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EP-4741412-A1 - GLYCOPEPTIDE, METHOD FOR ANALYZING GLYCOPEPTIDE, AND METHOD FOR ANALYZING CANCER

EP4741412A1EP 4741412 A1EP4741412 A1EP 4741412A1EP-4741412-A1

Abstract

It is an object of the present invention to provide a material for systematically searching for and identifying epitopes specific to various diseases including cancer. In addition, it is another object of the present invention to establish a method for analyzing diseases using such epitopes specific to various diseases including cancer. The present invention relates to a glycopeptide, in which one glycan consisting of 6 or less sugars binds to each of one or two or more threonines in a peptide consisting of the amino acid sequence TTPPTTATPIR, and the glycan is an O-linked glycan. Moreover, the present invention relates to a method for analyzing a glycopeptide, comprising analyzing whether or not an epitope glycopeptide is contained in a sample containing a glycopeptide obtained by protease digestion of the serum or the plasma, wherein the glycopeptide is a peptide consisting of the amino acid sequence TTPPTTATPIR, in which one glycan consisting of 6 or less sugars binds to each of one or two or more threonines, and the glycan is an O-linked glycan.

Inventors

  • NISHIMURA, SHIN-ICHIRO
  • YOKOI, Yasuhiro

Assignees

  • ENU Pharma, Inc.

Dates

Publication Date
20260513
Application Date
20240703

Claims (20)

  1. A glycopeptide, in which one glycan consisting of 6 or less sugars binds to each of one or two or more threonines in a peptide consisting of the amino acid sequence TTPPTTATPIR, and the glycan is an O-linked glycan.
  2. The glycopeptide according to claim 1, wherein the glycan is selected from the group consisting of the following (1) to (4): (1) GalNAcα1, (2) Galβ1,3GalNAcα1, (3) Neu5Acα2,6GalNAcα1, and (4) Neu5Acα2,3Galβ1,3GalNAcα1.
  3. The glycopeptide according to claim 1, wherein the binding position(s) of the glycan is one, two or three sites selected from the group consisting of the threonine at position 2, the threonine at position 5, and the threonine at position 6 from the N-terminal side.
  4. The glycopeptide according to claim 1, wherein the binding position(s) of the glycan is the threonine at position 2, the threonine at position 5, the threonine at position 6, the threonines at positions 2 and 5, the threonines at positions 2 and 6, the threonines at positions 5 and 6, or the threonines at positions 2, 5 and 6, from the N-terminal side.
  5. The glycopeptide according to claim 1, wherein the glycans binding to each of the two or more threonines are homologous or heterologous.
  6. The glycopeptide according to claim 2, wherein the glycopeptide having the glycan (2) is a glycopeptide represented by any of the following structural formulae:
  7. The glycopeptide according to claim 2, wherein the glycopeptide having the glycan (4) is a glycopeptide represented by any of the following structural formulae:
  8. The glycopeptide according to claim 2, wherein the glycopeptide having the glycan (1) is a glycopeptide represented by any of the following structural formulae:
  9. The glycopeptide according to claim 2, wherein the glycopeptide having the glycan (3) is a glycopeptide represented by any of the following structural formulae:
  10. The glycopeptide according to any one of claims 1 to 9, which is used as a tumor marker.
  11. The glycopeptide according to any one of claims 1 to 9, which is used as an immunogen for antibody production.
  12. A method for analyzing a glycopeptide, comprising: analyzing whether or not a glycopeptide is contained in a sample containing a glycopeptide obtained by protease digestion of the serum or the plasma, wherein the glycopeptide is a peptide consisting of the amino acid sequence TTPPTTATPIR, in which one glycan consisting of 6 or less sugars binds to each of one or two or more threonines, and the glycan is an O-linked glycan.
  13. The method for analyzing a glycopeptide according to claim 12, comprising analyzing whether or not Glycopeptide 15 and/or Glycopeptide 20 represented by the following structural formulae are contained in the sample:
  14. The method for analyzing a glycopeptide according to claim 13, comprising further analyzing whether or not one type, or two or more types selected from the group consisting of Glycopeptide 5, Glycopeptide 9, Glycopeptide 10 and Glycopeptide 16 represented by the following structural formulae are contained in the sample:
  15. A method for analyzing a glycopeptide, comprising analyzing whether or not one type, or two or more types selected from the group consisting of Glycopeptide 5, Glycopeptide 9, Glycopeptide 10 and Glycopeptide 16 represented by the following structural formulae are contained in a sample containing a glycopeptide obtained by protease digestion of the serum or the plasma:
  16. The method for analyzing a glycopeptide according to any one of claims 12 to 15, wherein the analyzing comprises performing the mass spectrometry of the glycopeptide.
  17. The method for analyzing a glycopeptide according to claim 16, wherein the analyzing comprises performing the mass spectrometry of the glycopeptide according to tandem mass spectrometry.
  18. The method for analyzing a glycopeptide according to any one of claims 12 to 15, which is used in detection of digestive system cancer and/or detection of the stage of digestive system cancer progression.
  19. The method for analyzing a glycopeptide according to claim 18, wherein the digestive system cancer is liver cancer or pancreatic cancer.
  20. The method for analyzing a glycopeptide according to any one of claims 12 to 15, wherein the sample is obtained by removing a portion of a serum protein or a plasma protein.

Description

Technical Field The present invention relates to a glycopeptide, a method for analyzing a glycopeptide, and a method for analyzing cancer. Background Art Fibronectin is a glycoprotein essential for cancer proliferation and growth, invasion, and further, formation of the cancer microenvironment, and such fibronectin has been known to be the most important extracellular matrix component. In addition, fibronectin has also been 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 are being developed based on the molecular mechanism of inhibiting disease-specific binding between fibronectin (a glycoprotein molecule), and integrin that is highly expressed on the surface of cancer cells (e.g., Non-Patent Document 2). It has been known that, in fibronectin in cancer patients and fetuses, three domains, namely, 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 (i.e., the expression levels thereof are low in adults) are generated by alternative splicing (e.g., Non-Patent Document 3). Research and development are actively underway regarding the discovery of drugs targeting fibronectin isomers containing such domains consisting of regions significantly expressed in cancer, in particular, drug delivery systems (DDS) for cancer therapy (e.g., Non-Patent Document 4). Moreover, in Non-Patent Documents 5 and 6, cancer diagnostic drugs using antibodies targeting each of ED-A and ED-B as biomarkers (targets) are studied. At present, clinical trials of antibody conjugates, to which cytokines such as IL-2 have bound, are continuously ongoing, but no practical applications have been achieved yet. Non-Patent Document 7 discloses that the mouse monoclonal antibody FDC-6, which was established by isolation and immunization of fibronectin generated from human hepatocellular carcinoma cells (HUH-7), reacts with fibronectin derived from cancer patients and fetuses, but does not react with fibronectin from adults. Such fibronectin is defined as oncofetal fibronectin (onfFN). FDC-6 is a mouse antibody that binds to the glycopeptide epitopes 2114Val-Thr (GalNAcα)-His-Pro-Gly-Tyr2119 and 2114Val-Thr (Galβ1,3GalNAcα)-His-Pro-Gly-Tyr2119 in the IIICS domain of onfFN (Non-Patent Documents 8 and 9). It has been reported that OnfFNs containing these glycosylation regions are deeply involved in the TGF-β-induced "epithelial-mesenchymal transition (EMT)" process in cancer progression and metastasis (Non-Patent Document 10). However, the significance and specific function of these glycosylation regions in cancer biology, as well as the potential for developing novel cancer diagnostic and therapeutic methods based on FDC-6 or this epitope, still remain unknown. Prior Art Documents Non-Patent Documents Non-Patent Document 1: R. O. Hynes, Fibronectin, Springer, New York, 1990Non-Patent Document 2: H. Kessler, et al., A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins. Sci. Rep. 2017, 7, 39805Non-Patent Document 3: J. E. Schwarzbauer, Alternative splicing of fibronectin: Three variants, three functions. BioEssays 1991, 13, 527-533Non-Patent Document 4: D. P. Reinhardt, et al., Fibronectin-targeted drug delivery in cancer. Adv. Drug Deliv. Rev. 2016, 97, 101-110Non-Patent Document 5: D. Neri, et al., The extra-domain A of fibronectin is a vascular marker of solid tumors and metastasis. Cancer Res. 2007, 67, 10948-10957Non-Patent Document 6: L. J. Dubois, et al., Human fibronectin extra domain B as a biomarker for targeted therapy in cancer. Mol. Oncol. 2020, 14, 1555-1568Non-Patent Document 7: Hakomori, 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, 6517-6521Non-Patent Document 8: Hakomori, S.-i. et al., The oncofetal structure of human fibronectin defined by monoclonal antibody FDC-6: Unique structural requirement for the antigenic specificity proved by a glycosylhexapeptide. J. Biol. Chem 1988, 263, 3314-3322Non-Patent Document 9: Hakomori, 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, 10472-10476Non-Patent Document 10: Hakomori, S.-i. et al., Involvement of O-glycosylation defining oncofetal fibronectin in epithelial-mesenchymal-transition process. Pro. Natl. Acad. Sci. USA 2011, 108, 17690-17695 Summary of Invention Objects to be Solved by the Invention As mentioned above, various studies having, as a target for cancer diagnostic and therapeutic drugs, fibronectin, a multifunctional, high-molecular-weigh