal fibronectin). ２１５１ Thr-Thr-Pro-Pro-Thr-Thr-Ala-Thr-Pro-Ile-Arg ２１６１We have found that post-translational modification alters the glycan structure in this peptide region. Furthermore, among the above-mentioned synthetic glycopeptides, we have discovered glycopeptides (glycopeptides 15 and 20, as well as glycopeptides 5, 9, 10, and 16) as specific "dynamic epitopes" within the fibronectin molecule, a glycoprotein with diverse functions, including regulating cancer cell adhesion, growth, invasion, and metastasis, that sensitively reflect the "stage of cancer progression." We have quantitatively demonstrated that the blood levels of fibronectin containing these dynamic epitopes generated by cancer-specific glycosylation rapidly change after a specific early stage in liver cancer patients. This has realized a novel, unprecedented, ultrasensitive biomarker technology that enables early detection and precise (quantitative) diagnosis of cancer progression. These glycopeptides are useful as immunogens for antibody production and as cancer vaccines.

[0067] Another embodiment relates to a method for analyzing glycopeptides, in the case of liver cancer, comprising analyzing whether a sample containing glycopeptides obtained by protease digestion of serum or plasma (a protease digest of serum or plasma) contains one or more types selected from the group consisting of glycopeptide 5, glycopeptide 9, glycopeptide 10, glycopeptide 11, and glycopeptide 16. Still another embodiment relates to a method for analyzing glycopeptides, in the case of pancreatic cancer, comprising analyzing whether a sample containing glycopeptides obtained by protease digestion of serum or plasma (a protease digest of serum or plasma) contains one or more types selected from the group consisting of glycopeptide 5, glycopeptide 8, glycopeptide 9, glycopeptide 10, glycopeptide 11, and glycopeptide 16. For example, this analytical method can confirm that a patient does not have cancer or evaluate the effectiveness of cancer treatment by confirming that a sample containing glycopeptides obtained by protease digestion of serum or plasma (a protease digest of serum or plasma) contains one or more types selected from the group consisting of glycopeptide 5, glycopeptide 9, glycopeptide 10, glycopeptide 11, and glycopeptide 16.

[0068] The features of the present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0069] In the following examples, the threonine (T2) located at the second position from the N-terminus may be referred to as Thr2152, the threonine (T5) located at the fifth position from the N-terminus as Thr2155, and the threonine (T6) located at the sixth position from the N-terminus as Thr2156.

[0070] Example: Synthesis of Peptide 1. Peptide 1 was synthesized by Fmoc solid-phase peptide synthesis. Specifically, using Trityl-OH-ChemMatrix resin (0.41 mmole / g, 25 mg, 10.25 μmol), the reaction proceeded sequentially under microwave irradiation (50°C) in a DMF (103 μL) solution containing Fmoc amino acid derivatives (41.0 μmol, 4 eq.), HBTU (41.0 μmol, 4 eq.), HOBt (41.0 μmol, 4 eq.), and DIEA (61.5 μmol, 6 eq.), with each step lasting 9 minutes. The Fmoc group was deprotected by treatment with 20% piperidine / DMF under microwave irradiation (50°C) for 3 minutes. The target peptide 1 was synthesized by repeating these two steps: elongation of various Fmoc amino acids and deprotection.

[0071] <Synthesis of glycopeptides 2 to 21> Fmoc glycoamino acid derivatives with glycans represented by the following structures were prepared. Glycoamino acid 24 was synthesized with reference to L. Corcilius, RJ Payne, Org. Lett. 2013, 15, 5794-5797; D. Liu, et al. J. Am. Chem. Soc. 2021, 143, 20216-20223.

[0072] For glycosylation of the second, fifth, and sixth threonines (Thr2152, Thr2155, and Thr2156) in glycopeptides 2–21, the Fmoc glycoamino acid derivatives bearing the above-mentioned glycans were used instead of the usual Fmoc amino acid derivatives under the following conditions: Fmoc glycoamino acid derivative (41.0 μmol, 2 eq.), HBTU (41.0 μmol, 2 eq.), HOBt (41.0 μmol, 2 eq.), and DIEA (61.5 μmol, 4 eq.) in DMF (51 μL), microwave irradiation at 50°C for 9 min. After condensation of all amino acid and glycoamino acid residues, the reaction mixture was added with TFA:HO:TIS (v:v:v = 95:2.5:2.5) and shaken at room temperature for 2 h. The reaction solution was filtered, and the crude product was precipitated by adding tert-butylmethyl ether (20 mL). The precipitate was then centrifuged (7,000 × g, 4°C, 20 min) and the supernatant was removed. The precipitate was dissolved in 50% aqueous acetonitrile and lyophilized to obtain glycopeptide intermediates. Of the lyophilized products, glycopeptide intermediates 2–6 and 12–16, which did not contain sialic acid residues, were dissolved in methanol (5 mL). The solution was then adjusted to pH 12.5 with 1N aqueous sodium hydroxide and shaken at room temperature for 2 hours to deprotect the acetyl groups in the glycan region. The reaction solution was neutralized with 30% aqueous acetic acid. The solvent was then evaporated, and the resulting syrupy residue was purified by reverse-phase high-performance liquid chromatography to obtain the desired glycopeptides 2–6 and 12–16.

[0073] Glycopeptides 7–11 containing the ST antigen (Neu5Acα2,3Galβ1,3GalNAcα1) were derived from glycopeptides 2–6 by enzymatic transglycosylation. Specifically, glycopeptides 2–5 (5 mM), Pasteurella multocida recombinant α-2,3-sialyltransferase (100 mU / mL), and CMP-Neu5Ac (30 mM) were dissolved in 100 mM HEPES (pH 7.0, 20 mM MgCl2, 2% BSA) at the final concentrations shown in parentheses. The reaction mixture was incubated at 37°C for 48 hours (glycopeptides 2–4) or 96 hours (glycopeptide 5). The mixture was then directly purified by reversed-phase high-performance liquid chromatography to yield the desired glycopeptides 7–10. Glycopeptide 11 was obtained by reacting glycopeptide 6 (1 mM), rat-derived recombinant α-2,3-sialyltransferase (20 mU / mL), and CMP-Neu5Ac (5 mM) in 50 mM HEPES (pH 7.0, 10 mM MgCl2, 0.1% BSA) at the final concentrations shown in the parentheses for 24 hours at room temperature, followed by direct purification by reversed-phase high-performance liquid chromatography (RPLC) as described above (Nishimura, S.-I. et al., Convergent solid-phase synthesis of macromolecular MUC1 models truly mimicking serum glycoprotein biomarkers of interstitial lung diseases. J. Am. Chem. Soc. 2016, 138, 8392-8395).

[0074] For glycopeptides 17–21 containing the STn antigen (Neu5Acα2,6GalNAcα1), the glycopeptide intermediate obtained by lyophilization after solid-phase synthesis was dissolved in methanol (4 mL), adjusted to pH 11.5 with 1N aqueous sodium hydroxide, and shaken at room temperature for 2 hours to deprotect the acetyl groups in the glycan region. The reaction mixture was then adjusted to pH 12 with 50 mM aqueous NaOH and shaken for an additional 5 hours to hydrolyze the methyl groups. The reaction solution was neutralized with 30% aqueous acetic acid. The solvent was then evaporated, and the resulting syrupy residue was purified by reversed-phase high-performance liquid chromatography to yield the desired glycopeptides 17–21.

[0075] Below, the synthesis schemes of glycopeptides 11 and 21 are shown as representative schemes.

[0076] Reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MALDI-TOFMS) of peptide 1 and glycopeptides 2–21. Separation and analysis of glycopeptides by RP-HPLC were performed on a reversed-phase C18 Inertsil ODS-3 column (250 × 4.6 mm, ID GL Sciences Inc., Tokyo, Japan) using a system consisting of a Hitachi L-7100 intelligent pump and an L-7405 UV detector. Specifically, the eluent A was 0.1% trifluoroacetic acid in water, and the eluent B was acetonitrile containing 0.1% trifluoroacetic acid (A / B = 95 / 5) at a flow rate of 1.0 mL / min for 50 min. The eluent was then eluted with a gradient program in which the proportion of eluent B increased from 5% to 30% over 50 min [0 min: A / B = 95 / 5 → 50 min: A / B = 70 / 30].

[0077] The molecular weights of the glycopeptides were measured by MALDI-TOF / MS using a Bruker Ultraflex III mass spectrometer in reflector positive and negative mode with 2,5-dihydrobenzoic acid (DHB) as the matrix. The results of MALDI-TOF / MS mass spectrometry for peptide 1 and glycopeptides 2 to 21 are shown in Figures 1 to 21.

[0078] <SRM channel settings for peptide 1 and glycopeptides 2 to 21> The SRM (selective reaction monitoring) parameters for peptide 1 and glycopeptides 2 to 21 were set using an LC-MS / MS (4000 QTRAP triple quadrupole mass spectrometer (AB Sciex Pte. Ltd.)) and Exigent NanoLC Expert. TM The SRM parameters were determined for each glycopeptide using a system consisting of a 400 (AB Sciex Pte. Ltd.) and optimized as follows: (a) parent ion selection in the first quadrupole (Q1), (b) optimization of collision-induced dissociation (CID) in the second quadrupole (Q2), and (c) evaluation and selection of fragment ions specific to each glycopeptide in the third quadrupole (Q3) (e.g., S.-I. Nishimura, et al., Selective reaction monitoring approach using structure-defined synthetic glycopeptides for validating glycopeptide biomarkers pre-determined by bottom-up glycoproteomics. RSC Adv 2022, 12, 21385-008121393).

[0079] The optimization process for SRM parameters is shown below using glycopeptide 16 as an example. The combination of Enhanced Mass (EMS) and Enhanced Resolution (ER) modes allowed for accurate Q1 and the determination of Q1 charge state. For example, the parent ion of glycopeptide 16 (2152 / 2155 / 2156Tn) exhibited multiple isotopologues centered at m / z 589.3 (theoretical value: m / z 589.30) at 0.3 Da intervals, indicating that this ion was a triply charged ion (Figure 22(a) is an actual spectrum of glycopeptide 16 obtained in ER mode). Furthermore, for Q2, fragment ions were observed in Enhanced Product Ion (EPI) mode, with collision energy (CE) gradually varied from 25 volts (0.8 min) to 130 volts (5 min), and Q3 was optimized (Figure 22(b)). For the Q3 candidate of glycopeptide 16, six fragment ions were observed: m / z 186.0 ([dehydrated GalNAc+H] + ), 204.0 ([GalNAc+H] + ), 680.2 ([M-2GalNAc+2H] 2+ ), 781.4 ([M-GalNAc+2H] 2+ ), 1156.0 ([Naked+H] + ), and 1358.9 ([M-2GalNAc+H] + ) was identified.

[0080] In a quadrupole mass analyzer, four parallel rod-shaped electrodes function as mass filters, selectively passing only ions within a specific mass range by adjusting the radio frequency (RF) and direct current (DC) voltages. Therefore, once the target ions Q1 and fragment ions Q3 are determined, the voltages must be set to allow only these ions to pass through each filter. Four parameters must be optimized: Declustering Potential (DP), Entrance Potential (EP), Collision Energy (CE), and Collision Cell Exit Potential (CXP). DP controls the voltage required to guide all ions generated by the ion generator to Q0. EP controls the voltage required to guide ions from Q0 to Q1. CE controls the voltage required to fragment target ions. CXP controls the voltage required to guide the generated fragment ions into Q3. These voltages are adjusted to maximize detection sensitivity. Of the four parameters, CE has the greatest impact on the sensitivity of parent ions and fragment ions (Figure 23).

[0081] The SRM parameters of peptide 1 and glycopeptides 2 to 21 determined by the above-mentioned method are as follows:

[0082] <Creation of a calibration curve using peptide 1 and glycopeptides 2 to 21 (synthetic (glyco)peptide preparations)> The concentrations of the standard solutions prepared using the synthetic (glyco)peptide preparations (peptide 1 and glycopeptides 2 to 21) used to create the calibration curve were determined by amino acid analysis. TMSynthetic glycopeptide preparations, peptide 1 and glycopeptides 2-21, were eluted using a nanoLC column (400) under the following conditions: Eluent A: 0.1% aqueous trifluoroacetic acid solution; Eluent B: acetonitrile containing 0.1% trifluoroacetic acid (A / B = 95 / 5) at a flow rate of 5.0 μL / min for 30 minutes, followed by a gradient program in which the proportion of eluent B increased from 5% to 30% over 30 minutes [0 min: A / B = 95 / 5 → 30 min: A / B = 70 / 30]. Calibration curves were created by plotting the peak areas of extracted ion chromatograms (XICs) for 0, 2.5, 5, 10, 25, 50, 100, 250, and 500 nM solutions of each glycopeptide under the above nanoLC elution conditions and SRM (MS / MS) parameters. From the results, we decided to adopt the SRM (MS / MS) parameters that provided the calibration curve with the highest detection sensitivity. For example, for peptide 1, CE (25 V) was the optimal channel, generating a fragment ion (Q3) at m / z 477.5. Similarly, calibration curves were created for peptide 1 and all glycopeptides 2 to 21 used as standards (Figures 24 to 44).

[0083] Quantitative Analysis of Fibronectin (FN)-Derived Glycopeptide Fragments (Peptide 1 and Glycopeptides 2-21) in the Peripheral Blood of Liver Cancer Patients, Pancreatic Cancer Patients, and Healthy Control Subjects Serum Sample Information Forty human serum samples were collected and stored at Okayama University Hospital (serum from healthy subjects, entries 1-5, and serum from pancreatic cancer patients, stage IVb, entries 26-30) and Hokkaido University Hospital (serum from hepatocellular carcinoma (HCC) patients, entries 6-25) after necessary procedures, including explanation of the study details and informed consent regarding the significance of the study, were completed and the subjects agreed to the study. The implementation of this example has already been approved by the Ethics Committee of the Graduate School of Advanced Life Science, Hokkaido University.

[0084]

[0085]

[0086] <<Pretreatment conditions for serum samples>> The 14 most common serum proteins with the highest blood concentrations were removed from serum (10 μL) using a multiple-affinity removal system ("MARS", Agilent Technologies spin columns) (N. Zolotarjova, et al., Combination of affinity depletion of abundant proteins and reversed-phase fractionation in proteomic analysis of human plasma / serum. J. Chromatogr. A2008, 1189, 332-338; E. Jankovska, et al., Affinity depletion versus relative protein enrichment: A side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage. Clin. Proteomics2019, 16, 9). The mixture was then centrifuged at 12,000 g for 5 minutes using an Amicon Ultra 100K (molecular weight cutoff, Merck). The resulting residue was dissolved in 50 mM ammonium carbonate (100 μL). 100 mM dithiothreitol (DTT, 10 μL) was added and the mixture was incubated at 37°C for 1 hour. 123 mM iodoacetamide (IAA, 7 μL) was added and incubated at room temperature for 30 minutes in a cool, dark place. 100 mM DTT (10 μL) was added to the reaction mixture to inactivate the IAA. Trypsin (Promega, sequencing-grade modified trypsin, trypsin / protein = 1 / 50) was then added and the mixture was digested at 37°C for 16 hours.The mixture containing glycopeptides and peptide fragments generated by trypsin digestion was purified using a GL-Tip GC (GL-Science) (Y. Uchida, et al. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS / MS: Application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood-brain barrier in ddY, FVB, and C57BL / 6J mice. Fluids Barriers CNS 2013, 10, 21). The solvent was then completely evaporated, and the mixture was dissolved in 0.1% formic acid (10 μL) to the original serum concentration, and then subjected to LC-MS / MS (SRM) analysis. Furthermore, the concentration of the tryptic digest of serum proteins was confirmed to be unchanged by spiking a synthetic glycopeptide sample of known concentration into the reaction system.

[0087] Absolute quantification of FN-derived glycopeptides in tryptic digests. Based on calibration curves (Figures 24-44) constructed using the optimized SRM channels for the synthesized peptide 1 and glycopeptides 2-21, the blood concentrations (nM) of target (glyco)peptide fragments 1-21 in tryptic digests from serum samples (five samples per group) of liver cancer patients (Liver), pancreatic cancer patients (Pan, stage 4 only), and healthy subjects were determined (Table 4 below). These results are presented in box plots (Figures 45-65 and Figures 68-76). Figures 68-76 compare blood glycopeptide concentrations between healthy subjects and stage 4 liver cancer and pancreatic cancer patients.

[0088]

[0089] Glycopeptide fragments 5, 9, 10, and 16 were all found at high concentrations (100-300 nM) in the serum of healthy individuals and patients with early stage liver cancer (stage 1). This suggests that the peptide regions generated by trypsin digestion within the IIICS domain are 2151 Thr-Thr-Pro-Pro-Thr-Thr-Ala-Thr-Pro-Ile-Arg 2161 is ubiquitously expressed on fibronectin, and three of the target sites (Thr2152, Thr2155, and Thr2156) were frequently modified by four cancer-specific O-glycans. Furthermore, glycopeptide fragments 2, 3, 4, 6, 7, 8, 12, 13, 17, 18, and 19 were rarely detected in patients with liver cancer. Glycosylation within this region occurred at at least two of the target sites (5, 10, 15, and 20) or all three of the target sites (11 and 16) (total of 70% of the total glycopeptides). In particular, glycopeptide fragments 5, 10, 15, and 20, in which both Thr2155 and Thr2156 were modified, accounted for 40% of the total.

[0090] Furthermore, a statistical significance test (unpaired two-tailed t-test) was performed on glycopeptides 5, 9, 15, 16, and 20, which showed a significant correlation with the stage of liver cancer. As shown in Figure 66, it was found that position-specific changes in glycan structure occurred with cancer progression.

[0091] The profile of changes in the concentrations (expression levels) of glycopeptide fragments 5, 9, 15, 16, and 20 derived from the IIICS domain of fibronectin in liver cancer revealed that the trends of increases and decreases in 5, 9, and 16 were correlated with those of 15 and 20, and that abrupt changes occurred at the boundary between healthy controls, stage 1 cancer patients, and stage 2 or later cancer patients. Of the three glycosylation sites reported to be present within this region (Thr2152, Thr2155, and Thr2156), focusing on glycopeptide fragments 5, 10, 15, and 20, in which Thr2155 and Thr2156 are simultaneously modified, suggests that post-translational modifications in this peptide region cause significant structural changes as cancer progresses from stage 1 to stage 2 or later, as shown in Figure 67.

[0092] As described above, this embodiment has realized a new technology for diagnosing liver cancer, particularly the progression from early stage (stage 1) to stage 2 or later, using tandem mass spectrometry (SRM) with human serum to rapidly, quantitatively, and accurately determine the stage of liver cancer. The fibronectin-derived glycopeptides produced by precision synthesis are novel substances that make this technology possible, and they also represent cancer-specific "dynamic epitopes." Antibodies that specifically recognize these are also important new target molecules for the development of new drugs that inhibit cancer spread and metastasis. Therefore, the significance and impact of this invention in creating new technologies and new industries is extremely great.

[0093] Furthermore, significant differences were found in the concentrations of glycopeptides 8, 9, and 10 in stage 4 pancreatic cancer patients compared to healthy individuals. This suggests that measuring the concentrations of these glycopeptides may enable the diagnosis of pancreatic cancer. Thus, this technology is also effective in diagnosing cancers other than liver cancer.

[0094] This invention is the world's first rapid and precise diagnostic technology using LC-MS / MS mass spectrometry (Selective reaction monitoring method, SRM) that can determine the stage of cancer by quantitative analysis of specific glycopeptide fragments in tryptic digests of blood proteins, and it is expected that this technology will be put into practical use in society at an early stage.

[0095] SEQ ID NO: 1 TTPPTTATPIR

Claims

1. A glycopeptide comprising the amino acid sequence: TPPTTATPIR, in which one or more threonines are each bound to one glycan consisting of six or less sugars, and the glycan is an O-linked glycan.

2. The glycopeptide according to claim 1, wherein the sugar chain is selected from the group consisting of the following (1) to (4): (1) GalNAcα1 (2) Galβ1,3GalNAcα1 (3) Neu5Acα2,6GalNAcα1 (4) Neu5Acα2,3Galβ1,3GalNAcα1 3. The glycopeptide according to claim 1, wherein the sugar chain is attached at one, two or three positions selected from the group consisting of the second threonine from the N-terminus, the fifth threonine and the sixth threonine.

4. The glycopeptide described in claim 1, wherein the binding positions of the glycan are, from the N-terminus, the second threonine, the fifth threonine, the sixth threonine, the second and fifth threonines, the second and sixth threonines, the fifth and sixth threonines, or the second, fifth and sixth threonines.

5. The glycopeptide according to claim 1, wherein the glycan bound to each of the two or more threonines is the same or different.

6. The glycopeptide according to claim 2, wherein the glycopeptide having the glycan of (2) is a glycopeptide represented by the following structural formula:

7. The glycopeptide according to claim 2, wherein the glycopeptide having the glycan of (4) is a glycopeptide represented by the following structural formula:

8. The glycopeptide according to claim 2, wherein the glycopeptide having the glycan of (1) is a glycopeptide represented by the following structural formula:

9. The glycopeptide according to claim 2, wherein the glycopeptide having the glycan of (3) is a glycopeptide represented by the following structural formula:

10. The glycopeptide described in any one of claims 1 to 9, for use as a tumor marker.

11. A glycopeptide described in any one of claims 1 to 9, which is used as an immunogen for producing an antibody.

12. A method for analyzing glycopeptides, comprising: analyzing whether or not a glycopeptide is contained in a sample containing glycopeptides obtained by protease digestion of serum or plasma, wherein the glycopeptide is a peptide consisting of the amino acid sequence: TPPTTATPIR, in which one or more threonines are each bound to one glycan consisting of six or less sugars, and the glycan is an O-linked glycan.

13. A method for analyzing glycopeptides according to claim 12, comprising analyzing whether the sample contains glycopeptide 15 and / or glycopeptide 20 represented by the following structural formula:

14. The method for analyzing glycopeptides described in claim 13, further comprising analyzing whether the sample contains one or more types selected from the group consisting of glycopeptide 5, glycopeptide 9, glycopeptide 10 and glycopeptide 16, which are represented by the following structural formulas.

15. A method for analyzing glycopeptides, comprising analyzing whether a sample containing glycopeptides obtained by protease digestion of serum or plasma contains one or more types selected from the group consisting of glycopeptide 5, glycopeptide 9, glycopeptide 10 and glycopeptide 16, which are represented by the following structural formulas.

16. The method for analyzing glycopeptides according to any one of claims 12 to 15, wherein the analysis comprises performing mass spectrometry of the glycopeptide.

17. The method of claim 16, wherein the analysis comprises performing mass analysis of the glycopeptide by tandem mass spectrometry.

18. A method for analyzing a glycopeptide described in any one of claims 12 to 15, which is used for detecting digestive system cancer and / or detecting the stage of digestive system cancer.

19. The method for analyzing glycopeptides described in claim 18, wherein the digestive system cancer is liver cancer or pancreatic cancer.

20. The method for analyzing glycopeptides according to any one of claims 12 to 15, wherein the sample is obtained by removing a portion of serum proteins or plasma proteins.

21. A method for analyzing cancer, comprising the method for analyzing glycopeptides according to any one of claims 12 to 15.

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