WO2021054420A1 - Sugar chain-presenting particles and production method thereof - Google Patents

Sugar chain-presenting particles and production method thereof Download PDF

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WO2021054420A1
WO2021054420A1 PCT/JP2020/035375 JP2020035375W WO2021054420A1 WO 2021054420 A1 WO2021054420 A1 WO 2021054420A1 JP 2020035375 W JP2020035375 W JP 2020035375W WO 2021054420 A1 WO2021054420 A1 WO 2021054420A1
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sugar chain
sugar
pattern
cancer
terminal
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PCT/JP2020/035375
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Japanese (ja)
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西村　紳一郎
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国立大学法人北海道大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to sugar chain-presenting particles that mimic exosomes, a method for producing the same, and a method for using the same.
  • the present invention relates to sugar chain-presenting particles that present a cancer sugar chain pattern on the particle surface and a method for producing the same.
  • DDS drug delivery system
  • Non-Patent Documents 1, 2 and the like various adhesion molecules constituting the stroma (extracellular matrix) and the surrounding stroma.
  • Various inorganic / metal nanoparticles cores of gold nanoparticles, quantum dots, silica-based fine particles, etc.
  • PEG polyethylene glycol
  • Non-Patent Document 3 Surprisingly, in a survey of 232 papers published in the last decade, only about 0.7% (mean) of the dose of nanopharmaceutical reached the targeted cancer tissue. That is. Most nanoparticulate drugs administered intravascularly are said to rapidly accumulate in the liver, be taken up by phagocytic cells such as macrophages, decompose and excrete, and this is due to various proteins such as albumin in the blood.
  • Non-Patent Document 4 cancer cells are based on many recent reports that cancer cell-derived exosomes (extracellular nanoparticles) determine the infiltration / metastasis of individual cancer cells and the organ orientation (metastasis destination). Research on exosomes of origin has been actively conducted, and there are cases in which new drug developments that utilize the functions of exosomes and various contained microRNAs are aimed at (for example, Non-Patent Document 5).
  • Exosomes are membrane vesicles formed inside cells that are released extracellularly, and function as a tool for signal transmission between cells.
  • the size of exosomes, the heterogeneity of loaded molecules, and the difficulty in purifying exosomes derived from cancer cells / tissues make it almost unclear about the specific mechanism of organ orientation at the molecular level.
  • Patent Document 1 WO2017 / 131242A1 / US2020138973 (A1)
  • Non-Patent Document 1 Prabhakar et al., Cancer Res. 2013, 73, 2412-2417
  • Non-Patent Document 2 Danhier, J. Control. Release 2016, 244, 108-121
  • Non-Patent Document 3 W.C.W. Chan et al., Nat. Mater. 2016, 1, 1-12
  • Non-Patent Document 4 K. A. Dawson et al., Nat. Biotech. 2012, 7, 779-786
  • Non-Patent Document 5 R. Kalluri, J. Clin. Invest. 2016, 126, 1208-1215
  • Non-Patent Document 6 S.-I. Nishimura et al., J. Am. Chem. Soc.
  • Non-Patent Document 7 S.-I. Nishimura et al., ACS Chem. Biol. 2015, 10, 2073-2086;
  • Non-Patent Document 8 M. Colombo, et al., Annu. Rev. Cell Dev. Biol. 2014, 30, 255-289
  • Non-Patent Document 9 S.-I. Nishimura et al., Angew. Chem. Int. Engl. Ed. 2005, 44, 91-96
  • Patent Document 1 and Non-Patent Documents 1 to 9 is incorporated herein by reference in particular.
  • Exosomes released from many cells are present in the blood.
  • An object of the present invention is to artificially construct particles that imitate exosomes, which are tools for information transmission between cells.
  • the purpose is to provide a new technology that utilizes the pharmacokinetics of exosomes released from information senders.
  • the present invention focused on the fact that the surface of an exosome presents sugar chains in a pattern similar to that of cells that release them. That is, for example, in the case of metastasis of cancer cells, exosomes released from cancer cells are considered to play a leading role in guiding the pharmacokinetics of cancer cells. Various cells and organs that take up exosomes are likely to first encounter bulky glycocalyx on the surface of the exosome membrane. In the present invention, focusing on this fact, the pharmacokinetics of cancer cell-derived exosomes, particularly migration and excretion in the body, and distribution or accumulation in organs and tissues (organ orientation) are sugar chain patterns on the surface of exosome membranes. It was found that the invention was led and determined by the above, and the present invention was completed.
  • human cancer cell-derived exosome mimicking fine particles can be produced by presenting all sugar chains of N-type sugar chains of cultured human cancer cells on the surface of nanosome membrane by the glycoblotting method, and these can be directly used in mice.
  • intravenously administering and observing the pharmacokinetics in real time it became clear that the pharmacokinetics and organ orientation of human cancer cell-derived exosomes can be easily determined.
  • particles imitating exosomes that presented an artificially prepared cancer-specific sugar chain pattern based on the results reproduced the intended pharmacokinetics after administration this technology was used in advance for individual cancers.
  • We have completed the present invention by demonstrating that drug discovery based on the new concept of molecular design of tailor-made DDS adapted to organ orientation in metastasis is possible.
  • the present invention is as follows.
  • Sugar chain presentation particles that are nanoparticles having sugar chains on the surface.
  • the average particle size of the sugar chain presentation particles is in the range of 10 to 100 nm.
  • At least a part of the surface of the nanoparticles is coated with phospholipids.
  • the sugar chain contained on the surface of the nanoparticles is a sugar chain pattern requested by cancer cells or a sugar chain pattern determined based on the profile of this sugar chain (hereinafter referred to as a cancer sugar chain pattern).
  • Sugar chain presentation particles are nanoparticles having sugar chains on the surface.
  • the cancer sugar chain pattern is (1) Sugar chain pattern A, whose pharmacokinetics mainly depend on terminal high mannose type sugar chains, (2) Sugar chain pattern B, whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains, (3) Sugar chain pattern C, whose pharmacokinetics mainly depends on terminal ⁇ 2,6 sialic acid type sugar chains, (4) The sugar chain presentation particle according to [1], which is a sugar chain pattern selected from the group consisting of a sugar chain pattern D whose pharmacokinetics mainly depends on terminal ⁇ 2,3 sialic acid type sugar chains.
  • the cancer sugar chain pattern A 45 mol% or more of the sugar chains are terminal hymannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%.
  • the terminal hymannose type sugar chain of the sugar chain is less than 45%, and the terminal sialic acid type sugar chain is 0% or more and less than 2%.
  • the cancer sugar chain pattern C 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal ⁇ 2,6 sialic acid type sugar chains is the content of the terminal ⁇ 2,3 sialic acid type sugar chains.
  • the phospholipid that covers at least a part of the surface of the nanoparticles is a sulfide bond of an alkanethiol having a phosphorylcholine group, and the sugar chain on the surface of the nanoparticles is a sulfide bond of an alkanethiol having an immobilized sugar chain.
  • the surface of the nanoparticles is any one of [1] to [3], which is a nanoparticle coated with a monomolecular film of an alkanethiol sulfide bond having a phosphorylcholine group and an alkanethiol sulfide bond having a sugar chain immobilized.
  • the sugar chain presenting particles according to. [5]
  • the sulfide bond of the alkanethiol having a phosphorylcholine group is represented by the following general formula (A).
  • n3 is an integer in the range of 2 to 30, and the -S-end is a carrier site where a sulfide bond is attached to the nanoparticles.
  • the sugar chain-presenting particles according to [4], wherein the sulfide bond of alkanethiol on which the sugar chain is immobilized is represented by the following general formula (B).
  • n1 is an integer of 2-30
  • n2 is an integer of 2 to 30, a bearing portion -S- terminal sulfide bound to the nanoparticles
  • R 10 is contained sugar It is a part.
  • [6] The sugar chain-presenting particle according to any one of [1] to [5], which further has a drug site.
  • the sugar chain presentation particle kit according to [8], wherein the different cancer sugar chain patterns are any two or more sugar chain patterns of patterns A to D according to [2].
  • a cancer therapeutic agent containing the sugar chain-presenting particles according to [6] or [7] as an active ingredient.
  • a method for determining a sugar chain pattern of a cancer cell which comprises profiling the sugar chain of the cancer cell from the cancer cell collected from a subject and determining the sugar chain pattern based on the profiled sugar chain.
  • the glycan pattern is determined by the profiled glycan.
  • the cancer sugar chain pattern A 45 mol% or more of the sugar chains are terminal hymannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%.
  • the terminal hymannose type sugar chain of the sugar chain is less than 45%, and the terminal sialic acid type sugar chain is 0% or more and less than 2%.
  • the cancer sugar chain pattern C 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal ⁇ 2,6 sialic acid type sugar chains is the content of the terminal ⁇ 2,3 sialic acid type sugar chains.
  • the cancer sugar chain pattern D 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of ⁇ 2,3 sialic acid type sugar chains is higher than the content of ⁇ 2,6 sialic acid type sugar chains.
  • the determination method according to [14]. [16] When the sugar chain profile of cancer cells is sugar chain pattern A, the cancer cells possessed by the subject show type 1 pharmacokinetics, suggesting that the cancer cells have a low metastasis tendency. When the glycan profile is glycan pattern B, the cancer cells possessed by the subject show type 2 pharmacokinetics, suggesting that the cancer cells tend to metastasize to the liver and spleen.
  • the glycan profile is glycan pattern C
  • the cancer cells possessed by the subject show type 3 pharmacokinetics, suggesting that the cancer cells tend to metastasize to the axilla and supraclavicular lymph nodes.
  • the glycan profile is glycan pattern D
  • the cancer cells possessed by the subject show type 4 pharmacokinetics, suggesting that the cancer cells tend to metastasize to the lungs, liver, spleen, brain and kidneys.
  • the determination method according to [14] or [15].
  • [17] The sugar chain-presenting particles according to any one of [1] to [7], which comprises presenting a cancer sugar chain pattern on the surface of nanoparticles having at least a part of the surface coated with phospholipids. Production method.
  • the presented cancer sugar chain pattern is a cancer sugar chain pattern obtained by cutting out a sugar chain of a cancer cell collected from a subject, or a profile of a sugar chain of a cancer cell collected from a subject.
  • the production method according to [17] which is a sugar chain pattern determined based on the obtained profile.
  • the nanoparticles in which at least a part of the surface is coated with phospholipids include the crosslinked precursor X represented by the following general formula (D), the phospholipid precursor represented by the following general formula (E), and colloidal nanoparticles.
  • n1 is an integer of 2 to 30, and n2 is an integer of 2 to 30.
  • n3 is an integer in the range of 2 to 30.
  • the aminooxy group of the cross-linking precursor X represented by the general formula (D) introduced into the surface-modified nanoparticles and the sugar chain of the cancer cell collected from the subject are cut out.
  • the cancer sugar chain pattern or the sugar chain of the cancer cell collected from the subject is profiled, and the reducing terminal of the sugar chain contained in the sugar chain pattern determined based on the obtained profile is used as a glyco.
  • the sugar chain pattern determined based on the profile is a sugar chain pattern in which the type and content of the sugar chain component are the same as the profile, or a part of the type and content of the sugar chain component is the same as the profile.
  • sugar chain-presenting particles having a cancer sugar chain pattern which can predict the metastasis destination of cancer cells in the primary lesion and show specific pharmacokinetics.
  • intravenously administering the sugar chain-presenting particles to mice for example, the process of cancer metastasis led by the cancer sugar chain pattern can be visualized in real time.
  • FIG. 1 shows the results of in vivo imaging of a method for producing sugar chain-presenting particles in which the sugar chain pattern of cancer cells is presented on the surface and their pharmacokinetics.
  • FIG. 2 shows the N-type sugar chain profile of cultured human cancer cells.
  • B Structure of major sugar chain components of each cancer cell (top 10 structures of expression level), pie chart is glycotyping analysis ) Is the result.
  • FIG. 3 shows the experimental results of the pharmacokinetics of sugar chain-presenting particles, which are nanoparticles that present the sugar chain pattern of cancer cells.
  • FIG. 3 shows the experimental results of the pharmacokinetics of sugar chain-presenting particles, which are nanoparticles that present the sugar chain pattern of cancer cells.
  • C Organ directivity of sugar chain-presenting particles: The relative fluorescence intensity in each organ was shown when the fluorescence intensity in the brain of the controlled particles having no sugar chain was 1.0. However, the axillary and supraclavicular lymph node tissues were not removed by autopsy.
  • FIG. 4 shows the results of an experiment in which the egg white sugar chain of Ruddy duck is modified into the sugar chain pattern of human cancer cells.
  • A Multi-branched sugar chain structure found in Ruddy duck egg white and modification method by sialic acid transfer enzyme
  • FIG. 4 shows the results of an experiment in which the egg white sugar chain of Ruddy duck is modified into the sugar chain pattern of human cancer cells.
  • FIG. 5 shows the results of examining the pharmacokinetics and organ directivity of nanoparticles presenting sugar chains of egg white and yolk, modified sugar chain patterns, and sugar chain patterns of ⁇ 2,6 type sialic acid type sugar chains. ..
  • A Nanoparticles presenting two types of sugar chains obtained by modifying the egg white sugar chains of Button Quail, Chicken, and Ruddy duck, and modifying the egg white sugar chains of Ruddy duck, and further presenting sugar chains derived from chicken egg yellow sugar peptide.
  • Glyco-presenting particles (GNS-2,6S-A2), which are nanoparticles, and controls are pharmacodynamics up to 3 hours after intravenous administration (numbers are zeta potential), and (b) major mice dissected after 3 hours.
  • Distribution of sugar chain-presenting particles (GNS-Button Quail, GNS-Chicken, GNS-Ruddy duck, GNS-2,3S-Ruddy duck, GNS-2,6S-Ruddy duck, GNS-2,6S-A2) in organs Status.
  • FIG. 5 shows the results of examining the pharmacokinetics and organ directivity of nanoparticles presenting sugar chains of egg white and yolk, modified sugar chain patterns, and sugar chain patterns of ⁇ 2,6 type sialic acid type sugar chains. ..
  • FIG. 6 shows the experimental results of the pharmacokinetics of sugar chain-presenting particles prepared by mixing Japanese Quail egg white sugar chain and chicken egg brown sugar peptide-derived sugar chain (2,6S-A2).
  • A Japanese Quail Egg white-derived sugar chains (about 6 nmol) were mixed with 0.6 nmol, 1.2 nmol, and 2.4 nmol sugar chains derived from 2,6S-A2, respectively. It is the result of the chain profile.
  • the present invention relates to sugar chain-presenting particles, which are nanoparticles having sugar chains on the surface, and the sugar chain-presenting particles are: (1) The average particle size of the sugar chain presentation particles is in the range of 10 to 100 nm. (2) At least a part of the surface of the nanoparticles is coated with phospholipids. (3) The sugar chain on the surface of the nanoparticles is a sugar chain pattern derived from cancer cells or a pattern imitating a sugar chain pattern determined based on the profile of the sugar chain (cancer sugar chain pattern).
  • the sugar chain presenting particles of the present invention have a phospholipid coating and a cancer sugar chain pattern on the surface of the nanoparticles.
  • the sugar chain pattern is the presence or absence of a specific type of sugar chain or the presence of a specific type of sugar chain in a group of sugar chains consisting of two or more different sugar chains based on the surface sugar chain profile. Means a pattern specified by quantity.
  • the sugar chain pattern is specified by the types of sugar chain components and their contents.
  • the sugar chain-presenting particles that capture and present the sugar chains of the cancer cells as they are are the sugar chain-presenting particles having a sugar chain pattern derived from the cancer cells.
  • the sugar chain-presenting particles in the present invention are also sugar chain-presenting particles that profile the sugar chains of cancer cells, determine the sugar chain pattern based on the profile, and have the determined sugar chain pattern.
  • the sugar chain pattern determined based on the profile is a sugar chain pattern in which the type and content of the sugar chain component are the same as the profile, or a part of the type and content of the sugar chain component is the same as the profile. It may be either a sugar chain pattern.
  • the amount of some types of the sugar chain component contained in the profile can be reduced or deleted. More specifically, when the sugar chain components contained in the profile are classified into several types, the sugar chain components belonging to one or two or more types having a high content are contained, and the one or two or more types having a low content are included. It can be a sugar chain pattern in which the amount of the sugar chain component belonging to is reduced or deleted. Examples of the types of sugar chain components include terminal hymannose type sugar chain, terminal galactose type sugar chain, terminal N-acetylglucosamine type sugar chain, terminal ⁇ 2,6 sialic acid type sugar chain, and terminal ⁇ 2,3 sialic acid type sugar.
  • the sugar chain components in the profile are, for example, a terminal hymannose type sugar chain, a terminal galactose type sugar chain, and a terminal N-acetylglucosamine type sugar chain in descending order of content, the terminal N-acetylglucosamine type having a smaller content
  • the sugar chain pattern in which the amount of sugar chains is reduced or deleted can be used as the sugar chain pattern determined from the profile.
  • the sugar chain pattern in which the content ratio of the sugar chain components belonging to the same type is changed can be used as the sugar chain pattern determined from the profile. Since the pharmacokinetics of the sugar chain-presenting particles change depending on the type of sugar chain, the sugar chain pattern can be determined by appropriately changing the profile in consideration of the change in the pharmacokinetics.
  • the sugar chain pattern in the present invention may include four cancer sugar chain patterns A to D that can be classified according to the pharmacokinetics of the sugar chain presenting particles of the present invention.
  • (1) Sugar chain pattern A whose pharmacokinetics mainly depend on terminal high mannose type sugar chains
  • (2) Sugar chain pattern B whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains
  • Sugar chain pattern C whose pharmacokinetics mainly depends on terminal ⁇ 2,6 sialic acid type sugar chains
  • Sugar chain pattern D in which pharmacokinetics mainly depend on terminal ⁇ 2,3 sialic acid type sugar chains.
  • the pharmacokinetics means the temporal movement of the sugar chain-presenting particles in the body of the animal to which the sugar chain-presenting particles are administered via the circulatory system and the like, and the temporal distribution of the sugar chain-presenting particles to each organ or tissue. ..
  • the property of being distributed or accumulated over time in each organ or tissue in the pharmacokinetics may mean particularly organ orientation.
  • the cancer cells targeted in the present invention are cancer cells that release exosomes, which are targets of mimicking the sugar chain presentation particles of the present invention, and include cancer cells collected from a living body and cancer cells cultured after collection. Is done.
  • the cancer cells may be a single cell that has been purely cultured, or a mixture of a plurality of cancer cells having different grades of malignancy.
  • the existing sugar chain pattern of cancer cells that is used as standard can also be used as the sugar chain pattern in the sugar chain presentation particles of the present invention.
  • standard human cancer cells include MCF7 (breast cancer), MDA-MB-231 (breast cancer), A549 (lung cancer), HepG2 (liver cancer), A375 (melanoma), HCT116 (colon cancer), Hela (uterine cancer), MNNG / NOS (osteosarcoma), AGC (gastric cancer), MIAPaCa-2 (pancreatic cancer), A431 (skin cancer), SKOV (ovarian cancer) and the like can be mentioned.
  • the sugar chain presenting particle means a particle having a function (information transmission-like function between cells) that imitates the function of an exosome, which is a particle used for information transmission between cells.
  • Exosomes contain nucleic acids, proteins, etc. inside them, and are transmitted to receiving cells via exosomes secreted from cells. Exosomes function as communication tools between cells.
  • the sugar chain presenting particles produced in the present invention have a function of transmitting information to receiving cells and tissues and organs in which they are aggregated, similar to the original exosomes.
  • the nanoparticles can be metal nanoparticles or semiconductor nanoparticles.
  • the material of the metal nanoparticles is not particularly limited, but may be gold, platinum, silver, or iron magnetic material.
  • the metal nanoparticles are gold nanoparticles, platinum nanoparticles, and the silver nanoparticles are iron magnetic materials. It can be nanoparticles. In particular, as the metal nanoparticles, gold nanoparticles, platinum nanoparticles, and silver nanoparticles are preferable from the viewpoint of safety to the living body.
  • the semiconductor nanoparticles can also be quantum dots. Quantum dots are small lumps of about 10 and several nanometers in which hundreds to thousands of semiconductor atoms are gathered, and are fluorescent nanoparticles. The wavelength (color) of fluorescence emitted differs depending on the particle size. Commercially available products can be used, and by using quantum dots for the nanoparticles of the complex of the present invention, it is possible to obtain a fluorescent complex, and it is also possible to monitor the dynamics in vivo.
  • the nanoparticles can have a particle size in the range of 0.1 to 100 nm, preferably in the range of 1 to 50 nm, more preferably in the range of 5 to 40 nm, still more preferably in the range of 5 to 30 nm, even more preferably 10. It is in the range of ⁇ 30 nm.
  • the sugar chain-presenting particles having a phospholipid coating and a cancer sugar chain pattern on the surface of the nanoparticles have an average particle size in the range of 10 to 100 nm, preferably in the range of 10 to 60 nm, and more preferably in the range of 12 to 50 nm. More preferably, it is in the range of 15 to 40 nm, and further preferably, it is in the range of 15 to 30 nm.
  • the average particle size of the nanoparticles and the sugar chain presentation particles can be measured by a dynamic light scattering method.
  • a fiber optical dynamic light scattering photometer for particle size distribution measurement
  • a fiber optical dynamic light scattering photometer FDLS-3000 manufactured by Otsuka Electronics Co., Ltd.
  • the surface of the nanoparticles is coated with phospholipids.
  • Completely coated metal nanoparticles with a phospholipid alkane thiol mixed monolayer having an average particle size of about 20 nm do not form protein corona due to non-specific adsorption even in blood, and accumulate in specific organs when intravenously administered to mice. It is a stable nanoparticle that stays uniformly throughout the body even after 3 hours after administration (Non-Patent Documents 6-7, Patent Document 1).
  • the sulfide bond of an alkanethiol having a phosphorylcholine group can be a phospholipid pseudo substance represented by the general formula (A).
  • n3 is an integer in the range of 2 to 30, and is a carrier site where the —S— terminal is sulfide-bonded to the nanoparticles.
  • n3 is an integer in the range of 2 to 30, preferably 5 to 20, and more preferably 7 to 15.
  • the amount of phospholipid or phospholipid pseudo substance supported on one nanoparticles is preferably 80% or more of the metal element (reaction point) on the surface of the nanoparticles.
  • the sugar chain presenting particles of the present invention have a cancer sugar chain pattern on the surface of the nanoparticles.
  • This cancer sugar chain pattern is a sugar chain pattern that presents a sugar chain derived from a cancer cell that releases exosomes as a sender of information, or a cancer sugar that is determined by profiling the sugar chain of a cancer cell. It is a chain pattern.
  • the sugar chain pattern derived from cancer cells is a sugar chain pattern derived from cancer cells collected from a specific cancer treatment patient, which enhances the predictability of the pharmacokinetics of exosomes released from the cells. It is preferable for this.
  • Existing cancer cell-derived sugar chain patterns that are used as standard can also be used for prediction.
  • the sugar chain pattern derived from cancer cells can be presented by cutting out the sugar chains of cancer cells and capturing them on the surface of nanoparticles.
  • the sugar chains of the cells are presented as they are on the surface of the nanoparticles, but they can be used as sugar chain components within the range of the cancer sugar chain pattern determined in the cells. It does not matter if there are fluctuations.
  • the above cancer cell-derived sugar chain profile can be identified according to the general protocol of the Glycoblotting method (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537). .. Glycotyping analysis using internal standard compounds as an index can clarify the relationship between their expression levels and major structural motifs.
  • the existing method S.-I. Nishimura eta l., J. Am. Chem. Soc. 2011, 133, 12507-12517; S.-I. Nishimura et al., ACS Chem. Biol It can be supported on nanoparticles by .2015, 10, 2073-2086; S.-I. Nishimura, WO2017 / 131242A1). See Example 1 (A) for details.
  • the sugar chain patterns of the cancer cells examined were at least (1) the pharmacokinetics mainly terminal high mannose type.
  • Glycan pattern A which depends on glycans
  • Sugar chain pattern B whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains
  • Sugar chain pattern C whose pharmacokinetics mainly depends on terminal ⁇ 2,6 sialic acid type sugar chains
  • the pharmacokinetics of sugar chain-presenting particles having cancer sugar chain patterns A to D are as follows.
  • the sugar chain-presenting particles having the cancer sugar chain pattern A show type 1 pharmacokinetics in which the excretion of the particles from the body is promoted as compared with the sugar chain-presenting particles having no sugar chain.
  • the sugar chain-presenting particles having the cancer sugar chain pattern B show type 2 pharmacokinetics accumulated in the liver and spleen as compared with the sugar chain-presenting particles having no sugar chain.
  • the sugar chain-presenting particles having the cancer sugar chain pattern C show type 3 pharmacokinetics accumulated in the axilla and the supraclavicular lymph nodes as compared with the sugar chain-presenting particles having no sugar chain.
  • the sugar chain-presenting particles having the cancer sugar chain pattern D are distributed (distributed and accumulated) in organs such as lung, liver, spleen, brain, and kidney as compared with the sugar chain-presenting particles having no sugar chain. Shows the pharmacokinetics of.
  • the terminal high mannose type sugar chain constituting the sugar chain pattern means a sugar chain in which the end of the sugar chain is branched into two or more and all the sugars in the branched portion are mannose. Examples of terminal high mannose type sugar chains are shown below.
  • the terminal galactose-type sugar chain constituting the sugar chain pattern means a sugar chain in which one sugar chain alone or a plurality of terminal sugars are galactose. Examples of terminal galactose-type sugar chains are shown below.
  • the terminal N-acetylglucosamine type sugar chain constituting the sugar chain pattern means a sugar chain in which one sugar chain alone or a plurality of terminal sugars are N-acetylglucosamine. Examples of terminal N-acetylglucosamine type sugar chains are shown below.
  • the terminal sialic acid type sugar chain constituting the sugar chain pattern means a sugar chain in which one sugar chain alone or a plurality of terminal sugars are sialic acid.
  • the terminal sialic acid type sugar chain includes a complex type sugar chain containing Neu5Ac ⁇ 2,6Gal unit at the end and a complex type sugar chain containing Neu5Ac ⁇ 2,3Gal unit at the end.
  • a complex type sugar chain containing a Neu5Ac ⁇ 2,6Gal unit at the end is referred to as a terminal ⁇ 2,6 sialic acid type sugar chain
  • a complex type sugar chain containing a Neu5Ac ⁇ 2,3Gal unit at the end is referred to as a terminal ⁇ 2,3 sialic acid.
  • Examples of terminal sialic acid type sugar chains are shown below.
  • sugar chain-presenting particles that present sugar chains derived from each cancer cell were prepared, and the pharmacokinetics when these were intravenously administered to mice were compared with the sugar chain-presenting particles that did not present sugar chains. As a control, it was observed in real time by a near-infrared fluorescence spectrum (see Example 1).
  • an artificial sugar chain that artificially determines the sugar chain pattern based on the sugar chain profile of the above-mentioned human cultured cancer cells and presents the sugar chain within the range of the determined sugar chain pattern.
  • Presented particles were prepared.
  • the sugar chain pattern of exosomes derived from cancer cells leads the pharmacokinetics of exosomes and finally the organ orientation of cancer cells. It was proved to be decided (see Example 2).
  • Cancer sugar chain pattern A In the cancer sugar chain pattern A, 45 mol% or more of the sugar chains contained in the sugar chains are terminal high mannose type sugar chains. Hereinafter, unless otherwise specified,% of the sugar chain means mol%.
  • the content of the terminal high mannose type sugar chain (HM) in the sugar chain presentation particles of the present invention having the cancer sugar chain pattern A shown in the examples is as follows.
  • the terminal sialic acid type sugar chain is 0% in each case.
  • the cancer sugar chain pattern A can include a terminal galactose type sugar chain, a terminal N-acetylglucosamine type sugar chain, a terminal sialic acid type sugar chain, and the like as sugar chain components in addition to the terminal hymannose type sugar chain.
  • the terminal sialic acid type sugar chain is 0% or more and less than 2%.
  • the terminal sialic acid type sugar chain is 2% or more, it can be determined to be sugar chain pattern C or sugar chain pattern D, and the effect of the terminal sialic acid type sugar chain is prioritized (described later).
  • the terminal sialic acid type sugar chain is less than 2% and the terminal hymannose type sugar chain is 45% or more, the types of the terminal galactose type sugar chain and the terminal N-acetylglucosamine type sugar chain which are other sugar chain components. Regardless of the amount and amount, the sugar chain-presenting particles show type 1 pharmacokinetics and do not show distribution to specific organs (organ orientation).
  • the terminal high mannose type sugar chain can be in the range of 50% to 95%, for example.
  • the cancer sugar chain pattern B is a cancer sugar chain pattern in which the terminal high mannose type sugar chain is less than 45% and the terminal sialic acid type sugar chain is 0% or more and less than 2%.
  • Sugar chain-presenting particles having a sugar chain pattern in which the terminal hymannose-type sugar chain is less than 45% and the terminal sialic acid-type sugar chain is 0% or more and less than 2% exhibit type 2 pharmacokinetics.
  • the terminal galactose type sugar chain and the terminal N-acetylglucosamine type sugar chain can be included, but the terminal hymannox type sugar chain is 45%.
  • the sugar chain pattern B Can be decided.
  • the sugar chain-presenting particles of the cancer sugar chain pattern B show type 2 pharmacokinetics.
  • the content of ⁇ G) is shown in Table 2.
  • the terminal sialic acid type sugar chain is 0% in each case.
  • the example of GNS-Chicken of Example 2 having a cancer sugar chain pattern B was accumulated in the liver and spleen 180 minutes after intravenous administration. This phenomenon was also the same in the example of GNS-Ruddy Duck of Example 2 (see FIGS. 5b and c). That is, it can be seen that the sugar chain-presenting particles having the cancer sugar chain pattern B exhibit type 2 pharmacokinetics accumulated in the liver and spleen as compared with the control particles having no sugar chain.
  • the cancer sugar chain pattern C is a cancer sugar chain pattern in which 2 to 100% of the sugar chains are terminal sialic acid type sugar chains. Further, the terminal sialic acid type sugar chain includes a terminal ⁇ 2,6 sialic acid type sugar chain and a terminal ⁇ 2,3 sialic acid type sugar chain, and the cancer sugar chain pattern C is an ⁇ 2,6 sialic acid type sugar chain. Is higher than the content of ⁇ 2,3 sialic acid type sugar chains.
  • the content of the terminal sialic acid type sugar chain can be in the range of, for example, 3 to 100%, 4 to 80%, or 5 to 60%.
  • the sugar chain other than the terminal sialic acid type sugar chain may be a terminal hymannose type sugar chain, a terminal galactose type sugar chain, and / or a terminal N-acetylglucosamine type sugar chain.
  • the cancer sugar chain pattern D is a cancer sugar chain pattern in which 2 to 100% of the sugar chains are terminal sialic acid type sugar chains.
  • the cancer sugar chain pattern D is a case where the content of the ⁇ 2,3 sialic acid type sugar chain is larger than the content of the ⁇ 2,6 sialic acid type sugar chain.
  • the content of the terminal sialic acid type sugar chain can be in the range of, for example, 3 to 100%, 4 to 80%, or 5 to 60%.
  • the sugar chain other than the terminal sialic acid type sugar chain may be a terminal hymannose type sugar chain, a terminal galactose type sugar chain, and / or a terminal N-acetylglucosamine type sugar chain.
  • ⁇ 2,3 sialic acid type sugar chain ( ⁇ 2,3), ⁇ 2,6 sialic acid type sugar chain ( ⁇ 2,3) of the sugar chain presentation particles of the present invention having the cancer sugar chain patterns of patterns C and D shown in Examples.
  • Table 3 shows the contents of 6) and the terminal high mannose type sugar chain (HM). Terminal galactose-type sugar chains (Gal) and terminal N-acetylglucosamine-type sugar chains (NAc-G) are not shown.
  • NS which is a particle having no sugar chain shown in FIGS. 3b and 3c
  • fluorescence from the particle was observed almost entirely throughout the body 180 minutes after intravenous administration.
  • the particles were, for example, 180 minutes after intravenous administration, as compared with NS. It accumulated in the axilla and supraclavicular lymph nodes. From this, the sugar chain-presenting particles having the cancer sugar chain pattern C show the type 3 pharmacokinetics accumulated in the axilla and the supraclavicular lymph nodes as compared with the sugar chain-presenting particles having no sugar chain. I understand.
  • Example 3 having the cancer sugar chain pattern of pattern D
  • the particles were lung, liver, spleen, brain, and kidney 180 minutes after intravenous administration. It was distributed (dispersed accumulation) to such organs. From this, the sugar chain-presenting particles having the cancer sugar chain pattern D are distributed (distributed and accumulated) in organs such as lung, liver, spleen, brain, and kidney as compared with the sugar chain-presenting particles having no sugar chain. It can be seen that it shows the pharmacokinetics of type 4 that is produced.
  • GNS-A549 of Example 1 particles were distributed (dispersed accumulation) in organs such as lung, liver, spleen, brain, and kidney 180 minutes after intravenous administration (particularly, FIGS. 3b and c). This phenomenon was similar to the example of 2,3-Ruddy Duck of Example 2 having the cancer sugar chain pattern D.
  • Example 1 it is not specified whether the terminal sialic acid type sugar chain is an ⁇ 2,3 sialic acid type sugar chain or an ⁇ 2,6 sialic acid type sugar chain, but from the result of this pharmacokinetics, it is not specified.
  • GNS-A549 and GNS-HepG2 of Example 1 were judged to be sugar chain pattern D and are shown in Table 4.
  • Whether it is an ⁇ 2,6 sialic acid type sugar chain or an ⁇ 2,3 sialic acid type sugar chain is determined by its function in the present specification, but it can also be specified by an analysis method such as chemical / enzymatic decomposition characteristics. Is.
  • the cancer sugar chain pattern can be the sugar chain itself of cancer cells that are commercially available and generalized for research purposes as described above, or cancer cells of specific cancer treatment patients. Further, the cancer sugar chain pattern can be a sugar chain pattern determined based on the profile obtained by profiling the sugar chain of the cancer cell.
  • the sugar chain pattern determined based on the profile is a sugar chain pattern in which the type and content of the sugar chain component are the same as the profile, or a part of the type and content of the sugar chain component is the same as the profile. It may be either a sugar chain pattern. When a part of the type and content of the sugar chain component is the same as the profile, the amount of some types of the sugar chain component contained in the profile can be reduced or deleted.
  • the sugar chain components contained in the profile are classified into several types, the sugar chain components belonging to one or two or more types having a high content are contained, and the one or two or more types having a low content are included.
  • It can be a sugar chain pattern in which the amount of the sugar chain component belonging to is reduced or deleted.
  • the sugar chain pattern determined based on the profile obtained by profiling the sugar chain of the cancer cell is the above-mentioned cancer sugar chain pattern derived from the cancer cell, the type of the sugar chain component, and each sugar chain component. Although the content is different, it is a sugar chain pattern that shows the same pharmacokinetics as the cancer sugar chain pattern derived from cancer cells.
  • sugar chain of a cancer cell When the sugar chain of a cancer cell is profiled, when the sugar chain component contained in the profile is classified into several types, the sugar chain component belonging to one or more types having a high content is contained as it is, and the content is high. By reducing or deleting the amount of sugar chain components belonging to a small number of 1 or 2 or more types, it is possible to obtain a sugar chain pattern showing the same pharmacokinetics as the cancer sugar chain pattern derived from cancer cells in which the sugar chain is profiled. ..
  • the types of sugar chain components include terminal hymannose type sugar chain, terminal galactose type sugar chain, terminal N-acetylglucosamine type sugar chain, terminal ⁇ 2,6 sialic acid type sugar chain, and terminal ⁇ 2,3 sialic acid type sugar. You can raise a chain.
  • a sugar chain pattern has a type of sugar chain component and a content of each sugar chain component that are different from the cancer sugar chain pattern derived from cancer cells, but the pharmacokinetics mainly depend on the terminal hymannose type sugar chain. When it is the chain pattern A, it shows the pharmacokinetics of type 1. Similarly, a sugar chain pattern has a type of sugar chain component and a content of each sugar chain component that are different from the cancer sugar chain pattern derived from cancer cells, but the pharmacokinetics are mainly terminal galactose-type sugar chains or terminals. When it is a sugar chain pattern B that depends on an N-acetylglucosamine type sugar chain, it shows type 2 pharmacokinetics.
  • a certain sugar chain pattern has a type of sugar chain component and a content of each sugar chain component different from the cancer sugar chain pattern derived from cancer cells, but the pharmacokinetics are mainly terminal ⁇ 2,6 sialic acid type sugar chains. If it is a dependent sugar chain pattern C, it shows type 3 pharmacokinetics.
  • a certain sugar chain pattern has a type of sugar chain component and a content of each sugar chain component different from those of cancer cell-derived cancer sugar chain patterns, but the pharmacokinetics are mainly terminal ⁇ 2,3 sialic acid type sugar chains. When it is the dependent sugar chain pattern D, it shows the pharmacokinetics of type 4.
  • the sugar chain presenting particles of the present invention profile the sugar chain of the cancer cell, for example, in the prevention or treatment of the primary tumor from the cancer cell to the metastasis site, and the cancer sugar chain is based on the obtained profile. Which of the patterns A to D can be determined, and sugar chain presentation particles having the selected sugar chain pattern can be prepared and provided by a known method.
  • the selected sugar chain pattern can be artificially modified by treating an existing sugar chain known to contain a specific sugar chain with, for example, an enzyme, and is carried out as an existing sugar chain.
  • sugar chains contained in egg whites of quail, chickens, ducks and the like and chicken egg yolks can be mentioned. It is also possible to use the sugar chain of the existing material as it is or by processing it as appropriate as a specific sugar chain pattern.
  • the sugar chain can be used as a material for providing a desired cancer sugar chain pattern by treating the sugar chain with an enzyme.
  • sugar chains of quail, chicken, duck egg white and chicken egg yolk used in Example 2 are safe and inexpensive biological materials having a clear sugar chain structure and expression level profile thereof, these sugar chains can be used as they are.
  • sugar chain pattern can be not only derived from a living body but also a mixture of artificially synthesized sugar chain components.
  • the artificial synthesis of the sugar chain component can be carried out by a known method.
  • the sugar chain can be presented on the particle surface by a known direct blotting method (Non-Patent Document 9).
  • the presentation of sugar chains on the particle surface is carried out, for example, by reacting the prepared nanosomes with a mixture containing cancer sugar chains by the methods shown in Examples 1 (C) and 2 (B).
  • Sugar chain presentation particles can be prepared.
  • the sugar chain on the surface of the nanoparticles can be a sulfide bond of an alkanethiol that presents the sugar chain represented by the following general formula (B).
  • n1 is an integer of 2 to 30
  • n2 is an integer of 2 to 30
  • the -S-terminal is a carrier site where a sulfide bond is attached to nanoparticles
  • R 10 is a sugar chain-containing site.
  • R 10 is a cancer oligosaccharide patterns, sugar or cancer cells, components of the sugar chain according to the sugar chain pattern A ⁇ D which are determined based on the sugar chain profile And their composition ratio.
  • sugar chain-presenting particles of the present invention can be schematically represented by, for example, the following general formula (10).
  • sugar chain-presenting particles of the present invention have pharmacokinetics according to each pattern of sugar chains, they can be accumulated in a specific tissue or organ by utilizing the properties. Therefore, by selecting the pattern of the sugar chain, it is possible to obtain sugar chain presentation particles that can be accumulated in a specific tissue or organ, and DDS can also be provided by utilizing this property.
  • the sugar chain presenting particles of the present invention can further have a drug site.
  • the drug at the drug site is not particularly limited, and may be, for example, an anticancer agent or an anti-inflammatory agent.
  • a drug-containing group represented by the general formula (C) can be supported on the surface of the nanoparticles as a drug site.
  • n1 is an integer of 2 to 30
  • n2 is an integer of 2 to 30
  • the -S-terminal is a carrier site where a sulfide bond is attached to nanoparticles
  • R 20 is a drug-containing site. is there.
  • n1 and n2 in the formula for the drug-containing group represented by the general formula (C) the distance between the drug-containing group and the nanoparticles can be maintained within a desired range.
  • n1 is an integer in the range of 2 to 30, preferably 5 to 16, and more preferably 4 to 15.
  • n2 is an integer in the range of 2 to 30, preferably 5 to 20, more preferably 7 to 15.
  • sugar chain-presenting particles of the present invention having a drug site can be schematically represented by, for example, the following general formula (11).
  • the residues supported on the nanoparticles (NP) of the general formula (11) are the sugar chain-containing group represented by the general formula (B), the phospholipid group represented by the general formula (A), and the general formula (A) from the top. It is a drug-containing group represented by (C).
  • the actual complex contains one or more sugar chain-containing groups represented by the general formula (B), phospholipid groups represented by the general formula (A), and drug-containing groups represented by the general formula (C). It is supported on nanoparticles NP as a sulfide conjugate.
  • the sugar chain-presenting particles of the present invention When the sugar chain-presenting particles of the present invention have a drug site, they include cancer preventive agents and therapeutic agents containing the sugar chain-presenting particles as an active ingredient.
  • the cancer preventive agent and the therapeutic agent of the present invention preferably have a cancer sugar chain pattern in which the sugar chain presenting particles capture the sugar chain derived from the cancer cell and present it as it is. It may be a sugar chain presentation particle having a cancer sugar chain pattern B, C or D determined based on the obtained profile. These sugar chain-presenting particles show type 2 to 4 pharmacokinetics according to each sugar chain pattern, and are useful for the prevention and treatment of specific metastatic cancers.
  • sugar chain presentation particles having cancer sugar chain pattern B it is useful for prevention and treatment of metastatic cancer to the liver and spleen.
  • sugar chain-presenting particles having a cancer sugar chain pattern C it is useful for the prevention and treatment of metastatic cancer to the axilla and supraclavicular lymph nodes.
  • sugar chain presenting particles having cancer sugar chain pattern D it is useful for prevention and treatment of metastatic cancer to organs such as lung, liver, spleen, brain and kidney.
  • the present invention includes a sugar chain presentation particle kit containing two or more types of sugar chain presentation particles having different cancer sugar chain patterns.
  • Each sugar chain presentation particle in the kit may be stored in a separate container or as a mixture in one container.
  • the sugar chain-presenting particles included in the kit are any of the above-mentioned sugar chain-presenting particles of the present invention.
  • the different cancer sugar chain patterns in the kit are any two or more sugar chain patterns of the above sugar chain patterns A to D.
  • the sugar chain patterns A to D are selected by determining the sugar chain pattern by, for example, profiling the sugar chains of cancer cells collected from a cancer patient.
  • the sugar chain presentation particles which are the sugar chain patterns are selected and used in the determined sugar chain pattern, but a plurality of sugar chain presentation particles may be used in combination depending on the type of cancer. Can also be used.
  • two types of sugar chain patterns B and C may be used in combination as sugar chain presentation particles that can be accumulated in both the liver and spleen (type 2) and the axilla and supraclavicular lymph nodes (type 3). Absent.
  • the sugar chain presentation particles can be sugar chain presentation particles further having a drug site.
  • the drug include a preventive drug and a therapeutic drug for canes.
  • the sugar chain-presenting particles of the present invention can be formulated by a method known to those skilled in the art, using the sugar chain-presenting particles as an active ingredient.
  • it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injectable suspension.
  • pharmacologically acceptable carriers or vehicles specifically sterile water or saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavors, excipients, vehicles, preservatives.
  • the sterile composition for injection can be formulated according to normal formulation practices using a vehicle such as distilled water for injection.
  • Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizers such as.
  • Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol and nonionic surfactants such as polysorbate 80 (TM), HCO-60 may be used in combination.
  • oily liquid examples include sesame oil and soybean oil, and benzyl benzoate and benzyl alcohol may be used in combination as solubilizing agents. It may also be blended with buffers such as phosphate buffers, sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol and antioxidants.
  • buffers such as phosphate buffers, sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol and antioxidants.
  • the prepared injection solution is usually filled in a suitable ampoule. Liposomes can also be used to encapsulate the drug for cell delivery.
  • the sugar chain presentation particles and the sugar chain presentation particle kit of the present invention are suitable for cancer patients or patients suspected of having cancer for the prevention or treatment of metastatic cancer to organs, depending on the purpose of use. It can be administered by the administration route.
  • the administration is oral or parenteral, preferably parenteral administration, and specific examples thereof include injection type, nasal administration type, pulmonary administration type, and transdermal administration type.
  • injection type for example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
  • the dose and administration method of the sugar chain presentation particles and the sugar chain presentation particle kit of the present invention can be appropriately selected depending on the age, weight, sex, nature or severity of the symptom to be treated, and the like.
  • the dose of the pharmaceutical composition containing the sugar chain-presenting particles and the sugar chain-presenting particle kit of the present invention can be selected, for example, in the range of 0.0001 mg to 1,000 mg per 1 kg of body weight at a time. Alternatively, the dose can be selected in the range of 0.01 to 100,000 mg / body per patient, but is not necessarily limited to these values.
  • the dose and administration method vary depending on the age, weight, sex, symptoms, etc. of the patient, but can be appropriately selected by the person concerned.
  • the present invention includes the above-mentioned method for producing sugar chain-presenting particles of the present invention.
  • the sugar chain-presenting particles of the present invention can be prepared by presenting a desired cancer sugar chain pattern on the surface of nanoparticles in which at least a part of the surface is coated with phospholipid. The method for preparing nanoparticles in which at least a part of the surface is coated with phospholipid will be described later.
  • the specific sugar chain pattern is a sugar chain pattern determined based on a profile obtained by profiling the sugar chain of cancer cells collected from a subject.
  • the sugar chain pattern determined based on the profile is a sugar chain pattern in which the type and content of the sugar chain component are the same as the profile, or a part of the type and content of the sugar chain component is the same as the profile. It may be either a sugar chain pattern.
  • the amount of some types of the sugar chain component contained in the profile can be reduced or deleted. More specifically, when the sugar chain components contained in the profile are classified into several types, the sugar chain components belonging to one or two or more types having a high content are contained, and the one or two or more types having a low content are included. It can be a sugar chain pattern in which the amount of the sugar chain component belonging to is reduced or deleted.
  • the sugar chain of a cancer cell is profiled, when the sugar chain component contained in the profile is classified into several types, the sugar chain component belonging to one or more types having a high content is contained as it is, and the content is high.
  • sugar chain components belonging to a small number of 1 or 2 or more types By reducing or deleting the amount of sugar chain components belonging to a small number of 1 or 2 or more types, it is possible to obtain a sugar chain pattern showing the same pharmacokinetics as the cancer sugar chain pattern derived from cancer cells in which the sugar chain is profiled. ..
  • the types of sugar chain components include terminal hymannose type sugar chain, terminal galactose type sugar chain, terminal N-acetylglucosamine type sugar chain, terminal ⁇ 2,6 sialic acid type sugar chain, and terminal ⁇ 2,3 sialic acid type sugar. You can raise a chain.
  • FIG. 1 shows a conceptual diagram of a method for producing sugar chain-presenting particles of the present invention (a method for producing an exosome model in which a sugar chain pattern of cancer cells is presented on the surface and its pharmacokinetics / organ-oriented in vivo imaging).
  • (A) is suitable for the basic structure of sugar chain presentation particles: alkanethiol having a phosphorylcholine group as a head on the surface of metal nanoparticles such as quantum dots, gold nanoparticles, and gold-coated magnetic nanoparticles as a core.
  • Anti-aggregating nanoparticles completely coated with a self-assembled monolayer of alcanthiol containing an amount of aminooxylinker.
  • (B) is the basic principle of the glycoblotting method, in which the reducing end of the sugar chain (compound containing an aldehyde group or a ketone group) and the aminooxy group on the surface of the nanoparticles specifically react to form an oxime bond.
  • (C) shows a method for presenting a cancer cell-derived sugar chain pattern on the surface of nanoparticles and in vivo imaging. A mixture containing sugar chains prepared from collected or cultured cancer cells is captured and presented on the surface of nanoparticles by a glycoblotting method utilizing a chemical reaction specific to sugar chains having a reducing end (equivalent to an aldehyde group). Let me.
  • the nanoparticles whose surface is coated with phospholipids are composed of a crosslinked precursor X represented by the following general formula (D), a phospholipid precursor represented by the following general formula (E), and colloidal nanoparticles. It can be carried out by mixing to obtain surface-modified nanoparticles in which the crosslinked precursor X and the phospholipid are supported on the surface of the nanoparticles. This method is described in Patent Document 1, and the entire description of Patent Document 1 is incorporated herein by reference in particular.
  • n1 is an integer of 2 to 30, and n2 is an integer of 2 to 30.
  • n3 is an integer in the range of 2 to 30.
  • n1 is an integer in the range of 2 to 30, preferably 5 to 20, and more preferably 7 to 15.
  • n2 is an integer in the range of 2 to 30, preferably 5 to 20, more preferably 7 to 15.
  • the general formula (E) is the same as the phospholipid pseudo substance group represented by the general formula (A) except that the terminal is an SH group.
  • n3 is an integer in the range of 2 to 30, preferably 5 to 20, and more preferably 7 to 15.
  • Both the crosslinked precursor X represented by the general formula (D) and the phospholipid pseudo substance precursor represented by the general formula (E) are commercially available and prepared by the method described in the references. You can also do it. (References: T. Ohyanagi, et. Al., J. Am. Chem. Soc. 2011, 133, 12507-12517)
  • the mixing ratio of the cross-linking precursor X represented by the general formula (D), the phospholipid pseudo substance precursor represented by the general formula (E), and the nanoparticles is the cross-linking precursor X represented by the general formula (D) and the general formula. It can be appropriately determined in consideration of the desired carrying amount of the phospholipid pseudo substance precursor represented by (E) with respect to the nanoparticles.
  • the crosslinked precursor X represented by the general formula (D) shown in the following scheme has n1 of 6, n2 of 9, and the phospholipid pseudomaterial precursor represented by the general formula (E) of n3. ..
  • the molar ratio AO / PC of the aminooxylinker (AO) to the phosphorylcholine linker (PC) is arbitrary and can be, for example, in the range of 100/1 to 1/100.
  • the ratio of the sugar chain structure-containing group represented by the general formula (B) to the phospholipid pseudomaterial group represented by the general formula (A) is, for example, a molar ratio in the range of 1: 100 to 100: 1. It is possible, and preferably in the range of 1:10 to 10: 1, so that the ratio can be similar to this.
  • the nanoparticles are the same as those described for the sugar chain presentation particles.
  • colloidal quantum dots QDs (QD: Quantum Dot)
  • QDs QD: Quantum Dot
  • Colloidal quantum dots have protecting groups on the surface of luminescent semiconductor nanoparticles with a diameter range of, for example, 1 to 20 nm.
  • the colloidal quantum dots shown in the above scheme have a trialkylphosphate group on the surface. Even when the nanoparticles are metal nanoparticles, colloidal metal nanoparticles can be used as a raw material. Colloidal quantum dots and colloidal metal nanoparticles are commercially available.
  • the desired cancer sugar chain pattern is presented on the surface of the obtained surface-modified nanoparticles.
  • the aminooxy group of the cross-linking precursor X represented by the general formula (D) introduced into the surface-modified nanoparticles obtained above has a reducing end (equivalent to an aldehyde group). It can be carried out by a glycoblotting method that specifically reacts with.
  • the sugar chain-presenting particles of the present invention having a drug site present or carry a desired cancer sugar chain pattern and drug-containing site on the surface of the surface-modified nanoparticles.
  • the sugar chain and the drug-containing site can be presented or supported sequentially or simultaneously.
  • the drug-containing site can be supported.
  • the drug-containing precursor Z represented by the following general formula (G) is mixed with the surface-modified nanoparticles presenting the sugar chain and linked to the cross-linked precursor X on the surface-modified nanoparticles, and the general formula (C) is used. It can be obtained by further forming the indicated drug-containing groups.
  • R 20 is a drug-containing site.
  • the sugar chain-containing site and the drug-containing site in the production method of the present invention have the same meaning as the sugar chain-containing site and the drug-containing site in the particles of the present invention, respectively.
  • the drug-containing precursor Z represented by the general formula (G) can be synthesized, for example, according to the method described in Patent Document 1 (WO2017 / 131242A1 / US2020138973 (A1)).
  • the present invention includes profiling sugar chains on the surface of cancer cells from cancer cells collected from a subject and determining a sugar chain pattern based on the profiled sugar chains.
  • the profile of sugar chains on the surface of cancer cells can be performed by a known analytical method as described above.
  • the obtained sugar chain pattern was determined by the profiled sugar chain.
  • Sugar chain pattern A whose pharmacokinetics mainly depend on terminal high mannose type sugar chains
  • Sugar chain pattern B whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains
  • Sugar chain pattern C whose pharmacokinetics mainly depend on terminal ⁇ 2,6 sialic acid type sugar chains
  • Sugar chain pattern D whose pharmacokinetics mainly depend on terminal ⁇ 2,3 sialic acid type sugar chains This can be done by specifying which sugar chain pattern is selected from the group consisting of.
  • the sugar chain pattern is, for example, In the cancer sugar chain pattern A, 45 mol% or more of the sugar chains are terminal hymannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%. In the cancer sugar chain pattern B, the terminal hymannose type sugar chain of the sugar chain is less than 45%, and the terminal sialic acid type sugar chain is 0% or more and less than 2%. In the cancer sugar chain pattern C, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal ⁇ 2,6 sialic acid type sugar chains is the content of the terminal ⁇ 2,3 sialic acid type sugar chains.
  • the cancer sugar chain pattern D 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal ⁇ 2,3 sialic acid type sugar chains is the content of the terminal ⁇ 2,6 sialic acid type sugar chains. More than the amount.
  • the determined sugar chain pattern when the determined sugar chain pattern is sugar chain pattern A, it shows type 1 pharmacokinetics, suggesting that the tendency of cancer cells to metastasize is low.
  • the determined sugar chain pattern when the determined sugar chain pattern is sugar chain pattern B, it shows type 2 pharmacokinetics, suggesting that cancer cells tend to metastasize to the liver and spleen.
  • the determined glycan pattern When the determined glycan pattern is glycan pattern C, it exhibits type 3 pharmacokinetics, suggesting that cancer cells are prone to metastasis to the axilla and supraclavicular lymph nodes.
  • the determined glycan pattern When the determined glycan pattern is glycan pattern D, it exhibits type 4 pharmacokinetics, suggesting that cancer cells are prone to metastasis to lung, liver, spleen, brain and kidney.
  • the sugar chain pattern of cancer cells possessed by the subject is a sugar chain pattern exhibiting any of the pharmacokinetics of types 1 to 4.
  • medical personnel can diagnose the metastasis of the targeted cancer cells.
  • Example 1 Profile of post-translational sugar chain modification status (sugar chain expression pattern and their expression level) of all proteins present in cancer cells and on the cell membrane surface
  • Glycan profile of human cancer cells By using 4 types of cultured human cancer cells (MCF7, MDA-MB-231, A549, HepG2), about 5 x 10 5 each (about 100 ⁇ g as total protein amount), the total sugar of these cancer cells
  • the chain profile was analyzed in detail according to the general protocol of cell glycoblotting (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537) (Fig. 2a).
  • Fig. 2a the general protocol of cell glycoblotting
  • Fig. 2b the relationship between their expression levels and the main structural motifs was clarified by glicotyping analysis using the internal standard compound as an index
  • PC-SH 11-mercaptoundecylphosphorylcholine
  • AOHEG-SH 1,11'-dithio bis
  • quantum dots 200 ⁇ L, 1 ⁇ M TOPO-coated QD800 in decane
  • a mixed solvent of MeOH (200 ⁇ L) and i-PrOH (400 ⁇ L) are added to a mixed solvent of MeOH (200 ⁇ L) and i-PrOH (400 ⁇ L), and the mixture is centrifuged at room temperature for 5 minutes to obtain TOPO-coated QD800. Insolubilize and precipitate to remove the supernatant.
  • TOPO-coated QD800 was solubilized by adding n-hexane (400 ⁇ L) to the residue, and PC-SH (32 ⁇ L, 100 mM / MeOH, Medicinal Chemistry Pharmaceuticals), AOHEG-SH (2 ⁇ L, 10 mM / MilliQ,) were added to this solution.
  • Cancer sugar chain pattern presentation nanoparticles are separated by ultrafiltration (YM50, Thermo Fischer), washed with Milli Q (500 ⁇ L), dissolved in physiological saline (200 ⁇ L, 0.9% NaCl aqueous solution) and used for animal experiments. ..
  • the average particle size of the obtained cancer sugar chain pattern-presenting nanoparticles was 15.1 to 28.0 nm as a result of measurement with a fiber optical dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Denshi).
  • cancer sugar chain pattern-presenting nanoparticles (sugar chain-presenting particles)
  • Cancer sugar chain pattern-presenting nanoparticles (100 ⁇ L, 1 ⁇ M / 0.9% NaCl aqueous solution) were intravenously administered to mice aged 5 weeks or older (male, BALB / c).
  • the in vivo dynamics of nanoparticles up to 3 hours after administration were observed in real time using a near-infrared fluorescence spectrum using an IVIS imaging system (Summit Pharmaceuticals International) (exposure time 1 sec, excitation wavelength 710 nm, detection wavelength 820 nm). ..
  • the mice were dissected and the main organs were removed and the fluorescence intensity of each was observed.
  • the pharmacokinetics of the cancer sugar chain pattern-presenting nanoparticles is described as the pharmacokinetics of the movement and excretion of the particles in the body after administration, and each organ of the particles.
  • the distribution and accumulation state in tissues are described as organ-oriented.
  • Nanoparticles (GNS-MCF7, GNS-MDA-MB-231, GNS-A549, GNS-HepG2) that presented sugar chains of four types of cultured human cancer cells (MCF7, MDA-MB-231, A549, HepG2)
  • Fig. 3 shows the pharmacokinetics of each of the controls up to 3 hours after intravenous administration of mice and the distribution state in each organ when dissected 3 hours later.
  • GNS-MCF7 As shown in FIGS. 2a and 2b, as sugar chain components, about 94% of the total sugar chains are 7 types of high mannose type sugar chains and about 6% are sugar chain patterns containing 2 types of sugar chains having galactose ends.
  • MCF7 which is a non-metastatic breast cancer cell
  • Fig. 3a Almost no accumulation in organs was observed in the mice dissected after 3 hours, and a part of them was mixed in the feces in the gastrointestinal tract (Fig. 3b).
  • the sugar chain-presenting particles presenting the high-mannose-type sugar chain (oligosaccharide having only mannose at the end) of the sugar chain pattern A have an excretion mechanism to the outside of the body and exhibit type 1 pharmacokinetics. It was revealed.
  • GNS- MDA-MB-231 As sugar chain components, a sugar chain pattern containing 5 types of double- or triple-chain terminal sialic acid-type sugar chains at 18% of the total sugar chains and 5 types of high-mannose-type sugar chains at 82% (Figs. 2a and 2b). ), And when the sugar chain pattern of MDA-MB-231, which is a metastatic breast cancer cell, is presented, it is completely different from the pharmacokinetics of GNS-MCF7, and it reaches the axillary and supraclavicular lymph nodes 3 hours after intravenous administration. Accumulation was particularly remarkable (indicated by arrows), showing type 3 pharmacokinetics.
  • GNS-A549 As sugar chain components, 5 types of double- or triple-chain terminal sialic acid-type sugar chains are 19% of the total sugar chains, sugar chains having a galactose terminal are 11%, and sugar chains having N-acetylglucosamine at the ends. It is a sugar chain pattern containing 8% and 62% of 7 types of high mannose type sugar chains (Figs. 2a and 2b). When the sugar chain pattern of A549 is presented, it is widely spread throughout the body even 3 hours after intravenous administration. It is distributed, and the accumulation in the lung, liver, and spleen is very remarkable, and the distribution can be confirmed in the brain and kidney, showing type 4 pharmacokinetics.
  • GNS-A549 which accounts for 19% of the total sugar chain of Neu5Ac ⁇ 2,6Gal or Neu5Ac ⁇ 2,3Gal units, clearly accumulates more in the lungs, liver, and spleen than 6% GNS-HepG2.
  • the organ directivity (accumulation) 3 hours after administration of nanoparticles presenting these two types of cancer cell-derived sugar chains was higher in the case of GNS-HepG2 than in the case of GNS-MDA-MB-231.
  • Example 2 Sugar chain presentation particles MDA-MB-231 cells and A549 cells having an artificially prepared sugar chain pattern with different binding modes of terminal sialic acid and galactose ( ⁇ 2,6 bond and ⁇ 2,3 bond) and All HepG2 cells are human cancer cells that significantly express double- or triple-chain terminal sialic acid-type sugar chains as sugar chain components.
  • the pharmacokinetics and organ orientation were different for MDA-MB-231 cells, A549 cells and HepG2 cells.
  • the binding mode of sialic acid and galactose determines the difference in the pharmacokinetics of these sugar chain pattern-presenting nanoparticles after intravenous administration. It was. Therefore, in this example, nanoparticles in which only one of the individual terminal sialic acid-type sugar chains linked in advance by ⁇ 2,6 bond and ⁇ 2,3 bond are artificially presented are prepared, and the pharmacokinetics and organs of each are prepared. The directivity was examined, and the difference in pharmacokinetics due to the difference in terminal sialic acid type sugar chains was confirmed.
  • the non-reducing end which is the main sugar chain in the egg white of Ruddy duck, is a multi-branched sugar chain structure of galactose
  • two types of sialic acid transferases with known substrate specificity Pasteurella multocida-derived ⁇ 2,3- (N) -Sialyltransferase and human ⁇ 2,6- (N) -sialyltransferase added sialic acid to terminal galactose and modified it to induce a sugar chain pattern containing only either Neu5Ac ⁇ 2,3Gal or Neu5Ac ⁇ 2,6Gal (Fig. 4a). ..
  • Ruddy duck egg white (17 mg / 50 ⁇ L in Milli Q, equivalent to about 1 mM LacNAc unit), CMP-Neu5Ac (10 ⁇ L, 200 mM / milliQ, Yamasa), ⁇ 2,3- (N) -sialyltransferase (5 ⁇ L, 1000 mU / mL, Sigma Aldrich), HEPES-NaOH buffer (10 ⁇ L, 1 M / milliQ, pH 8.0) and milliQ (10 ⁇ L) are added to bring the total volume to 100 ⁇ L, and the mixture is reacted at 37 ° C. for 20 hours.
  • Ruddy duck egg white (17 mg / 50 ⁇ L in Milli Q, equivalent to about 1 mM LacNAc unit), CMP-Neu5Ac (10 ⁇ L, 200 mM / milliQ, Yamasa), ⁇ 2,6- (N) -sialyltransferase (10 ⁇ L, 516 mU / mL, Medicinal Chemistry Pharmaceuticals), phosphate buffer (10 ⁇ L, 1 M / milliQ, pH6.5) and milliQ (10 ⁇ L) are added to make a total volume of 100 ⁇ L, and the mixture is reacted at 37 ° C. for 20 hours.
  • step (1-2) Sugar chain profiles of Button Quail, Chicken, Ruddy duck egg white protein and sialic acid-modified Ruddy duck egg white :
  • step (A) the equivalent amount of 500 ⁇ g of 5 kinds of egg white / sialic acid-modified egg white total protein equivalent was added to 1,4-dithithreiol (DTT, 20 ⁇ L, 120 mM / MilliQ) at 60 ° C. for 30 minutes, and then iodoacetamide (IAA, Reaction with 40 ⁇ L, 123 mM / MilliQ) in a cool dark place at room temperature for 1 hour.
  • DTT 1,4-dithithreiol
  • IAA iodoacetamide
  • Ruddy duck egg white also contains no sialic acid as a sugar chain component, and 3 to 5 multi-branched sugar chains with galactose residues at the non-reducing ends account for about 65% of the total. It was confirmed that the rest contained about 25% of high mannose type sugar chains and about 10% of sugar chains ending with N-acetylglucosamine. It was also revealed that there are no fucose residues in the sugar chains of these three species of bird egg white.
  • Ruddy duck egg whites are treated with ⁇ 2,3- (N) -sialyltransferase (recombinant Pasteurella multocida) and ⁇ 2,6- (N) -sialyltransferase (recombinant human) in the presence of CMP-sialic acid according to the scheme shown in Fig. 4a. 2,3S-Ruddy duck and 2,6S-Ruddy duck with a large amount of acid added could be induced.
  • Sugar chain pattern presentation nanoparticles were prepared. The average particle size of the obtained sugar chain pattern-presenting nanoparticles was 14.4 to 24.4 nm as a result of measurement with a fiber optical dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Denshi).
  • Nanometers presenting these sugar chains were intravenously administered to mice by the method described in Example 1 (2-4) to examine their pharmacokinetics and organ directivity with a near-infrared fluorescence microscope (exposure time 1 sec, It was observed at an excitation wavelength of 710 nm and a detection wavelength of 820 nm) (Figs. 5a to 5c).
  • the sugar chain structure and its profile are clear, and the sugar chain pattern obtained by modifying the sugar chains of quail, chicken, duck egg white and chicken egg yolk, which are safe and inexpensive biological materials. It can be seen that the sugar chain presenting particles of the present invention can be produced by presenting quail on nanoparticles.
  • GNS-ButtonQuail prepared from ButtonQuail egg white, in which high mannose-type sugar chains account for about 50% of the total, has a slightly prolonged retention in mice (Fig. 5b).
  • the pharmacokinetics after 3 hours (Fig. 5c) are very similar to those of GNS-MCF7 (Fig. 3c), and most of them are excreted outside the body (type 1 pharmacokinetics).
  • GNS-Chicken and GNS-Ruddy duck which presented the sugar chain pattern of Chicken and Ruddy duck, have a remarkably high degree of accumulation in the liver and spleen (Figs. 5b and 5c) (type 2 pharmacokinetics).
  • GNS-Chicken and GNS-Ruddy duck sialic acid-free galactose and N-acetylglucosamine-terminated major sugar chains with a high degree of branching and mammalian liver lectins (mammalian hepatic) that are highly expressed in these organs and cells It seems that it largely depends on the specific interaction with the asialoglycoprotein receptor represented by lectin) (for example, Y.C. Lee et al., Acc. Chem. Res. 1997, 28, 321-327). etc).
  • Ruddy duck GNS-2 prepared from two types of nanoparticles (GNS-2,3S-Ruddy duck and GNS-2,6S-Ruddy duck) and chicken egg yellow sugar peptide showing a sugar chain pattern derived from egg white.
  • the main terminal sialic acid type sugar chain of MDA-MB-231 cells is a double or triple chain type sugar chain containing Neu5Ac ⁇ 2,6Gal unit at the end. Neu5Ac ⁇ 2,6Gal unit on the surface of the exosome membrane due to the difference between GNS-MCF7 (Figs. 3b and 3c) and GNS-Button Quail's pharmacokinetics and organ orientation (Figs.
  • GNS-2,3S-Ruddy duck pharmacokinetics / organ orientation Figs. 5b and 5c
  • GNS-A549 and GNS-HepG2 are very similar (type 4 pharmacokinetics), so A549 cells.
  • the major terminal sialic acid-type sugar chain structure of HepG2 cells is a double-stranded or triple-chain complex sugar chain containing Neu5Ac ⁇ 2,3Gal unit at the end, and the organs of these two cancer cell-derived exosome models. Due to the difference in directivity (Fig.
  • Example 3 In Example 2, the ratio of the terminal sialic acid-type sugar chain component and the hymannose-type sugar chain component whose binding position to galactose was controlled by enzymatic modification of the egg white sugar chain of Ruddy duck to the total sugar chain was the pharmacokinetics and organs. It became clear that it has a great influence on the directionality. Therefore, the content of high mannose type sugar chains is about 70%, which is even higher than Button Quail egg white (50%), and the sugar chain pattern is simpler.
  • Japanese Quail egg white-derived sugar chains S.-I. Nishimura et al. , J. Agri. Food Chem.
  • Nanoparticles (GNSs) were prepared. Nanoparticles presenting an artificially prepared sugar chain pattern were prepared by mixing two types of sugar chains (patterns).
  • the present invention can construct sugar chain-presenting particles derived from cancer cells, and these particles are useful for the prevention and treatment of cancer.

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Abstract

The present invention relates to sugar chain-presenting particles which are nanoparticles having sugar chains on the surfaces thereof, wherein: (1) the average particle diameter of the sugar chain-presenting particles is in a range of 10-100 nm; (2) at least a portion of the surfaces of the nanoparticles is coated with phospholipids; and (3) the sugar chains on the surfaces of the nanoparticles have sugar chain patterns derived from a cancer cell or sugar chain patterns (hereinafter, referred to as cancer sugar chain patterns) determined on the basis of the profile of this sugar chain. The present invention relates to a method for determining sugar chain patterns of a cancer cell, the method comprising profiling sugar chains of the cancer cell from a cancer cell taken from a subject, and determining sugar chain patterns on the basis of the profiled sugar chains. According to the present invention, sugar chain-presenting particles are constructed which are tools for transferring information between cells, and a method for using the same is provided.

Description

糖鎖提示粒子およびその製造方法Sugar chain presentation particles and their production method
　本発明は、エキソソーム(Exosome)を模倣した糖鎖提示粒子およびその製造方法、その利用方法に関する。本発明は、がん糖鎖パターンを粒子表面に提示する糖鎖提示粒子およびその製造方法に関する。
関連出願の相互参照
　本出願は、２０１９年９月２０日出願の日本特願２０１９－１７１１３５号の優先権を主張し、その全記載は、ここに特に開示として援用される。 The present invention relates to sugar chain-presenting particles that mimic exosomes, a method for producing the same, and a method for using the same. The present invention relates to sugar chain-presenting particles that present a cancer sugar chain pattern on the particle surface and a method for producing the same.
Cross-reference to related applications This application claims the priority of Japanese Patent Application No. 2019-171135 filed on September 20, 2019, the entire description of which is incorporated herein by reference in particular.
　医薬品の治療効果を高めるためにはその疾患の原因となる、あるいは病態の増悪因子となる標的分子が高発現する特定の臓器や組織にその医薬品を選択的に送達する技術（ドラッグデリバリーシステム、以下DDSと略す）が重要である。現在、PEG修飾リポソームを活用した抗腫瘍ナノ医薬（anticancer nanomedicine）が承認・上市されている。これらのナノ医薬の薬効は基本的にがん組織において顕著なEPR（enhanced permeability and retention）効果に依存するとされてきたが、そのEPR効果はがんの種類、部位、進行度、さらにがん細胞と周辺の間質（細胞外マトリックス）を構成する様々な接着分子など多くの因子によって大きく影響されることが明らかとなっている（非特許文献１、２など）。EPR効果に有利な、より高度な分子設計が可能な無機・金属ナノ微粒子（金ナノ微粒子、量子ドット、シリカ系微粒子等をコアとする）をポリエチレングリコール（PEG）修飾化合物等により被覆した様々な人工細胞外微粒子によるDDSの研究開発が活発に進められてきた。 In order to enhance the therapeutic effect of a drug, a technology for selectively delivering the drug to a specific organ or tissue in which a target molecule that causes the disease or is an exacerbating factor of the pathological condition is highly expressed (drug delivery system, hereinafter DDS) is important. Currently, anti-tumor nanomedicine utilizing PEG-modified liposomes has been approved and put on the market. It has been said that the efficacy of these nanopharmaceuticals basically depends on the remarkable EPR (enhanced performance and retention) effect in cancer tissues, but the EPR effect depends on the type, site, progression, and cancer cells of the cancer. It has been clarified that it is greatly influenced by many factors such as various adhesion molecules constituting the stroma (extracellular matrix) and the surrounding stroma ( Non-Patent Documents 1, 2 and the like). Various inorganic / metal nanoparticles (cores of gold nanoparticles, quantum dots, silica-based fine particles, etc.) capable of more advanced molecular design, which are advantageous for the EPR effect, coated with polyethylene glycol (PEG) -modified compounds, etc. Research and development of DDS using artificial extracellular particles has been actively promoted.
　しかし、最近これらのリポソームや金属ナノ微粒子を含むほとんどのナノ医薬が実際には投与した実験動物の生体内で期待したような体内動態（例えば、がん組織での取り込み能）を実現できていなかったという調査結果が報告されている（非特許文献３）。驚いたことに、過去１０年間に発表された２３２篇の論文を対象にした調査では標的としたがん組織に到達したナノ医薬は投与量の約０．７％（平均値）に過ぎなかったという。血管内に投与したほとんどのナノ微粒子型医薬品は速やかに肝臓に集積してマクロファージなどの貪食細胞に取り込まれて分解・***されるというが、これは血液中のアルブミンをはじめとする様々なタンパク質の非特異吸着により形成するナノ微粒子表面の「タンパク質コロナ」の性質に大きく依存するという（非特許文献４など）。
　一方、個々のがん細胞の浸潤・転移、さらに臓器指向性（転移先）はがん細胞由来のエキソソーム（細胞外ナノ微粒子）が決定しているという最近の多くの報告を根拠としてがん細胞由来エキソソームの研究が盛んに行われており、エキソソーム及び内包される様々なマイクロRNAの機能を利用する新たな医薬品開発を指向する事例も見られる（例えば、非特許文献５）。 However, recently, most nanopharmaceuticals containing these liposomes and metal nanoparticles have not been able to achieve the pharmacokinetics (for example, ability to be taken up in cancer tissues) expected in vivo of experimental animals to which they have actually been administered. The results of the investigation have been reported (Non-Patent Document 3). Surprisingly, in a survey of 232 papers published in the last decade, only about 0.7% (mean) of the dose of nanopharmaceutical reached the targeted cancer tissue. That is. Most nanoparticulate drugs administered intravascularly are said to rapidly accumulate in the liver, be taken up by phagocytic cells such as macrophages, decompose and excrete, and this is due to various proteins such as albumin in the blood. It is said that it largely depends on the properties of "protein corona" on the surface of nanoparticles formed by non-specific adsorption (Non-Patent Document 4, etc.).
On the other hand, cancer cells are based on many recent reports that cancer cell-derived exosomes (extracellular nanoparticles) determine the infiltration / metastasis of individual cancer cells and the organ orientation (metastasis destination). Research on exosomes of origin has been actively conducted, and there are cases in which new drug developments that utilize the functions of exosomes and various contained microRNAs are aimed at (for example, Non-Patent Document 5).
　エキソソームは、細胞内に形成された膜小胞が、細胞外に放出されたものであり、細胞間の情報伝達のツールとして機能している。しかし、エキソソームのサイズや積載分子の不均一性、さらにがん細胞・組織に由来するエキソソームの精製が困難なため具体的な分子レベルでの臓器指向性メカニズムについてはほとんど不明である。 Exosomes are membrane vesicles formed inside cells that are released extracellularly, and function as a tool for signal transmission between cells. However, the size of exosomes, the heterogeneity of loaded molecules, and the difficulty in purifying exosomes derived from cancer cells / tissues make it almost unclear about the specific mechanism of organ orientation at the molecular level.
特許文献１：WO2017/131242A1/US2020138973(A1) Patent Document 1: WO2017 / 131242A1 / US2020138973 (A1)
非特許文献１：Prabhakar et al., Cancer Res. 2013, 73, 2412-2417
非特許文献２：Danhier, J. Control. Release 2016, 244, 108-121
非特許文献３：W. C. W. Chan et al., Nat. Mater. 2016, 1, 1-12
非特許文献４：K. A. Dawson et al., Nat. Biotech. 2012, 7, 779-786
非特許文献５：R. Kalluri, J. Clin. Invest. 2016, 126, 1208-1215
非特許文献６：S.-I. Nishimura et al., J. Am. Chem. Soc. 2011, 133, 12507-12517
非特許文献７：S.-I. Nishimura et al., ACS Chem. Biol. 2015, 10, 2073-2086;
非特許文献８：M. Colombo, et al., Annu. Rev. Cell Dev. Biol. 2014, 30, 255-289
非特許文献９：S.-I. Nishimura et al., Angew. Chem. Int. Engl. Ed. 2005, 44, 91-96
特許文献１及び非特許文献１～９の全記載は、ここに特に開示として援用される。 Non-Patent Document 1: Prabhakar et al., Cancer Res. 2013, 73, 2412-2417
Non-Patent Document 2: Danhier, J. Control. Release 2016, 244, 108-121
Non-Patent Document 3: W.C.W. Chan et al., Nat. Mater. 2016, 1, 1-12
Non-Patent Document 4: K. A. Dawson et al., Nat. Biotech. 2012, 7, 779-786
Non-Patent Document 5: R. Kalluri, J. Clin. Invest. 2016, 126, 1208-1215
Non-Patent Document 6: S.-I. Nishimura et al., J. Am. Chem. Soc. 2011, 133, 12507-12517
Non-Patent Document 7: S.-I. Nishimura et al., ACS Chem. Biol. 2015, 10, 2073-2086;
Non-Patent Document 8: M. Colombo, et al., Annu. Rev. Cell Dev. Biol. 2014, 30, 255-289
Non-Patent Document 9: S.-I. Nishimura et al., Angew. Chem. Int. Engl. Ed. 2005, 44, 91-96
The entire description of Patent Document 1 and Non-Patent Documents 1 to 9 is incorporated herein by reference in particular.
　血中には多くの細胞から放出されたエキソソームが存在し、例えば、がん患者において、がん細胞・組織に由来するエキソソームのみ精製することが困難なため具体的な分子レベルでの臓器指向性メカニズムについてはほとんど不明であった。 Exosomes released from many cells are present in the blood. For example, in cancer patients, it is difficult to purify only exosomes derived from cancer cells / tissues, so organ orientation at a specific molecular level. Little was known about the mechanism.
　本発明の目的は、細胞間の情報伝達のツールであるエキソソームを模倣した粒子を人工的に構築することにある。そして、情報の送り手から放出されるエキソソームの体内動態を利用する新しい技術を提供することにある。 An object of the present invention is to artificially construct particles that imitate exosomes, which are tools for information transmission between cells. The purpose is to provide a new technology that utilizes the pharmacokinetics of exosomes released from information senders.
　より具体的には、がん細胞由来エキソソームのモデルとして極めて有効であり、転移機能解析を対象とする種々の分子を担持した糖鎖提示粒子の体内投与後の動態や臓器指向性の予測法の提供、さらにヒトでの臨床を可能とする、がん転移予防や治療のための新たなナノ微粒子ＤＤＳの開発を実現するための技術の提供を目的とする。さらに本発明は、がん細胞の転移性を示唆することができる糖鎖パターンの決定方法も提供することを目的とする。 More specifically, it is extremely effective as a model for cancer cell-derived exosomes, and is a method for predicting the dynamics and organ orientation of sugar chain-presenting particles carrying various molecules for metastatic function analysis after in-vivo administration. The purpose of this study is to provide technology for realizing the development of new nanoparticle DDS for cancer metastasis prevention and treatment, which enables clinical practice in humans. Furthermore, it is an object of the present invention to provide a method for determining a sugar chain pattern that can suggest metastasis of cancer cells.
　本発明は、エキソソームの表面が、これを放出する細胞の糖鎖と同様のパターンにて糖鎖を提示していることに着目した。すなわち、例えば、がん細胞の転移の場合、がん細胞から放出されるエキソソームが、がん細胞の体内動態を導く先導的な役割を果たすと考えられる。エキソソームを取り込む様々な細胞や臓器は最初にエキソソーム膜表面の嵩高い糖衣（Ｇｌｙｃｏｃａｌｉｘ）と遭遇する可能性が高い。本発明では、この事実に着目して、がん細胞由来のエキソソームにおける体内動態、特に体内での移動及び排出並びに臓器や組織への分布あるいは集積（臓器指向性）はエキソソーム膜表面の糖鎖パターンによって先導および決定されていることを見出し、本発明を完成するに至った。 The present invention focused on the fact that the surface of an exosome presents sugar chains in a pattern similar to that of cells that release them. That is, for example, in the case of metastasis of cancer cells, exosomes released from cancer cells are considered to play a leading role in guiding the pharmacokinetics of cancer cells. Various cells and organs that take up exosomes are likely to first encounter bulky glycocalyx on the surface of the exosome membrane. In the present invention, focusing on this fact, the pharmacokinetics of cancer cell-derived exosomes, particularly migration and excretion in the body, and distribution or accumulation in organs and tissues (organ orientation) are sugar chain patterns on the surface of exosome membranes. It was found that the invention was led and determined by the above, and the present invention was completed.
　具体的には、ヒト培養がん細胞のN型糖鎖をグライコブロッティング法により全ての糖鎖をナノソーム膜表面に提示することでヒトがん細胞由来エキソソーム模倣微粒子を作製でき、これらを直接マウスに静脈内投与して体内動態をリアルタイムで観察することで、ヒトがん細胞由来エキソソームの体内動態と臓器指向性が簡単に判定できることが明らかとなった。さらに、その結果をもとに人工的に作製したがん特異的糖鎖パターンを提示したエキソソームを模倣した粒子が投与後に意図した通りの体内動態を再現したことから本技術により予め個々のがん転移における臓器指向性に適応したテイラーメイドDDSを分子設計するという新しい概念に基づく創薬が可能となることが実証でき、本発明を完成した。 Specifically, human cancer cell-derived exosome mimicking fine particles can be produced by presenting all sugar chains of N-type sugar chains of cultured human cancer cells on the surface of nanosome membrane by the glycoblotting method, and these can be directly used in mice. By intravenously administering and observing the pharmacokinetics in real time, it became clear that the pharmacokinetics and organ orientation of human cancer cell-derived exosomes can be easily determined. Furthermore, since particles imitating exosomes that presented an artificially prepared cancer-specific sugar chain pattern based on the results reproduced the intended pharmacokinetics after administration, this technology was used in advance for individual cancers. We have completed the present invention by demonstrating that drug discovery based on the new concept of molecular design of tailor-made DDS adapted to organ orientation in metastasis is possible.
　本発明は以下の通りである。
［１］
糖鎖を表面に有するナノ粒子である糖鎖提示粒子であって、
（１）糖鎖提示粒子の平均粒子径は１０～１００ｎｍの範囲であり、
（２）ナノ粒子の表面の少なくとも一部はリン脂質で被覆されており、
（３）ナノ粒子表面に有する糖鎖は、がん細胞依頼の糖鎖パターンまたはこの糖鎖のプロファイルに基づいて決定された糖鎖パターン(以下、がん糖鎖パターンと呼ぶ)である、
糖鎖提示粒子。
［２］
がん糖鎖パターンは、
（１）体内動態が主に末端ハイマンノース型糖鎖に依存する糖鎖パターンＡ、
（２）体内動態が主に末端ガラクトース型糖鎖又は末端N-アセチルグルコサミン型糖鎖に依存する糖鎖パターンＢ、
（３）体内動態が主に末端α２，６シアル酸型糖鎖に依存する糖鎖パターンＣ、
（４）体内動態が主に末端α２，３シアル酸型糖鎖に依存する糖鎖パターンＤからなる群から選択される糖鎖パターンである、［１］に記載の糖鎖提示粒子。
［３］
がん糖鎖パターンＡは、糖鎖の４５モル％以上が末端ハイマンノース型糖鎖であり、末端シアル酸型糖鎖は０％以上、２％未満であり、
がん糖鎖パターンＢは、糖鎖の末端ハイマンノース型糖鎖が４５％未満であり、かつ末端シアル酸型糖鎖が０％以上、２％未満であり、
がん糖鎖パターンＣは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつ末端α２，６シアル酸型糖鎖の含有量が末端α２，３シアル酸型糖鎖の含有量より多く、
がん糖鎖パターンＤは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつα２，３シアル酸型糖鎖の含有量がα２，６シアル酸型糖鎖の含有量より多い、［２］に記載の糖鎖提示粒子。
［４］
ナノ粒子表面の少なくとも一部を被覆するリン脂質は、ホスホリルコリン基を有するアルカンチオールのスルフィド結合体であり、ナノ粒子表面に有する糖鎖は、糖鎖を固定化したアルカンチオールのスルフィド結合体であり、ナノ粒子表面はホスホリルコリン基を有するアルカンチオールのスルフィド結合体および糖鎖を固定化したアルカンチオールのスルフィド結合体の単分子膜で被覆されたナノ粒子である［１］～［３］のいずれかに記載の糖鎖提示粒子。
［５］
前記ホスホリルコリン基を有するアルカンチオールのスルフィド結合体が、下記一般式（Ａ）で示され、
Figure JPOXMLDOC01-appb-C000006
 （一般式（Ａ）中、ｎ３は２～３０の範囲の整数であり、－Ｓ－末端がナノ粒子にスルフィド結合する担持部位である。）
前記糖鎖を固定化したアルカンチオールのスルフィド結合体が、下記一般式（Ｂ）で示される、［４］に記載の糖鎖提示粒子。
Figure JPOXMLDOC01-appb-C000007
 （一般式（Ｂ）中、ｎ１は２～３０の整数であり、ｎ２は２～３０の整数であり、－Ｓ－末端がナノ粒子にスルフィド結合する担持部位であり、Ｒ１０は糖鎖含有部位である。）
［６］
薬剤部位をさらに有する、［１］～［５］のいずれか１項に記載の糖鎖提示粒子。
［７］
前記単分子膜は、下記一般式（Ｃ）で示される、薬剤部位を有するアルカンチオールのスルフィド結合体をさらに含む、［４］または［５］に記載の糖鎖提示粒子。
Figure JPOXMLDOC01-appb-C000008
 一般式（Ｃ）中、ｎ１は２～３０の整数であり、ｎ２は２～３０の整数であり、－Ｓ－末端がナノ粒子にスルフィド結合する担持部位であり、Ｒ２０は薬剤含有部位である。
［８］
異なるがん糖鎖パターンを有する２種類以上の糖鎖提示粒子を含有し、糖鎖提示粒子は、［１］～［７］のいずれか１項に記載の糖鎖提示粒子である、糖鎖提示粒子キット。
［９］
異なるがん糖鎖パターンが、［２］に記載のパターンＡ～Ｄのいずれか２種以上の糖鎖パターンである、［８］に記載の糖鎖提示粒子キット。
［１０］
糖鎖提示粒子は、［６］または［７］に記載の薬物部位をさらに有する糖鎖提示粒子である、［８］に記載の糖鎖提示粒子キット。
［１１］
［６］または［７］に記載の糖鎖提示粒子を有効成分として含有する、がん転移予防薬。
［１２］
［６］または［７］に記載の糖鎖提示粒子を有効成分として含有する、がん治療薬。
［１３］
被検者から採取したがん細胞から、がん細胞の糖鎖をプロファイルし、プロファイルした糖鎖に基づいて糖鎖パターンを決定することを含む、がん細胞の糖鎖パターン決定方法。
［１４］
糖鎖パターンの決定は、プロファイルした糖鎖が、
（１）体内動態が主に末端ハイマンノース型糖鎖に依存する糖鎖パターンＡ、
（２）体内動態が主に末端ガラクトース型糖鎖又は末端N-アセチルグルコサミン型糖鎖に依存する糖鎖パターンＢ、
（３）体内動態が主に末端α２，６シアル酸型糖鎖に依存する糖鎖パターンＣ、及び
（４）体内動態が主に末端α２，３シアル酸型糖鎖に依存する糖鎖パターンＤからなる群から選択される何れの糖鎖パターンであるかを特定することで行う、［１３］に記載の決定方法。
［１５］
がん糖鎖パターンＡは、糖鎖の４５モル％以上が末端ハイマンノース型糖鎖であり、末端シアル酸型糖鎖は０％以上、２％未満であり、
がん糖鎖パターンＢは、糖鎖の末端ハイマンノース型糖鎖が４５％未満であり、かつ末端シアル酸型糖鎖が０％以上、２％未満であり、
がん糖鎖パターンＣは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつ末端α２，６シアル酸型糖鎖の含有量が末端α２，３シアル酸型糖鎖の含有量より多く、
がん糖鎖パターンＤは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつα２，３シアル酸型糖鎖の含有量がα２，６シアル酸型糖鎖の含有量より多い、［１４］に記載の決定方法。
［１６］
がん細胞の糖鎖プロファイルが糖鎖パターンＡの場合、被検者が有するがん細胞はタイプ１の体内動態を示し、がん細胞の転移傾向が低いことを示唆し、
糖鎖プロファイルが糖鎖パターンＢの場合、被検者が有するがん細胞はタイプ２の体内動態を示し、がん細胞が肝臓及び脾臓への転移傾向があることを示唆し、
糖鎖プロファイルが糖鎖パターンＣの場合、被検者が有するがん細胞はタイプ３の体内動態を示し、がん細胞が腋窩及び鎖骨上リンパ節への転移傾向があることを示唆し、
糖鎖プロファイルが糖鎖パターンＤの場合、被検者が有するがん細胞はタイプ４の体内動態を示し、がん細胞が肺、肝臓、脾臓、脳及び腎臓への転移傾向があることを示唆する、［１４］又は［１５］に記載の決定方法。
［１７］
表面の少なくとも一部をリン脂質で被覆されたナノ粒子の表面に、がん糖鎖パターンを提示することを含む、［１］～［７］のいずれか１項に記載の糖鎖提示粒子の製造方法。
［１８］
前記提示されるがん糖鎖パターンは、被検者から採取したがん細胞の糖鎖を切り出したがん糖鎖パターン、又は、被検者から採取したがん細胞の糖鎖をプロファイルし、得られたプロファイルに基づいて決定された糖鎖パターンである、［１７］に記載の製造方法。
［１９］
前記表面の少なくとも一部をリン脂質で被覆されたナノ粒子は、下記一般式（Ｄ）で示される架橋前駆体Ｘ及び下記一般式（Ｅ）で示されるリン脂質前駆体とコロイド状ナノ粒子とを混合して、ナノ粒子の表面に架橋前駆体Ｘ及びリン脂質を担持した表面修飾ナノ粒子を得ることで実施できる、［１７］又は［１８］に記載の製造方法。
Figure JPOXMLDOC01-appb-C000009
 （一般式（Ｄ）中、ｎ１は２～３０の整数であり、ｎ２は２～３０の整数である。)
Figure JPOXMLDOC01-appb-C000010
 (一般式（Ｅ）中、ｎ３は２～３０の範囲の整数である。)
［２０］
がん糖鎖パターンの提示は、表面修飾ナノ粒子に導入された一般式（Ｄ）で示される架橋前駆体Ｘが有するアミノオキシ基と、被検者から採取したがん細胞の糖鎖を切り出したがん糖鎖パターン、又は、被検者から採取したがん細胞の糖鎖をプロファイルし、得られたプロファイルに基づいて決定された糖鎖パターンに含まれる糖鎖が有する還元末端とをグライコブロッティング法により反応させることで行う、［１９］に記載の製造方法。
［２１］
プロファイルに基づいて決定される糖鎖パターンは、糖鎖成分の種類と含有量をプロファイルと同一とした糖鎖パターンであるか、あるいは糖鎖成分の種類と含有量の一部をプロファイルと同一とした糖鎖パターンである［１８］～［２０］のいずれか１項に記載の製造方法。 The present invention is as follows.
[1]
Sugar chain presentation particles that are nanoparticles having sugar chains on the surface.
(1) The average particle size of the sugar chain presentation particles is in the range of 10 to 100 nm.
(2) At least a part of the surface of the nanoparticles is coated with phospholipids.
(3) The sugar chain contained on the surface of the nanoparticles is a sugar chain pattern requested by cancer cells or a sugar chain pattern determined based on the profile of this sugar chain (hereinafter referred to as a cancer sugar chain pattern).
Sugar chain presentation particles.
[2]
The cancer sugar chain pattern is
(1) Sugar chain pattern A, whose pharmacokinetics mainly depend on terminal high mannose type sugar chains,
(2) Sugar chain pattern B, whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains,
(3) Sugar chain pattern C, whose pharmacokinetics mainly depends on terminal α2,6 sialic acid type sugar chains,
(4) The sugar chain presentation particle according to [1], which is a sugar chain pattern selected from the group consisting of a sugar chain pattern D whose pharmacokinetics mainly depends on terminal α2,3 sialic acid type sugar chains.
[3]
In the cancer sugar chain pattern A, 45 mol% or more of the sugar chains are terminal hymannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%.
In the cancer sugar chain pattern B, the terminal hymannose type sugar chain of the sugar chain is less than 45%, and the terminal sialic acid type sugar chain is 0% or more and less than 2%.
In the cancer sugar chain pattern C, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal α2,6 sialic acid type sugar chains is the content of the terminal α2,3 sialic acid type sugar chains. More than the amount,
In the cancer sugar chain pattern D, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of α2,3 sialic acid type sugar chains is higher than the content of α2,6 sialic acid type sugar chains. Many sugar chain-presenting particles according to [2].
[4]
The phospholipid that covers at least a part of the surface of the nanoparticles is a sulfide bond of an alkanethiol having a phosphorylcholine group, and the sugar chain on the surface of the nanoparticles is a sulfide bond of an alkanethiol having an immobilized sugar chain. , The surface of the nanoparticles is any one of [1] to [3], which is a nanoparticle coated with a monomolecular film of an alkanethiol sulfide bond having a phosphorylcholine group and an alkanethiol sulfide bond having a sugar chain immobilized. The sugar chain presenting particles according to.
[5]
The sulfide bond of the alkanethiol having a phosphorylcholine group is represented by the following general formula (A).
Figure JPOXMLDOC01-appb-C000006
 (In the general formula (A), n3 is an integer in the range of 2 to 30, and the -S-end is a carrier site where a sulfide bond is attached to the nanoparticles.)
The sugar chain-presenting particles according to [4], wherein the sulfide bond of alkanethiol on which the sugar chain is immobilized is represented by the following general formula (B).
Figure JPOXMLDOC01-appb-C000007
 In (formula (B), n1 is an integer of 2-30, n2 is an integer of 2 to 30, a bearing portion -S- terminal sulfide bound to the nanoparticles, R 10 is contained sugar It is a part.)
[6]
The sugar chain-presenting particle according to any one of [1] to [5], which further has a drug site.
[7]
The sugar chain-presenting particles according to [4] or [5], wherein the monolayer further contains a sulfide bond of an alkanethiol having a drug site, which is represented by the following general formula (C).
Figure JPOXMLDOC01-appb-C000008
 In the general formula (C), n1 is an integer of 2 to 30, n2 is an integer of 2 to 30, the -S-terminal is a carrier site where a sulfide bond is attached to nanoparticles, and R 20 is a drug-containing site. is there.
[8]
A sugar chain containing two or more types of sugar chain-presenting particles having different cancer sugar chain patterns, and the sugar chain-presenting particles are the sugar chain-presenting particles according to any one of [1] to [7]. Presented particle kit.
[9]
The sugar chain presentation particle kit according to [8], wherein the different cancer sugar chain patterns are any two or more sugar chain patterns of patterns A to D according to [2].
[10]
The sugar chain presentation particle kit according to [8], wherein the sugar chain presentation particle is a sugar chain presentation particle further having the drug site according to [6] or [7].
[11]
A cancer metastasis preventive agent containing the sugar chain-presenting particles according to [6] or [7] as an active ingredient.
[12]
A cancer therapeutic agent containing the sugar chain-presenting particles according to [6] or [7] as an active ingredient.
[13]
A method for determining a sugar chain pattern of a cancer cell, which comprises profiling the sugar chain of the cancer cell from the cancer cell collected from a subject and determining the sugar chain pattern based on the profiled sugar chain.
[14]
The glycan pattern is determined by the profiled glycan.
(1) Sugar chain pattern A, whose pharmacokinetics mainly depend on terminal high mannose type sugar chains,
(2) Sugar chain pattern B, whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains,
(3) Sugar chain pattern C whose pharmacokinetics mainly depend on terminal α2,6 sialic acid type sugar chains, and (4) Sugar chain pattern D whose pharmacokinetics mainly depend on terminal α2,3 sialic acid type sugar chains The determination method according to [13], which is performed by specifying which sugar chain pattern is selected from the group consisting of.
[15]
In the cancer sugar chain pattern A, 45 mol% or more of the sugar chains are terminal hymannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%.
In the cancer sugar chain pattern B, the terminal hymannose type sugar chain of the sugar chain is less than 45%, and the terminal sialic acid type sugar chain is 0% or more and less than 2%.
In the cancer sugar chain pattern C, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal α2,6 sialic acid type sugar chains is the content of the terminal α2,3 sialic acid type sugar chains. More than the amount,
In the cancer sugar chain pattern D, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of α2,3 sialic acid type sugar chains is higher than the content of α2,6 sialic acid type sugar chains. Many, the determination method according to [14].
[16]
When the sugar chain profile of cancer cells is sugar chain pattern A, the cancer cells possessed by the subject show type 1 pharmacokinetics, suggesting that the cancer cells have a low metastasis tendency.
When the glycan profile is glycan pattern B, the cancer cells possessed by the subject show type 2 pharmacokinetics, suggesting that the cancer cells tend to metastasize to the liver and spleen.
When the glycan profile is glycan pattern C, the cancer cells possessed by the subject show type 3 pharmacokinetics, suggesting that the cancer cells tend to metastasize to the axilla and supraclavicular lymph nodes.
When the glycan profile is glycan pattern D, the cancer cells possessed by the subject show type 4 pharmacokinetics, suggesting that the cancer cells tend to metastasize to the lungs, liver, spleen, brain and kidneys. The determination method according to [14] or [15].
[17]
The sugar chain-presenting particles according to any one of [1] to [7], which comprises presenting a cancer sugar chain pattern on the surface of nanoparticles having at least a part of the surface coated with phospholipids. Production method.
[18]
The presented cancer sugar chain pattern is a cancer sugar chain pattern obtained by cutting out a sugar chain of a cancer cell collected from a subject, or a profile of a sugar chain of a cancer cell collected from a subject. The production method according to [17], which is a sugar chain pattern determined based on the obtained profile.
[19]
The nanoparticles in which at least a part of the surface is coated with phospholipids include the crosslinked precursor X represented by the following general formula (D), the phospholipid precursor represented by the following general formula (E), and colloidal nanoparticles. The production method according to [17] or [18], which can be carried out by mixing the above to obtain surface-modified nanoparticles in which the crosslinked precursor X and the phospholipid are supported on the surface of the nanoparticles.
Figure JPOXMLDOC01-appb-C000009
 (In the general formula (D), n1 is an integer of 2 to 30, and n2 is an integer of 2 to 30.)
Figure JPOXMLDOC01-appb-C000010
 (In the general formula (E), n3 is an integer in the range of 2 to 30.)
[20]
To present the cancer sugar chain pattern, the aminooxy group of the cross-linking precursor X represented by the general formula (D) introduced into the surface-modified nanoparticles and the sugar chain of the cancer cell collected from the subject are cut out. The cancer sugar chain pattern or the sugar chain of the cancer cell collected from the subject is profiled, and the reducing terminal of the sugar chain contained in the sugar chain pattern determined based on the obtained profile is used as a glyco. The production method according to [19], which is carried out by reacting by a blotting method.
[21]
The sugar chain pattern determined based on the profile is a sugar chain pattern in which the type and content of the sugar chain component are the same as the profile, or a part of the type and content of the sugar chain component is the same as the profile. The production method according to any one of [18] to [20], which is a sugar chain pattern.
　本発明によれば、原発巣のがん細胞の転移先を予測できる、特定の体内動態を示す、がん糖鎖パターンを有する糖鎖提示粒子を提供できる。この糖鎖提示粒子は、例えば、マウスに静脈内投与することで、がん糖鎖パターンが先導するがん転移のプロセスをリアルタイムで可視化することができる。さらに本発明によれば、かん転移の予防、がん治療において有効なＤＤＳ技術を提供することもできる。さらに本発明によれば、がん細胞の転移性を示唆することができる糖鎖パターンの決定方法も提供することができる。 According to the present invention, it is possible to provide sugar chain-presenting particles having a cancer sugar chain pattern, which can predict the metastasis destination of cancer cells in the primary lesion and show specific pharmacokinetics. By intravenously administering the sugar chain-presenting particles to mice, for example, the process of cancer metastasis led by the cancer sugar chain pattern can be visualized in real time. Further, according to the present invention, it is possible to provide an effective DDS technique in the prevention of canker metastasis and the treatment of cancer. Furthermore, according to the present invention, it is possible to provide a method for determining a sugar chain pattern that can suggest metastasis of cancer cells.
図１は、がん細胞の糖鎖パターンを表面に提示した糖鎖提示粒子の作製法とその体内動態のin vivoイメージングの結果である。FIG. 1 shows the results of in vivo imaging of a method for producing sugar chain-presenting particles in which the sugar chain pattern of cancer cells is presented on the surface and their pharmacokinetics. 図２は、ヒト培養がん細胞のN型糖鎖プロファイルを示す。（a）MALDI-TOFMSによる糖鎖構造解析、IS=internal standard、（b）各がん細胞の主要な糖鎖成分の構造（発現量の上位10構造）、円グラフはグライコタイピング分析（glycotyping analysis）の結果である。FIG. 2 shows the N-type sugar chain profile of cultured human cancer cells. (A) Glycan structure analysis by MALDI-TOFMS, IS = internal standard, (b) Structure of major sugar chain components of each cancer cell (top 10 structures of expression level), pie chart is glycotyping analysis ) Is the result. 図３は、がん細胞の糖鎖パターンを提示するナノ粒子である糖鎖提示粒子の体内動態の実験結果を示す。（a）4種類のヒトがん細胞糖鎖を提示した糖鎖提示粒子とコントロールを静脈内投与後3時間までの体内動態（数字はゼータ電位）、（b）3時間経過後に解剖した主要な臓器での糖鎖提示粒子の分布状態。FIG. 3 shows the experimental results of the pharmacokinetics of sugar chain-presenting particles, which are nanoparticles that present the sugar chain pattern of cancer cells. (A) Glycan-presenting particles presenting four types of human cancer cell sugar chains and controls were dissected up to 3 hours after intravenous administration (numbers are zeta potentials), and (b) major dissected after 3 hours. Distribution of sugar chain-presenting particles in organs. 図３は、がん細胞の糖鎖パターンを提示するナノ粒子である糖鎖提示粒子の体内動態の実験結果を示す。（c）糖鎖提示粒子の臓器指向性：糖鎖を有しないコントロールとした粒子の脳での蛍光強度を1.0とした際の各臓器での相対的な蛍光強度を示した。ただし、腋窩および鎖骨上リンパ節組織は解剖により摘出していない。FIG. 3 shows the experimental results of the pharmacokinetics of sugar chain-presenting particles, which are nanoparticles that present the sugar chain pattern of cancer cells. (C) Organ directivity of sugar chain-presenting particles: The relative fluorescence intensity in each organ was shown when the fluorescence intensity in the brain of the controlled particles having no sugar chain was 1.0. However, the axillary and supraclavicular lymph node tissues were not removed by autopsy. 図４は、Ruddy duckの卵白糖鎖からヒトがん細胞の糖鎖パターンに改変する実験の結果を示す。（a）Ruddy duckの卵白に見られた多分岐型糖鎖の構造とシアル酸転移酵素による改変法、（b）Button Quail、Chicken、Ruddy duck卵白の糖鎖の階層構造およびRuddy duck卵白から改変した２種類の糖鎖パターン（2,3S-Ruddy duckと2,6S-Ruddy duck）。FIG. 4 shows the results of an experiment in which the egg white sugar chain of Ruddy duck is modified into the sugar chain pattern of human cancer cells. (A) Multi-branched sugar chain structure found in Ruddy duck egg white and modification method by sialic acid transfer enzyme, (b) Hierarchical structure of sugar chain in Button Quail, Chicken, Ruddy duck egg white and modification from Ruddy duck egg white Two types of sugar chain patterns (2,3S-Ruddyduck and 2,6S-Ruddyduck). 図４は、Ruddy duckの卵白糖鎖からヒトがん細胞の糖鎖パターンに改変する実験の結果を示す。（c）糖鎖提示粒子に提示する糖鎖のうち主要な糖鎖成分の割合（発現量の上位10の成分）、パイ（pie）グラフはグライコタイピング分析（glycotyping analysis）の結果を示す。FIG. 4 shows the results of an experiment in which the egg white sugar chain of Ruddy duck is modified into the sugar chain pattern of human cancer cells. (C) The ratio of the main sugar chain components (the top 10 components in the expression level) among the sugar chains presented to the sugar chain presentation particles, and the pie graph show the results of glycotyping analysis. 図５は、卵白および卵黄の糖鎖及び改変した糖鎖パターン、さらに、α２，６型シアル酸型糖鎖の糖鎖パターンを提示したナノ粒子の体内動態や臓器指向性を調べた結果を示す。（a）Button Quail、Chicken、Ruddy duckの卵白糖鎖、及びRuddy duckの卵白糖鎖を改変しシアリル化した2種の糖鎖を提示したナノ粒子、さらに鶏卵黄糖ペプチド由来糖鎖を提示したナノ粒子（GNS-2,6S-A2）である糖鎖提示粒子とコントロールを静脈内投与後3時間までの体内動態（数字はゼータポテンシャル）、（b）3時間経過後に解剖したマウスの主要な臓器での糖鎖提示粒子（GNS-Button Quail、GNS-Chicken、GNS-Ruddy duck、GNS-2,3S-Ruddy duck、GNS-2,6S-Ruddy duck、GNS-2,6S-A2）の分布状態。FIG. 5 shows the results of examining the pharmacokinetics and organ directivity of nanoparticles presenting sugar chains of egg white and yolk, modified sugar chain patterns, and sugar chain patterns of α2,6 type sialic acid type sugar chains. .. (A) Nanoparticles presenting two types of sugar chains obtained by modifying the egg white sugar chains of Button Quail, Chicken, and Ruddy duck, and modifying the egg white sugar chains of Ruddy duck, and further presenting sugar chains derived from chicken egg yellow sugar peptide. Glyco-presenting particles (GNS-2,6S-A2), which are nanoparticles, and controls are pharmacodynamics up to 3 hours after intravenous administration (numbers are zeta potential), and (b) major mice dissected after 3 hours. Distribution of sugar chain-presenting particles (GNS-Button Quail, GNS-Chicken, GNS-Ruddy duck, GNS-2,3S-Ruddy duck, GNS-2,6S-Ruddy duck, GNS-2,6S-A2) in organs Status. 図５は、卵白および卵黄の糖鎖及び改変した糖鎖パターン、さらに、α２，６型シアル酸型糖鎖の糖鎖パターンを提示したナノ粒子の体内動態や臓器指向性を調べた結果を示す。（c）ナノソームの臓器指向性：コントロールとした糖鎖を有しないナノ粒子の脳での近赤外蛍光強度を1.0とした際の各臓器での相対的な蛍光強度を示した。ただし、腋窩および鎖骨上リンパ節組織は解剖により摘出していない。FIG. 5 shows the results of examining the pharmacokinetics and organ directivity of nanoparticles presenting sugar chains of egg white and yolk, modified sugar chain patterns, and sugar chain patterns of α2,6 type sialic acid type sugar chains. .. (C) Organ directivity of nanosomes: The relative fluorescence intensity in each organ was shown when the near-infrared fluorescence intensity in the brain of nanoparticles having no controlled sugar chain was set to 1.0. However, the axillary and supraclavicular lymph node tissues were not removed by autopsy. 図６は、Japanese Quail卵白糖鎖と鶏卵黄糖ペプチド由来糖鎖（2,6S-A2）を混合して作製した糖鎖提示粒子の体内動態の実験結果を示す。（a）Japanese Quail卵白由来の糖鎖（約6 nmol）に2,6S-A2由来の糖鎖をそれぞれ0.6 nmol、1.2 nmol、および2.4 nmol添加して混合し、これを提示したナノ粒子の糖鎖プロファイルの結果である。FIG. 6 shows the experimental results of the pharmacokinetics of sugar chain-presenting particles prepared by mixing Japanese Quail egg white sugar chain and chicken egg brown sugar peptide-derived sugar chain (2,6S-A2). (A) Japanese Quail Egg white-derived sugar chains (about 6 nmol) were mixed with 0.6 nmol, 1.2 nmol, and 2.4 nmol sugar chains derived from 2,6S-A2, respectively. It is the result of the chain profile.
＜糖鎖提示粒子＞
　本発明は、糖鎖を表面に有するナノ粒子である糖鎖提示粒子に関し、この糖鎖提示粒子は、
（１）糖鎖提示粒子の平均粒子径は１０～１００ｎｍの範囲であり、
（２）ナノ粒子の表面の少なくとも一部はリン脂質で被覆されており、
（３）ナノ粒子表面に有する糖鎖は、がん細胞由来の糖鎖パターン、またはこの糖鎖のプロファイルに基づいて決定された糖鎖パターンを模したパターン（がん糖鎖パターン）である。 <Sugar chain presentation particles>
The present invention relates to sugar chain-presenting particles, which are nanoparticles having sugar chains on the surface, and the sugar chain-presenting particles are:
(1) The average particle size of the sugar chain presentation particles is in the range of 10 to 100 nm.
(2) At least a part of the surface of the nanoparticles is coated with phospholipids.
(3) The sugar chain on the surface of the nanoparticles is a sugar chain pattern derived from cancer cells or a pattern imitating a sugar chain pattern determined based on the profile of the sugar chain (cancer sugar chain pattern).
　本発明の糖鎖提示粒子は、ナノ粒子の表面にリン脂質の被覆及びがん糖鎖パターンを有する。本願明細書において糖鎖パターンとは、表面の糖鎖プロファイルに基づく２以上の異なる糖鎖からなる糖鎖の集団において、特定の種類の糖鎖の存在の有無または特定の種類の糖鎖の存在量により特定されるパターンを意味する。糖鎖パターンは、糖鎖成分の種類とそれらの含有量により特定される。 The sugar chain presenting particles of the present invention have a phospholipid coating and a cancer sugar chain pattern on the surface of the nanoparticles. In the present specification, the sugar chain pattern is the presence or absence of a specific type of sugar chain or the presence of a specific type of sugar chain in a group of sugar chains consisting of two or more different sugar chains based on the surface sugar chain profile. Means a pattern specified by quantity. The sugar chain pattern is specified by the types of sugar chain components and their contents.
　したがって、本発明において、がん細胞の糖鎖をそのまま捕捉し提示した糖鎖提示粒子は、がん細胞に由来する糖鎖パターンを有する糖鎖提示粒子である。また、がん細胞の糖鎖をプロファイルし、プロファイルに基づいて糖鎖パターンを決定し、この決定された糖鎖パターンを有する糖鎖提示粒子も、本発明における糖鎖提示粒子である。プロファイルに基づいて決定される糖鎖パターンは、糖鎖成分の種類と含有量をプロファイルと同一とした糖鎖パターンであるか、あるいは糖鎖成分の種類と含有量の一部をプロファイルと同一とした糖鎖パターンであるか、のいずれでもよい。糖鎖成分の種類と含有量の一部をプロファイルと同一とする場合、プロファイルに含まれる糖鎖成分の一部の種類を減量または欠損させることができる。より詳細には、プロファイルに含まれる糖鎖成分をいくつかの型に分類した場合、含有量が多い１又は２以上の型に属する糖鎖成分を含み、含有量が少ない１又は２以上の型に属する糖鎖成分を減量または欠損させた糖鎖パターンであることができる。糖鎖成分の型としては、例えば、末端ハイマンノース型糖鎖、末端ガラクトース型糖鎖、末端Ｎ－アセチルグルコサミン型糖鎖、末端α２，６シアル酸型糖鎖、末端α２，３シアル酸型糖鎖を挙げることができる。プロファイルにおける糖鎖成分が、例えば、含有量が多い順に末端ハイマンノース型糖鎖、末端ガラクトース型糖鎖及び末端Ｎ－アセチルグルコサミン型糖鎖であった場合、含有量の少ない末端Ｎ－アセチルグルコサミン型糖鎖を減量するか、または欠損させた糖鎖パターンをプロファイルから決定した糖鎖パターンとすることができる。あるいは、１つの型に複数の糖鎖成分が含まれる場合、同じ型に属する糖鎖成分の含有割合を変化させた糖鎖パターンをプロファイルから決定した糖鎖パターンとすることもできる。糖鎖の型によって糖鎖提示粒子の体内動態が変化することから、体内動態の変化を考慮して、プロファイルを適宜変更して糖鎖パターンを決定することもできる。 Therefore, in the present invention, the sugar chain-presenting particles that capture and present the sugar chains of the cancer cells as they are are the sugar chain-presenting particles having a sugar chain pattern derived from the cancer cells. In addition, the sugar chain-presenting particles in the present invention are also sugar chain-presenting particles that profile the sugar chains of cancer cells, determine the sugar chain pattern based on the profile, and have the determined sugar chain pattern. The sugar chain pattern determined based on the profile is a sugar chain pattern in which the type and content of the sugar chain component are the same as the profile, or a part of the type and content of the sugar chain component is the same as the profile. It may be either a sugar chain pattern. When a part of the type and content of the sugar chain component is the same as the profile, the amount of some types of the sugar chain component contained in the profile can be reduced or deleted. More specifically, when the sugar chain components contained in the profile are classified into several types, the sugar chain components belonging to one or two or more types having a high content are contained, and the one or two or more types having a low content are included. It can be a sugar chain pattern in which the amount of the sugar chain component belonging to is reduced or deleted. Examples of the types of sugar chain components include terminal hymannose type sugar chain, terminal galactose type sugar chain, terminal N-acetylglucosamine type sugar chain, terminal α2,6 sialic acid type sugar chain, and terminal α2,3 sialic acid type sugar. You can raise a chain. When the sugar chain components in the profile are, for example, a terminal hymannose type sugar chain, a terminal galactose type sugar chain, and a terminal N-acetylglucosamine type sugar chain in descending order of content, the terminal N-acetylglucosamine type having a smaller content The sugar chain pattern in which the amount of sugar chains is reduced or deleted can be used as the sugar chain pattern determined from the profile. Alternatively, when one type contains a plurality of sugar chain components, the sugar chain pattern in which the content ratio of the sugar chain components belonging to the same type is changed can be used as the sugar chain pattern determined from the profile. Since the pharmacokinetics of the sugar chain-presenting particles change depending on the type of sugar chain, the sugar chain pattern can be determined by appropriately changing the profile in consideration of the change in the pharmacokinetics.
　本発明における糖鎖パターンには、本発明の糖鎖提示粒子の体内動態によって分類できるＡからＤの４つのがん糖鎖パターンがあり得る。
（１）体内動態が主に末端ハイマンノース型糖鎖に依存する糖鎖パターンＡ、
（２）体内動態が主に末端ガラクトース型糖鎖又は末端Ｎ－アセチルグルコサミン型糖鎖に依存する糖鎖パターンＢ、
（３）体内動態が主に末端α２，６シアル酸型糖鎖に依存する糖鎖パターンＣ、
（４）体内動態が主に末端α２，３シアル酸型糖鎖に依存する糖鎖パターンＤ。 The sugar chain pattern in the present invention may include four cancer sugar chain patterns A to D that can be classified according to the pharmacokinetics of the sugar chain presenting particles of the present invention.
(1) Sugar chain pattern A, whose pharmacokinetics mainly depend on terminal high mannose type sugar chains,
(2) Sugar chain pattern B, whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains,
(3) Sugar chain pattern C, whose pharmacokinetics mainly depends on terminal α2,6 sialic acid type sugar chains,
(4) Sugar chain pattern D in which pharmacokinetics mainly depend on terminal α2,3 sialic acid type sugar chains.
　体内動態とは、糖鎖提示粒子を投与された動物の体内における、糖鎖提示粒子の循環器系などを介しての経時的な動き、及び各臓器や組織への経時的な分布を意味する。本願明細書では、体内動態の中の各臓器や組織へ経時的に分布あるいは集積する性質を特に臓器指向性を意味することがある。 The pharmacokinetics means the temporal movement of the sugar chain-presenting particles in the body of the animal to which the sugar chain-presenting particles are administered via the circulatory system and the like, and the temporal distribution of the sugar chain-presenting particles to each organ or tissue. .. In the specification of the present application, the property of being distributed or accumulated over time in each organ or tissue in the pharmacokinetics may mean particularly organ orientation.
　本発明において対象となるがん細胞は、本発明の糖鎖提示粒子の模倣対象であるエキソソームを放出するがん細胞であり、生体から採取したがん細胞、採集後に培養したがん細胞が含まれる。がん細胞は、純粋培養した単一の細胞であっても、悪性度等の異なる複数のがん細胞の混合物であってもかまわない。 The cancer cells targeted in the present invention are cancer cells that release exosomes, which are targets of mimicking the sugar chain presentation particles of the present invention, and include cancer cells collected from a living body and cancer cells cultured after collection. Is done. The cancer cells may be a single cell that has been purely cultured, or a mixture of a plurality of cancer cells having different grades of malignancy.
　また、標準的に利用されている既存のがん細胞の糖鎖パターンも、本発明の糖鎖提示粒子において糖鎖パターンとして使用することができる。例えば、標準的なヒトがん細胞としては、ＭＣＦ７（乳がん）、ＭＤＡ－ＭＢ－２３１（乳がん）、Ａ５４９（肺がん）、ＨｅｐＧ２（肝臓がん）、Ａ３７５（メラノーマ）、ＨＣＴ１１６（大腸がん）、Ｈｅｌａ（子宮がん）、ＭＮＮＧ／ＮＯＳ（骨肉腫）、ＡＧＣ（胃がん）、ＭＩＡＰａＣａ－２（膵臓がん）、Ａ４３１（皮膚がん）、ＳＫＯＶ（卵巣がん）等を挙げることができる。 In addition, the existing sugar chain pattern of cancer cells that is used as standard can also be used as the sugar chain pattern in the sugar chain presentation particles of the present invention. For example, standard human cancer cells include MCF7 (breast cancer), MDA-MB-231 (breast cancer), A549 (lung cancer), HepG2 (liver cancer), A375 (melanoma), HCT116 (colon cancer), Hela (uterine cancer), MNNG / NOS (osteosarcoma), AGC (gastric cancer), MIAPaCa-2 (pancreatic cancer), A431 (skin cancer), SKOV (ovarian cancer) and the like can be mentioned.
　糖鎖提示粒子とは、細胞間の情報伝達に使われている粒子であるエキソソームの機能を模倣した機能(細胞間の情報伝達様機能)を有する粒子との意味である。エキソソームはその内部に核酸、タンパク質などを含んでおり、細胞から分泌したエキソソームを介して受け取り側の細胞に伝達される。エキソソームは細胞間のコミュニケーションツールとしての機能がある。本発明において作製された糖鎖提示粒子は、本来のエキソソームと同様に、受け取り側の細胞やこれが集合した組織や臓器に対して情報を伝達する機能を有する。 The sugar chain presenting particle means a particle having a function (information transmission-like function between cells) that imitates the function of an exosome, which is a particle used for information transmission between cells. Exosomes contain nucleic acids, proteins, etc. inside them, and are transmitted to receiving cells via exosomes secreted from cells. Exosomes function as communication tools between cells. The sugar chain presenting particles produced in the present invention have a function of transmitting information to receiving cells and tissues and organs in which they are aggregated, similar to the original exosomes.
　ナノ粒子は、金属ナノ粒子又は半導体ナノ粒子であることができる。金属ナノ粒子の材質には、特に制限はないが、金、白金、銀、鉄磁性体であることができ、金属ナノ粒子は、金ナノ粒子、白金ナノ粒子、銀ナノ粒子は、鉄磁性体ナノ粒子であることができる。特に、金属ナノ粒子は、金ナノ粒子、白金ナノ粒子、銀ナノ粒子が、生体に対する安全性という観点から好ましい。 The nanoparticles can be metal nanoparticles or semiconductor nanoparticles. The material of the metal nanoparticles is not particularly limited, but may be gold, platinum, silver, or iron magnetic material. The metal nanoparticles are gold nanoparticles, platinum nanoparticles, and the silver nanoparticles are iron magnetic materials. It can be nanoparticles. In particular, as the metal nanoparticles, gold nanoparticles, platinum nanoparticles, and silver nanoparticles are preferable from the viewpoint of safety to the living body.
　半導体ナノ粒子の材質には、特に制限はない。半導体ナノ粒子は、量子ドットであることもできる。量子ドットとは半導体原子が数百個から数千個集まった１０数ｎｍ程度の小さな塊であり、蛍光性ナノ粒子である。粒子径により発光する蛍光の波長（色）が異なる。市販品を利用でき、本発明の複合体のナノ粒子に量子ドットを用いると、蛍光発光性の複合体とすることができ、生体内での動態をモニタリングすることも可能である。 There are no particular restrictions on the material of the semiconductor nanoparticles. The semiconductor nanoparticles can also be quantum dots. Quantum dots are small lumps of about 10 and several nanometers in which hundreds to thousands of semiconductor atoms are gathered, and are fluorescent nanoparticles. The wavelength (color) of fluorescence emitted differs depending on the particle size. Commercially available products can be used, and by using quantum dots for the nanoparticles of the complex of the present invention, it is possible to obtain a fluorescent complex, and it is also possible to monitor the dynamics in vivo.
　ナノ粒子は、粒子径が０．１～１００ｎｍの範囲であることができ、好ましくは、１～５０ｎｍの範囲、より好ましくは５～４０ｎｍの範囲、さらに好ましくは５～３０ｎｍの範囲、一層好ましく１０～３０ｎｍの範囲である。ナノ粒子の表面にリン脂質の被覆及びがん糖鎖パターンを有する糖鎖提示粒子は、平均粒子径は１０～１００ｎｍの範囲であり、好ましくは１０～６０ｎｍの範囲、より好ましく１２～５０ｎｍの範囲、さらに好ましく１５～４０ｎｍの範囲、一層好ましく１５～３０ｎｍの範囲である。ナノ粒子及び糖鎖提示粒子の平均粒子径は、動的光散乱法により測定することができる。測定装置としては、ファイバー光学動的光散乱光度計 (粒径分布測定用)を用いることができる。より具体的にはファイバー光学動的光散乱光度計ＦＤＬＳ－３０００（大塚電子製）を用いることができる。 The nanoparticles can have a particle size in the range of 0.1 to 100 nm, preferably in the range of 1 to 50 nm, more preferably in the range of 5 to 40 nm, still more preferably in the range of 5 to 30 nm, even more preferably 10. It is in the range of ~ 30 nm. The sugar chain-presenting particles having a phospholipid coating and a cancer sugar chain pattern on the surface of the nanoparticles have an average particle size in the range of 10 to 100 nm, preferably in the range of 10 to 60 nm, and more preferably in the range of 12 to 50 nm. More preferably, it is in the range of 15 to 40 nm, and further preferably, it is in the range of 15 to 30 nm. The average particle size of the nanoparticles and the sugar chain presentation particles can be measured by a dynamic light scattering method. As the measuring device, a fiber optical dynamic light scattering photometer (for particle size distribution measurement) can be used. More specifically, a fiber optical dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Electronics Co., Ltd.) can be used.
　本発明の糖鎖提示粒子は、ナノ粒子の表面がリン脂質で被覆されている。平均粒径２０ｎｍ程度のリン脂質アルカンチオール混合単分子膜による完全被覆型金属ナノ微粒子は、血液中でも非特異吸着によるタンパク質コロナを形成しないこと、さらにマウスに静脈内投与した際に特定の臓器に集積せず投与後３時間に至っても全身を一様に滞留する安定なナノ微粒子である（非特許文献６－７、特許文献１）。 In the sugar chain presentation particles of the present invention, the surface of the nanoparticles is coated with phospholipids. Completely coated metal nanoparticles with a phospholipid alkane thiol mixed monolayer having an average particle size of about 20 nm do not form protein corona due to non-specific adsorption even in blood, and accumulate in specific organs when intravenously administered to mice. It is a stable nanoparticle that stays uniformly throughout the body even after 3 hours after administration (Non-Patent Documents 6-7, Patent Document 1).
　前記ホスホリルコリン基を有するアルカンチオールのスルフィド結合体は、一般式（Ａ）で示されるリン脂質疑似物質であることができる。
Figure JPOXMLDOC01-appb-C000011
 　一般式（Ａ）中、ｎ３は２～３０の範囲の整数であり、－Ｓ－末端がナノ粒子にスルフィド結合する担持部位である。 The sulfide bond of an alkanethiol having a phosphorylcholine group can be a phospholipid pseudo substance represented by the general formula (A).
Figure JPOXMLDOC01-appb-C000011
 In the general formula (A), n3 is an integer in the range of 2 to 30, and is a carrier site where the —S— terminal is sulfide-bonded to the nanoparticles.
　リン脂質疑似物質は、ナノ粒子の表面を修飾し、本発明の糖鎖提示粒子表面への、蛋白質コロナの非特異的吸着を防止する機能を付与する。この観点からｎ３は２～３０、好ましくは５～２０、より好ましくは７～１５の範囲の整数である。 The phospholipid pseudo substance modifies the surface of the nanoparticles and imparts a function of preventing non-specific adsorption of protein corona on the surface of the sugar chain-presenting particles of the present invention. From this point of view, n3 is an integer in the range of 2 to 30, preferably 5 to 20, and more preferably 7 to 15.
　１個のナノ粒子に対するリン脂質又はリン脂質疑似物質の担持量は、ナノ粒子の表面における金属元素（反応点）の８０％以上あることが好ましい。 The amount of phospholipid or phospholipid pseudo substance supported on one nanoparticles is preferably 80% or more of the metal element (reaction point) on the surface of the nanoparticles.
　本発明の糖鎖提示粒子は、ナノ粒子の表面にがん糖鎖パターンを有する。このがん糖鎖パターンは、情報の送り手として、エキソソームを放出するがん細胞由来の糖鎖を提示した糖鎖パターンであるか、あるいはがん細胞の糖鎖をプロファイルし決定したがん糖鎖パターンである。がん細胞由来の糖鎖パターンは、特定のがん治療患者から採取したがん細胞由来の糖鎖の糖鎖パターンであることが、その細胞から放出されるエキソソームの体内動態の予測性を高めるためには好ましい。標準的に利用されている既存のがん細胞由来の糖鎖パターンを予測に利用することもできる。がん細胞由来の糖鎖パターンは、がん細胞の糖鎖を切り出し、これをナノ粒子の表面に捕捉することで、提示することができる。細胞からの糖鎖の切り出しやナノ粒子への捕捉において、細胞の糖鎖がそのままナノ粒子表面に提示されることが好ましいが、細胞において決定されるがん糖鎖パターンの範囲において糖鎖成分に変動があってもかまわない。 The sugar chain presenting particles of the present invention have a cancer sugar chain pattern on the surface of the nanoparticles. This cancer sugar chain pattern is a sugar chain pattern that presents a sugar chain derived from a cancer cell that releases exosomes as a sender of information, or a cancer sugar that is determined by profiling the sugar chain of a cancer cell. It is a chain pattern. The sugar chain pattern derived from cancer cells is a sugar chain pattern derived from cancer cells collected from a specific cancer treatment patient, which enhances the predictability of the pharmacokinetics of exosomes released from the cells. It is preferable for this. Existing cancer cell-derived sugar chain patterns that are used as standard can also be used for prediction. The sugar chain pattern derived from cancer cells can be presented by cutting out the sugar chains of cancer cells and capturing them on the surface of nanoparticles. When cutting out sugar chains from cells or capturing them into nanoparticles, it is preferable that the sugar chains of the cells are presented as they are on the surface of the nanoparticles, but they can be used as sugar chain components within the range of the cancer sugar chain pattern determined in the cells. It does not matter if there are fluctuations.
　上記のがん細胞由来の糖鎖プロファイルは、グライコブロッティング法の一般的なプロトコル（S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537）に従って特定することができる。内部標準化合物を指標としてグライコタイピング解析によって主要な構造モチーフとのそれら発現量の関係を明らかにすることができる。また、糖鎖は、既存の方法（S.-I. Nishimura eta l., J. Am. Chem. Soc. 2011, 133, 12507-12517; S.-I. Nishimura et al., ACS Chem. Biol. 2015, 10, 2073-2086; S.-I. Nishimura, WO2017/131242A1）によりナノ粒子に担持することができる。詳細は実施例１（Ａ）を参照。 The above cancer cell-derived sugar chain profile can be identified according to the general protocol of the Glycoblotting method (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537). .. Glycotyping analysis using internal standard compounds as an index can clarify the relationship between their expression levels and major structural motifs. For sugar chains, the existing method (S.-I. Nishimura eta l., J. Am. Chem. Soc. 2011, 133, 12507-12517; S.-I. Nishimura et al., ACS Chem. Biol It can be supported on nanoparticles by .2015, 10, 2073-2086; S.-I. Nishimura, WO2017 / 131242A1). See Example 1 (A) for details.
　がん細胞の糖鎖の糖鎖パターンを有する糖鎖提示粒子の体内動態を分析した結果、検討対象としたがん細胞の糖鎖パターンは、少なくとも
（１）体内動態が主に末端ハイマンノース型糖鎖に依存する糖鎖パターンＡ、
（２）体内動態が主に末端ガラクトース型糖鎖又は末端N-アセチルグルコサミン型糖鎖に依存する糖鎖パターンＢ、
（３）体内動態が主に末端α２，６シアル酸型糖鎖に依存する糖鎖パターンＣ、
（４）体内動態が主に末端α２，３シアル酸型糖鎖に依存する糖鎖パターンＤからなる群から選択される糖鎖パターンであることが判明し、かつがん糖鎖パターンを有する糖鎖提示粒子の体内動態は、がん糖鎖パターンに依存して決まることが明らかになった（実施例１、２及び３参照）。 As a result of analyzing the pharmacokinetics of the sugar chain-presenting particles having the sugar chain pattern of the sugar chains of the cancer cells, the sugar chain patterns of the cancer cells examined were at least (1) the pharmacokinetics mainly terminal high mannose type. Glycan pattern A, which depends on glycans,
(2) Sugar chain pattern B, whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains,
(3) Sugar chain pattern C, whose pharmacokinetics mainly depends on terminal α2,6 sialic acid type sugar chains,
(4) A sugar that is found to be a sugar chain pattern selected from the group consisting of a sugar chain pattern D whose pharmacokinetics mainly depends on terminal α2,3 sialic acid type sugar chains, and has a cancer sugar chain pattern. It was revealed that the pharmacokinetics of the chain-presenting particles depend on the cancer sugar chain pattern (see Examples 1, 2 and 3).
　がん糖鎖パターンＡ～Ｄを有する糖鎖提示粒子の体内動態は以下の通りである。
　がん糖鎖パターンＡを有する糖鎖提示粒子は、糖鎖を有さない糖鎖提示粒子と比べて、粒子の体外への***が促進されるタイプ１の体内動態を示す。
　がん糖鎖パターンＢを有する糖鎖提示粒子は、糖鎖を有さない糖鎖提示粒子と比べて、肝臓や脾臓へ集積されるタイプ２の体内動態を示す。
　がん糖鎖パターンＣを有する糖鎖提示粒子は、糖鎖を有さない糖鎖提示粒子と比べて、腋窩や鎖骨上リンパ節へ集積されるタイプ３の体内動態を示す。
　がん糖鎖パターンＤを有する糖鎖提示粒子は、糖鎖を有さない糖鎖提示粒子と比べて、肺、肝臓、脾臓、脳、腎臓などの臓器へ分布（分散集積）されるタイプ４の体内動態を示す。 The pharmacokinetics of sugar chain-presenting particles having cancer sugar chain patterns A to D are as follows.
The sugar chain-presenting particles having the cancer sugar chain pattern A show type 1 pharmacokinetics in which the excretion of the particles from the body is promoted as compared with the sugar chain-presenting particles having no sugar chain.
The sugar chain-presenting particles having the cancer sugar chain pattern B show type 2 pharmacokinetics accumulated in the liver and spleen as compared with the sugar chain-presenting particles having no sugar chain.
The sugar chain-presenting particles having the cancer sugar chain pattern C show type 3 pharmacokinetics accumulated in the axilla and the supraclavicular lymph nodes as compared with the sugar chain-presenting particles having no sugar chain.
The sugar chain-presenting particles having the cancer sugar chain pattern D are distributed (distributed and accumulated) in organs such as lung, liver, spleen, brain, and kidney as compared with the sugar chain-presenting particles having no sugar chain. Shows the pharmacokinetics of.
Figure JPOXMLDOC01-appb-C000012
 　上記糖鎖パターンを構成する末端ハイマンノース型糖鎖とは、糖鎖の末端が2以上に枝分かれをし、枝分かれした部分の糖が全てマンノースである糖鎖を意味する。末端ハイマンノース型糖鎖の例を以下に示す。
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000012
 The terminal high mannose type sugar chain constituting the sugar chain pattern means a sugar chain in which the end of the sugar chain is branched into two or more and all the sugars in the branched portion are mannose. Examples of terminal high mannose type sugar chains are shown below.
Figure JPOXMLDOC01-appb-C000013
　上記糖鎖パターンを構成する末端ガラクトース型糖鎖とは、１つの糖鎖の単独又は複数の末端の糖がガラクトースである糖鎖を意味する。末端ガラクトース型糖鎖の例を以下に示す。
Figure JPOXMLDOC01-appb-C000014
 The terminal galactose-type sugar chain constituting the sugar chain pattern means a sugar chain in which one sugar chain alone or a plurality of terminal sugars are galactose. Examples of terminal galactose-type sugar chains are shown below.
Figure JPOXMLDOC01-appb-C000014
　上記糖鎖パターンを構成する末端N-アセチルグルコサミン型糖鎖とは、１つの糖鎖の単独又は複数の末端の糖がN-アセチルグルコサミンである糖鎖を意味する。末端N-アセチルグルコサミン型糖鎖の例を以下に示す。
Figure JPOXMLDOC01-appb-C000015
 The terminal N-acetylglucosamine type sugar chain constituting the sugar chain pattern means a sugar chain in which one sugar chain alone or a plurality of terminal sugars are N-acetylglucosamine. Examples of terminal N-acetylglucosamine type sugar chains are shown below.
Figure JPOXMLDOC01-appb-C000015
　上記糖鎖パターンを構成する末端シアル酸型糖鎖とは、１つの糖鎖の単独又は複数の末端の糖がシアル酸である糖鎖を意味する。末端シアル酸型糖鎖には、末端にＮｅｕ５Ａｃα２，６Ｇａｌユニットを含む複合型糖鎖と、末端にＮｅｕ５Ａｃα２，３Ｇａｌユニットを含む複合型糖鎖とがある。本願明細書では、末端にＮｅｕ５Ａｃα２，６Ｇａｌユニットを含む複合型糖鎖を末端α２，６シアル酸型糖鎖と表記し、末端にＮｅｕ５Ａｃα２，３Ｇａｌユニットを含む複合型糖鎖を末端α２，３シアル酸型糖鎖と表記する。末端シアル酸型糖鎖の例を以下に示す。
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000017
 The terminal sialic acid type sugar chain constituting the sugar chain pattern means a sugar chain in which one sugar chain alone or a plurality of terminal sugars are sialic acid. The terminal sialic acid type sugar chain includes a complex type sugar chain containing Neu5Acα2,6Gal unit at the end and a complex type sugar chain containing Neu5Acα2,3Gal unit at the end. In the present specification, a complex type sugar chain containing a Neu5Acα2,6Gal unit at the end is referred to as a terminal α2,6 sialic acid type sugar chain, and a complex type sugar chain containing a Neu5Acα2,3Gal unit at the end is referred to as a terminal α2,3 sialic acid. Notated as type sugar chain. Examples of terminal sialic acid type sugar chains are shown below.
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000017
　本発明では、転移性および転移による再発が見られる臓器（臓器指向性）についての情報が既に存在するヒト培養がん細胞４種（ＭＣＦ７，ＭＤＡ－ＭＢ－２３１，Ａ５４９，ＨｅｐＧ２）を用いて、（１）がん細胞内および膜表面に存在する全てのタンパク質の翻訳後糖鎖修飾の状態（Ｎ型糖鎖）をプロファイルした。次いで（２）それらのがん細胞の糖鎖を直接グライコブロッティング法（非特許文献９）でナノソーム（量子ドットから調製した蛍光性ナノ微粒子：非特許文献６－７、特許文献１）に捕捉して、それぞれのがん細胞由来の糖鎖を提示する糖鎖提示粒子を作製し、さらにこれらをマウスに静脈内投与した際の体内動態を、糖鎖を提示していない糖鎖提示粒子を比較対照として近赤外蛍光スペクトルによりリアルタイムで観察した(実施例１参照)。 In the present invention, four types of cultured human cancer cells (MCF7, MDA-MB-231, A549, HepG2) for which information on metastatic and metastatic recurrence is already present (organ-oriented) are used. (1) The state of post-translational sugar chain modification (N-type sugar chain) of all proteins present in cancer cells and on the surface of the membrane was profiled. Next, (2) the sugar chains of those cancer cells were directly captured by nanosomes (fluorescent nanoparticles prepared from quantum dots: Non-Patent Documents 6-7, Patent Document 1) by the glycoblotting method (Non-Patent Document 9). Then, sugar chain-presenting particles that present sugar chains derived from each cancer cell were prepared, and the pharmacokinetics when these were intravenously administered to mice were compared with the sugar chain-presenting particles that did not present sugar chains. As a control, it was observed in real time by a near-infrared fluorescence spectrum (see Example 1).
　その結果を受けて、（３）人工的に上記のヒト培養がん細胞の糖鎖プロファイルに基づき糖鎖パターンを決定し、決定した糖鎖パターンの範囲で糖鎖を提示する人工的な糖鎖提示粒子を作製した。これらを同様の手法でマウスに静脈内投与して体内動態を観察した結果、がん細胞由来のエキソソームの糖鎖パターンがエキソソームの体内動態を先導して最終的にがん細胞の臓器指向性を決定していることが証明された（実施例２参照）。 Based on the results, (3) an artificial sugar chain that artificially determines the sugar chain pattern based on the sugar chain profile of the above-mentioned human cultured cancer cells and presents the sugar chain within the range of the determined sugar chain pattern. Presented particles were prepared. As a result of intravenously administering these to mice by the same method and observing the pharmacokinetics, the sugar chain pattern of exosomes derived from cancer cells leads the pharmacokinetics of exosomes and finally the organ orientation of cancer cells. It was proved to be decided (see Example 2).
（１）がん糖鎖パターンＡ
　がん糖鎖パターンＡは、糖鎖に含まれる糖鎖の４５モル％以上が末端ハイマンノース型糖鎖である。以下、特にことわらない限り、糖鎖の％はモル％を意味する。実施例で示したがん糖鎖パターンＡを有する本発明の糖鎖提示粒子における末端ハイマンノース型糖鎖（ＨＭ）の含有率は以下の通りである。末端シアル酸型糖鎖は何れの場合も０％である。 (1) Cancer sugar chain pattern A
In the cancer sugar chain pattern A, 45 mol% or more of the sugar chains contained in the sugar chains are terminal high mannose type sugar chains. Hereinafter, unless otherwise specified,% of the sugar chain means mol%. The content of the terminal high mannose type sugar chain (HM) in the sugar chain presentation particles of the present invention having the cancer sugar chain pattern A shown in the examples is as follows. The terminal sialic acid type sugar chain is 0% in each case.
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
　がん糖鎖パターンＡは、末端ハイマンノース型糖鎖以外に糖鎖成分として、末端ガラクトース型糖鎖、末端N-アセチルグルコサミン型糖鎖及び末端シアル酸型糖鎖等を含むことができるが、末端シアル酸型糖鎖は０％以上、２％未満である。末端シアル酸型糖鎖が２％以上になると、糖鎖パターンＣまたは糖鎖パターンＤと決定でき、末端シアル酸型糖鎖の効果が優先的になる（後述する）。末端シアル酸型糖鎖が２％未満であり、末端ハイマンノース型糖鎖が４５％以上であれば、その他の糖鎖成分である末端ガラクトース型糖鎖、末端N-アセチルグルコサミン型糖鎖の種類と量に関わらず、糖鎖提示粒子は、タイプ１の体内動態を示し、特定の臓器への分布（臓器指向性）は示さない。末端ハイマンノース型糖鎖は、例えば、５０％～９５％の範囲であることができる。 The cancer sugar chain pattern A can include a terminal galactose type sugar chain, a terminal N-acetylglucosamine type sugar chain, a terminal sialic acid type sugar chain, and the like as sugar chain components in addition to the terminal hymannose type sugar chain. The terminal sialic acid type sugar chain is 0% or more and less than 2%. When the terminal sialic acid type sugar chain is 2% or more, it can be determined to be sugar chain pattern C or sugar chain pattern D, and the effect of the terminal sialic acid type sugar chain is prioritized (described later). If the terminal sialic acid type sugar chain is less than 2% and the terminal hymannose type sugar chain is 45% or more, the types of the terminal galactose type sugar chain and the terminal N-acetylglucosamine type sugar chain which are other sugar chain components. Regardless of the amount and amount, the sugar chain-presenting particles show type 1 pharmacokinetics and do not show distribution to specific organs (organ orientation). The terminal high mannose type sugar chain can be in the range of 50% to 95%, for example.
　図３ａに示すように、糖鎖を有さない糖鎖提示粒子であるＮＳ（コントロール）の例では、静脈投与後１８０分後にもほぼ全身に粒子からの蛍光が観察されたのに比べて、例えば、がん糖鎖パターンＡを有する実施例１のＧＮＳ－ＭＣＦ－７の例は、静脈投与後１８０分後には、ほとんどの粒子が体外に排出されていた。この現象は、実施例２のＧＮＳ－Ｂｕｔｔｏｎ　Ｑｕａｉｌ（図５参照）及び実施例３の０／６／３２でも同様であった。がん糖鎖パターンＡを有する糖鎖提示粒子は、糖鎖を有さない糖鎖提示粒子と比べて、粒子の体外への***が促進されるタイプ１の体内動態を示すことが分かる。 As shown in FIG. 3a, in the example of NS (control), which is a sugar chain-presenting particle having no sugar chain, fluorescence from the particle was observed almost entirely throughout the body 180 minutes after intravenous administration, as compared with that. For example, in the example of GNS-MCF-7 of Example 1 having the cancer sugar chain pattern A, most of the particles were excreted from the body 180 minutes after intravenous administration. This phenomenon was the same in GNS-Buton Quail of Example 2 (see FIG. 5) and 0/6/32 of Example 3. It can be seen that the sugar chain-presenting particles having the cancer sugar chain pattern A exhibit type 1 pharmacokinetics in which the excretion of the particles from the body is promoted as compared with the sugar chain-presenting particles having no sugar chain.
（２）がん糖鎖パターンＢ
　がん糖鎖パターンＢは、末端ハイマンノース型糖鎖が４５％未満であり、かつ末端シアル酸型糖鎖が０％以上、２％未満であるがん糖鎖パターンである。末端ハイマンノース型糖鎖が４５％未満であり、かつ末端シアル酸型糖鎖が０％以上、２％未満である糖鎖パターンを有する糖鎖提示粒子は、タイプ２の体内動態を示す。末端ハイマンノース型糖鎖及び末端シアル酸型糖鎖以外の糖鎖成分として、末端ガラクトース型糖鎖及び末端N-アセチルグルコサミン型糖鎖を含むことができるが、末端ハイマンノース型糖鎖が４５％未満であり、かつ末端シアル酸型糖鎖は０％以上、２％未満である限り、末端ガラクトース型糖鎖及び末端N-アセチルグルコサミン型糖鎖を量比が変わっても、糖鎖パターンＢと決定することができる。がん糖鎖パターンＢの糖鎖提示粒子はタイプ２の体内動態を示す。 (2) Cancer sugar chain pattern B
The cancer sugar chain pattern B is a cancer sugar chain pattern in which the terminal high mannose type sugar chain is less than 45% and the terminal sialic acid type sugar chain is 0% or more and less than 2%. Sugar chain-presenting particles having a sugar chain pattern in which the terminal hymannose-type sugar chain is less than 45% and the terminal sialic acid-type sugar chain is 0% or more and less than 2% exhibit type 2 pharmacokinetics. As sugar chain components other than the terminal hymannose type sugar chain and the terminal sialic acid type sugar chain, the terminal galactose type sugar chain and the terminal N-acetylglucosamine type sugar chain can be included, but the terminal hymannox type sugar chain is 45%. As long as it is less than 0% and the terminal sialic acid type sugar chain is 0% or more and less than 2%, even if the amount ratio of the terminal galactose type sugar chain and the terminal N-acetylglucosamine type sugar chain is changed, the sugar chain pattern B Can be decided. The sugar chain-presenting particles of the cancer sugar chain pattern B show type 2 pharmacokinetics.
　実施例で示したがん糖鎖パターンＢを有する本発明の糖鎖提示粒子の末端ハイマンノース型糖鎖（ＨＭ）、末端ガラクトース型糖鎖（Ｇａｌ）及び末端N-アセチルグルコサミン型糖鎖（ＮＡｃ－Ｇ）の含有率は表２の通りである。末端シアル酸型糖鎖は何れの場合も０％である。 Terminal hymannose type sugar chain (HM), terminal galactose type sugar chain (Gal) and terminal N-acetylglucosamine type sugar chain (NAc) of the sugar chain presentation particles of the present invention having the cancer sugar chain pattern B shown in the examples. The content of −G) is shown in Table 2. The terminal sialic acid type sugar chain is 0% in each case.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
　図５ｂ及びｃに示すように、例えば、がん糖鎖パターンＢを有する実施例２のＧＮＳ－Ｃｈｉｃｋｅｎの例は、静脈投与後１８０分後には、肝臓や脾臓へ集積されていた。この現象は、実施例２のＧＮＳ－Ｒｕｄｄｙ　Ｄｕｃｋの例（図５ｂ及びｃ参照）でも同様であった。即ち、がん糖鎖パターンＢを有する糖鎖提示粒子は、糖鎖を有さないコントロール粒子と比べて、肝臓や脾臓へ集積されるタイプ２の体内動態を示すことが分かる。 As shown in FIGS. 5b and 5c, for example, the example of GNS-Chicken of Example 2 having a cancer sugar chain pattern B was accumulated in the liver and spleen 180 minutes after intravenous administration. This phenomenon was also the same in the example of GNS-Ruddy Duck of Example 2 (see FIGS. 5b and c). That is, it can be seen that the sugar chain-presenting particles having the cancer sugar chain pattern B exhibit type 2 pharmacokinetics accumulated in the liver and spleen as compared with the control particles having no sugar chain.
（３）がん糖鎖パターンＣ
　がん糖鎖パターンＣは、糖鎖の２～１００％が末端シアル酸型糖鎖であるがん糖鎖パターンである。さらに、末端シアル酸型糖鎖には、末端α２，６シアル酸型糖鎖と末端α２，３シアル酸型糖鎖があるが、がん糖鎖パターンＣは、α２，６シアル酸型糖鎖の含有量がα２，３シアル酸型糖鎖の含有量より多い場合である。末端シアル酸型糖鎖の含有量は、例えば、３～１００％、４～８０％、または５～６０％の範囲であることができる。末端シアル酸型糖鎖以外の糖鎖は、末端ハイマンノース型糖鎖、末端ガラクトース型糖鎖及び／又は末端N-アセチルグルコサミン型糖鎖であってもかまわない。 (3) Cancer sugar chain pattern C
The cancer sugar chain pattern C is a cancer sugar chain pattern in which 2 to 100% of the sugar chains are terminal sialic acid type sugar chains. Further, the terminal sialic acid type sugar chain includes a terminal α2,6 sialic acid type sugar chain and a terminal α2,3 sialic acid type sugar chain, and the cancer sugar chain pattern C is an α2,6 sialic acid type sugar chain. Is higher than the content of α2,3 sialic acid type sugar chains. The content of the terminal sialic acid type sugar chain can be in the range of, for example, 3 to 100%, 4 to 80%, or 5 to 60%. The sugar chain other than the terminal sialic acid type sugar chain may be a terminal hymannose type sugar chain, a terminal galactose type sugar chain, and / or a terminal N-acetylglucosamine type sugar chain.
（４）がん糖鎖パターンＤ
　がん糖鎖パターンＤは、糖鎖の２～１００％が末端シアル酸型糖鎖であるがん糖鎖パターンである。がん糖鎖パターンＤは、α２，３シアル酸型糖鎖の含有量がα２，６シアル酸型糖鎖の含有量より多い場合である。末端シアル酸型糖鎖の含有量は、例えば、３～１００％、４～８０％、または５～６０％の範囲であることができる。末端シアル酸型糖鎖以外の糖鎖は、末端ハイマンノース型糖鎖、末端ガラクトース型糖鎖及び／又は末端N-アセチルグルコサミン型糖鎖であってもかまわない。 (4) Cancer sugar chain pattern D
The cancer sugar chain pattern D is a cancer sugar chain pattern in which 2 to 100% of the sugar chains are terminal sialic acid type sugar chains. The cancer sugar chain pattern D is a case where the content of the α2,3 sialic acid type sugar chain is larger than the content of the α2,6 sialic acid type sugar chain. The content of the terminal sialic acid type sugar chain can be in the range of, for example, 3 to 100%, 4 to 80%, or 5 to 60%. The sugar chain other than the terminal sialic acid type sugar chain may be a terminal hymannose type sugar chain, a terminal galactose type sugar chain, and / or a terminal N-acetylglucosamine type sugar chain.
　実施例で示したパターンＣ及びＤのがん糖鎖パターンを有する本発明の糖鎖提示粒子のα２，３シアル酸型糖鎖（α２，３）、α２，６シアル酸型糖鎖（α２，６）及び末端ハイマンノース型糖鎖（ＨＭ）の含有率は表３の通りである。末端ガラクトース型糖鎖（Ｇａｌ）及び末端N-アセチルグルコサミン型糖鎖（ＮＡｃ－Ｇ）は表示せず。 Α2,3 sialic acid type sugar chain (α2,3), α2,6 sialic acid type sugar chain (α2,3) of the sugar chain presentation particles of the present invention having the cancer sugar chain patterns of patterns C and D shown in Examples. Table 3 shows the contents of 6) and the terminal high mannose type sugar chain (HM). Terminal galactose-type sugar chains (Gal) and terminal N-acetylglucosamine-type sugar chains (NAc-G) are not shown.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
　図３ｂ及びｃに示す、糖鎖を有さない粒子であるＮＳ（コントロール）は、静脈投与後１８０分後にもほぼ全身に粒子からの蛍光が観察された。それに対して、パターンＣのがん糖鎖パターンを有する実施例２の２，６－Ｒｕｄｄｙ　Ｄｕｃｋ（図５ｂ及びｃ参照）では、ＮＳに比べて、例えば、静脈投与後１８０分後には、粒子は腋窩や鎖骨上リンパ節へ集積された。このことから、がん糖鎖パターンＣを有する糖鎖提示粒子は、糖鎖を有さない糖鎖提示粒子と比べて、腋窩や鎖骨上リンパ節へ集積されるタイプ３の体内動態を示すことが分かる。 In NS (control), which is a particle having no sugar chain shown in FIGS. 3b and 3c, fluorescence from the particle was observed almost entirely throughout the body 180 minutes after intravenous administration. On the other hand, in the 2,6-Ruddy Duck (see FIGS. 5b and c) of Example 2 having the cancer sugar chain pattern of pattern C, the particles were, for example, 180 minutes after intravenous administration, as compared with NS. It accumulated in the axilla and supraclavicular lymph nodes. From this, the sugar chain-presenting particles having the cancer sugar chain pattern C show the type 3 pharmacokinetics accumulated in the axilla and the supraclavicular lymph nodes as compared with the sugar chain-presenting particles having no sugar chain. I understand.
　パターンＤのがん糖鎖パターンを有する実施例３の２，３－Ｒｕｄｄｙ　Ｄｕｃｋ（図５ｂ及びｃ参照）の例は、静脈投与後１８０分後には、粒子は肺、肝臓、脾臓、脳、腎臓などの臓器へ分布（分散集積）された。このことから、がん糖鎖パターンＤを有する糖鎖提示粒子は、糖鎖を有さない糖鎖提示粒子と比べて、肺、肝臓、脾臓、脳、腎臓などの臓器へ分布（分散集積）されるタイプ４の体内動態を示すことが分かる。 In the example of 2,3-Ruddy Duck (see FIGS. 5b and c) of Example 3 having the cancer sugar chain pattern of pattern D, the particles were lung, liver, spleen, brain, and kidney 180 minutes after intravenous administration. It was distributed (dispersed accumulation) to such organs. From this, the sugar chain-presenting particles having the cancer sugar chain pattern D are distributed (distributed and accumulated) in organs such as lung, liver, spleen, brain, and kidney as compared with the sugar chain-presenting particles having no sugar chain. It can be seen that it shows the pharmacokinetics of type 4 that is produced.
　次に、α２，６シアル酸型糖鎖とα２，３シアル酸型糖鎖の特定について説明する。実施例１のＧＮＳ－ＭＤＡ－ＭＢ－２３１の例は、静脈投与後１８０分後には、粒子は腋窩や鎖骨上リンパ節へ集積された（特に、図３ｂ及びｃ）。この現象は、がん糖鎖パターンＣを有する実施例２の２，６－Ｒｕｄｄｙ　Ｄｕｃｋの例と同様であった。実施例１においては、末端シアル酸型糖鎖がα２，３シアル酸型糖鎖であるか、α２，６シアル酸型糖鎖であるか特定をしていないが、この体内動態の結果から、実施例１のＧＮＳ－ＭＤＡ－ＭＢ－２３１は糖鎖パターンＣと判断して表４に表示した。 Next, the identification of the α2,6 sialic acid type sugar chain and the α2,3 sialic acid type sugar chain will be described. In the example of GNS-MDA-MB-231 of Example 1, particles accumulated in the axilla and supraclavicular lymph nodes 180 minutes after intravenous administration (particularly, FIGS. 3b and 3c). This phenomenon was similar to the example of 2,6-Ruddy Duck of Example 2 having the cancer sugar chain pattern C. In Example 1, it is not specified whether the terminal sialic acid type sugar chain is an α2,3 sialic acid type sugar chain or an α2,6 sialic acid type sugar chain, but from the result of this pharmacokinetics, it is not specified. GNS-MDA-MB-231 of Example 1 was determined to be sugar chain pattern C and is shown in Table 4.
　実施例１のＧＮＳ－Ａ５４９の例は、静脈投与後１８０分後には、粒子は肺、肝臓、脾臓、脳、腎臓などの臓器へ分布（分散集積）された（特に、図３ｂ及びｃ）。この現象は、がん糖鎖パターンＤを有する実施例２の２，３－Ｒｕｄｄｙ　Ｄｕｃｋの例と同様であった。実施例１においては、末端シアル酸型糖鎖がα２，３シアル酸型糖鎖であるか、α２，６シアル酸型糖鎖であるか特定をしていないが、この体内動態の結果から、実施例１のＧＮＳ－Ａ５４９及びＧＮＳ－ＨｅｐＧ２は糖鎖パターンＤと判断して表４に表示した。α２，６シアル酸型糖鎖かα２，３シアル酸型糖鎖であるかの特定は、本明細書ではその機能において判断したが、化学的・酵素分解特性などの分析方法においても特定は可能である。 In the example of GNS-A549 of Example 1, particles were distributed (dispersed accumulation) in organs such as lung, liver, spleen, brain, and kidney 180 minutes after intravenous administration (particularly, FIGS. 3b and c). This phenomenon was similar to the example of 2,3-Ruddy Duck of Example 2 having the cancer sugar chain pattern D. In Example 1, it is not specified whether the terminal sialic acid type sugar chain is an α2,3 sialic acid type sugar chain or an α2,6 sialic acid type sugar chain, but from the result of this pharmacokinetics, it is not specified. GNS-A549 and GNS-HepG2 of Example 1 were judged to be sugar chain pattern D and are shown in Table 4. Whether it is an α2,6 sialic acid type sugar chain or an α2,3 sialic acid type sugar chain is determined by its function in the present specification, but it can also be specified by an analysis method such as chemical / enzymatic decomposition characteristics. Is.
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000021
　がん糖鎖パターンは、前述のように研究用途に市販され一般化しているがん細胞または特定のがん治療患者のがん細胞の糖鎖そのものであることができる。さらにがん糖鎖パターンは、がん細胞の糖鎖をプロファイルし、得られたプロファイルに基づいて決定された糖鎖パターンであることができる。プロファイルに基づいて決定される糖鎖パターンは、糖鎖成分の種類と含有量をプロファイルと同一とした糖鎖パターンであるか、あるいは糖鎖成分の種類と含有量の一部をプロファイルと同一とした糖鎖パターンであるか、のいずれでもよい。糖鎖成分の種類と含有量の一部をプロファイルと同一とする場合、プロファイルに含まれる糖鎖成分の一部の種類を減量または欠損させることができる。より詳細には、プロファイルに含まれる糖鎖成分をいくつかの型に分類した場合、含有量が多い１又は２以上の型に属する糖鎖成分を含み、含有量が少ない１又は２以上の型に属する糖鎖成分を減量または欠損させた糖鎖パターンであることができる。がん細胞の糖鎖をプロファイルし、得られたプロファイルに基づいて決定された糖鎖パターンとは、がん細胞由来の上記のがん糖鎖パターンと糖鎖成分の種類と各糖鎖成分の含有量が異なるが、がん細胞由来のがん糖鎖パターンと同一の体内動態を示す糖鎖パターンである。がん細胞の糖鎖をプロファイルした場合、プロファイルに含まれる糖鎖成分をいくつかの型に分類した場合、含有量が多い１又は２以上の型に属する糖鎖成分をそのまま含み、含有量が少ない１又は２以上の型に属する糖鎖成分を減量または欠損させることで、糖鎖をプロファイルしたがん細胞由来のがん糖鎖パターンと同一の体内動態を示す糖鎖パターンとすることができる。糖鎖成分の型としては、例えば、末端ハイマンノース型糖鎖、末端ガラクトース型糖鎖、末端Ｎ－アセチルグルコサミン型糖鎖、末端α２，６シアル酸型糖鎖、末端α２，３シアル酸型糖鎖を挙げることができる。 The cancer sugar chain pattern can be the sugar chain itself of cancer cells that are commercially available and generalized for research purposes as described above, or cancer cells of specific cancer treatment patients. Further, the cancer sugar chain pattern can be a sugar chain pattern determined based on the profile obtained by profiling the sugar chain of the cancer cell. The sugar chain pattern determined based on the profile is a sugar chain pattern in which the type and content of the sugar chain component are the same as the profile, or a part of the type and content of the sugar chain component is the same as the profile. It may be either a sugar chain pattern. When a part of the type and content of the sugar chain component is the same as the profile, the amount of some types of the sugar chain component contained in the profile can be reduced or deleted. More specifically, when the sugar chain components contained in the profile are classified into several types, the sugar chain components belonging to one or two or more types having a high content are contained, and the one or two or more types having a low content are included. It can be a sugar chain pattern in which the amount of the sugar chain component belonging to is reduced or deleted. The sugar chain pattern determined based on the profile obtained by profiling the sugar chain of the cancer cell is the above-mentioned cancer sugar chain pattern derived from the cancer cell, the type of the sugar chain component, and each sugar chain component. Although the content is different, it is a sugar chain pattern that shows the same pharmacokinetics as the cancer sugar chain pattern derived from cancer cells. When the sugar chain of a cancer cell is profiled, when the sugar chain component contained in the profile is classified into several types, the sugar chain component belonging to one or more types having a high content is contained as it is, and the content is high. By reducing or deleting the amount of sugar chain components belonging to a small number of 1 or 2 or more types, it is possible to obtain a sugar chain pattern showing the same pharmacokinetics as the cancer sugar chain pattern derived from cancer cells in which the sugar chain is profiled. .. Examples of the types of sugar chain components include terminal hymannose type sugar chain, terminal galactose type sugar chain, terminal N-acetylglucosamine type sugar chain, terminal α2,6 sialic acid type sugar chain, and terminal α2,3 sialic acid type sugar. You can raise a chain.
　ある糖鎖パターンが、がん細胞由来のがん糖鎖パターンと異なる糖鎖成分の種類と各糖鎖成分の含有量を有するが、体内動態が主に末端ハイマンノース型糖鎖に依存する糖鎖パターンＡである場合、タイプ１の体内動態を示す。
　同様に、ある糖鎖パターンが、がん細胞由来のがん糖鎖パターンと異なる糖鎖成分の種類と各糖鎖成分の含有量を有するが、体内動態が主に末端ガラクトース型糖鎖又は末端N-アセチルグルコサミン型糖鎖に依存する糖鎖パターンＢである場合、タイプ２の体内動態を示す。
　ある糖鎖パターンが、がん細胞由来のがん糖鎖パターンと異なる糖鎖成分の種類と各糖鎖成分の含有量を有するが、体内動態が主に末端α２，６シアル酸型糖鎖に依存する糖鎖パターンＣである場合、タイプ３の体内動態を示す。
　ある糖鎖パターンが、がん細胞由来のがん糖鎖パターンと異なる糖鎖成分の種類と各糖鎖成分の含有量を有するが、体内動態が主に末端α２，３シアル酸型糖鎖に依存する糖鎖パターンＤである場合、タイプ４の体内動態を示す。 A sugar chain pattern has a type of sugar chain component and a content of each sugar chain component that are different from the cancer sugar chain pattern derived from cancer cells, but the pharmacokinetics mainly depend on the terminal hymannose type sugar chain. When it is the chain pattern A, it shows the pharmacokinetics of type 1.
Similarly, a sugar chain pattern has a type of sugar chain component and a content of each sugar chain component that are different from the cancer sugar chain pattern derived from cancer cells, but the pharmacokinetics are mainly terminal galactose-type sugar chains or terminals. When it is a sugar chain pattern B that depends on an N-acetylglucosamine type sugar chain, it shows type 2 pharmacokinetics.
A certain sugar chain pattern has a type of sugar chain component and a content of each sugar chain component different from the cancer sugar chain pattern derived from cancer cells, but the pharmacokinetics are mainly terminal α2,6 sialic acid type sugar chains. If it is a dependent sugar chain pattern C, it shows type 3 pharmacokinetics.
A certain sugar chain pattern has a type of sugar chain component and a content of each sugar chain component different from those of cancer cell-derived cancer sugar chain patterns, but the pharmacokinetics are mainly terminal α2,3 sialic acid type sugar chains. When it is the dependent sugar chain pattern D, it shows the pharmacokinetics of type 4.
　本発明の糖鎖提示粒子は、例えば、原発巣のがん細胞からの転移部位への予防や治療においては、がん細胞の糖鎖をプロファイルし、得られたプロファイルに基づいてがん糖鎖パターンＡ～Ｄのいずれであるかを決定し、選択した糖鎖パターンを有する糖鎖提示粒子を公知の方法を用いて調製し、提供することができる。 The sugar chain presenting particles of the present invention profile the sugar chain of the cancer cell, for example, in the prevention or treatment of the primary tumor from the cancer cell to the metastasis site, and the cancer sugar chain is based on the obtained profile. Which of the patterns A to D can be determined, and sugar chain presentation particles having the selected sugar chain pattern can be prepared and provided by a known method.
　選択した糖鎖パターンは、特定の糖鎖を含むことが知られている既存の糖鎖を例えば、酵素を用いて処理することで人工的に改変することができ、既存の糖鎖としては実施例２に示すように、うずら、鶏、アヒルなどの卵白および鶏卵黄に含まれる糖鎖を挙げることができる。既存材料の糖鎖をそのままあるいは適宜加工して特定の糖鎖パターンとして利用することもできる。例えば、実施例２に示すように、糖鎖を、酵素処理することで、所望のがん糖鎖パターンを提供する材料として用いることができる。あるいは、組成の分かっている複数の糖鎖を含む材料を適当な比率で混合することで、所望のがん糖鎖パターンを提供する材料として用いることができる（実施例２参照）。 The selected sugar chain pattern can be artificially modified by treating an existing sugar chain known to contain a specific sugar chain with, for example, an enzyme, and is carried out as an existing sugar chain. As shown in Example 2, sugar chains contained in egg whites of quail, chickens, ducks and the like and chicken egg yolks can be mentioned. It is also possible to use the sugar chain of the existing material as it is or by processing it as appropriate as a specific sugar chain pattern. For example, as shown in Example 2, the sugar chain can be used as a material for providing a desired cancer sugar chain pattern by treating the sugar chain with an enzyme. Alternatively, by mixing a material containing a plurality of sugar chains having a known composition in an appropriate ratio, it can be used as a material that provides a desired cancer sugar chain pattern (see Example 2).
　実施例２で用いたうずら、鶏、アヒルの卵白および鶏卵黄の糖鎖は、糖鎖構造およびその発現量プロファイルが明確で安全・安価な生体由来素材であるので、これらの糖鎖をそのまま、あるいは原料として改変した後にナノ粒子に提示して糖鎖提示粒子とすることで、がん細胞由来のエキソソームと同様の体内動態を再現できる。糖鎖パターンは、生体由来のものだけではなく、人工的に合成した糖鎖成分の混合物であることもできる。糖鎖成分の人工的合成は、公知の方法で行うことができる。 Since the sugar chains of quail, chicken, duck egg white and chicken egg yolk used in Example 2 are safe and inexpensive biological materials having a clear sugar chain structure and expression level profile thereof, these sugar chains can be used as they are. Alternatively, by modifying it as a raw material and then presenting it on nanoparticles to obtain sugar chain-presenting particles, it is possible to reproduce the same pharmacokinetics as exosomes derived from cancer cells. The sugar chain pattern can be not only derived from a living body but also a mixture of artificially synthesized sugar chain components. The artificial synthesis of the sugar chain component can be carried out by a known method.
　糖鎖の粒子表面への提示は、公知の直接グライコブロッティング法（非特許文献９）により実施できる。糖鎖の粒子表面への提示は、例えば、実施例１（Ｃ）及び実施例２（Ｂ）で示す方法で、作製したナノソームにがん糖鎖を含む混合物を反応させることで、実施して糖鎖提示粒子を調製できる。 The sugar chain can be presented on the particle surface by a known direct blotting method (Non-Patent Document 9). The presentation of sugar chains on the particle surface is carried out, for example, by reacting the prepared nanosomes with a mixture containing cancer sugar chains by the methods shown in Examples 1 (C) and 2 (B). Sugar chain presentation particles can be prepared.
　前記ナノ粒子の表面の糖鎖は、下記一般式（Ｂ）で示される糖鎖を提示するアルカンチオールのスルフィド結合体であることができる。
Figure JPOXMLDOC01-appb-C000022
 　一般式（Ｂ）中、ｎ１は２～３０の整数であり、ｎ２は２～３０の整数であり、－Ｓ－末端がナノ粒子にスルフィド結合する担持部位であり、Ｒ１０は糖鎖含有部位である。糖鎖含有部位Ｒ１０の糖鎖は、がん糖鎖パターンであり、がん細胞の糖鎖や、糖鎖のプロファイルに基づいて決定された糖鎖パターンＡ～Ｄに応じた糖鎖の成分及びそれらの組成比を有する。 The sugar chain on the surface of the nanoparticles can be a sulfide bond of an alkanethiol that presents the sugar chain represented by the following general formula (B).
Figure JPOXMLDOC01-appb-C000022
 In the general formula (B), n1 is an integer of 2 to 30, n2 is an integer of 2 to 30, the -S-terminal is a carrier site where a sulfide bond is attached to nanoparticles, and R 10 is a sugar chain-containing site. Is. Sugar carbohydrate containing moieties R 10 is a cancer oligosaccharide patterns, sugar or cancer cells, components of the sugar chain according to the sugar chain pattern A ~ D which are determined based on the sugar chain profile And their composition ratio.
　本発明の糖鎖提示粒子は、例えば、下記一般式（１０）で模式的に示すことができる。
Figure JPOXMLDOC01-appb-C000023
 The sugar chain-presenting particles of the present invention can be schematically represented by, for example, the following general formula (10).
Figure JPOXMLDOC01-appb-C000023
　一般式（１０）のナノ粒子（ＮＰ）に担持されている残基は、糖鎖含有部位として、Ｒ１０を有する一般式（Ｂ）で示される糖鎖含有基及び一般式（Ａ）で示されるリン脂質基である。いずれも各基の末端のスルフィド（－Ｓ－）を介してナノ粒子（ＮＰ）に担持される。実際の複合体では、一般式（Ａ）で示されるリン脂質基は１個以上、ナノ粒子ＮＰに担持されている。また、Ｒ１０には、糖鎖パターンを構成する糖鎖成分からなる複数の糖鎖成分含有基が含まれる。 Residues which are carried on the nanoparticles (NP) of the general formula (10), as a sugar chain-containing site, represented by sugar chain-containing group and the general formula represented by the general formula with R 10 (B) (A) Is a phospholipid group. Both are supported on nanoparticles (NP) via sulfides (-S-) at the ends of each group. In the actual complex, one or more phospholipid groups represented by the general formula (A) are supported on the nanoparticles NP. Further, R 10 contains a plurality of sugar chain component-containing groups composed of sugar chain components constituting the sugar chain pattern.
　本発明の糖鎖提示粒子は、各パターンの糖鎖に応じた体内動態を有するので、その性質を利用して、特定の組織や臓器に集積させることができる。そのため、糖鎖が有するパターンを選択することで、特定の組織や臓器に集積させることができる糖鎖提示粒子とすることができ、この性質を利用してＤＤＳを提供することもできる。 Since the sugar chain-presenting particles of the present invention have pharmacokinetics according to each pattern of sugar chains, they can be accumulated in a specific tissue or organ by utilizing the properties. Therefore, by selecting the pattern of the sugar chain, it is possible to obtain sugar chain presentation particles that can be accumulated in a specific tissue or organ, and DDS can also be provided by utilizing this property.
　本発明の糖鎖提示粒子は、薬剤部位をさらに有することができる。薬剤部位の薬剤には特に制限はないが、例えば、抗がん剤、抗炎症剤、であることができる。 The sugar chain presenting particles of the present invention can further have a drug site. The drug at the drug site is not particularly limited, and may be, for example, an anticancer agent or an anti-inflammatory agent.
　本発明の糖鎖提示粒子においては、薬剤部位として、例えば、一般式（Ｃ）で示される薬剤含有基が、ナノ粒子の表面に担持されていることができる。
Figure JPOXMLDOC01-appb-C000024
 　一般式（Ｃ）中、ｎ１は２～３０の整数であり、ｎ２は２～３０の整数であり、－Ｓ－末端がナノ粒子にスルフィド結合する担持部位であり、Ｒ２０は薬剤含有部位である。 In the sugar chain presentation particles of the present invention, for example, a drug-containing group represented by the general formula (C) can be supported on the surface of the nanoparticles as a drug site.
Figure JPOXMLDOC01-appb-C000024
 In the general formula (C), n1 is an integer of 2 to 30, n2 is an integer of 2 to 30, the -S-terminal is a carrier site where a sulfide bond is attached to nanoparticles, and R 20 is a drug-containing site. is there.
　一般式（Ｃ）で示される薬剤含有基は、式中のｎ１及びｎ２を選択することで、薬剤含有基とナノ粒子との間の距離を所望の範囲に保つことができる。ｎ１は２～３０、好ましくは５～１６、より好ましくは４～１５の範囲の整数である。ｎ２は２～３０、好ましくは５～２０、より好ましくは７～１５の範囲の整数である。 By selecting n1 and n2 in the formula for the drug-containing group represented by the general formula (C), the distance between the drug-containing group and the nanoparticles can be maintained within a desired range. n1 is an integer in the range of 2 to 30, preferably 5 to 16, and more preferably 4 to 15. n2 is an integer in the range of 2 to 30, preferably 5 to 20, more preferably 7 to 15.
　本発明の糖鎖提示粒子で薬剤部位を有する粒子は、例えば、下記一般式（１１）で模式的に示すことができる。
Figure JPOXMLDOC01-appb-C000025
 The sugar chain-presenting particles of the present invention having a drug site can be schematically represented by, for example, the following general formula (11).
Figure JPOXMLDOC01-appb-C000025
　一般式（１１）のナノ粒子（ＮＰ）に担持されている残基は、上から一般式（Ｂ）で示される糖鎖含有基、一般式（Ａ）で示されるリン脂質基、及び一般式（Ｃ）で示される薬剤含有基である。実際の複合体は、一般式（Ｂ）で示される糖鎖含有基、一般式（Ａ）で示されるリン脂質基、及び一般式（Ｃ）で示される薬剤含有基は、それぞれ１個以上、スルフィド結合体としてナノ粒子ＮＰに担持されている。 The residues supported on the nanoparticles (NP) of the general formula (11) are the sugar chain-containing group represented by the general formula (B), the phospholipid group represented by the general formula (A), and the general formula (A) from the top. It is a drug-containing group represented by (C). The actual complex contains one or more sugar chain-containing groups represented by the general formula (B), phospholipid groups represented by the general formula (A), and drug-containing groups represented by the general formula (C). It is supported on nanoparticles NP as a sulfide conjugate.
　本発明の糖鎖提示粒子は薬剤部位を有する場合、この糖鎖提示粒子を有効成分として含有する、がん予防薬や治療薬を包含する。本発明のがん予防薬や治療薬は、糖鎖提示粒子ががん細胞由来の糖鎖を捕捉しそのままに提示したがん糖鎖パターンであることが好ましいが、この糖鎖をプロファイルし、得られたプロファイルに基づいて決定したがん糖鎖パターンＢ、ＣまたはＤを有する糖鎖提示粒子であってもかまわない。この糖鎖提示粒子は、各糖鎖パターンに応じたタイプ２～４の体内動態を示し、特定の転移性がんの予防や治療に有用である。 When the sugar chain-presenting particles of the present invention have a drug site, they include cancer preventive agents and therapeutic agents containing the sugar chain-presenting particles as an active ingredient. The cancer preventive agent and the therapeutic agent of the present invention preferably have a cancer sugar chain pattern in which the sugar chain presenting particles capture the sugar chain derived from the cancer cell and present it as it is. It may be a sugar chain presentation particle having a cancer sugar chain pattern B, C or D determined based on the obtained profile. These sugar chain-presenting particles show type 2 to 4 pharmacokinetics according to each sugar chain pattern, and are useful for the prevention and treatment of specific metastatic cancers.
　がん糖鎖パターンＢを有する糖鎖提示粒子の場合、肝臓や脾臓への転移性がんの予防や治療に有用であり、
がん糖鎖パターンＣを有する糖鎖提示粒子の場合、腋窩や鎖骨上リンパ節への転移性がんの予防や治療に有用であり、
がん糖鎖パターンＤを有する糖鎖提示粒子の場合、肺、肝臓、脾臓、脳、腎臓などの臓器への転移性がんの予防や治療に有用である。 In the case of sugar chain presentation particles having cancer sugar chain pattern B, it is useful for prevention and treatment of metastatic cancer to the liver and spleen.
In the case of sugar chain-presenting particles having a cancer sugar chain pattern C, it is useful for the prevention and treatment of metastatic cancer to the axilla and supraclavicular lymph nodes.
In the case of sugar chain presenting particles having cancer sugar chain pattern D, it is useful for prevention and treatment of metastatic cancer to organs such as lung, liver, spleen, brain and kidney.
（糖鎖提示粒子キット）
　本発明は、異なるがん糖鎖パターンを有する２種類以上の糖鎖提示粒子を含有する、糖鎖提示粒子キットを包含する。キットにおける各糖鎖提示粒子は、別々の容器に保存されているか、1つの容器に混合物として保存されていてもよい。キットに含まれる糖鎖提示粒子は、上記本発明の糖鎖提示粒子のいずれかである。キットにおける異なるがん糖鎖パターンは、上記糖鎖パターンＡ～Ｄのいずれか２種以上の糖鎖パターンである。糖鎖パターンＡ～Ｄの選択は、例えば、がん患者から採取されたがん細胞の糖鎖をプロファイルすることで、糖鎖パターンを決定し、選択される。 (Sugar chain presentation particle kit)
The present invention includes a sugar chain presentation particle kit containing two or more types of sugar chain presentation particles having different cancer sugar chain patterns. Each sugar chain presentation particle in the kit may be stored in a separate container or as a mixture in one container. The sugar chain-presenting particles included in the kit are any of the above-mentioned sugar chain-presenting particles of the present invention. The different cancer sugar chain patterns in the kit are any two or more sugar chain patterns of the above sugar chain patterns A to D. The sugar chain patterns A to D are selected by determining the sugar chain pattern by, for example, profiling the sugar chains of cancer cells collected from a cancer patient.
　本発明の糖鎖提示粒子キットは、決定された糖鎖パターンにおいて、その糖鎖パターンである糖鎖提示粒子を選択して使用することになるが、がんの種類によっては、複数を併用して使用することもできる。例えば、糖鎖パターンＢとＣの２種を併用して、肝臓及び脾臓（タイプ２）並びに腋窩及び鎖骨上リンパ節（タイプ３）の両方へ集積され得る糖鎖提示粒子として使用してもかまわない。 In the sugar chain presentation particle kit of the present invention, the sugar chain presentation particles which are the sugar chain patterns are selected and used in the determined sugar chain pattern, but a plurality of sugar chain presentation particles may be used in combination depending on the type of cancer. Can also be used. For example, two types of sugar chain patterns B and C may be used in combination as sugar chain presentation particles that can be accumulated in both the liver and spleen (type 2) and the axilla and supraclavicular lymph nodes (type 3). Absent.
　本発明の糖鎖提示粒子キットは、糖鎖提示粒子が、薬物部位をさらに有する糖鎖提示粒子であることができる。薬物としては、かんの予防薬、治療薬が挙げられる。 In the sugar chain presentation particle kit of the present invention, the sugar chain presentation particles can be sugar chain presentation particles further having a drug site. Examples of the drug include a preventive drug and a therapeutic drug for canes.
　本発明の糖鎖提示粒子は、糖鎖提示粒子を有効成分として、当業者に公知の方法で製剤化することが可能である。例えば、水もしくはそれ以外の薬学的に許容し得る液との無菌性溶液、又は懸濁液剤の注射剤の形で非経口的に使用できる。例えば、薬理学上許容される担体もしくは媒体、具体的には、滅菌水や生理食塩水、植物油、乳化剤、懸濁剤、界面活性剤、安定剤、香味剤、賦形剤、ベヒクル、防腐剤、結合剤などと適宜組み合わせて、一般に認められた製薬実施に要求される単位用量形態で混和することによって製剤化することが考えられる。これら製剤における有効成分量は指示された範囲の適当な用量が得られるようにするものである。 The sugar chain-presenting particles of the present invention can be formulated by a method known to those skilled in the art, using the sugar chain-presenting particles as an active ingredient. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injectable suspension. For example, pharmacologically acceptable carriers or vehicles, specifically sterile water or saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavors, excipients, vehicles, preservatives. , It is conceivable to formulate by appropriately combining with a binder and the like and mixing in a unit dose form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations ensures that an appropriate dose in the indicated range is available.
　注射のための無菌組成物は注射用蒸留水のようなベヒクルを用いて通常の製剤実施に従って処方することができる。注射用の水溶液としては、例えば生理食塩水、ブドウ糖やその他の補助薬を含む等張液、例えばＤ－ソルビトール、Ｄ－マンノース、Ｄ－マンニトール、塩化ナトリウムが挙げられ、適当な溶解補助剤、例えばアルコール、具体的にはエタノール、ポリアルコール、例えばプロピレングリコール、ポリエチレングリコール、非イオン性界面活性剤、例えばポリソルベート８０（ＴＭ）、ＨＣＯ－６０と併用してもよい。 The sterile composition for injection can be formulated according to normal formulation practices using a vehicle such as distilled water for injection. Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizers such as. Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol and nonionic surfactants such as polysorbate 80 (TM), HCO-60 may be used in combination.
　油性液としてはゴマ油、大豆油があげられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコールと併用してもよい。また、緩衝剤、例えばリン酸塩緩衝液、酢酸ナトリウム緩衝液、無痛化剤、例えば、塩酸プロカイン、安定剤、例えばベンジルアルコール、フェノール、酸化防止剤と配合してもよい。調製された注射液は通常、適当なアンプルに充填させる。また、リポソームも細胞送達のために該薬剤をカプセル化するために使用可能である。 Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate and benzyl alcohol may be used in combination as solubilizing agents. It may also be blended with buffers such as phosphate buffers, sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol and antioxidants. The prepared injection solution is usually filled in a suitable ampoule. Liposomes can also be used to encapsulate the drug for cell delivery.
　本発明の糖鎖提示粒子及び糖鎖提示粒子キットは、使用目的に応じて、臓器への転移性がんの予防や治療のために、がん患者またはがんが疑われる患者に、適宜の投与経路で、投与することができる。 The sugar chain presentation particles and the sugar chain presentation particle kit of the present invention are suitable for cancer patients or patients suspected of having cancer for the prevention or treatment of metastatic cancer to organs, depending on the purpose of use. It can be administered by the administration route.
　投与は、経口又は非経口であり、好ましくは非経口投与であり、具体的には、注射剤型、経鼻投与剤型、経肺投与剤型、経皮投与型などが挙げられる。注射剤型の例としては、例えば、静脈内注射、筋肉内注射、腹腔内注射、皮下注射などにより全身又は局部的に投与することができる。 The administration is oral or parenteral, preferably parenteral administration, and specific examples thereof include injection type, nasal administration type, pulmonary administration type, and transdermal administration type. As an example of the injection type, for example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
　本発明の糖鎖提示粒子及び糖鎖提示粒子キットの用量および投与方法は、患者の年齡、体重、性別、治療すべき症状の性質もしくは重篤度等により適宜選択することができる。本発明の糖鎖提示粒子及び糖鎖提示粒子キットを含有する医薬組成物の投与量としては、例えば、一回につき体重１ｋｇあたり０．０００１ｍｇから１，０００ｍｇの範囲で選ぶことが可能である。あるいは、患者あたり０．０１～１００，０００ｍｇ／ｂｏｄｙの範囲で投与量を選ぶことができるが、これらの数値に必ずしも制限されるものではない。投与量、投与方法は、患者の年齢、体重、性別、症状などにより変動するが、当事者であれば適宜選択することが可能である。 The dose and administration method of the sugar chain presentation particles and the sugar chain presentation particle kit of the present invention can be appropriately selected depending on the age, weight, sex, nature or severity of the symptom to be treated, and the like. The dose of the pharmaceutical composition containing the sugar chain-presenting particles and the sugar chain-presenting particle kit of the present invention can be selected, for example, in the range of 0.0001 mg to 1,000 mg per 1 kg of body weight at a time. Alternatively, the dose can be selected in the range of 0.01 to 100,000 mg / body per patient, but is not necessarily limited to these values. The dose and administration method vary depending on the age, weight, sex, symptoms, etc. of the patient, but can be appropriately selected by the person concerned.
＜糖鎖提示粒子の製造方法＞
　本発明は、上記本発明の糖鎖提示粒子の製造方法を包含する。
　本発明の糖鎖提示粒子は、表面の少なくとも一部をリン脂質で被覆されたナノ粒子の表面に、所望のがん糖鎖パターンを提示することで調製できる。表面の少なくとも一部をリン脂質で被覆されたナノ粒子の調製方法は後述する。 <Manufacturing method of sugar chain presentation particles>
The present invention includes the above-mentioned method for producing sugar chain-presenting particles of the present invention.
The sugar chain-presenting particles of the present invention can be prepared by presenting a desired cancer sugar chain pattern on the surface of nanoparticles in which at least a part of the surface is coated with phospholipid. The method for preparing nanoparticles in which at least a part of the surface is coated with phospholipid will be described later.
　所望のがん糖鎖パターンとしては、前述のように、被検者から採取したがん細胞の糖鎖を切り出しそのまま利用するがん糖鎖パターン、又は、既存材料の糖鎖をそのままあるいは適宜改変して特定の糖鎖パターンに調整したものであってもかまわない。特定の糖鎖パターンとは、被検者から採取したがん細胞の糖鎖をプロファイルし、得られたプロファイルに基づいて決定された糖鎖パターンである。プロファイルに基づいて決定される糖鎖パターンは、糖鎖成分の種類と含有量をプロファイルと同一とした糖鎖パターンであるか、あるいは糖鎖成分の種類と含有量の一部をプロファイルと同一とした糖鎖パターンであるか、のいずれでもよい。糖鎖成分の種類と含有量の一部をプロファイルと同一とする場合、プロファイルに含まれる糖鎖成分の一部の種類を減量または欠損させることができる。より詳細には、プロファイルに含まれる糖鎖成分をいくつかの型に分類した場合、含有量が多い１又は２以上の型に属する糖鎖成分を含み、含有量が少ない１又は２以上の型に属する糖鎖成分を減量または欠損させた糖鎖パターンであることができる。がん細胞の糖鎖をプロファイルした場合、プロファイルに含まれる糖鎖成分をいくつかの型に分類した場合、含有量が多い１又は２以上の型に属する糖鎖成分をそのまま含み、含有量が少ない１又は２以上の型に属する糖鎖成分を減量または欠損させることで、糖鎖をプロファイルしたがん細胞由来のがん糖鎖パターンと同一の体内動態を示す糖鎖パターンとすることができる。糖鎖成分の型としては、例えば、末端ハイマンノース型糖鎖、末端ガラクトース型糖鎖、末端Ｎ－アセチルグルコサミン型糖鎖、末端α２，６シアル酸型糖鎖、末端α２，３シアル酸型糖鎖を挙げることができる。 As the desired cancer sugar chain pattern, as described above, the cancer sugar chain pattern obtained by cutting out the sugar chain of the cancer cell collected from the subject and using it as it is, or the sugar chain of the existing material as it is or appropriately modified. It may be adjusted to a specific sugar chain pattern. The specific sugar chain pattern is a sugar chain pattern determined based on a profile obtained by profiling the sugar chain of cancer cells collected from a subject. The sugar chain pattern determined based on the profile is a sugar chain pattern in which the type and content of the sugar chain component are the same as the profile, or a part of the type and content of the sugar chain component is the same as the profile. It may be either a sugar chain pattern. When a part of the type and content of the sugar chain component is the same as the profile, the amount of some types of the sugar chain component contained in the profile can be reduced or deleted. More specifically, when the sugar chain components contained in the profile are classified into several types, the sugar chain components belonging to one or two or more types having a high content are contained, and the one or two or more types having a low content are included. It can be a sugar chain pattern in which the amount of the sugar chain component belonging to is reduced or deleted. When the sugar chain of a cancer cell is profiled, when the sugar chain component contained in the profile is classified into several types, the sugar chain component belonging to one or more types having a high content is contained as it is, and the content is high. By reducing or deleting the amount of sugar chain components belonging to a small number of 1 or 2 or more types, it is possible to obtain a sugar chain pattern showing the same pharmacokinetics as the cancer sugar chain pattern derived from cancer cells in which the sugar chain is profiled. .. Examples of the types of sugar chain components include terminal hymannose type sugar chain, terminal galactose type sugar chain, terminal N-acetylglucosamine type sugar chain, terminal α2,6 sialic acid type sugar chain, and terminal α2,3 sialic acid type sugar. You can raise a chain.
　本発明の糖鎖提示粒子の製造方法の概念図（がん細胞の糖鎖パターンを表面に提示したエキソソームモデルの作製法とその体内動態・臓器指向性のin vivoイメージング）を図１に示す。（a）は、糖鎖提示粒子の基本的な構造：量子ドット、金ナノ微粒子、金被覆磁性ナノ微粒子などの金属ナノ微粒子をコアとしてその表面を、ホスホリルコリン基を頭部に持つアルカンチオールと適当量のアミノオキシリンカーを含むアルカンチオールの自己組織化混合単分子膜で完全被覆した抗凝集性ナノ微粒子である。（b）は、グライコブロッティング法の基本原理であり、糖鎖の還元末端（アルデヒド基やケトン基を含む化合物）とナノ粒子表面のアミノオキシ基が特異的に反応してオキシム結合を形成する。（c）は、ナノ粒子表面へのがん細胞由来糖鎖パターンの提示法とｉｎ　ｖｉｖｏイメージングを示す。採取した、あるいは培養したがん細胞から調製した糖鎖を含む混合物から還元末端（アルデヒド基と等価）を持つ糖鎖に特異的な化学反応を活用したグライコブロッティング法によりナノ粒子表面に捕捉し提示させる。 FIG. 1 shows a conceptual diagram of a method for producing sugar chain-presenting particles of the present invention (a method for producing an exosome model in which a sugar chain pattern of cancer cells is presented on the surface and its pharmacokinetics / organ-oriented in vivo imaging). (A) is suitable for the basic structure of sugar chain presentation particles: alkanethiol having a phosphorylcholine group as a head on the surface of metal nanoparticles such as quantum dots, gold nanoparticles, and gold-coated magnetic nanoparticles as a core. Anti-aggregating nanoparticles completely coated with a self-assembled monolayer of alcanthiol containing an amount of aminooxylinker. (B) is the basic principle of the glycoblotting method, in which the reducing end of the sugar chain (compound containing an aldehyde group or a ketone group) and the aminooxy group on the surface of the nanoparticles specifically react to form an oxime bond. (C) shows a method for presenting a cancer cell-derived sugar chain pattern on the surface of nanoparticles and in vivo imaging. A mixture containing sugar chains prepared from collected or cultured cancer cells is captured and presented on the surface of nanoparticles by a glycoblotting method utilizing a chemical reaction specific to sugar chains having a reducing end (equivalent to an aldehyde group). Let me.
　表面の少なくとも一部をリン脂質で被覆されたナノ粒子は、下記一般式（Ｄ）で示される架橋前駆体Ｘ及び下記一般式（Ｅ）で示されるリン脂質前駆体とコロイド状ナノ粒子とを混合して、ナノ粒子の表面に架橋前駆体Ｘ及びリン脂質を担持した表面修飾ナノ粒子を得ることで実施できる。この方法は、特許文献１に記載され、特許文献１の全記載は、ここに特に開示として援用される。 The nanoparticles whose surface is coated with phospholipids are composed of a crosslinked precursor X represented by the following general formula (D), a phospholipid precursor represented by the following general formula (E), and colloidal nanoparticles. It can be carried out by mixing to obtain surface-modified nanoparticles in which the crosslinked precursor X and the phospholipid are supported on the surface of the nanoparticles. This method is described in Patent Document 1, and the entire description of Patent Document 1 is incorporated herein by reference in particular.
Figure JPOXMLDOC01-appb-C000026
 （一般式（Ｄ）中、ｎ１は２～３０の整数であり、ｎ２は２～３０の整数である。)
Figure JPOXMLDOC01-appb-C000026
 (In the general formula (D), n1 is an integer of 2 to 30, and n2 is an integer of 2 to 30.)
Figure JPOXMLDOC01-appb-C000027
 (一般式（Ｅ）中、ｎ３は２～３０の範囲の整数である。)
Figure JPOXMLDOC01-appb-C000027
 (In the general formula (E), n3 is an integer in the range of 2 to 30.)
　一般式（Ｄ）中、ｎ１は２～３０、好ましくは５～２０、より好ましくは７～１５の範囲の整数である。ｎ２は２～３０、好ましくは５～２０、より好ましくは７～１５の範囲の整数である。一般式（Ｅ）は、末端がＳＨ基であること以外は一般式（Ａ）で示されるリン脂質疑似物質基と同様である。一般式（Ｅ）中、ｎ３は２～３０、好ましくは５～２０、より好ましくは７～１５の範囲の整数である。一般式（Ｄ）で示される架橋前駆体Ｘ及び一般式（Ｅ）で示されるリン脂質疑似物質前駆体は、いずれも市販品を入手可能であり、また、参考文献に記載の方法で調製することもできる。(参考文献：T. Ohyanagi, et. al., J. Am. Chem. Soc. 2011, 133, 12507-12517) In the general formula (D), n1 is an integer in the range of 2 to 30, preferably 5 to 20, and more preferably 7 to 15. n2 is an integer in the range of 2 to 30, preferably 5 to 20, more preferably 7 to 15. The general formula (E) is the same as the phospholipid pseudo substance group represented by the general formula (A) except that the terminal is an SH group. In the general formula (E), n3 is an integer in the range of 2 to 30, preferably 5 to 20, and more preferably 7 to 15. Both the crosslinked precursor X represented by the general formula (D) and the phospholipid pseudo substance precursor represented by the general formula (E) are commercially available and prepared by the method described in the references. You can also do it. (References: T. Ohyanagi, et. Al., J. Am. Chem. Soc. 2011, 133, 12507-12517)
　一般式（Ｄ）で示される架橋前駆体Ｘ、一般式（Ｅ）で示されるリン脂質疑似物質前駆体及びナノ粒子の混合比率は、一般式（Ｄ）で示される架橋前駆体Ｘ及び一般式（Ｅ）で示されるリン脂質疑似物質前駆体のナノ粒子に対する所望の担持量を考慮して適宜決定することができる。 The mixing ratio of the cross-linking precursor X represented by the general formula (D), the phospholipid pseudo substance precursor represented by the general formula (E), and the nanoparticles is the cross-linking precursor X represented by the general formula (D) and the general formula. It can be appropriately determined in consideration of the desired carrying amount of the phospholipid pseudo substance precursor represented by (E) with respect to the nanoparticles.
　下記スキーム中に示す一般式（Ｄ）で示される架橋前駆体Ｘはｎ１が６であり、ｎ２が９であり、一般式（Ｅ）で示されるリン脂質疑似物質前駆体のｎ３が９である。アミノオキシリンカー（ＡＯ）とホスホリルコリンリンカー（ＰＣ）のモル比ＡＯ／ＰＣは任意であり、例えば、１００／１～１／１００の範囲であることができる。一般式（Ｂ）で示される糖鎖構造含有基と一般式（Ａ）で示されるリン脂質疑似物質基との比率が、例えば、モル比率が１：１００～１００：１の範囲であることができ、好ましくは１：１０～１０：１の範囲であることから、これに準じた比であることができる。 The crosslinked precursor X represented by the general formula (D) shown in the following scheme has n1 of 6, n2 of 9, and the phospholipid pseudomaterial precursor represented by the general formula (E) of n3. .. The molar ratio AO / PC of the aminooxylinker (AO) to the phosphorylcholine linker (PC) is arbitrary and can be, for example, in the range of 100/1 to 1/100. The ratio of the sugar chain structure-containing group represented by the general formula (B) to the phospholipid pseudomaterial group represented by the general formula (A) is, for example, a molar ratio in the range of 1: 100 to 100: 1. It is possible, and preferably in the range of 1:10 to 10: 1, so that the ratio can be similar to this.
Figure JPOXMLDOC01-appb-C000028
Figure JPOXMLDOC01-appb-C000028
　ナノ粒子は、糖鎖提示粒子において説明したものと同様である。上記スキームでは、金属ナノ粒子の原料としてコロイド状の量子ドット（ＱＤｓ（ＱＤ：Ｑｕａｎｔｕｍ　Ｄｏｔ））を用いた。コロイド状量子ドットは、直径の範囲が、例えば、１～２０ｎｍの発光性の半導体ナノ粒子の表面に保護基を有する。上記スキームに示すコロイド状量子ドットは表面にトリアルキルリン酸基を有する。ナノ粒子が金属ナノ粒子の場合も、コロイド状の金属ナノ粒子を原料として用いることができる。コロイド状量子ドット及びコロイド状金属ナノ粒子は市販品を入手可能である。 The nanoparticles are the same as those described for the sugar chain presentation particles. In the above scheme, colloidal quantum dots (QDs (QD: Quantum Dot)) were used as raw materials for metal nanoparticles. Colloidal quantum dots have protecting groups on the surface of luminescent semiconductor nanoparticles with a diameter range of, for example, 1 to 20 nm. The colloidal quantum dots shown in the above scheme have a trialkylphosphate group on the surface. Even when the nanoparticles are metal nanoparticles, colloidal metal nanoparticles can be used as a raw material. Colloidal quantum dots and colloidal metal nanoparticles are commercially available.
　得られた表面修飾ナノ粒子の表面に、所望のがん糖鎖パターンを提示する。糖鎖の提示は、上記で得られた表面修飾ナノ粒子に導入された一般式（Ｄ）で示される架橋前駆体Ｘが有するアミノオキシ基が、還元末端（アルデヒド基と等価）を持つ糖鎖に特異的に反応するグライコブロッティング法により行うことができる。 The desired cancer sugar chain pattern is presented on the surface of the obtained surface-modified nanoparticles. In the presentation of the sugar chain, the aminooxy group of the cross-linking precursor X represented by the general formula (D) introduced into the surface-modified nanoparticles obtained above has a reducing end (equivalent to an aldehyde group). It can be carried out by a glycoblotting method that specifically reacts with.
　薬剤部位を有する本発明の糖鎖提示粒子は、前記表面修飾ナノ粒子の表面に、所望のがん糖鎖パターン及び薬剤含有部位を提示または担持する。糖鎖及び薬剤含有部位の提示又は担持は、逐次または同時に行うことができる。例えば、上述の通りグライコブロッティング法により糖鎖を提示した後に、薬剤含有部位の担持を行うことができる。糖鎖を提示した表面修飾ナノ粒子に下記一般式（Ｇ）で示される薬剤含有前駆体Ｚを混合して、前記表面修飾ナノ粒子上の架橋前駆体Ｘと連結させて一般式（Ｃ）で示される薬剤含有基をさらに形成することで得ることができる。 The sugar chain-presenting particles of the present invention having a drug site present or carry a desired cancer sugar chain pattern and drug-containing site on the surface of the surface-modified nanoparticles. The sugar chain and the drug-containing site can be presented or supported sequentially or simultaneously. For example, as described above, after the sugar chain is presented by the Glycoblotting method, the drug-containing site can be supported. The drug-containing precursor Z represented by the following general formula (G) is mixed with the surface-modified nanoparticles presenting the sugar chain and linked to the cross-linked precursor X on the surface-modified nanoparticles, and the general formula (C) is used. It can be obtained by further forming the indicated drug-containing groups.
Figure JPOXMLDOC01-appb-C000029
 （一般式（Ｇ）中、Ｒ２０は薬剤含有部位である。）
Figure JPOXMLDOC01-appb-C000029
 (In the general formula (G), R 20 is a drug-containing site.)
　本発明の製造方法における糖鎖含有部位及び薬剤含有部位は、本発明の粒子における糖鎖含有部位及び薬剤含有部位とそれぞれ同義である。一般式（Ｇ）で示される薬剤含有前駆体Ｚは、例えば、特許文献１（ＷＯ２０１７／１３１２４２Ａ１／ＵＳ２０２０１３８９７３（Ａ１））に記載の方法に準じて合成できる。 The sugar chain-containing site and the drug-containing site in the production method of the present invention have the same meaning as the sugar chain-containing site and the drug-containing site in the particles of the present invention, respectively. The drug-containing precursor Z represented by the general formula (G) can be synthesized, for example, according to the method described in Patent Document 1 (WO2017 / 131242A1 / US2020138973 (A1)).
(がん細胞の糖鎖パターン決定方法)
　本発明は、被検者から採取したがん細胞から、がん細胞の表面にある糖鎖をプロファイルし、プロファイルした糖鎖に基づいて糖鎖パターンを決定することを含む。
　がん細胞の表面にある糖鎖のプロファイルは、上述のように、公知の分析法により行うことができる。 (Method for determining sugar chain pattern of cancer cells)
The present invention includes profiling sugar chains on the surface of cancer cells from cancer cells collected from a subject and determining a sugar chain pattern based on the profiled sugar chains.
The profile of sugar chains on the surface of cancer cells can be performed by a known analytical method as described above.
　得られた糖鎖パターンの決定は、プロファイルした糖鎖が、
（１）体内動態が主に末端ハイマンノース型糖鎖に依存する糖鎖パターンＡ、
（２）体内動態が主に末端ガラクトース型糖鎖又は末端N-アセチルグルコサミン型糖鎖に依存する糖鎖パターンＢ、
（３）体内動態が主に末端α２，６シアル酸型糖鎖に依存する糖鎖パターンＣ、及び
（４）体内動態が主に末端α２，３シアル酸型糖鎖に依存する糖鎖パターンＤからなる群から選択される何れの糖鎖パターンであるかを特定することで行うことができる。 The obtained sugar chain pattern was determined by the profiled sugar chain.
(1) Sugar chain pattern A, whose pharmacokinetics mainly depend on terminal high mannose type sugar chains,
(2) Sugar chain pattern B, whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains,
(3) Sugar chain pattern C whose pharmacokinetics mainly depend on terminal α2,6 sialic acid type sugar chains, and (4) Sugar chain pattern D whose pharmacokinetics mainly depend on terminal α2,3 sialic acid type sugar chains This can be done by specifying which sugar chain pattern is selected from the group consisting of.
　上記糖鎖パターンは、より具体的には、例えば、
がん糖鎖パターンＡは、糖鎖の４５モル％以上が末端ハイマンノース型糖鎖であり、末端シアル酸型糖鎖は０％以上、２％未満であり、
がん糖鎖パターンＢは、糖鎖の末端ハイマンノース型糖鎖が４５％未満であり、かつ末端シアル酸型糖鎖が０％以上、２％未満であり、
がん糖鎖パターンＣは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつ末端α２，６シアル酸型糖鎖の含有量が末端α２，３シアル酸型糖鎖の含有量より多く、
がん糖鎖パターンＤは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつ末端α２，３シアル酸型糖鎖の含有量が末端α２，６シアル酸型糖鎖の含有量より多い。 More specifically, the sugar chain pattern is, for example,
In the cancer sugar chain pattern A, 45 mol% or more of the sugar chains are terminal hymannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%.
In the cancer sugar chain pattern B, the terminal hymannose type sugar chain of the sugar chain is less than 45%, and the terminal sialic acid type sugar chain is 0% or more and less than 2%.
In the cancer sugar chain pattern C, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal α2,6 sialic acid type sugar chains is the content of the terminal α2,3 sialic acid type sugar chains. More than the amount,
In the cancer sugar chain pattern D, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal α2,3 sialic acid type sugar chains is the content of the terminal α2,6 sialic acid type sugar chains. More than the amount.
　本発明の決定方法において、決定された糖鎖パターンが糖鎖パターンＡの場合、タイプ１の体内動態を示し、がん細胞の転移傾向が低いことを示唆する。決定された糖鎖パターンが糖鎖パターンＢの場合、タイプ２の体内動態を示し、がん細胞が肝臓及び脾臓への転移傾向があることを示唆する。決定された糖鎖パターンが糖鎖パターンＣの場合、タイプ３の体内動態を示し、がん細胞が腋窩及び鎖骨上リンパ節への転移傾向があることを示唆する。決定された糖鎖パターンが糖鎖パターンＤの場合、タイプ４の体内動態を示し、がん細胞が肺、肝臓、脾臓、脳及び腎臓への転移傾向があることを示唆する。 In the determination method of the present invention, when the determined sugar chain pattern is sugar chain pattern A, it shows type 1 pharmacokinetics, suggesting that the tendency of cancer cells to metastasize is low. When the determined sugar chain pattern is sugar chain pattern B, it shows type 2 pharmacokinetics, suggesting that cancer cells tend to metastasize to the liver and spleen. When the determined glycan pattern is glycan pattern C, it exhibits type 3 pharmacokinetics, suggesting that cancer cells are prone to metastasis to the axilla and supraclavicular lymph nodes. When the determined glycan pattern is glycan pattern D, it exhibits type 4 pharmacokinetics, suggesting that cancer cells are prone to metastasis to lung, liver, spleen, brain and kidney.
　本発明の決定方法で決定されたがん細胞の糖鎖パターンに基づいて、被検者が有するがん細胞の糖鎖パターンが、タイプ１～４の何れの体内動態を示す糖鎖パターンであるかを勘案して、医療関係者により、対象となったがん細胞の転移性を診断することができる。 Based on the sugar chain pattern of cancer cells determined by the determination method of the present invention, the sugar chain pattern of cancer cells possessed by the subject is a sugar chain pattern exhibiting any of the pharmacokinetics of types 1 to 4. With this in mind, medical personnel can diagnose the metastasis of the targeted cancer cells.
　以下、本発明を実施例に基づいて更に詳細に説明する。但し、実施例は本発明の例示であって、本発明は実施例に限定される意図ではない。 Hereinafter, the present invention will be described in more detail based on examples. However, the examples are examples of the present invention, and the present invention is not intended to be limited to the examples.
実施例１
（１）がん細胞内および細胞膜表面に存在する全てのタンパク質の翻訳後糖鎖修飾の状態（糖鎖発現パターン及びそれらの発現量）のプロファイル Example 1
(1) Profile of post-translational sugar chain modification status (sugar chain expression pattern and their expression level) of all proteins present in cancer cells and on the cell membrane surface
ヒトがん細胞の糖鎖プロファイル：
　4種類のヒト培養がん細胞（MCF7, MDA-MB-231, A549, HepG2）を、それぞれ約5 x 105個（全タンパク質量として約100μg）用いることで、これらのがん細胞の全糖鎖プロファイルを細胞のグライコブロッティング法の一般的なプロトコル（S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537）に従って詳細に解析した（図2a）。次いで、それらの糖鎖構造について内部標準化合物を指標としてグライコタイピング解析によって主要な構造モチーフとのそれら発現量の関係を明らかにした（図2b）。 Glycan profile of human cancer cells :
By using 4 types of cultured human cancer cells (MCF7, MDA-MB-231, A549, HepG2), about 5 x 10 5 each (about 100 μg as total protein amount), the total sugar of these cancer cells The chain profile was analyzed in detail according to the general protocol of cell glycoblotting (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537) (Fig. 2a). Next, regarding their sugar chain structures, the relationship between their expression levels and the main structural motifs was clarified by glicotyping analysis using the internal standard compound as an index (Fig. 2b).
（２）がん糖鎖パターン提示ナノ粒子(糖鎖提示粒子)の作製法およびマウスへの静脈内投与後のリアルタイムイメージングによる体内動態の解析（原理とプロトコルについては図1を参照） (2) Method for producing cancer sugar chain pattern-presenting nanoparticles (sugar chain-presenting particles) and analysis of pharmacokinetics by real-time imaging after intravenous administration to mice (see Fig. 1 for the principle and protocol).
（２－１）がん細胞由来糖鎖の調製法：
　ヒト培養がん細胞MCF7（ATCC）、A549（JCBR）、HepG2（RIKEN）それぞれ1 x 106個を10% fetal bovine serum（FBS）を含むD-MEM High-glucose培地（Wako）中、37℃、5% CO2の条件下で48時間培養する。また、MDA-MB-231（RIKEN）については1 x 106個を10% fetal bovine serum（FBS）を含むLeibovitz’s L-15培地（Wako）中、37℃で48時間培養することでそれぞれのがん細胞を2～3 x 106個（全タンパク質量として約500μgに相当）とする。細胞を氷冷した1 mLの0.2 Mリン酸緩衝液（pH7.4）で剥がしとり10 mM EDTAを含むリン酸緩衝液（pH7.4）1 mLに懸濁させて10,000 g、4℃で10分間遠心して上清を除く。残渣に0.1%SDS、1%Triton-X100および100 mM炭酸水素アンモニウムから成る溶液100μLを加えて可溶化し、総タンパク質量をBCA（bicinchoninic acid）法により定量する。タンパク質500μg相当量を1,4-dithithreiol（DTT, 20μL, 120 mM/MilliQ）と60℃で30分、次いでiodoacetamide（IAA, 40μL, 123 mM/MilliQ）と冷暗所、室温にて1時間反応させる。タンパク質混合液に400Uのトリプシン（Sigma Aldrich）を加えて37℃で一晩処理後90℃で10分間加熱することで加水分解を停止させる。この反応液に2UのPNGaseF（Roche Applied Science）を加えて37℃で一晩反応させて減圧下遠心型濃縮器（SpeedVac）で溶媒を留去して乾固させ、分析用試料を作製した。 (2-1) Method for preparing sugar chains derived from cancer cells :
Human cultured cancer cells MCF7 (ATCC), A549 (JCBR), HepG2 (RIKEN) 1 x 10 6 each in D-MEM High-glucose medium (Wako) containing 10% fetal bovine serum (FBS) at 37 ° C. Incubate for 48 hours under 5% CO 2 conditions. For MDA-MB-231 (RIKEN), 1 x 10 6 pieces were cultured in Leibovitz's L-15 medium (Wako) containing 10% fetal bovine serum (FBS) at 37 ° C for 48 hours. The number of cells is 2-3 x 10 6 (equivalent to about 500 μg of total protein). Peel the cells in 1 mL of ice-cooled 0.2 M phosphate buffer (pH 7.4) and suspend in 1 mL of phosphate buffer (pH 7.4) containing 10 mM EDTA, 10,000 g, 10 at 4 ° C. Centrifuge for minutes to remove supernatant. To the residue is added 100 μL of a solution consisting of 0.1% SDS, 1% Triton-X100 and 100 mM ammonium hydrogencarbonate to solubilize the residue, and the total amount of protein is quantified by the BCA (bicinchoninic acid) method. Equivalent to 500 μg of protein is reacted with 1,4-dithithreiol (DTT, 20 μL, 120 mM / MilliQ) at 60 ° C. for 30 minutes, then with iodoacetamide (IAA, 40 μL, 123 mM / MilliQ) in a cool, dark place at room temperature for 1 hour. Add 400 U of trypsin (Sigma Aldrich) to the protein mixture, treat overnight at 37 ° C, and heat at 90 ° C for 10 minutes to stop hydrolysis. 2U of PNGase F (Roche Applied Science) was added to this reaction solution, and the mixture was reacted overnight at 37 ° C., and the solvent was distilled off with a centrifugal concentrator (SpeedVac) under reduced pressure to dry it to prepare a sample for analysis.
（２－２）糖鎖提示用ナノ粒子の作製法：
　常法（S.-I. Nishimura eta l., J. Am. Chem. Soc. 2011, 133, 12507-12517; S.-I. Nishimura et al., ACS Chem. Biol. 2015, 10, 2073-2086; S.-I. Nishimura, WO2017/131242A1）に従って、量子ドット（1μM TOPO-coated QD800 in decane, Thermo Fischer）を11-mercaptoundecylphosphorylcholine（以下PC-SHと略す）と11,11'-dithio bis[undec-11-yl 12-(aminooxyacetyl)amino hexa(ethyleneglycol)（以下AOHEG-SHと略す）の2種のアルカンチオール誘導体からなる混合単分子膜により完全被覆したナノ粒子（PC-SH/AOHEG-SH=80/1）を作成した。具体的には量子ドット（200μL, 1μM TOPO-coated QD800 in decane）をMeOH（200μL）とi-PrOH（400μL）の混合溶媒に添加して室温で15,000 g、5分間遠心してTOPO-coated QD800を不溶化・沈殿させて上清を除く。残渣にn-hexane（400μL）を加えてTOPO-coated QD800を可溶化し、この溶液にPC-SH（32μL, 100 mM/MeOH, Medicinal Chemistry Pharmaceuticals）、AOHEG-SH（2μL, 10 mM/MilliQ, Mediicinal Chemistry Pharmaceuticals）、さらにNaBH4（1μL, 12 wt% in 14 N NaOH）とMilli Q（200μL）を加え室温で30分間vortex mixerにより激しく撹拌する。静置して有機層を除去した後、水層のナノ粒子を限外ろ過（YM50、Thermo Fischer）により分取、Milli Q（500μL）で3回洗浄して精製した（100μL, 2μM/MilliQとして速やかにがん細胞の糖鎖ブロッティングに供する）。同時にPC-SHのみで単分子膜被覆した糖鎖を持たないナノ粒子（PC-SH/AOHEG-SH=100/0）をコントロールとして作製した。 (2-2) Method for producing nanoparticles for presenting sugar chains :
Conventional method (S.-I. Nishimura eta l., J. Am. Chem. Soc. 2011, 133, 12507-12517; S.-I. Nishimura et al., ACS Chem. Biol. 2015, 10, 2073- 2086; S.-I. Nishimura, WO2017 / 131242A1), quantum dots (1 μM TOPO-coated QD800 in decane, Thermo Fischer) are 11-mercaptoundecylphosphorylcholine (hereinafter abbreviated as PC-SH) and 11,11'-dithio bis [ Nanoparticles (PC-SH / AOHEG-SH) completely coated with a mixed monomolecular membrane consisting of two alkanethiol derivatives of undec-11-yl 12- (aminooxyacetyl) amino hexa (ethyleneglycol) (hereinafter abbreviated as AOHEG-SH). = 80/1) was created. Specifically, quantum dots (200 μL, 1 μM TOPO-coated QD800 in decane) are added to a mixed solvent of MeOH (200 μL) and i-PrOH (400 μL), and the mixture is centrifuged at room temperature for 5 minutes to obtain TOPO-coated QD800. Insolubilize and precipitate to remove the supernatant. TOPO-coated QD800 was solubilized by adding n-hexane (400 μL) to the residue, and PC-SH (32 μL, 100 mM / MeOH, Medicinal Chemistry Pharmaceuticals), AOHEG-SH (2 μL, 10 mM / MilliQ,) were added to this solution. Mediicinal Chemistry Pharmaceuticals), NaBH 4 (1 μL, 12 wt% in 14 N NaOH) and Milli Q (200 μL) are added, and the mixture is vigorously stirred at room temperature for 30 minutes with a vortex mixer. After allowing to stand to remove the organic layer, the nanoparticles in the aqueous layer were separated by ultrafiltration (YM50, Thermo Fischer), washed 3 times with Milli Q (500 μL) and purified (as 100 μL, 2 μM / Milli Q). Immediately use for sugar chain blotting of cancer cells). At the same time, nanoparticles (PC-SH / AOHEG-SH = 100/0) without sugar chains coated with a monolayer only with PC-SH were prepared as controls.
（２－３）本発明の糖鎖提示粒子であるがん糖鎖パターン提示ナノ粒子の作製法：
（A）の工程で種々のがん細胞から調製した糖鎖を含む混合物（20μL, 全タンパク質量として約500μg相当）と50 mM酢酸緩衝液（200μL, pH4.0）を（B）の工程で作製したナノ粒子溶液（100μL, 2μM/MilliQ: PC-SH/AOHEG-SH=80/1）に加えて37℃で1.5時間反応させる（時々緩やかに撹拌する程度）。がん糖鎖パターン提示ナノ粒子は限外ろ過（YM50、Thermo Fischer）により分取、Milli Q（500μL）で洗浄し、生理食塩水（200μL, 0.9％NaCl水溶液）に溶解させて動物実験に供する。得られたがん糖鎖パターン提示ナノ粒子の平均粒子径は、ファイバー光学動的光散乱光度計FDLS-3000（大塚電子製）で測定した結果、15.1～28.0 nmであった。 (2-3) Method for producing cancer sugar chain pattern-presenting nanoparticles, which are sugar chain-presenting particles of the present invention :
A mixture containing sugar chains (20 μL, equivalent to about 500 μg in total protein amount) prepared from various cancer cells in step (A) and 50 mM acetate buffer (200 μL, pH 4.0) were added in step (B). Add to the prepared nanoparticle solution (100 μL, 2 μM / MilliQ: PC-SH / AOHEG-SH = 80/1) and react at 37 ° C for 1.5 hours (sometimes with gentle stirring). Cancer sugar chain pattern presentation nanoparticles are separated by ultrafiltration (YM50, Thermo Fischer), washed with Milli Q (500 μL), dissolved in physiological saline (200 μL, 0.9% NaCl aqueous solution) and used for animal experiments. .. The average particle size of the obtained cancer sugar chain pattern-presenting nanoparticles was 15.1 to 28.0 nm as a result of measurement with a fiber optical dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Denshi).
（２－４）がん糖鎖パターン提示ナノ粒子(糖鎖提示粒子)の体内動態と臓器指向性：
　がん糖鎖パターン提示ナノ粒子（100μL, 1μM/0.9％NaCl水溶液）を5週齢以上のマウス（male, BALB/c）に静脈内投与した。投与後3時間までのナノ粒子の生体内動態をIVISイメージングシステム（Summit Pharmaceuticals International）を用いて近赤外蛍光スペクトルによりリアルタイムで観察した（露出時間1 sec、励起波長710 nm、検出波長820 nm）。静脈内投与から3時間経過後、マウスを解剖し主要な臓器を摘出してそれぞれの蛍光強度を観察した。以下、実施例においては、がん糖鎖パターン提示ナノ粒子(糖鎖提示粒子)の体内動態は、投与後の体内での粒子の移動及び排出の状態を体内動態と記載し、粒子の各臓器や組織への分布や集積状態を特に臓器指向性と記載する。 (2-4) Pharmacokinetics and organ orientation of cancer sugar chain pattern-presenting nanoparticles (sugar chain-presenting particles) :
Cancer sugar chain pattern-presenting nanoparticles (100 μL, 1 μM / 0.9% NaCl aqueous solution) were intravenously administered to mice aged 5 weeks or older (male, BALB / c). The in vivo dynamics of nanoparticles up to 3 hours after administration were observed in real time using a near-infrared fluorescence spectrum using an IVIS imaging system (Summit Pharmaceuticals International) (exposure time 1 sec, excitation wavelength 710 nm, detection wavelength 820 nm). .. Three hours after the intravenous administration, the mice were dissected and the main organs were removed and the fluorescence intensity of each was observed. Hereinafter, in the examples, the pharmacokinetics of the cancer sugar chain pattern-presenting nanoparticles (sugar chain-presenting particles) is described as the pharmacokinetics of the movement and excretion of the particles in the body after administration, and each organ of the particles. The distribution and accumulation state in tissues are described as organ-oriented.
　4種類のヒト培養がん細胞（MCF7, MDA-MB-231, A549, HepG2）の糖鎖を提示したナノ粒子（GNS-MCF7, GNS-MDA-MB-231, GNS-A549, GNS-HepG2）のそれぞれについて、およびコントロールのマウス静脈内投与後3時間までの体内動態と3時間後に解剖した際の各臓器での分布状態を図3に示した。 Nanoparticles (GNS-MCF7, GNS-MDA-MB-231, GNS-A549, GNS-HepG2) that presented sugar chains of four types of cultured human cancer cells (MCF7, MDA-MB-231, A549, HepG2) Fig. 3 shows the pharmacokinetics of each of the controls up to 3 hours after intravenous administration of mice and the distribution state in each organ when dissected 3 hours later.
(i) コントロール（糖鎖を有しないナノ粒子）
　これらの実験により、PC-SHのみを用いて単分子膜被覆したコントロールは静脈内投与後3時間を経過しても特定の臓器指向性を示さず（各臓器への相対的指向性は0.8～2.5）、すい臓以外の臓器（全身）にほぼ一様に分布することが再確認された（S.-I. Nishimura et al., J. Am. Chem. Soc. 2011, 133, 12507-12517）。 (i) Control (nanoparticles without sugar chains)
According to these experiments, the monolayer-coated control using only PC-SH did not show specific organ orientation 3 hours after intravenous administration (relative orientation to each organ was 0.8 to 0). 2.5), it was reconfirmed that it is distributed almost uniformly in organs other than the pancreas (whole body) (S.-I. Nishimura et al., J. Am. Chem. Soc. 2011, 133, 12507-12517). ..
(ii) GNS-MCF7
　図2aと2bに示すとおり、糖鎖成分として、全糖鎖の約94％が7種類のハイマンノース型糖鎖で約6％がガラクトース末端を持つ2種類の糖鎖を含む糖鎖パターンである、転移性の見られない乳がん細胞であるMCF7の糖鎖パターンを提示した場合はそのほとんどが静脈内投与後特定の臓器に集積すること無く約15分以内に速やかに体外に排出された（図3a）。3時間後に解剖したマウスにおいても臓器での集積はほとんど観察されず、その一部が胃腸内の糞便に混入していた（図3b）。すなわち、糖鎖パターンＡのハイマンノース型糖鎖（末端がマンノースのみのオリゴ糖）を提示する糖鎖提示粒子は、体外への***機構を有しており、タイプ１の体内動態を示すことが明らかになった。 (ii) GNS-MCF7
As shown in FIGS. 2a and 2b, as sugar chain components, about 94% of the total sugar chains are 7 types of high mannose type sugar chains and about 6% are sugar chain patterns containing 2 types of sugar chains having galactose ends. When the sugar chain pattern of MCF7, which is a non-metastatic breast cancer cell, was presented, most of them were rapidly excreted from the body within about 15 minutes without accumulating in a specific organ after intravenous administration (Fig.). 3a). Almost no accumulation in organs was observed in the mice dissected after 3 hours, and a part of them was mixed in the feces in the gastrointestinal tract (Fig. 3b). That is, the sugar chain-presenting particles presenting the high-mannose-type sugar chain (oligosaccharide having only mannose at the end) of the sugar chain pattern A have an excretion mechanism to the outside of the body and exhibit type 1 pharmacokinetics. It was revealed.
(iii) GNS- MDA-MB-231
　糖鎖成分として、2本鎖あるいは3本鎖の5種類の末端シアル酸型糖鎖を全糖鎖の18％、5種類のハイマンノース型糖鎖を82％含む糖鎖パターン（図2aと2b）であり、転移性の乳がん細胞であるMDA-MB-231の糖鎖パターンを提示した場合はGNS-MCF7の体内動態とは全く異なり、静脈内投与後3時間において腋窩および鎖骨上リンパ節への集積が特に顕著であり（矢印で表示）、タイプ３の体内動態を示した。また、投与後1～２時間あたりまでは肺、心臓、肝臓、脾臓、腎臓などにもナノ粒子の分布が確認できたが3時間後までにはそのほとんどが体外に排出された。以上の結果はナノ粒子の表面における糖鎖成分であるNeu5Acα2,6GalあるいはNeu5Acα2,3Galユニットを末端に含む糖鎖が乳がん細胞の転移性と臓器指向性を決定していることを示唆している。 (iii) GNS- MDA-MB-231
As sugar chain components, a sugar chain pattern containing 5 types of double- or triple-chain terminal sialic acid-type sugar chains at 18% of the total sugar chains and 5 types of high-mannose-type sugar chains at 82% (Figs. 2a and 2b). ), And when the sugar chain pattern of MDA-MB-231, which is a metastatic breast cancer cell, is presented, it is completely different from the pharmacokinetics of GNS-MCF7, and it reaches the axillary and supraclavicular lymph nodes 3 hours after intravenous administration. Accumulation was particularly remarkable (indicated by arrows), showing type 3 pharmacokinetics. In addition, the distribution of nanoparticles was confirmed in the lungs, heart, liver, spleen, kidneys, etc. up to about 1 to 2 hours after administration, but most of them were excreted from the body by 3 hours. The above results suggest that sugar chains containing Neu5Acα2,6Gal or Neu5Acα2,3Gal units, which are sugar chain components on the surface of nanoparticles, determine the metastasis and organ orientation of breast cancer cells.
(iv) GNS-A549
　糖鎖成分として、2本鎖あるいは3本鎖の5種類の末端シアル酸型糖鎖を全糖鎖の19％、ガラクトース末端を持つ糖鎖を11％、N-アセチルグルコサミンを末端に持つ糖鎖を8％、さらに7種類のハイマンノース型糖鎖を62％含む糖鎖パターンである（図2aと2b）、A549の糖鎖パターンを提示した場合は静脈内投与後3時間においても全身に広く分布しており、特に肺、肝臓、脾臓での蓄積が非常に顕著で脳と腎臓にも分布が確認でき、タイプ４の体内動態を示した。 (iv) GNS-A549
As sugar chain components, 5 types of double- or triple-chain terminal sialic acid-type sugar chains are 19% of the total sugar chains, sugar chains having a galactose terminal are 11%, and sugar chains having N-acetylglucosamine at the ends. It is a sugar chain pattern containing 8% and 62% of 7 types of high mannose type sugar chains (Figs. 2a and 2b). When the sugar chain pattern of A549 is presented, it is widely spread throughout the body even 3 hours after intravenous administration. It is distributed, and the accumulation in the lung, liver, and spleen is very remarkable, and the distribution can be confirmed in the brain and kidney, showing type 4 pharmacokinetics.
(v) GNS-HepG2
　糖成分として、2本鎖あるいは3本鎖の2種類の末端シアル酸型糖鎖を全糖鎖の6％、ガラクトース末端を持つ糖鎖を9％、N-アセチルグルコサミンを末端に持つ糖鎖を13％、さらに7種類のハイマンノース型糖鎖を72％含む糖鎖パターン（図2aと2b）である、HepG2の糖鎖パターンを提示したナノ粒子の場合は、GNS-A549と非常に良く似たタイプ４の体内動態を示した。しかし、Neu5Acα2,6GalあるいはNeu5Acα2,3Galユニットの全糖鎖に占める割合が19％のGNS-A549は6％のGNS-HepG2よりも肺、肝臓、および脾臓への集積量がいずれも明らかに多いことも明らかとなった。しかし、これら2種のがん細胞由来糖鎖を提示したナノ粒子の投与後3時間での臓器指向性（蓄積性）は、GNS-HepG2の場合は、GNS-MDA-MB-231の場合と大きく異なることから、これらの違いは末端のシアル酸とガラクトースの結合様式（α2,6結合とα2,3結合の違い）が静脈内投与後のナノ粒子の臓器指向性に影響していることが考えられた。 (v) GNS-HepG2
As sugar components, 6% of all sugar chains are double-stranded or triple-chain terminal sialic acid-type sugar chains, 9% are sugar chains with galactose ends, and sugar chains with N-acetylglucosamine at the ends. In the case of nanoparticles presenting the HepG2 sugar chain pattern, which is a sugar chain pattern containing 13% and 72% of 7 types of high mannose type sugar chains (Figs. 2a and 2b), it is very similar to GNS-A549. The pharmacokinetics of type 4 was shown. However, GNS-A549, which accounts for 19% of the total sugar chain of Neu5Acα2,6Gal or Neu5Acα2,3Gal units, clearly accumulates more in the lungs, liver, and spleen than 6% GNS-HepG2. Was also revealed. However, the organ directivity (accumulation) 3 hours after administration of nanoparticles presenting these two types of cancer cell-derived sugar chains was higher in the case of GNS-HepG2 than in the case of GNS-MDA-MB-231. These differences indicate that the binding mode of terminal sialic acid and galactose (difference between α2,6 binding and α2,3 binding) affects the organ orientation of nanoparticles after intravenous administration. it was thought.
Figure JPOXMLDOC01-appb-T000030
Figure JPOXMLDOC01-appb-T000030
実施例２
（１）末端のシアル酸とガラクトースの結合様式（α2,6結合とα2,3結合）が相違する人工的に作製した糖鎖パターンを有する糖鎖提示粒子
　MDA-MB-231細胞とA549細胞およびHepG2細胞はいずれも、糖鎖成分として、2本鎖あるいは3本鎖の末端シアル酸型糖鎖を有意に発現するヒトがん細胞である。しかし、実施例１の結果では、体内動態と臓器指向性がMDA-MB-231細胞とA549細胞およびHepG2細胞について異なっていた。このことからシアル酸とガラクトースの結合様式（α2,6結合とα2,3結合の違い）が静脈内投与後のこれら糖鎖パターン提示ナノ粒子の体内動態の違いを決定していることが予想された。そこで本実施例では、予めα2,6結合とα2,3結合で連結された個々の末端シアル酸型糖鎖のどちらかのみを人工的に提示したナノ粒子を作製し、それぞれの体内動態及び臓器指向性を検討し、末端シアル酸型糖鎖の違いによる体内動態の違いを確認した。 Example 2
(1) Sugar chain presentation particles MDA-MB-231 cells and A549 cells having an artificially prepared sugar chain pattern with different binding modes of terminal sialic acid and galactose (α2,6 bond and α2,3 bond) and All HepG2 cells are human cancer cells that significantly express double- or triple-chain terminal sialic acid-type sugar chains as sugar chain components. However, in the results of Example 1, the pharmacokinetics and organ orientation were different for MDA-MB-231 cells, A549 cells and HepG2 cells. From this, it is expected that the binding mode of sialic acid and galactose (difference between α2,6 binding and α2,3 binding) determines the difference in the pharmacokinetics of these sugar chain pattern-presenting nanoparticles after intravenous administration. It was. Therefore, in this example, nanoparticles in which only one of the individual terminal sialic acid-type sugar chains linked in advance by α2,6 bond and α2,3 bond are artificially presented are prepared, and the pharmacokinetics and organs of each are prepared. The directivity was examined, and the difference in pharmacokinetics due to the difference in terminal sialic acid type sugar chains was confirmed.
　Ruddy duckの卵白において主要な糖鎖である非還元末端がガラクトースの多分岐型糖鎖構造であるので、基質特異性が既知の2種類のシアル酸転移酵素Pasteurella multocida由来α2,3-(N)-sialyltransferaseおよびhuman α2,6-(N)-sialyltransferaseによってシアル酸を末端のガラクトースに付加し改変させて、Neu5Acα2,3GalあるいはNeu5Acα2,6Galのどちらかのみを含む糖鎖パターンを誘導した（図4a）。 Since the non-reducing end, which is the main sugar chain in the egg white of Ruddy duck, is a multi-branched sugar chain structure of galactose, two types of sialic acid transferases with known substrate specificity, Pasteurella multocida-derived α2,3- (N) -Sialyltransferase and human α2,6- (N) -sialyltransferase added sialic acid to terminal galactose and modified it to induce a sugar chain pattern containing only either Neu5Acα2,3Gal or Neu5Acα2,6Gal (Fig. 4a). ..
（１－１）Ruddy duckの卵白糖鎖プロファイルのシアル酸転移酵素による改変法：
　Ruddy duck卵白（17 mg/50μL in Milli Q, 約1 mM LacNAc unitに相当）にCMP-Neu5Ac（10μL, 200 mM/milliQ, Yamasa）、α2,3-(N)-sialyltransferase（5μL, 1000 mU/mL, Sigma Aldrich）、HEPES-NaOH緩衝液（10μL, 1 M/milliQ, pH8.0）さらにmilliQ（10μL）を加えて総容量を100μLとし、37℃で20時間反応させる。同様に、Ruddy duck卵白（17 mg/50μL in Milli Q, 約1 mM LacNAc unitに相当）にCMP-Neu5Ac（10μL, 200 mM/milliQ, Yamasa）、α2,6-(N)-sialyltransferase（10μL, 516 mU/mL, Medicinal Chemistry Pharmaceuticals）、リン酸緩衝液（10μL, 1 M/milliQ, pH6.5）さらにmilliQ（10μL）を加えて総容量100μLとし、37℃で20時間反応させる。 (1-1) Modification method of Ruddy duck's egg white sugar chain profile with sialic acid transferase :
Ruddy duck egg white (17 mg / 50 μL in Milli Q, equivalent to about 1 mM LacNAc unit), CMP-Neu5Ac (10 μL, 200 mM / milliQ, Yamasa), α2,3- (N) -sialyltransferase (5 μL, 1000 mU / mL, Sigma Aldrich), HEPES-NaOH buffer (10 μL, 1 M / milliQ, pH 8.0) and milliQ (10 μL) are added to bring the total volume to 100 μL, and the mixture is reacted at 37 ° C. for 20 hours. Similarly, Ruddy duck egg white (17 mg / 50 μL in Milli Q, equivalent to about 1 mM LacNAc unit), CMP-Neu5Ac (10 μL, 200 mM / milliQ, Yamasa), α2,6- (N) -sialyltransferase (10 μL, 516 mU / mL, Medicinal Chemistry Pharmaceuticals), phosphate buffer (10 μL, 1 M / milliQ, pH6.5) and milliQ (10 μL) are added to make a total volume of 100 μL, and the mixture is reacted at 37 ° C. for 20 hours.
（１－２）Button Quail、Chicken、Ruddy duck卵白タンパク質およびシアル酸修飾Ruddy duck卵白の糖鎖プロファイル：
　上記(A)の工程に従って5種類の卵白・シアル酸修飾卵白総タンパク質換算500μg相当量を1,4-dithithreiol（DTT, 20μL, 120 mM/MilliQ）と60℃で30分、次いでiodoacetamide（IAA, 40μL, 123 mM/MilliQ）と冷暗所、室温にて1時間反応させる。タンパク質混合液に400Uのトリプシン（Sigma Aldrich）を加えて37℃で一晩処理後90℃で10分間加熱することで加水分解を停止させる。この反応液に2UのPNGaseF（Roche Applied Science）を加えて37℃で一晩反応させて減圧下遠心型濃縮器（SpeedVac）で溶媒を留去して乾固させて糖鎖プロファイリング用の試料を作製した（図4bおよび4c）。 (1-2) Sugar chain profiles of Button Quail, Chicken, Ruddy duck egg white protein and sialic acid-modified Ruddy duck egg white :
According to the above step (A), the equivalent amount of 500 μg of 5 kinds of egg white / sialic acid-modified egg white total protein equivalent was added to 1,4-dithithreiol (DTT, 20 μL, 120 mM / MilliQ) at 60 ° C. for 30 minutes, and then iodoacetamide (IAA, Reaction with 40 μL, 123 mM / MilliQ) in a cool dark place at room temperature for 1 hour. Add 400 U of trypsin (Sigma Aldrich) to the protein mixture, treat overnight at 37 ° C, and heat at 90 ° C for 10 minutes to stop hydrolysis. Add 2 U of PNGase F (Roche Applied Science) to this reaction solution, react overnight at 37 ° C, distill off the solvent in a centrifuge under reduced pressure (SpeedVac), and dry to obtain a sample for sugar chain profiling. Prepared (Figs. 4b and 4c).
　図4bと4cから明らかなようにButton Quail卵白の糖鎖は糖鎖成分として約50％がハイマンノース型糖鎖でN-アセチルグルコサミンを末端とする糖鎖が40％さらにわずかのガラクトース末端が見られるのみでシアル酸は全く存在しない。Chicken卵白についても糖鎖成分として、シアル酸は検出されず、N－アセチルグルコサミンを末端とする3～5本鎖の多分岐型の糖鎖が約40％とガラクトース末端が30％で残りがハイマンノース型糖鎖であった。Ruddy duckの卵白にも、糖鎖成分としてシアル酸は全く見られず非還元末端にガラクトース残基が存在する3～5本鎖の多分岐型の糖鎖が全体の約65％を占めており、残りはハイマンノース型糖鎖が25％およびN-アセチルグルコサミンを末端とする糖鎖が10％程度存在することが確認できた。また、これら3種の鳥卵白の糖鎖にはフコース残基が全く存在しないことも明らかになった。 As is clear from FIGS. 4b and 4c, about 50% of the sugar chains of Button Quail egg white are high mannose type sugar chains, and 40% of sugar chains ending with N-acetylglucosamine are found to have a slight galactose end. There is no sialic acid at all. Sialic acid was not detected as a sugar chain component in chicken egg white, and about 40% of 3 to 5 multi-branched sugar chains ending with N-acetylglucosamine and 30% of galactose ends were high. It was a mannose-type sugar chain. Ruddy duck egg white also contains no sialic acid as a sugar chain component, and 3 to 5 multi-branched sugar chains with galactose residues at the non-reducing ends account for about 65% of the total. It was confirmed that the rest contained about 25% of high mannose type sugar chains and about 10% of sugar chains ending with N-acetylglucosamine. It was also revealed that there are no fucose residues in the sugar chains of these three species of bird egg white.
　図4aのスキームに従って Ruddy duckの卵白をCMP-シアル酸の存在下α2,3-(N)-sialyltransferase（recombinant Pasteurella multocida）およびα2,6-(N)-sialyltransferase（recombinant human）で処理してシアル酸が多数付加した2,3S-Ruddy duckと2,6S-Ruddy duckが誘導できた。 Ruddy duck egg whites are treated with α2,3- (N) -sialyltransferase (recombinant Pasteurella multocida) and α2,6- (N) -sialyltransferase (recombinant human) in the presence of CMP-sialic acid according to the scheme shown in Fig. 4a. 2,3S-Ruddy duck and 2,6S-Ruddy duck with a large amount of acid added could be induced.
　また、グライコタイピング法（S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537）による糖鎖プロファイルの結果、2,3S-Ruddy duckと2,6S-Ruddy duckはヒトがん細胞A549やHepG2の糖鎖プロファイルとも非常に良く似ていた（図2bと4c）。 In addition, as a result of the sugar chain profile by the glico typing method (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537), 2,3S-Ruddy duck and 2,6S-Ruddy duck It was also very similar to the sugar chain profiles of human cancer cells A549 and HepG2 (Figs. 2b and 4c).
（２）人工的に作製した糖鎖パターンを提示した糖鎖提示粒子の臓器指向性
　実施例２(１)の方法で調製したButton Quail、Chicken、およびRuddy duckの卵白糖鎖、Ruddy duckの卵白糖鎖を改変した2種類の糖鎖パターンの糖鎖試料（いずれも総タンパク質換算500μg相当量から調製）、さらに鶏卵黄の糖ペプチド（500μg、17 nmol; sialylglycopeptide, SGP, Tokyo Chemical Industry、糖鎖部位においてα２，６結合型でシアル酸とガラクトースが結合）を実施例１(２－１)の工程に従ってPNGase処理して粗生成物を調製した。これらの糖鎖を含む混合物を実施例１(２－３)の工程に従ってナノ粒子溶液（100μL, 2μM/MilliQ: PC-SH/AOHEG-SH=80/1）と混合・反応させた後に精製して糖鎖パターン提示ナノ粒子（GNSs）を作製した。得られた糖鎖パターン提示ナノ粒子の平均粒子径は、ファイバー光学動的光散乱光度計FDLS-3000(大塚電子製)で測定した結果 14.4～24.4nmであった。これらの糖鎖を提示したナノ粒子を実施例１(２－４)に記載した方法でマウスに静脈内投与してそれらの体内動態と臓器指向性を近赤外蛍光顕微鏡（露出時間1 sec、励起波長710 nm、検出波長820 nm）で観察した（図5a～5c）。 (2) Organ-Orientation of Sugar Chain Presenting Particles Presenting Artificially Prepared Sugar Chain Pattern Button Quail, Chicken, and Ruddy duck Eggs White Sugar Chains, Ruddy duck Eggs Prepared by the Method of Example 2 (1) Two types of sugar chain patterns with modified white sugar chains (both prepared from the equivalent amount of 500 μg in terms of total protein), and sialylglycopeptide, SGP, Tokyo Chemical Industry, sugar chains of chicken egg yolk (500 μg, 17 nmol; sialylglycopeptide, SGP, Tokyo Chemical Industry) A crude product was prepared by subjecting sialic acid and galactose to α2,6 bond type at the site and performing PNGase treatment according to the step of Example 1 (2-1). A mixture containing these sugar chains was mixed and reacted with a nanoparticle solution (100 μL, 2 μM / MilliQ: PC-SH / AOHEG-SH = 80/1) according to the step of Example 1 (2-3), and then purified. Sugar chain pattern presentation nanoparticles (GNSs) were prepared. The average particle size of the obtained sugar chain pattern-presenting nanoparticles was 14.4 to 24.4 nm as a result of measurement with a fiber optical dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Denshi). Nanometers presenting these sugar chains were intravenously administered to mice by the method described in Example 1 (2-4) to examine their pharmacokinetics and organ directivity with a near-infrared fluorescence microscope (exposure time 1 sec, It was observed at an excitation wavelength of 710 nm and a detection wavelength of 820 nm) (Figs. 5a to 5c).
　図5に示したとおり、糖鎖構造およびそのプロファイルが明確であり、安全・安価な生体由来素材であるうずら、鶏、アヒルの卵白および鶏卵黄の糖鎖を改変して得られた糖鎖パターンをナノ粒子に提示することで、本発明の糖鎖提示粒子を製造できることが分かる。 As shown in Fig. 5, the sugar chain structure and its profile are clear, and the sugar chain pattern obtained by modifying the sugar chains of quail, chicken, duck egg white and chicken egg yolk, which are safe and inexpensive biological materials. It can be seen that the sugar chain presenting particles of the present invention can be produced by presenting quail on nanoparticles.
　具体的には、(i)ハイマンノース型糖鎖が全体の約50％を占めるButton Quail卵白から調製したGNS-Button Quailのマウス体内での滞留性はやや延長しているが（図5b）投与から3時間経過後の体内動態（図5c）はGNS-MCF7の体内動態（図3c）と酷似しておりほとんどが体外に***されている(タイプ１の体内動態)。 Specifically, (i) GNS-ButtonQuail prepared from ButtonQuail egg white, in which high mannose-type sugar chains account for about 50% of the total, has a slightly prolonged retention in mice (Fig. 5b). The pharmacokinetics after 3 hours (Fig. 5c) are very similar to those of GNS-MCF7 (Fig. 3c), and most of them are excreted outside the body (type 1 pharmacokinetics).
(ii)ChickenとRuddy duckの糖鎖パターンを提示したGNS-ChickenとGNS-Ruddy duckは肝臓と脾臓への集積度が際立って高いが（図5bと5c）(タイプ２の体内動態)、これはGNS-ChickenとGNS-Ruddy duckのシアル酸を持たないガラクトースやN-アセチルグルコサミンを末端とする分岐度の高い主要な糖鎖とこれらの臓器・細胞に高発現する哺乳類の肝レクチン（mammalian hepatic lectin）に代表されるアシアロ糖タンパク質レセプター等との特異的な相互作用に依存するところが大きいと思われる（例えばY. C. Lee et al., Acc. Chem. Res. 1997, 28, 321-327等）。 (ii) GNS-Chicken and GNS-Ruddy duck, which presented the sugar chain pattern of Chicken and Ruddy duck, have a remarkably high degree of accumulation in the liver and spleen (Figs. 5b and 5c) (type 2 pharmacokinetics). GNS-Chicken and GNS-Ruddy duck sialic acid-free galactose and N-acetylglucosamine-terminated major sugar chains with a high degree of branching and mammalian liver lectins (mammalian hepatic) that are highly expressed in these organs and cells It seems that it largely depends on the specific interaction with the asialoglycoprotein receptor represented by lectin) (for example, Y.C. Lee et al., Acc. Chem. Res. 1997, 28, 321-327). etc).
(iii)Ruddy duck卵白から誘導した糖鎖パターンを提示した2種のナノ粒子（GNS-2,3S-Ruddy duckとGNS-2,6S-Ruddy duck）および鶏卵黄糖ペプチドから調製したGNS-2,6S-A2による体内動態・臓器指向性の解析からMDA-MB-231細胞の主要な末端シアル酸型糖鎖はNeu5Acα2,6Galユニットを末端に含む2本鎖あるいは3本鎖の複合型糖鎖であること（図5bと5c）、さらにGNS-MCF7（図3bと3c）やGNS-Button Quailの体内動態・臓器指向性（図5bと5c）との違いからエキソソーム膜表面におけるNeu5Acα2,6Galユニットを末端に含む糖鎖の全糖鎖（あるいはハイマンノース型糖鎖）に対する割合（分布密度）が乳がん細胞の転移性と臓器指向性を決定していることが明らかになった（図5bに示すとおり、GNS-2,6S-Ruddy duckとGNS-2,6S-A2において腋窩および鎖骨上リンパ節への集積が確認できる）(タイプ３の体内動態)。 (iii) Ruddy duck GNS-2 prepared from two types of nanoparticles (GNS-2,3S-Ruddy duck and GNS-2,6S-Ruddy duck) and chicken egg yellow sugar peptide showing a sugar chain pattern derived from egg white. From the analysis of pharmacokinetics and organ orientation by 6S-A2, the main terminal sialic acid type sugar chain of MDA-MB-231 cells is a double or triple chain type sugar chain containing Neu5Acα2,6Gal unit at the end. Neu5Acα2,6Gal unit on the surface of the exosome membrane due to the difference between GNS-MCF7 (Figs. 3b and 3c) and GNS-Button Quail's pharmacokinetics and organ orientation (Figs. 5b and 5c). It was clarified that the ratio (distribution density) of sugar chains containing glycans to the total sugar chains (or high mannose type sugar chains) determines the metastasis and organ orientation of breast cancer cells (Fig. 5b). As per, GNS-2,6S-Ruddy duck and GNS-2,6S-A2 can be confirmed to accumulate in the axillary and supraclavicular lymph nodes) (type 3 pharmacokinetics).
　一方、(iv)GNS-2,3S-Ruddy duckの体内動態・臓器指向性（図5bと5c）とGNS-A549及びGNS-HepG2が酷似している（タイプ４の体内動態）ことからA549細胞とHepG2細胞の主要な末端シアル酸型糖鎖構造はNeu5Acα2,3Galユニットを末端に含む2本鎖あるいは3本鎖の複合型糖鎖であること、さらにこの2つのがん細胞由来エキソソームモデルの臓器指向性の違い（図3c）からNeu5Acα2,3Galユニットを末端に含む2本鎖あるいは3本鎖の複合型糖鎖の含有率が静脈内投与後のナノ粒子の臓器指向性のみならず滞留性（血中濃度や半減期）にも強く影響することが示唆された。 On the other hand, (iv) GNS-2,3S-Ruddy duck pharmacokinetics / organ orientation (Figs. 5b and 5c) and GNS-A549 and GNS-HepG2 are very similar (type 4 pharmacokinetics), so A549 cells. The major terminal sialic acid-type sugar chain structure of HepG2 cells is a double-stranded or triple-chain complex sugar chain containing Neu5Acα2,3Gal unit at the end, and the organs of these two cancer cell-derived exosome models. Due to the difference in directivity (Fig. 3c), the content of double-stranded or triple-stranded complex sugar chains containing Neu5Acα2,3Gal unit at the end is not only organ-directivity but also retention of nanoparticles after intravenous administration (Fig. 3c). It was suggested that it also has a strong effect on blood concentration and half-life.
Figure JPOXMLDOC01-appb-T000031
Figure JPOXMLDOC01-appb-T000031
実施例３
　実施例２において、Ruddy duckの卵白糖鎖の酵素による改変によりガラクトースとの結合位置が制御された末端シアル酸型糖鎖成分とハイマンノース型糖鎖成分の全糖鎖に対する比率が体内動態と臓器指向性に大きな影響を与えることが明らかになった。そこで、ハイマンノース型糖鎖の含有率が約70％とButton Quail卵白（50％）よりもさらに高く糖鎖パターンがよりシンプルなJapanese Quail卵白由来の糖鎖（S.-I. Nishimura et al., J. Agri. Food Chem. 2018, in press）と鶏卵黄糖ペプチド由来2,6S-A2糖鎖を任意の割合で混合して、ナノ粒子に提示し、本発明の糖鎖提示粒子を作製した。得られたがん糖鎖パターン提示ナノ粒子の平均粒子径は、ファイバー光学動的光散乱光度計FDLS-3000(大塚電子製)で測定した結果、27.3nmであった。 Example 3
In Example 2, the ratio of the terminal sialic acid-type sugar chain component and the hymannose-type sugar chain component whose binding position to galactose was controlled by enzymatic modification of the egg white sugar chain of Ruddy duck to the total sugar chain was the pharmacokinetics and organs. It became clear that it has a great influence on the directionality. Therefore, the content of high mannose type sugar chains is about 70%, which is even higher than Button Quail egg white (50%), and the sugar chain pattern is simpler. Japanese Quail egg white-derived sugar chains (S.-I. Nishimura et al. , J. Agri. Food Chem. 2018, in press) and 2,6S-A2 sugar chains derived from chicken egg white sugar peptide are mixed at an arbitrary ratio and presented to nanoparticles to prepare the sugar chain-presenting particles of the present invention. did. The average particle size of the obtained cancer sugar chain pattern-presenting nanoparticles was 27.3 nm as a result of measurement with a fiber optical dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Denshi).
（３－１）
　Japanese Quail卵白由来の糖鎖試料（約6 nmol; 総タンパク質換算100μg相当量から調製）に2,6S-A2糖鎖試料（17 nmol; SGP 500μgから調製）をそれぞれ0.6 nmol、1.2 nmol、および2.4 nmol添加して3種類の糖鎖を調製した。これらの糖鎖のプロファイルをグライコブロッティング法で解析した結果（図中の％は各ピーク面積から算出した2,6S-A2 糖鎖の全体に占める割合を示す）を図６a、表７に示した。これらの糖鎖パターンを実施例１(２－３)の工程に従ってナノ粒子（100μL, 1μM/MilliQ: PC-SH/AOHEG-SH=40/1）に提示して合計5種類の糖鎖パターン提示ナノ粒子（GNSs）を作製した。2種類の糖鎖（パターン）の混合により人工的に作製した糖鎖パターンを提示するナノ粒子を調製した。 (3-1)
Japanese Quail Glycan samples derived from egg white (about 6 nmol; prepared from 100 μg equivalent to total protein) and 2,6S-A2 glycan samples (17 nmol; prepared from SGP 500 μg) were 0.6 nmol, 1.2 nmol, and 2.4, respectively. Three types of sugar chains were prepared by adding nmol. The results of analyzing the profiles of these sugar chains by the Glycoblotting method (% in the figure indicates the ratio of the 2,6S-A2 sugar chains to the whole calculated from each peak area) are shown in FIGS. 6a and 7. .. These sugar chain patterns are presented to nanoparticles (100 μL, 1 μM / MilliQ: PC-SH / AOHEG-SH = 40/1) according to the step of Example 1 (2-3), and a total of 5 types of sugar chain patterns are presented. Nanoparticles (GNSs) were prepared. Nanoparticles presenting an artificially prepared sugar chain pattern were prepared by mixing two types of sugar chains (patterns).
Figure JPOXMLDOC01-appb-T000032
Figure JPOXMLDOC01-appb-T000032
　本発明はがん細胞由来の糖鎖提示粒子を構築でき、この粒子は、がんの予防や治療に有用である。 The present invention can construct sugar chain-presenting particles derived from cancer cells, and these particles are useful for the prevention and treatment of cancer.

Claims (21)

  1. 糖鎖を表面に有するナノ粒子である糖鎖提示粒子であって、
    （１）糖鎖提示粒子の平均粒子径は１０～１００ｎｍの範囲であり、
    （２）ナノ粒子の表面の少なくとも一部はリン脂質で被覆されており、
    （３）ナノ粒子表面に有する糖鎖は、がん細胞由来の糖鎖パターン、またはこの糖鎖のプロファイルに基づいて決定された糖鎖パターン(以下、がん糖鎖パターンと呼ぶ)である、
    糖鎖提示粒子。     Sugar chain presentation particles that are nanoparticles having sugar chains on the surface.
    (1) The average particle size of the sugar chain presentation particles is in the range of 10 to 100 nm.
    (2) At least a part of the surface of the nanoparticles is coated with phospholipids.
    (3) The sugar chain contained on the surface of the nanoparticles is a sugar chain pattern derived from cancer cells or a sugar chain pattern determined based on the profile of the sugar chain (hereinafter referred to as a cancer sugar chain pattern).
    Sugar chain presentation particles.
  2. がん糖鎖パターンは、
    （１）体内動態が主に末端ハイマンノース型糖鎖に依存する糖鎖パターンＡ、
    （２）体内動態が主に末端ガラクトース型糖鎖又は末端N-アセチルグルコサミン型糖鎖に依存する糖鎖パターンＢ、
    （３）体内動態が主に末端α２，６シアル酸型糖鎖に依存する糖鎖パターンＣ、
    （４）体内動態が主に末端α２，３シアル酸型糖鎖に依存する糖鎖パターンＤからなる群から選択される糖鎖パターンである、請求項１に記載の糖鎖提示粒子。     The cancer sugar chain pattern is
    (1) Sugar chain pattern A, whose pharmacokinetics mainly depend on terminal high mannose type sugar chains,
    (2) Sugar chain pattern B, whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains,
    (3) Sugar chain pattern C, whose pharmacokinetics mainly depends on terminal α2,6 sialic acid type sugar chains,
    (4) The sugar chain-presenting particle according to claim 1, which is a sugar chain pattern selected from the group consisting of a sugar chain pattern D whose pharmacokinetics mainly depends on terminal α2,3 sialic acid type sugar chains.
  3. がん糖鎖パターンＡは、糖鎖の４５モル％以上が末端ハイマンノース型糖鎖であり、末端シアル酸型糖鎖は０％以上、２％未満であり、
    がん糖鎖パターンＢは、糖鎖の末端ハイマンノース型糖鎖が４５％未満であり、かつ末端シアル酸型糖鎖が０％以上、２％未満であり、
    がん糖鎖パターンＣは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつ末端シアル酸型糖鎖の含有量が末端α２，３シアル酸型糖鎖の含有量より多く、
    がん糖鎖パターンＤは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつα2,3シアル酸型糖鎖の含有量がα２，６シアル酸型糖鎖の含有量より多い、請求項２に記載の糖鎖提示粒子。     In the cancer sugar chain pattern A, 45 mol% or more of the sugar chains are terminal hymannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%.
    In the cancer sugar chain pattern B, the terminal hymannose type sugar chain of the sugar chain is less than 45%, and the terminal sialic acid type sugar chain is 0% or more and less than 2%.
    In the cancer sugar chain pattern C, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal sialic acid type sugar chains is larger than the content of the terminal α2,3 sialic acid type sugar chains. ,
    In the cancer sugar chain pattern D, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of α2,3 sialic acid type sugar chains is higher than the content of α2,6 sialic acid type sugar chains. The sugar chain presenting particles according to claim 2, which are abundant.
  4. ナノ粒子表面の少なくとも一部を被覆するリン脂質は、ホスホリルコリン基を有するアルカンチオールのスルフィド結合体であり、ナノ粒子表面に有する糖鎖は、糖鎖を固定化したアルカンチオールのスルフィド結合体であり、ナノ粒子表面はホスホリルコリン基を有するアルカンチオールのスルフィド結合体および糖鎖を固定化したアルカンチオールのスルフィド結合体の単分子膜で被覆されている請求項１～３のいずれかに記載の糖鎖提示粒子。 The phospholipid that covers at least a part of the surface of the nanoparticles is a sulfide bond of an alkanethiol having a phosphorylcholine group, and the sugar chain on the surface of the nanoparticles is a sulfide bond of an alkanethiol having an immobilized sugar chain. The sugar chain according to any one of claims 1 to 3, wherein the surface of the nanoparticles is coated with a monomolecular film of a sulfide bond of an alkanethiol having a phosphorylcholine group and a sulfide bond of an alkanethiol on which a sugar chain is immobilized. Presented particles.
  5. 前記ホスホリルコリン基を有するアルカンチオールのスルフィド結合体が、下記一般式（Ａ）で示され、
    Figure JPOXMLDOC01-appb-C000001
     （一般式（Ａ）中、ｎ３は２～３０の範囲の整数であり、－Ｓ－末端がナノ粒子にスルフィド結合する担持部位である。）
    前記糖鎖を固定化したアルカンチオールのスルフィド結合体が、下記一般式（Ｂ）で示される、請求項４に記載の糖鎖提示粒子。
    Figure JPOXMLDOC01-appb-C000002
     （一般式（Ｂ）中、ｎ１は２～３０の整数であり、ｎ２は２～３０の整数であり、－Ｓ－末端がナノ粒子にスルフィド結合する担持部位であり、Ｒ１０は糖鎖含有部位である。）     The sulfide bond of the alkanethiol having a phosphorylcholine group is represented by the following general formula (A).
    Figure JPOXMLDOC01-appb-C000001
     (In the general formula (A), n3 is an integer in the range of 2 to 30, and the -S-end is a carrier site where a sulfide bond is attached to the nanoparticles.)
    The sugar chain-presenting particles according to claim 4, wherein the sulfide bond of alkanethiol on which the sugar chain is immobilized is represented by the following general formula (B).
    Figure JPOXMLDOC01-appb-C000002
     In (formula (B), n1 is an integer of 2-30, n2 is an integer of 2 to 30, a bearing portion -S- terminal sulfide bound to the nanoparticles, R 10 is contained sugar It is a part.)
  6. 薬剤部位をさらに有する、請求項１～５のいずれか１項に記載の糖鎖提示粒子。 The sugar chain-presenting particle according to any one of claims 1 to 5, further comprising a drug site.
  7. 前記単分子膜は、下記一般式（Ｃ）で示される、薬剤部位を有するアルカンチオールのスルフィド結合体をさらに含む、請求項４または５に記載の糖鎖提示粒子。
    Figure JPOXMLDOC01-appb-C000003
     一般式（Ｃ）中、ｎ１は２～３０の整数であり、ｎ２は２～３０の整数であり、－Ｓ－末端がナノ粒子にスルフィド結合する担持部位であり、Ｒ２０は薬剤含有部位である。     The sugar chain-presenting particle according to claim 4 or 5, wherein the monolayer further contains a sulfide bond of an alkanethiol having a drug site, which is represented by the following general formula (C).
    Figure JPOXMLDOC01-appb-C000003
     In the general formula (C), n1 is an integer of 2 to 30, n2 is an integer of 2 to 30, the -S-terminal is a carrier site where a sulfide bond is attached to nanoparticles, and R 20 is a drug-containing site. is there.
  8. 異なるがん糖鎖パターンを有する２種類以上の糖鎖提示粒子を含有し、糖鎖提示粒子は、請求項１～７のいずれか１項に記載の糖鎖提示粒子である、糖鎖提示粒子キット。 Sugar chain presentation particles containing two or more types of sugar chain presentation particles having different cancer sugar chain patterns, and the sugar chain presentation particles are the sugar chain presentation particles according to any one of claims 1 to 7. kit.
  9. 異なるがん糖鎖パターンが、請求項２に記載の糖鎖パターンＡ～Ｄのいずれか２種以上の糖鎖パターンである、請求項８に記載の糖鎖提示粒子キット。 The sugar chain presentation particle kit according to claim 8, wherein the different cancer sugar chain patterns are sugar chain patterns of any two or more of the sugar chain patterns A to D according to claim 2.
  10. 糖鎖提示粒子は、請求項６または７に記載の薬物部位をさらに有する糖鎖提示粒子である、請求項８に記載の糖鎖提示粒子キット。 The sugar chain presentation particle kit according to claim 8, wherein the sugar chain presentation particles are sugar chain presentation particles further having the drug site according to claim 6 or 7.
  11. 請求項６または７に記載の糖鎖提示粒子を有効成分として含有する、がん転移予防薬。 A cancer metastasis preventive agent containing the sugar chain-presenting particles according to claim 6 or 7 as an active ingredient.
  12. 請求項６または７に記載の糖鎖提示粒子を有効成分として含有する、がん治療薬。 A cancer therapeutic agent containing the sugar chain-presenting particles according to claim 6 or 7 as an active ingredient.
  13. 被検者から採取したがん細胞から、がん細胞の糖鎖をプロファイルし、プロファイルした糖鎖に基づいて糖鎖パターンを決定することを含む、がん細胞の糖鎖パターン決定方法。 A method for determining a sugar chain pattern of a cancer cell, which comprises profiling the sugar chain of the cancer cell from the cancer cell collected from a subject and determining the sugar chain pattern based on the profiled sugar chain.
  14. 糖鎖パターンの決定は、プロファイルした糖鎖が、
    （１）体内動態が主に末端ハイマンノース型糖鎖に依存する糖鎖パターンＡ、
    （２）体内動態が主に末端ガラクトース型糖鎖又は末端N-アセチルグルコサミン型糖鎖に依存する糖鎖パターンＢ、
    （３）体内動態が主に末端α２，６シアル酸型糖鎖に依存する糖鎖パターンＣ、及び
    （４）体内動態が主に末端α２，３シアル酸型糖鎖に依存する糖鎖パターンＤからなる群から選択される何れの糖鎖パターンであるかを特定することで行う、請求項１３に記載の決定方法。     The glycan pattern is determined by the profiled glycan.
    (1) Sugar chain pattern A, whose pharmacokinetics mainly depend on terminal high mannose type sugar chains,
    (2) Sugar chain pattern B, whose pharmacokinetics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains,
    (3) Sugar chain pattern C whose pharmacokinetics mainly depend on terminal α2,6 sialic acid type sugar chains, and (4) Sugar chain pattern D whose pharmacokinetics mainly depend on terminal α2,3 sialic acid type sugar chains The determination method according to claim 13, which is performed by specifying which sugar chain pattern is selected from the group consisting of.
  15. がん糖鎖パターンＡは、糖鎖の４５モル％以上が末端ハイマンノース型糖鎖であり、末端シアル酸型糖鎖は０％以上、２％未満であり、
    がん糖鎖パターンＢは、糖鎖の末端ハイマンノース型糖鎖が４５％未満であり、かつ末端シアル酸型糖鎖が０％以上、２％未満であり、
    がん糖鎖パターンＣは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつ末端α２，６シアル酸型糖鎖の含有量が末端α２，３シアル酸型糖鎖の含有量より多く、
    がん糖鎖パターンＤは、糖鎖の２～１００％が末端シアル酸型糖鎖であり、かつα2,3シアル酸型糖鎖の含有量がα２，６シアル酸型糖鎖の含有量より多い、請求項１４に記載の決定方法。     In the cancer sugar chain pattern A, 45 mol% or more of the sugar chains are terminal hymannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%.
    In the cancer sugar chain pattern B, the terminal hymannose type sugar chain of the sugar chain is less than 45%, and the terminal sialic acid type sugar chain is 0% or more and less than 2%.
    In the cancer sugar chain pattern C, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of the terminal α2,6 sialic acid type sugar chains is the content of the terminal α2,3 sialic acid type sugar chains. More than the amount,
    In the cancer sugar chain pattern D, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of α2,3 sialic acid type sugar chains is higher than the content of α2,6 sialic acid type sugar chains. The determination method according to claim 14, which is often the case.
  16. がん細胞の糖鎖プロファイルが糖鎖パターンＡの場合、被検者が有するがん細胞はタイプ１の体内動態を示し、がん細胞の転移傾向が低いことを示唆し、
    糖鎖プロファイルが糖鎖パターンＢの場合、被検者が有するがん細胞はタイプ２の体内動態を示し、がん細胞が肝臓及び脾臓への転移傾向があることを示唆し、
    糖鎖プロファイルが糖鎖パターンＣの場合、被検者が有するがん細胞はタイプ３の体内動態を示し、がん細胞が腋窩及び鎖骨上リンパ節への転移傾向があることを示唆し、
    糖鎖プロファイルが糖鎖パターンＤの場合、被検者が有するがん細胞はタイプ４の体内動態を示し、がん細胞が肺、肝臓、脾臓、脳及び腎臓への転移傾向があることを示唆する、請求項１４又は１５に記載の決定方法。     When the sugar chain profile of cancer cells is sugar chain pattern A, the cancer cells possessed by the subject show type 1 pharmacokinetics, suggesting that the cancer cells have a low metastasis tendency.
    When the glycan profile is glycan pattern B, the cancer cells possessed by the subject show type 2 pharmacokinetics, suggesting that the cancer cells tend to metastasize to the liver and spleen.
    When the glycan profile is glycan pattern C, the cancer cells possessed by the subject show type 3 pharmacokinetics, suggesting that the cancer cells tend to metastasize to the axilla and supraclavicular lymph nodes.
    When the sugar chain profile is sugar chain pattern D, the cancer cells possessed by the subject show type 4 pharmacokinetics, suggesting that the cancer cells tend to metastasize to the lungs, liver, spleen, brain and kidneys. The determination method according to claim 14 or 15.
  17. 表面の少なくとも一部をリン脂質で被覆されたナノ粒子の表面に、がん糖鎖パターンを提示することを含む、請求項１～７のいずれか１項に記載の糖鎖提示粒子の製造方法。 The method for producing a sugar chain-presenting particle according to any one of claims 1 to 7, which comprises presenting a cancer sugar chain pattern on the surface of nanoparticles having at least a part of the surface coated with phospholipid. ..
  18. 前記提示されるがん糖鎖パターンは、被検者から採取したがん細胞の糖鎖を切り出したがん糖鎖パターン、又は、被検者から採取したがん細胞の糖鎖をプロファイルし、得られたプロファイルに基づいて決定された糖鎖パターンである、請求項１７に記載の製造方法。 The presented cancer sugar chain pattern is a cancer sugar chain pattern obtained by cutting out a sugar chain of a cancer cell collected from a subject, or a profile of a sugar chain of a cancer cell collected from a subject. The production method according to claim 17, which is a sugar chain pattern determined based on the obtained profile.
  19. 前記表面の少なくとも一部をリン脂質で被覆されたナノ粒子は、下記一般式（Ｄ）で示される架橋前駆体Ｘ及び下記一般式（Ｅ）で示されるリン脂質前駆体とコロイド状ナノ粒子とを混合して、ナノ粒子の表面に架橋前駆体Ｘ及びリン脂質を担持した表面修飾ナノ粒子を得ることで実施できる、請求項１７又は１８に記載の製造方法。
    Figure JPOXMLDOC01-appb-C000004
     （一般式（Ｄ）中、ｎ１は２～３０の整数であり、ｎ２は２～３０の整数である。)
    Figure JPOXMLDOC01-appb-C000005
     (一般式（Ｅ）中、ｎ３は２～３０の範囲の整数である。)     The nanoparticles in which at least a part of the surface is coated with phospholipids include the crosslinked precursor X represented by the following general formula (D), the phospholipid precursor represented by the following general formula (E), and colloidal nanoparticles. The production method according to claim 17 or 18, which can be carried out by mixing the above to obtain surface-modified nanoparticles in which the crosslinked precursor X and the phospholipid are carried on the surface of the nanoparticles.
    Figure JPOXMLDOC01-appb-C000004
     (In the general formula (D), n1 is an integer of 2 to 30, and n2 is an integer of 2 to 30.)
    Figure JPOXMLDOC01-appb-C000005
     (In the general formula (E), n3 is an integer in the range of 2 to 30.)
  20. がん糖鎖パターンの提示は、表面修飾ナノ粒子に導入された一般式（Ｄ）で示される架橋前駆体Ｘが有するアミノオキシ基と、被検者から採取したがん細胞の糖鎖を切り出したがん糖鎖パターン、又は、被検者から採取したがん細胞の糖鎖をプロファイルし、得られたプロファイルに基づいて決定された糖鎖パターンに含まれる糖鎖が有する還元末端とをグライコブロッティング法により反応させることで行う、請求項１９に記載の製造方法。 To present the cancer sugar chain pattern, the aminooxy group of the cross-linking precursor X represented by the general formula (D) introduced into the surface-modified nanoparticles and the sugar chain of the cancer cell collected from the subject are cut out. The cancer sugar chain pattern or the sugar chain of the cancer cell collected from the subject is profiled, and the reducing terminal of the sugar chain contained in the sugar chain pattern determined based on the obtained profile is used as a glyco. The production method according to claim 19, wherein the reaction is carried out by a blotting method.
  21. プロファイルに基づいて決定される糖鎖パターンは、糖鎖成分の種類と含有量をプロファイルと同一とした糖鎖パターンであるか、あるいは糖鎖成分の種類と含有量の一部をプロファイルと同一とした糖鎖パターンである請求項１８～２０のいずれか１項に記載の製造方法。
          The sugar chain pattern determined based on the profile is a sugar chain pattern in which the type and content of the sugar chain component are the same as the profile, or a part of the type and content of the sugar chain component is the same as the profile. The production method according to any one of claims 18 to 20, which is a sugar chain pattern obtained.
PCT/JP2020/035375 2019-09-20 2020-09-18 Sugar chain-presenting particles and production method thereof WO2021054420A1 (en)

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