Glycan-presenting particles and method for producing the same

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

Technical Field

[0001] The present invention relates to a sugar chain-presenting particle that mimics an exosome, a method for producing the same, and a method for using the same. The present invention relates to a sugar chain-presenting particle that presents 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 specific reference.

Background Art

[0002] In order to enhance the therapeutic effect of a pharmaceutical, a technology for selectively delivering the pharmaceutical to a specific organ or tissue in which a target molecule that causes the disease or exacerbates the pathological condition is highly expressed (drug delivery system, hereinafter abbreviated as DDS) is important. Currently, anticancer nanomedicines utilizing PEG-modified liposomes have been approved and marketed. The efficacy of these nanomedicines has basically been considered to depend on the significant enhanced permeability and retention (EPR) effect in cancer tissues. However, it has been clarified that the EPR effect is greatly influenced by many factors such as the type, site, degree of progression of cancer, and various adhesion molecules that constitute the stroma (extracellular matrix) between cancer cells and the surrounding area (Non-Patent Documents 1, 2, etc.). Research and development of DDS using various artificial extracellular microparticles in which inorganic / metal nanoparticles (such as gold nanoparticles, quantum dots, and silica-based microparticles as cores) that enable more advanced molecular design advantageous for the EPR effect are coated with polyethylene glycol (PEG) modification compounds and the like have been actively promoted.

[0003] However, recently, it has been reported that most of these nanomedicines containing liposomes and metal nanoparticles have not actually achieved the expected in vivo behavior (e.g., uptake ability in cancer tissues) in the experimental animals administered (Non-Patent Document 3). Surprisingly, in an investigation of 232 papers published in the past 10 years, it was found that the nanomedicines that reached the targeted cancer tissues accounted for only about 0.7% (average value) of the administered dose. Most of the nanoparticle-based pharmaceuticals administered intravascularly rapidly accumulate in the liver and are taken up by phagocytic cells such as macrophages and then decomposed and excreted, which largely depends on the properties of the "protein corona" on the surface of the nanoparticles formed by the non-specific adsorption of various proteins including albumin in the blood (Non-Patent Document 4, etc.). On the other hand, based on many recent reports that the invasion, metastasis, and organ tropism (metastasis destination) of individual cancer cells are determined by exosomes (extracellular nanoparticles) derived from cancer cells, research on exosomes derived from cancer cells has been actively conducted, and there are also cases aiming at the development of new pharmaceuticals using the functions of exosomes and various microRNAs encapsulated therein (e.g., Non-Patent Document 5).

[0004] Exosomes are membrane vesicles formed inside cells and released extracellularly, and function as a tool for cell-to-cell communication. However, due to the size and heterogeneity of the cargo molecules of exosomes, and the difficulty in purifying exosomes derived from cancer cells and tissues, the organ tropism mechanism at the specific molecular level is almost unknown.

[0005] Patent Document 1: WO2017 / 131242A1 / US2020138973(A1)

[0006] 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 All descriptions of Patent Document 1 and Non-Patent Documents 1 to 9 are hereby incorporated by reference as specific disclosures.

Summary of the Invention

Problems to be Solved by the Invention

[0007] Exosomes released from many cells exist in the blood. For example, in cancer patients, it has been almost unclear about the organotropic mechanism at the specific molecular level because it is difficult to purify only exosomes derived from cancer cells and tissues.

[0008] An object of the present invention is to artificially construct particles that mimic exosomes, which are tools for intercellular information transmission. And it is to provide a new technique that utilizes the in vivo dynamics of exosomes released from the information sender.

[0009] More specifically, it is extremely effective as a model of cancer cell-derived exosomes, provides a method for predicting the behavior and organ tropism of sugar chain-presenting particles carrying various molecules for metastasis function analysis after in vivo administration, and further provides a technology for realizing the development of a new nano-particle DDS for cancer prevention and treatment that enables clinical applications in humans. Furthermore, the present invention also aims to provide a method for determining a sugar chain pattern that can suggest the metastatic potential of cancer cells.

Means for Solving the Problems

[0010] The present invention focused on the fact that the surface of exosomes presents sugar chains in a pattern similar to that of the sugar chains of the cells that release them. That is, for example, in the case of cancer cell metastasis, exosomes released from cancer cells are considered to play a leading role in guiding the in vivo behavior of cancer cells. Various cells and organs that take up exosomes are likely to first encounter the bulky glycocalyx on the surface of the exosome membrane. In the present invention, by paying attention to this fact, it was found that the in vivo behavior of cancer cell-derived exosomes, particularly their movement and excretion in the body, as well as their distribution or accumulation in organs and tissues (organ tropism), are guided and determined by the sugar chain pattern on the exosome membrane surface, leading to the completion of the present invention.

[0011] Specifically, human cancer cell-derived exosome-mimicking particles can be prepared by presenting all the sugar chains of N-type sugar chains of human cultured cancer cells on the surface of the nanosome membrane by the glycoblotting method. By directly intravenously administering these to mice and observing their in vivo behavior in real time, it became clear that the in vivo behavior and organ tropism of human cancer cell-derived exosomes can be easily determined. Furthermore, based on the results, it was demonstrated that drug discovery based on the new concept of molecularly designing a tailor-made DDS adapted to the organ tropism in individual cancer metastases in advance was possible because particles mimicking exosomes presenting an artificially prepared cancer-specific sugar chain pattern reproduced the intended in vivo behavior, thus completing the present invention.

[0012] The present invention is as follows. [1] A sugar chain-presenting particle which is a nanoparticle having a sugar chain on its surface, (1) The average particle diameter of the sugar chain-presenting particle is in the range of 10 to 100 nm, (2) At least a part of the surface of the nanoparticle is coated with a phospholipid, (3) The sugar chain on the nanoparticle surface is a sugar chain pattern determined based on the sugar chain pattern required by cancer cells or the profile of this sugar chain (hereinafter referred to as the cancer sugar chain pattern). Sugar chain-presenting particle. [2] The cancer sugar chain pattern is (1) Sugar chain pattern A whose in vivo dynamics mainly depend on terminal high-mannose type sugar chains, (2) Sugar chain pattern B whose in vivo dynamics mainly depend on terminal galactose type sugar chains or terminal N-acetylglucosamine type sugar chains, (3) Sugar chain pattern C whose in vivo dynamics mainly depend on terminal α2,6 sialic acid type sugar chains, (4) A sugar chain pattern selected from the group consisting of sugar chain pattern D whose in vivo dynamics mainly depend on terminal α2,3 sialic acid type sugar chains, the sugar chain-presenting particle according to [1]. [3] In cancer sugar chain pattern A, 45 mol% or more of the sugar chains are terminal high-mannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%, In cancer sugar chain pattern B, the terminal high-mannose type sugar chains of the sugar chains are less than 45%, and the terminal sialic acid type sugar chains are 0% or more and less than 2%, In cancer sugar chain pattern C, 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of terminal α2,6 sialic acid type sugar chains is more than the content of terminal α2,3 sialic acid type sugar chains, In 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 more than the content of α2,6 sialic acid type sugar chains, the sugar chain-presenting particle according to [2]. [4] The phospholipid that coats at least a part of the nanoparticle surface is a sulfide conjugate of an alkanethiol having a phosphorylcholine group, the sugar chain on the nanoparticle surface is a sulfide conjugate of an alkanethiol immobilized with the sugar chain, and the nanoparticle surface is coated with a monolayer of a sulfide conjugate of an alkanethiol having a phosphorylcholine group and a sulfide conjugate of an alkanethiol immobilized with the sugar chain. The sugar chain-presenting particle according to any one of [1] to [3]. [5] The sulfide conjugate of the alkanethiol having a phosphorylcholine group is represented by the following general formula (A): [Chemical formula] (In general formula (A), n3 is an integer in the range of 2 to 30, and the -S- terminal is a supporting site that forms a sulfide bond with the nanoparticle.) The sulfide conjugate of the alkanethiol immobilized with the sugar chain is represented by the following general formula (B), the sugar chain-presenting particle according to [4]. [Chemical formula] (In general formula (B), n1 is an integer of 2 to 30, n2 is an integer of 2 to 30, the -S- terminal is a supporting site that forms a sulfide bond with the nanoparticle, and R 10 is a sugar chain-containing site.) [6] The sugar chain-presenting particle according to any one of [1] to [5], further having a drug site. [7] The monolayer further contains a sulfide conjugate of an alkanethiol having a drug site, represented by the following general formula (C), the sugar chain-presenting particle according to [4] or [5]. [Chemical formula] In general formula (C), n1 is an integer of 2 to 30, n2 is an integer of 2 to 30, the -S- terminal is a supporting site that forms a sulfide bond with the nanoparticle, and R 20 is a drug-containing site. [8] A sugar chain presentation particle kit containing two or more types of sugar chain presentation particles having different cancer sugar chain patterns, wherein the sugar chain presentation particles are the sugar chain presentation particles described in any one of [1] to [7]. [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 pattern A to D described in [2].

[10] The sugar chain presentation particle kit according to [8], wherein the sugar chain presentation particles are sugar chain presentation particles further having the drug site described in [6] or [7].

[11] A cancer metastasis preventive drug containing, as an active ingredient, the sugar chain presentation particle described in [6] or [7].

[12] A cancer therapeutic drug containing, as an active ingredient, the sugar chain presentation particle described in [6] or [7].

[13] A method for determining the sugar chain pattern of cancer cells, comprising profiling the sugar chains of cancer cells collected from a subject and determining the sugar chain pattern based on the profiled sugar chains.

[14] The determination of the sugar chain pattern is performed by identifying which sugar chain pattern the profiled sugar chains are selected from the group consisting of: (1) Sugar chain pattern A in which the pharmacokinetics mainly depend on terminal high mannose type sugar chains, (2) Sugar chain pattern B in which the pharmacokinetics mainly depend on terminal galactose type sugar chains or terminal N-acetylglucosamine type sugar chains, (3) Sugar chain pattern C in which the pharmacokinetics mainly depend on terminal α2,6 sialic acid type sugar chains, and (4) Sugar chain pattern D in which the pharmacokinetics mainly depend on terminal α2,3 sialic acid type sugar chains, as described in

[13] .

[15] Cancer sugar chain pattern A has 45 mol% or more of the sugar chains being terminal high mannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%, Cancer sugar chain pattern B has less than 45% of the sugar chains being terminal high mannose type sugar chains and the terminal sialic acid type sugar chains are 0% or more and less than 2%, Cancer glycan pattern C is such that 2 to 100% of the glycans are terminal sialic acid-type glycans, and the content of terminal α2,6 sialic acid-type glycans is greater than the content of terminal α2,3 sialic acid-type glycans. Cancer glycan pattern D is a determination method described in

[14] , in which 2 to 100% of the glycans are terminal sialic acid-type glycans, and the content of α2,3 sialic acid-type glycans is greater than the content of α2,6 sialic acid-type glycans.

[16] When the glycan profile of cancer cells is glycan pattern A, the cancer cells possessed by the subject show type 1 in vivo behavior, suggesting that the cancer cells have a low tendency to metastasize. When the glycan profile is glycan pattern B, the cancer cells possessed by the subject show type 2 in vivo behavior, suggesting that the cancer cells have a tendency 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 in vivo behavior, suggesting that the cancer cells have a tendency 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 in vivo behavior, suggesting that the cancer cells have a tendency to metastasize to the lung, liver, spleen, brain, and kidney, which is the determination method described in

[14] or

[15] .

[17] A method for producing a glycan-presenting particle according to any one of [1] to [7], including presenting a cancer glycan pattern on the surface of a nanoparticle having at least a part of its surface coated with a phospholipid.

[18] The presented cancer glycan pattern is a cancer glycan pattern obtained by cleaving the glycans of cancer cells collected from a subject, or a glycan pattern determined based on the profile obtained by profiling the glycans of cancer cells collected from a subject, which is the production method described in

[17] .

[19] The nanoparticle having at least a part of its surface coated with a phospholipid can be obtained by mixing a crosslinking precursor X represented by the following general formula (D), a phospholipid precursor represented by the following general formula (E), and colloidal nanoparticles to support the crosslinking precursor X and the phospholipid on the surface of the nanoparticles, which is the production method described in

[17] or

[18] .

Chem.

Chem.

[20] The presentation of the cancer glycan pattern is carried out by reacting the aminooxy group of the cross-linking precursor X represented by general formula (D) introduced into the surface-modified nanoparticles with the cancer glycan pattern obtained by cleaving the glycans of cancer cells collected from a subject, or the reducing end of the glycans contained in the glycan pattern determined based on the profile obtained by profiling the glycans of cancer cells collected from a subject, by the glycoblotting method, the production method described in

[19] .

[21] The glycan pattern determined based on the profile is a glycan pattern in which the types and contents of glycan components are the same as those of the profile, or a glycan pattern in which some of the types and contents of glycan components are the same as those of the profile. The production method according to any one of

[18] to

[20] .

Advantages of the Invention

[0013] According to the present invention, it is possible to provide a glycan-presenting particle having a cancer glycan pattern that shows a specific in vivo behavior and can predict the metastasis destination of cancer cells in the primary tumor. This glycan-presenting particle can, for example, visualize the process of cancer metastasis led by the cancer glycan pattern in real time by intravenous administration to a mouse. Furthermore, according to the present invention, it is also possible to provide a DDS technology effective in preventing cancer metastasis and treating cancer. Furthermore, according to the present invention, it is also possible to provide a method for determining a glycan pattern that can suggest the metastatic property of cancer cells.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

Figure 3-1

Figure 3-2

Figure 4-1

Figure 4-2

Figure 5-1

Figure 5-2

Figure 6

Mode for Carrying Out the Invention

[0015] <Sugar chain-presenting particle> The present invention relates to sugar chain-presenting particles which are nanoparticles having sugar chains on their surfaces, and these sugar chain-presenting particles (1) The average particle diameter of the sugar chain-presenting particles is in the range of 10 to 100 nm, (2) At least a part of the surface of the nanoparticles is coated with a phospholipid, (3) The sugar chain on the nanoparticle surface is a pattern (cancer sugar chain pattern) that mimics the sugar chain pattern derived from cancer cells or a sugar chain pattern determined based on the profile of this sugar chain.

[0016] 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 means a pattern specified by the presence or absence of a specific type of sugar chain or the abundance of a specific type of sugar chain in a population of sugar chains composed of two or more different sugar chains based on the sugar chain profile on the surface. The sugar chain pattern is specified by the types of sugar chain components and their contents.

[0017] Therefore, in the present invention, the glycan-presenting particles that directly capture and present the glycans of cancer cells are glycan-presenting particles having a glycan pattern derived from cancer cells. Also, glycan-presenting particles that profile the glycans of cancer cells, determine a glycan pattern based on the profile, and have the determined glycan pattern are also the glycan-presenting particles in the present invention. The glycan pattern determined based on the profile may be either a glycan pattern in which the types and contents of glycan components are the same as those in the profile, or a glycan pattern in which some of the types and contents of glycan components are the same as those in the profile. When some of the types and contents of glycan components are the same as those in the profile, some of the types of glycan components included in the profile can be reduced or deleted. More specifically, when the glycan components included in the profile are classified into several types, it can be a glycan pattern that includes glycan components belonging to one or more types with a high content and reduces or deletes glycan components belonging to one or more types with a low content. Examples of the types of glycan components include terminal high-mannose type glycans, terminal galactose type glycans, terminal N-acetylglucosamine type glycans, terminal α2,6 sialic acid type glycans, and terminal α2,3 sialic acid type glycans. When the glycan components in the profile are, for example, terminal high-mannose type glycans, terminal galactose type glycans, and terminal N-acetylglucosamine type glycans in descending order of content, a glycan pattern in which the terminal N-acetylglucosamine type glycan with a low content is reduced or deleted can be determined as the glycan pattern from the profile. Alternatively, when a plurality of glycan components are included in one type, a glycan pattern in which the content ratio of the glycan components belonging to the same type is changed can also be determined as the glycan pattern from the profile. Since the in vivo behavior of glycan-presenting particles changes depending on the type of glycan, considering the change in in vivo behavior, the profile can be appropriately changed to determine the glycan pattern.

[0018] The glycan pattern in the present invention can include four cancer glycan patterns, A to D, which can be classified according to the in vivo behavior of the glycan-presenting particles of the present invention. (1) Glycan pattern A in which the in vivo behavior mainly depends on terminal high-mannose type glycans, (2) A sugar chain pattern B in which the in vivo dynamics mainly depend on terminal galactose-type sugar chains or terminal N-acetylglucosamine-type sugar chains, (3) A sugar chain pattern C in which the in vivo dynamics mainly depend on terminal α2,6-sialic acid-type sugar chains, (4) A sugar chain pattern D in which the in vivo dynamics mainly depend on terminal α2,3-sialic acid-type sugar chains.

[0019] The in vivo dynamics mean the time-dependent movement of the sugar chain-presenting particles through the circulatory system etc. in the body of an animal administered with the sugar chain-presenting particles, and the time-dependent distribution to each organ and tissue. In the present specification, the property of being distributed or accumulated in each organ and tissue over time among the in vivo dynamics may particularly mean organ tropism.

[0020] The cancer cells targeted in the present invention are cancer cells that release exosomes which are the mimicking targets of the sugar chain-presenting particles of the present invention, and include cancer cells collected from a living body and cancer cells cultured after collection. The cancer cells may be a purely cultured single cell or a mixture of a plurality of cancer cells with different malignancy degrees etc.

[0021] Also, the sugar chain patterns of existing cancer cells that are standardly used can also be used as sugar chain patterns in the sugar chain-presenting 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 (stomach cancer), MIAPaCa-2 (pancreatic cancer), A431 (skin cancer), SKOV (ovarian cancer), etc.

[0022] The sugar chain-presenting particle means a particle having a function (cell-cell information transmission-like function) that mimics the function of exosomes, which are particles used for cell-cell information transmission. Exosomes contain nucleic acids, proteins, etc. inside them and are transmitted to recipient cells via exosomes secreted from cells. Exosomes function as a cell-cell communication tool. The sugar chain-presenting particles produced in the present invention have a function of transmitting information to recipient cells, tissues, or organs aggregated therefrom, similar to the original exosomes.

[0023] The nanoparticles can be metal nanoparticles or semiconductor nanoparticles. The material of the metal nanoparticles is not particularly limited, and it can be gold, platinum, silver, or a ferromagnetic material. The metal nanoparticles can be gold nanoparticles, platinum nanoparticles, silver nanoparticles, or ferromagnetic nanoparticles. In particular, gold nanoparticles, platinum nanoparticles, and silver nanoparticles are preferable from the viewpoint of safety to living bodies.

[0024] The material of the semiconductor nanoparticles is not particularly limited. The semiconductor nanoparticles can also be quantum dots. A quantum dot is a small lump of about 10 to several tens of nm in which several hundreds to several thousands of semiconductor atoms are aggregated, and it is a fluorescent nanoparticle. The wavelength (color) of the fluorescence emitted depends on the particle size. Commercially available products can be used. When quantum dots are used for the nanoparticles of the composite of the present invention, a fluorescent composite can be obtained, and it is also possible to monitor the behavior in vivo.

[0025] 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, and even more preferably in the range of 10 to 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, more preferably in the range of 12 to 50 nm, still more preferably in the range of 15 to 40 nm, and even more preferably in the range of 15 to 30 nm. The average particle sizes of the nanoparticles and the sugar chain-presenting particles can be measured by the dynamic light scattering method. As the measuring device, a fiber optic dynamic light scattering photometer (for particle size distribution measurement) can be used. More specifically, a fiber optic dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Electronics Co., Ltd.) can be used.

[0026] In the sugar chain-presenting particles of the present invention, the surface of the nanoparticles is coated with a phospholipid. The completely coated metal nanoparticles with a phospholipid alkanethiol mixed monolayer having an average particle size of about 20 nm do not form a protein corona due to non-specific adsorption even in blood, and are stable nanoparticles that do not accumulate in specific organs when intravenously administered to mice and remain uniformly throughout the body even 3 hours after administration (Non-Patent Documents 6-7, Patent Document 1).

[0027] The sulfide conjugate of the alkanethiol having a phosphorylcholine group can be a phospholipid mimetic substance represented by the general formula (A).

Chemical formula

[0028] The phospholipid mimetic substance modifies the surface of the nanoparticles and imparts a function of preventing non-specific adsorption of the protein corona to the surface of the sugar chain-presenting particles of the present invention. From this viewpoint, n3 is an integer in the range of 2 to 30, preferably 5 to 20, and more preferably 7 to 15.

[0029] The amount of phospholipid or phospholipid mimetic substance supported on one nanoparticle is preferably 80% or more of the metal element (reaction point) on the surface of the nanoparticle.

[0030] The sugar chain-presenting particle of the present invention has a cancer sugar chain pattern on the surface of the nanoparticle. This cancer sugar chain pattern is a sugar chain pattern that presents a sugar chain derived from a cancer cell that releases exosomes as an information sender, or a cancer sugar chain pattern obtained by profiling and determining the sugar chain of a cancer cell. The sugar chain pattern derived from a cancer cell is preferably the sugar chain pattern of a sugar chain derived from a cancer cell collected from a specific cancer treatment patient in order to enhance the predictability of the in vivo behavior of exosomes released from the cell. Existing cancer cell-derived sugar chain patterns that are commonly used can also be used for prediction. The sugar chain pattern derived from a cancer cell can be presented by excising the sugar chain of the cancer cell and capturing it on the surface of the nanoparticle. In excising the sugar chain from the cell and capturing it on the nanoparticle, it is preferable that the sugar chain of the cell is presented as it is on the surface of the nanoparticle, but there may be variations in the sugar chain components within the range of the cancer sugar chain pattern determined in the cell.

[0031] The above cancer cell-derived sugar chain profile can be specified according to the general protocol of the glycoblotting method (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537). The relationship between the main structural motifs and their expression levels can be clarified by glycotyping analysis using an internal standard compound as an index. Also, the sugar chain can be supported on the nanoparticle by existing methods (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). For details, refer to Example 1(A).

[0032] As a result of analyzing the in vivo dynamics of carbohydrate-presenting particles having the carbohydrate pattern of cancer cell carbohydrates, the carbohydrate pattern of the cancer cells under investigation was at least (1) Carbohydrate pattern A in which the in vivo dynamics mainly depend on terminal high-mannose type carbohydrates, (2) Carbohydrate pattern B in which the in vivo dynamics mainly depend on terminal galactose type carbohydrates or terminal N-acetylglucosamine type carbohydrates, (3) Carbohydrate pattern C in which the in vivo dynamics mainly depend on terminal α2,6 sialic acid type carbohydrates, (4) It was found that the carbohydrate pattern is selected from the group consisting of carbohydrate pattern D in which the in vivo dynamics mainly depend on terminal α2,3 sialic acid type carbohydrates, and it was revealed that the in vivo dynamics of carbohydrate-presenting particles having the cancer carbohydrate pattern are determined depending on the cancer carbohydrate pattern (see Examples 1, 2 and 3).

[0033] The in vivo dynamics of carbohydrate-presenting particles having cancer carbohydrate patterns A to D are as follows. Carbohydrate-presenting particles having cancer carbohydrate pattern A show type 1 in vivo dynamics in which the excretion of the particles outside the body is promoted as compared with carbohydrate-presenting particles having no carbohydrates. Carbohydrate-presenting particles having cancer carbohydrate pattern B show type 2 in vivo dynamics in which they accumulate in the liver and spleen as compared with carbohydrate-presenting particles having no carbohydrates. Carbohydrate-presenting particles having cancer carbohydrate pattern C show type 3 in vivo dynamics in which they accumulate in the axilla and supraclavicular lymph nodes as compared with carbohydrate-presenting particles having no carbohydrates. Carbohydrate-presenting particles having cancer carbohydrate pattern D show type 4 in vivo dynamics in which they are distributed (dispersedly accumulated) in organs such as the lung, liver, spleen, brain, and kidney as compared with carbohydrate-presenting particles having no carbohydrates.

[0034]

Chemical formula

Chemical formula

[0035] The terminal galactose-type sugar chain that constitutes the above sugar chain pattern means a sugar chain in which one or a plurality of terminal sugars of one sugar chain are galactose. Examples of the terminal galactose-type sugar chain are shown below.

Chemical formula

[0036] The terminal N-acetylglucosamine-type sugar chain that constitutes the above sugar chain pattern means a sugar chain in which one or a plurality of terminal sugars of one sugar chain are N-acetylglucosamine. Examples of the terminal N-acetylglucosamine-type sugar chain are shown below.

Chemical formula

[0037] The terminal sialic acid-type sugar chain that constitutes the above sugar chain pattern means a sugar chain in which one or a plurality of terminal sugars of one sugar chain are sialic acid. The terminal sialic acid-type sugar chain includes a complex sugar chain containing a Neu5Acα2,6Gal unit at the terminal and a complex sugar chain containing a Neu5Acα2,3Gal unit at the terminal. In the present specification, the complex sugar chain containing a Neu5Acα2,6Gal unit at the terminal is referred to as a terminal α2,6 sialic acid-type sugar chain, and the complex sugar chain containing a Neu5Acα2,3Gal unit at the terminal is referred to as a terminal α2,3 sialic acid-type sugar chain. Examples of the terminal sialic acid-type sugar chain are shown below.

Chemical formula

Chemical formula

[0038] In the present invention, using four types of human cultured cancer cells (MCF7, MDA-MB-231, A549, HepG2) for which information already exists regarding organs (organ tropism) in which metastasis and recurrence due to metastasis are observed, (1) the state of post-translational sugar chain modification (N-type sugar chains) of all proteins present inside and on the membrane surface of the cancer cells was profiled. Next, (2) the sugar chains of these cancer cells were directly captured by a glyco-blotting method (Non-Patent Document 9) onto nanoparticles (fluorescent nanoparticles prepared from quantum dots: Non-Patent Documents 6-7, Patent Document 1) to produce sugar chain-presenting particles that present sugar chains derived from each cancer cell, and furthermore, the in vivo dynamics when these were intravenously administered to mice were observed in real time by near-infrared fluorescence spectrum using sugar chain-presenting particles that do not present sugar chains as a comparative control (see Example 1).

[0039] In response to the results, (3) an artificial sugar chain pattern was determined based on the sugar chain profile of the above human cultured cancer cells, and artificial sugar chain-presenting particles that present sugar chains within the range of the determined sugar chain pattern were produced. As a result of observing the in vivo dynamics by administering these to mice in the same manner, it was proven that the sugar chain pattern of exosomes derived from cancer cells guides the in vivo dynamics of exosomes and ultimately determines the organ tropism of cancer cells (see Example 2).

[0040] (1) Cancer sugar chain pattern A In cancer sugar chain pattern A, 45 mol% or more of the sugar chains contained in the sugar chain are terminal high-mannose type sugar chains. Hereinafter, unless otherwise specified, the % of the sugar chain means mol%. The content rate of terminal high-mannose type sugar chains (HM) in the sugar chain-presenting particles of the present invention having cancer sugar chain pattern A shown in the examples is as follows. The content rate of terminal sialic acid type sugar chains is 0% in any case.

[0041]

Table 1

[0042] Cancer glycan pattern A can include, as glycan components in addition to the terminal high-mannose type glycan, terminal galactose type glycan, terminal N-acetylglucosamine type glycan, terminal sialic acid type glycan, etc., but the terminal sialic acid type glycan is 0% or more and less than 2%. When the terminal sialic acid type glycan is 2% or more, it can be determined as glycan pattern C or glycan pattern D, and the effect of the terminal sialic acid type glycan becomes prioritized (described later). If the terminal sialic acid type glycan is less than 2% and the terminal high-mannose type glycan is 45% or more, regardless of the types and amounts of the other glycan components, namely the terminal galactose type glycan and the terminal N-acetylglucosamine type glycan, the glycan-presenting particles show the in vivo kinetics of type 1 and do not show the distribution to specific organs (organ tropism). The terminal high-mannose type glycan can be, for example, in the range of 50% to 95%.

[0043] As shown in Fig. 3a, in the example of NS (control), a glycan-presenting particle without a glycan, almost fluorescence from the particles was observed throughout the body even 180 minutes after intravenous administration. In contrast, in the example of GNS-MCF-7 of Example 1 having cancer glycan pattern A, most of the particles had been excreted outside the body 180 minutes after intravenous administration. This phenomenon was the same for GNS-Button Quail of Example 2 (see Fig. 5) and 0 / 6 / 32 of Example 3. It can be seen that the glycan-presenting particles having cancer glycan pattern A show the in vivo kinetics of type 1 in which the excretion of the particles outside the body is promoted as compared with the glycan-presenting particles without a glycan.

[0044] (2) Cancer glycan pattern B Cancer glycan pattern B is a cancer glycan pattern in which the terminal high mannose-type glycan is less than 45% and the terminal sialic acid-type glycan is 0% or more and less than 2%. A glycan-presenting particle having a glycan pattern in which the terminal high mannose-type glycan is less than 45% and the terminal sialic acid-type glycan is 0% or more and less than 2% exhibits type 2 pharmacokinetics. As glycan components other than the terminal high mannose-type glycan and the terminal sialic acid-type glycan, terminal galactose-type glycan and terminal N-acetylglucosamine-type glycan can be included. As long as the terminal high mannose-type glycan is less than 45% and the terminal sialic acid-type glycan is 0% or more and less than 2%, even if the quantitative ratios of the terminal galactose-type glycan and the terminal N-acetylglucosamine-type glycan change, it can be determined as glycan pattern B. The glycan-presenting particle of cancer glycan pattern B exhibits type 2 pharmacokinetics.

[0045] The contents of the terminal high mannose-type glycan (HM), terminal galactose-type glycan (Gal), and terminal N-acetylglucosamine-type glycan (NAc-G) of the glycan-presenting particle of the present invention having the cancer glycan pattern B shown in the examples are as shown in Table 2. The terminal sialic acid-type glycan is 0% in any case.

[0046]

Table 2

[0047] As shown in FIGS. 5b and 5c, for example, the example of GNS-Chicken of Example 2 having cancer glycan pattern B was accumulated in the liver and spleen 180 minutes after intravenous administration. This phenomenon was the same in the example of GNS-Ruddy Duck of Example 2 (see FIGS. 5b and 5c). That is, it can be seen that the glycan-presenting particle having cancer glycan pattern B exhibits type 2 pharmacokinetics of being accumulated in the liver and spleen as compared with the control particle having no glycan.

[0048] (3) Cancer glycan pattern C Cancer glycan pattern C is a cancer glycan pattern in which 2 to 100% of the glycans are terminal sialic acid-type glycans. Further, among the terminal sialic acid-type glycans, there are terminal α2,6 sialic acid-type glycans and terminal α2,3 sialic acid-type glycans, and cancer glycan pattern C is a case where the content of α2,6 sialic acid-type glycans is higher than the content of α2,3 sialic acid-type glycans. The content of the terminal sialic acid-type glycans can be, for example, in the range of 3 to 100%, 4 to 80%, or 5 to 60%. The glycans other than the terminal sialic acid-type glycans may be terminal high mannose-type glycans, terminal galactose-type glycans, and / or terminal N-acetylglucosamine-type glycans.

[0049] (4) Cancer glycan pattern D Cancer glycan pattern D is a cancer glycan pattern in which 2 to 100% of the glycans are terminal sialic acid-type glycans. Cancer glycan pattern D is a case where the content of α2,3 sialic acid-type glycans is higher than the content of α2,6 sialic acid-type glycans. The content of the terminal sialic acid-type glycans can be, for example, in the range of 3 to 100%, 4 to 80%, or 5 to 60%. The glycans other than the terminal sialic acid-type glycans may be terminal high mannose-type glycans, terminal galactose-type glycans, and / or terminal N-acetylglucosamine-type glycans.

[0050] The contents of the α2,3 sialic acid-type glycan (α2,3), α2,6 sialic acid-type glycan (α2,6), and terminal high mannose-type glycan (HM) of the glycan-presenting particles of the present invention having the cancer glycan patterns C and D shown in the examples are as shown in Table 3. The terminal galactose-type glycan (Gal) and terminal N-acetylglucosamine-type glycan (NAc-G) are not shown.

[0051]

Table 3

[0052] NS (control), a particle without a sugar chain shown in FIGS. 3b and c, showed fluorescence from the particles almost throughout the body even 180 minutes after intravenous administration. In contrast, in the case of 2,6-Ruddy Duck of Example 2 having the cancer sugar chain pattern of Pattern C (see FIGS. 5b and c), compared with NS, for example, 180 minutes after intravenous administration, the particles were accumulated in the axilla and supraclavicular lymph nodes. From this, it can be seen that the sugar chain-presenting particles having the cancer sugar chain pattern C show the type 3 in vivo dynamics of being accumulated in the axilla and supraclavicular lymph nodes as compared with the sugar chain-presenting particles without a sugar chain.

[0053] In the example of 2,3-Ruddy Duck of Example 3 having the cancer sugar chain pattern of Pattern D (see FIGS. 5b and c), 180 minutes after intravenous administration, the particles were distributed (dispersedly accumulated) in organs such as the lung, liver, spleen, brain, and kidney. From this, it can be seen that the sugar chain-presenting particles having the cancer sugar chain pattern D show the type 4 in vivo dynamics of being distributed (dispersedly accumulated) in organs such as the lung, liver, spleen, brain, and kidney as compared with the sugar chain-presenting particles without a sugar chain.

[0054] 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, 180 minutes after intravenous administration, the particles were accumulated in the axilla and supraclavicular lymph nodes (particularly, FIGS. 3b and c). This phenomenon was the same as that in the example of 2,6-Ruddy Duck of Example 2 having the cancer sugar chain pattern C. In Example 1, the terminal sialic acid-type sugar chain was not identified as being an α2,3 sialic acid-type sugar chain or an α2,6 sialic acid-type sugar chain, but from the results of this in vivo dynamics, GNS-MDA-MB-231 of Example 1 was judged to have the sugar chain pattern C and shown in Table 4.

[0055] In the example of GNS-A549 in Example 1, 180 minutes after intravenous administration, the particles were distributed (dispersed and accumulated) to organs such as the lungs, liver, spleen, brain, and kidneys (especially in FIGS. 3b and c). This phenomenon was the same as that in the example of 2,3-Ruddy Duck in Example 2 having the cancer sugar chain pattern D. In Example 1, although it was not specified whether the terminal sialic acid-type sugar chain was an α2,3 sialic acid-type sugar chain or an α2,6 sialic acid-type sugar chain, from the results of this pharmacokinetics, GNS-A549 and GNS-HepG2 in Example 1 were judged to have the sugar chain pattern D and are shown in Table 4. The identification of whether it is an α2,6 sialic acid-type sugar chain or an α2,3 sialic acid-type sugar chain was judged in terms of its function in this specification, but it can also be identified by analysis methods such as chemical and enzymatic degradation characteristics.

[0056]

Table 4

[0057] Cancer glycan patterns can be the glycans themselves of cancer cells that are commercially available for research purposes and have become generalized, or of cancer cells of specific cancer treatment patients. Further, a cancer glycan pattern can be a glycan pattern determined based on profiling the glycans of cancer cells. The glycan pattern determined based on the profile can be either a glycan pattern in which the types and contents of glycan components are the same as the profile, or a glycan pattern in which some of the types and contents of glycan components are the same as the profile. When some of the types and contents of glycan components are the same as the profile, some of the types of glycan components included in the profile can be reduced or deleted. More specifically, when the glycan components included in the profile are classified into several types, it can be a glycan pattern that includes glycan components belonging to one or more types with a high content and reduces or deletes glycan components belonging to one or more types with a low content. The glycan pattern determined based on profiling the glycans of cancer cells and the above cancer glycan pattern derived from cancer cells have different types and contents of glycan components, but is a glycan pattern that exhibits the same in vivo dynamics as the cancer glycan pattern derived from cancer cells. When profiling the glycans of cancer cells, if the glycan components included in the profile are classified into several types, by including as they are glycan components belonging to one or more types with a high content and reducing or deleting glycan components belonging to one or more types with a low content, it is possible to obtain a glycan pattern that exhibits the same in vivo dynamics as the cancer glycan pattern derived from the cancer cells whose glycans were profiled. Examples of the types of glycan components include terminal high-mannose type glycans, terminal galactose type glycans, terminal N-acetylglucosamine type glycans, terminal α2,6 sialic acid type glycans, and terminal α2,3 sialic acid type glycans.

[0058] When a certain glycan pattern has different types and contents of glycan components from the cancer glycan pattern derived from cancer cells, but is a glycan pattern A whose in vivo dynamics mainly depend on terminal high-mannose type glycans, it exhibits type 1 in vivo dynamics. Similarly, when a certain sugar chain pattern has types of sugar chain components and contents of each sugar chain component different from those of the cancer sugar chain pattern derived from cancer cells, and the pharmacokinetics mainly depend on the terminal galactose-type sugar chain or the terminal N-acetylglucosamine-type sugar chain, it shows the pharmacokinetics of type 2. When a certain sugar chain pattern has types of sugar chain components and contents of each sugar chain component different from those of the cancer sugar chain pattern derived from cancer cells, and the pharmacokinetics mainly depend on the terminal α2,6 sialic acid-type sugar chain, it shows the pharmacokinetics of type 3. When a certain sugar chain pattern has types of sugar chain components and contents of each sugar chain component different from those of the cancer sugar chain pattern derived from cancer cells, and the pharmacokinetics mainly depend on the terminal α2,3 sialic acid-type sugar chain, it shows the pharmacokinetics of type 4.

[0059] For example, in the prevention and treatment of the metastasis site from cancer cells in the primary tumor, the sugar chain presenting particles of the present invention can profile the sugar chains of cancer cells, determine which of the cancer sugar chain patterns A to D is based on the obtained profile, and prepare and provide sugar chain presenting particles having the selected sugar chain pattern using a known method.

[0060] The selected sugar chain pattern can be artificially modified, for example, by treating an existing sugar chain containing a specific sugar chain with an enzyme. Examples of the existing sugar chain include the sugar chains contained in egg white and egg yolk of quail, chicken, duck, etc. as shown in Example 2. The sugar chains of the existing material can be used as they are or after appropriate processing 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 enzyme treatment. Alternatively, a material containing a plurality of sugar chains with known compositions can be mixed at an appropriate ratio and used as a material for providing a desired cancer sugar chain pattern (see Example 2).

[0061] The sugar chains of quail, chicken, duck egg white, and chicken egg yolk used in Example 2 are biomaterial-derived materials with clear sugar chain structures and their expression level profiles, which are safe and inexpensive. By presenting these sugar chains as they are or after modification as raw materials on nanoparticles to form sugar chain-presenting particles, the in vivo dynamics similar to those of exosomes derived from cancer cells can be reproduced. The sugar chain pattern can also be a mixture of artificially synthesized sugar chain components in addition to those derived from living organisms. The artificial synthesis of sugar chain components can be carried out by known methods.

[0062] The presentation of sugar chains on the surface of the particles can be carried out by a known direct glycoblotting method (Non-Patent Document 9). The presentation of sugar chains on the surface of the particles can be carried out, for example, by reacting a mixture containing cancer sugar chains with the prepared nanoparticles by the methods shown in Example 1 (C) and Example 2 (B) to prepare sugar chain-presenting particles.

[0063] The sugar chains on the surface of the nanoparticles can be sulfide conjugates of alkanethiols that present sugar chains represented by the following general formula (B).

Chemical formula

[0064] The sugar chain-presenting particles of the present invention can be schematically represented, for example, by the following general formula (10).

Chemical formula

[0065] The residue carried on the nanoparticles (NP) of general formula (10) is, as a sugar chain-containing site, R 10It is a sugar chain-containing group represented by the general formula (B) and a phospholipid group represented by the general formula (A), both of which are supported on the nanoparticle (NP) via a sulfide (-S-) at the terminal of each group. In the actual complex, one or more phospholipid groups represented by the general formula (A) are supported on the nanoparticle NP. Also, R 10 includes a plurality of sugar chain component-containing groups composed of sugar chain components constituting the sugar chain pattern.

[0066] Since the sugar chain-presenting particles of the present invention have in vivo kinetics according to the sugar chains of each pattern, they can be accumulated in specific tissues or organs by utilizing their properties. Therefore, by selecting the pattern of the sugar chain, sugar chain-presenting particles that can be accumulated in specific tissues or organs can be obtained, and a DDS can also be provided by utilizing this property.

[0067] The sugar chain-presenting particles of the present invention can further have a drug site. There is no particular limitation on the drug at the drug site, and for example, it can be an anticancer agent or an anti-inflammatory agent.

[0068] In the sugar chain-presenting particles of the present invention, as the drug site, for example, a drug-containing group represented by the general formula (C) can be supported on the surface of the nanoparticle.

Chemical formula

[0069] By selecting n1 and n2 in the general formula (C), the distance between the drug-containing group and the nanoparticle can be maintained within a desired range. n1 is an integer in the range of 2 to 30, preferably 5 to 16, 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.

[0070] The particles having a drug site with the sugar chain-presenting particles of the present invention can be schematically represented by, for example, the following general formula (11). [Chemical formula]

[0071] The residues carried on the nanoparticles (NP) of the general formula (11) are, from the top, a sugar chain-containing group represented by the general formula (B), a phospholipid group represented by the general formula (A), and a drug-containing group represented by the general formula (C). In the actual complex, the sugar chain-containing group represented by the general formula (B), the phospholipid group represented by the general formula (A), and the drug-containing group represented by the general formula (C) are each one or more and are carried on the nanoparticle NP as a sulfide conjugate.

[0072] When the sugar chain-presenting particles of the present invention have a drug site, cancer preventive agents and therapeutic agents containing these sugar chain-presenting particles as an active ingredient are included. The cancer preventive agents and therapeutic agents of the present invention preferably have a cancer sugar chain pattern in which the sugar chain-presenting particles capture and present as they are the sugar chains derived from cancer cells. However, sugar chain-presenting particles having cancer sugar chain patterns B, C, or D determined based on the profile obtained by profiling this sugar chain may also be used. These sugar chain-presenting particles exhibit in vivo kinetics of types 2 to 4 according to each sugar chain pattern and are useful for the prevention and treatment of specific metastatic cancers.

[0073] In the case of sugar chain-presenting particles having cancer sugar chain pattern B, they are useful for the prevention and treatment of metastatic cancers to the liver and spleen. In the case of sugar chain-presenting particles having cancer sugar chain pattern C, they are useful for the prevention and treatment of metastatic cancers to the axilla and supraclavicular lymph nodes. In the case of sugar chain-presenting particles having cancer sugar chain pattern D, they are useful for the prevention and treatment of metastatic cancers to organs such as the lung, liver, spleen, brain, and kidney.

[0074] (Sugar Chain-Presenting Particle Kit) The present invention encompasses a glycan-presenting particle kit containing two or more types of glycan-presenting particles having different cancer glycan patterns. Each glycan-presenting particle in the kit may be stored in a separate container or may be stored as a mixture in one container. The glycan-presenting particles included in the kit are any of the glycan-presenting particles of the present invention described above. The different cancer glycan patterns in the kit are any two or more of the glycan patterns A to D described above. The selection of the glycan patterns A to D is determined and selected, for example, by profiling the glycans of cancer cells collected from a cancer patient.

[0075] In the glycan-presenting particle kit of the present invention, in a determined glycan pattern, the glycan-presenting particles having that glycan pattern are selected and used. However, depending on the type of cancer, multiple types may also be used in combination. For example, two types, glycan pattern B and C, may be used in combination as glycan-presenting particles that can accumulate in both the liver and spleen (type 2) and the axillary and supraclavicular lymph nodes (type 3).

[0076] The glycan-presenting particle kit of the present invention can be such that the glycan-presenting particles further have a drug site. Examples of the drug include cancer preventive drugs and therapeutic drugs.

[0077] The glycan-presenting particles of the present invention can be formulated by a method known to those skilled in the art using the glycan-presenting particles as an active ingredient. For example, it can be used parenterally in the form of a sterile solution or suspension injection with water or other pharmaceutically acceptable liquids. For example, it can be formulated by appropriately combining a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dosage form required for generally recognized pharmaceutical practice. The amount of the active ingredient in these formulations is such that an appropriate dosage within the indicated range can be obtained.

[0078] A sterile composition for injection can be formulated according to normal pharmaceutical practices using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Appropriate solubilizing agents, 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.

[0079] Examples of oily liquids include sesame oil and soybean oil, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Additionally, buffers, such as phosphate buffer, sodium acetate buffer, soothing agents, such as procaine hydrochloride, stabilizers, such as benzyl alcohol, phenol, and antioxidants, may be formulated. The prepared injection solution is usually filled into appropriate ampoules. Liposomes can also be used to encapsulate the drug for cell delivery.

[0080] The sugar chain-presenting particles and sugar chain-presenting particle kits of the present invention can be administered to cancer patients or patients suspected of having cancer by an appropriate administration route, depending on the purpose of use, for the prevention and treatment of metastatic cancer to organs.

[0081] The administration can be oral or parenteral, preferably parenteral administration, specifically, injection dosage forms, nasal administration dosage forms, pulmonary administration dosage forms, transdermal administration forms, etc. are included. Examples of injection dosage forms can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0082] The dosage and administration method of the sugar chain-presenting particles and the sugar chain-presenting particle kit of the present invention can be appropriately selected according to the age, weight, sex, nature or severity of the symptoms to be treated, etc. of the patient. As the dosage of the pharmaceutical composition containing the sugar chain-presenting particles and the sugar chain-presenting particle kit of the present invention, for example, it can be selected in the range of 0.0001 mg to 1,000 mg per 1 kg of body weight per administration. Alternatively, the dosage can be selected in the range of 0.01 to 100,000 mg / body per patient, but these numerical values are not necessarily limiting. The dosage and administration method vary depending on the age, weight, sex, symptoms, etc. of the patient, but can be appropriately selected by those involved.

[0083] <Method for producing sugar chain-presenting particles> The present invention includes the method for producing the sugar chain-presenting particles of the present invention described above. 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 at least partially coated with a phospholipid on the surface. A method for preparing nanoparticles at least partially coated with a phospholipid on the surface will be described later.

[0084] As the desired cancer glycan pattern, as described above, it may be a cancer glycan pattern in which the glycans of cancer cells collected from a subject are excised and used as they are, or a glycan of an existing material that is adjusted to a specific glycan pattern as it is or with appropriate modification. The specific glycan pattern is a glycan pattern determined based on profiling the glycans of cancer cells collected from a subject. The glycan pattern determined based on the profile may be a glycan pattern in which the types and contents of glycan components are the same as those of the profile, or a glycan pattern in which some of the types and contents of glycan components are the same as those of the profile. When some of the types and contents of glycan components are the same as those of the profile, some of the types of glycan components included in the profile can be reduced or deleted. More specifically, when the glycan components included in the profile are classified into several types, it can be a glycan pattern that includes glycan components belonging to one or more types with a high content and reduces or deletes glycan components belonging to one or more types with a low content. When profiling the glycans of cancer cells, when the glycan components included in the profile are classified into several types, by including the glycan components belonging to one or more types with a high content as they are and reducing or deleting the glycan components belonging to one or more types with a low content, it is possible to obtain a glycan pattern that exhibits the same in vivo dynamics as the cancer glycan pattern derived from cancer cells whose glycans have been profiled. Examples of the types of glycan components include terminal high-mannose type glycans, terminal galactose type glycans, terminal N-acetylglucosamine type glycans, terminal α2,6 sialic acid type glycans, and terminal α2,3 sialic acid type glycans.

[0085] A conceptual diagram of the method for producing the carbohydrate-presenting particles of the present invention (a method for producing an exosome model presenting a cancer cell carbohydrate pattern on its surface and in vivo imaging of its biodistribution and organ tropism) is shown in Fig. 1. (a) shows the basic structure of the carbohydrate-presenting particles: metal nanoparticles such as quantum dots, gold nanoparticles, and gold-coated magnetic nanoparticles are used as cores, and their surfaces are completely coated with an anti-aggregation nanoparticle self-assembled mixed monolayer of an alkanethiol having a phosphorylcholine group at the head and an alkanethiol containing an appropriate amount of an aminooxy linker. (b) shows the basic principle of the glycoblotting method, in which the reducing end of the carbohydrate (a compound containing an aldehyde group or a ketone group) specifically reacts with the aminooxy group on the nanoparticle surface to form an oxime bond. (c) shows a method for presenting a cancer cell-derived carbohydrate pattern on the nanoparticle surface and in vivo imaging. The carbohydrate pattern is captured and presented on the nanoparticle surface by the glycoblotting method that utilizes a chemical reaction specific to the carbohydrate having a reducing end (equivalent to an aldehyde group) from a mixture containing carbohydrates prepared from collected or cultured cancer cells.

[0086] A nanoparticle having at least a part of its surface coated with a phospholipid can be carried out by mixing a crosslinking precursor X represented by the following general formula (D), a phospholipid precursor represented by the following general formula (E), and colloidal nanoparticles to obtain a surface-modified nanoparticle carrying the crosslinking precursor X and the phospholipid on the surface of the nanoparticle. This method is described in Patent Document 1, and the entire description of Patent Document 1 is hereby incorporated by specific reference.

[0087] [Chemical formula] (In general formula (D), n1 is an integer from 2 to 30, and n2 is an integer from 2 to 30.)

[0088] [Chemical formula] (In general formula (E), n3 is an integer in the range from 2 to 30.)

[0089] In general formula (D), n1 is an integer in the range of 2 to 30, preferably 5 to 20, 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. General formula (E) is the same as the phospholipid mimetic group represented by general formula (A) except that the terminal is an SH group. In general formula (E), n3 is an integer in the range of 2 to 30, preferably 5 to 20, more preferably 7 to 15. The crosslinking precursor X represented by general formula (D) and the phospholipid mimetic precursor represented by general formula (E) are both commercially available products and can also be prepared by the methods described in the reference documents. (Reference documents: T. Ohyanagi, et. al., J. Am. Chem. Soc. 2011, 133, 12507-12517)

[0090] The mixing ratio of the crosslinking precursor X represented by general formula (D), the phospholipid mimetic precursor represented by general formula (E), and the nanoparticles can be appropriately determined in consideration of the desired loading amount of the crosslinking precursor X represented by general formula (D) and the phospholipid mimetic precursor represented by general formula (E) on the nanoparticles.

[0091] In the crosslinking precursor X represented by general formula (D) shown in the following scheme, n1 is 6, n2 is 9, and n3 of the phospholipid mimetic precursor represented by general formula (E) is 9. The molar ratio AO / PC of the aminooxy linker (AO) and 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 general formula (B) to the phospholipid mimetic group represented by general formula (A) can be, for example, in the range of a molar ratio of 1:100 to 100:1, preferably in the range of 1:10 to 10:1, and thus can be a ratio according to this.

[0092]

Chemical formula

[0093] The nanoparticles are the same as those described in the glycoprotein-presenting particles. In the above scheme, colloidal quantum dots (QDs) were used as raw materials for the metal nanoparticles. The colloidal quantum dots have a protecting group on the surface of luminescent semiconductor nanoparticles with a diameter in the range of, for example, 1 to 20 nm. The colloidal quantum dots shown in the above scheme have trialkyl phosphate groups on the surface. Even when the nanoparticles are metal nanoparticles, colloidal metal nanoparticles can be used as raw materials. Commercially available products of colloidal quantum dots and colloidal metal nanoparticles are available.

[0094] The desired cancer sugar chain pattern is presented on the surface of the obtained surface-modified nanoparticles. The presentation of the sugar chain can be carried out by a glycoblotting method in which the aminooxy group of the cross-linking precursor X represented by the general formula (D) introduced into the surface-modified nanoparticles obtained above specifically reacts with a sugar chain having a reducing end (equivalent to an aldehyde group).

[0095] The glycoprotein-presenting particles of the present invention having a drug site present or carry a desired cancer sugar chain pattern and a drug-containing site on the surface of the surface-modified nanoparticles. The presentation or loading of the sugar chain and the drug-containing site can be carried out sequentially or simultaneously. For example, after presenting the sugar chain by the glycoblotting method as described above, the loading of the drug-containing site can be carried out. It can be obtained by mixing the drug-containing precursor Z represented by the following general formula (G) with the surface-modified nanoparticles presenting the sugar chain and linking it with the cross-linking precursor X on the surface-modified nanoparticles to further form a drug-containing group represented by the general formula (C).

[0096] [Chemical formula] (In the general formula (G), R 20 is a drug-containing site.)

[0097] The sugar chain-containing site and the drug-containing site in the production method of the present invention are synonymous with 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)).

[0098] (Method for determining the sugar chain pattern of cancer cells) The present invention includes profiling sugar chains on the surface of cancer cells collected from a subject and determining a sugar chain pattern based on the profiled sugar chains. As described above, the profiling of sugar chains on the surface of cancer cells can be performed by a known analysis method.

[0099] The determination of the obtained sugar chain pattern is based on the fact that the profiled sugar chains are (1) Sugar chain pattern A whose in vivo kinetics mainly depend on terminal high-mannose type sugar chains, (2) Sugar chain pattern B whose in vivo kinetics mainly depend on terminal galactose type sugar chains or terminal N-acetylglucosamine type sugar chains, (3) Sugar chain pattern C whose in vivo kinetics mainly depend on terminal α2,6 sialic acid type sugar chains, and (4) It can be performed by specifying which sugar chain pattern is selected from the group consisting of sugar chain pattern D whose in vivo kinetics mainly depend on terminal α2,3 sialic acid type sugar chains.

[0100] More specifically, the above sugar chain patterns are, for example, Cancer sugar chain pattern A: 45 mol% or more of the sugar chains are terminal high-mannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%, Cancer sugar chain pattern B: less than 45% of the sugar chains are terminal high-mannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%, Cancer sugar chain pattern C: 2 to 100% of the sugar chains are terminal sialic acid type sugar chains, and the content of terminal α2,6 sialic acid type sugar chains is more than the content of terminal α2,3 sialic acid type sugar chains, Cancer glycan pattern D is such that 2 to 100% of the glycans are terminal sialic acid type glycans, and the content of terminal α2,3 sialic acid type glycans is more than the content of terminal α2,6 sialic acid type glycans.

[0101] In the determination method of the present invention, when the determined glycan pattern is glycan pattern A, it shows type 1 in vivo kinetics, suggesting that the cancer cells have a low tendency to metastasize. When the determined glycan pattern is glycan pattern B, it shows type 2 in vivo kinetics, suggesting that the cancer cells tend to metastasize to the liver and spleen. When the determined glycan pattern is glycan pattern C, it shows type 3 in vivo kinetics, suggesting that the cancer cells tend to metastasize to the axilla and supraclavicular lymph nodes. When the determined glycan pattern is glycan pattern D, it shows type 4 in vivo kinetics, suggesting that the cancer cells tend to metastasize to the lung, liver, spleen, brain, and kidney.

[0102] Based on the glycan pattern of cancer cells determined by the determination method of the present invention, considering which glycan pattern showing type 1 to 4 in vivo kinetics the glycan pattern of the cancer cells possessed by the subject is, medical personnel can diagnose the metastatic potential of the target cancer cells.

Example

[0103] Hereinafter, the present invention will be described in more detail based on examples. However, the examples are illustrative of the present invention and the present invention is not intended to be limited to the examples.

[0104] Example 1 (1) Profile of the state of post-translational glycan modification (glycan expression pattern and their expression levels) of all proteins present in cancer cells and on the cell membrane surface

[0105] Glycan profile of human cancer cells : Four types of human cultured cancer cells (MCF7, MDA-MB-231, A549, HepG2) were each about 5 x 10 5By using 100 μg (as total protein amount) of each, the total glycan profiles of these cancer cells were analyzed in detail according to the general protocol of the cell glycoblotting method (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537) (Figure 2a). Subsequently, regarding their glycan structures, the relationship between their expression levels and the major structural motifs was clarified by glycotyping analysis using internal standard compounds as indicators (Figure 2b).

[0106] (2) Preparation method of cancer glycan pattern-presenting nanoparticles (glycan-presenting particles) and analysis of in vivo dynamics by real-time imaging after intravenous administration to mice (For the principle and protocol, see Figure 1)

[0107] (2-1) Method for preparing glycans derived from cancer cells : 1 x 10 of each of the human cultured cancer cells MCF7 (ATCC), A549 (JCBR), and HepG2 (RIKEN) are cultured in D-MEM High-glucose medium (Wako) containing 10% fetal bovine serum (FBS) at 37°C under 5% CO2 for 48 hours. Also, for MDA-MB-231 (RIKEN), 1 x 10 6 cells are cultured in Leibovitz’s L-15 medium (Wako) containing 10% fetal bovine serum (FBS) at 37°C for 48 hours to obtain 2 - 3 x 10 6 cells of each cancer cell. 6Use (equivalent to about 500 μg in total protein amount). Detach the cells with 1 mL of ice-cold 0.2 M phosphate buffer (pH 7.4), suspend them in 1 mL of phosphate buffer (pH 7.4) containing 10 mM EDTA, centrifuge at 10,000 g for 10 minutes at 4°C, and remove the supernatant. Add 100 μL of a solution consisting of 0.1% SDS, 1% Triton-X100, and 100 mM ammonium bicarbonate to the residue for solubilization, and quantify the total protein amount by the BCA (bicinchoninic acid) method. React an amount equivalent to 500 μg of protein with 1,4-dithithreiol (DTT, 20 μL, 120 mM / MilliQ) at 60°C for 30 minutes, and then with iodoacetamide (IAA, 40 μL, 123 mM / MilliQ) in the cold dark place at room temperature for 1 hour. Add 400 U of trypsin (Sigma Aldrich) to the protein mixture, treat it at 37°C overnight, and then heat at 90°C for 10 minutes to stop the hydrolysis. Add 2 U of PNGaseF (Roche Applied Science) to this reaction solution, react it at 37°C overnight, evaporate the solvent with a centrifugal concentrator under reduced pressure (SpeedVac) to dryness, and prepare a sample for analysis.

[0108] (2-2) Method for producing nanoparticles for glycan presentation : According to the conventional method (S.-I. Nishimura et al., 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), nanoparticles (PC-SH / AOHEG-SH = 80 / 1) completely coated with a mixed monolayer consisting of two alkane thiol derivatives, 11-mercaptoundecylphosphorylcholine (hereinafter abbreviated as PC-SH) and 11,11'-dithio bis[undec-11-yl 12-(aminooxyacetyl)amino hexa(ethyleneglycol)] (hereinafter abbreviated as AOHEG-SH), were prepared using quantum dots (1 μM TOPO-coated QD800 in decane, Thermo Fischer). Specifically, quantum dots (200 μL, 1 μM TOPO-coated QD800 in decane) were added to a mixed solvent of MeOH (200 μL) and i-PrOH (400 μL), centrifuged at 15,000 g for 5 minutes at room temperature to insolubilize and precipitate TOPO-coated QD800, and the supernatant was removed. n-Hexane (400 μL) was added to the residue to solubilize TOPO-coated QD800, and PC-SH (32 μL, 100 mM / MeOH, Medicinal Chemistry Pharmaceuticals), AOHEG-SH (2 μL, 10 mM / MilliQ, Mediicinal Chemistry Pharmaceuticals), NaBH4 (1 μL, 12 wt% in 14 N NaOH), and Milli Q (200 μL) were added thereto, and the mixture was vigorously stirred with a vortex mixer at room temperature for 30 minutes. After allowing the mixture to stand and removing the organic layer, the nanoparticles in the aqueous layer were separated by ultrafiltration (YM50, Thermo Fischer) and purified by washing three times with Milli Q (500 μL) (quickly used for glycoprotein blotting of cancer cells as 100 μL, 2 μM / MilliQ).At the same time, nanoparticles without sugar chains coated with only PC-SH (PC-SH / AOHEG-SH = 100 / 0) were prepared as a control.

[0109] (2-3) Method for producing cancer glycan pattern presentation nanoparticles, which are glycan presentation particles of the present invention : (A) A mixture containing sugar chains prepared from various cancer cells (20 μL, equivalent to about 500 μg of total protein) and 50 mM acetate buffer (200 μL, pH 4.0) were added to the nanoparticle solution (100 μL, 2 μM / MilliQ: PC-SH / AOHEG-SH = 80 / 1) prepared in step (B) and reacted at 37 °C for 1.5 hours (with occasional gentle stirring). The cancer sugar chain pattern-presenting nanoparticles were separated by ultrafiltration (YM50, Thermo Fischer), washed with Milli Q (500 μL), and dissolved in physiological saline (200 μL, 0.9% NaCl aqueous solution) for animal experiments. The average particle size of the obtained cancer sugar chain pattern-presenting nanoparticles was 15.1 - 28.0 nm as measured by a fiber optic dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Electronics).

[0110] (2-4) In vivo dynamics and organ tropism of cancer glycan pattern presentation nanoparticles (glycan presentation particles) : Cancer sugar chain pattern-presenting nanoparticles (100 μL, 1 μM / 0.9% NaCl aqueous solution) were intravenously administered to mice (male, BALB / c) aged 5 weeks or older. The in vivo dynamics of the nanoparticles up to 3 hours after administration were observed in real time by 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 intravenous administration, the mice were dissected, and the main organs were removed to observe the fluorescence intensity of each. Hereinafter, in the examples, the in vivo dynamics of the cancer sugar chain pattern-presenting nanoparticles (sugar chain-presenting particles) are described as the movement and excretion states of the particles in the body after administration, and the distribution and accumulation states of the particles in each organ and tissue are described as organ tropism in particular.

[0111] For each of the nanoparticles (GNS-MCF7, GNS-MDA-MB-231, GNS-A549, GNS-HepG2) presenting the glycans of four types of human cultured cancer cells (MCF7, MDA-MB-231, A549, HepG2), the in vivo kinetics up to 3 hours after intravenous administration to control mice and the distribution states in each organ at the time of dissection 3 hours later are shown in Fig. 3.

[0112] (i) Control (nanoparticles without glycans) From these experiments, it was reconfirmed that the control coated with a monolayer using only PC-SH did not show specific organ tropism even 3 hours after intravenous administration (relative tropism to each organ was 0.8 - 2.5), and was distributed almost uniformly to organs other than the spleen (whole body) (S.-I. Nishimura et al., J. Am. Chem. Soc. 2011, 133, 12507 - 12517).

[0113] (ii) GNS-MCF7 As shown in Figs. 2a and 2b, when presenting the glycan pattern of MCF7, a non-metastatic breast cancer cell, as the glycan component, about 94% of the total glycans are seven types of high-mannose type glycans and about 6% are glycans with two types of glycans having galactose terminals, most of them were rapidly excreted outside the body within about 15 minutes without accumulating in specific organs after intravenous administration (Fig. 3a). Almost no accumulation in organs was observed even in the mice dissected 3 hours later, and a part of them was mixed in the feces in the gastrointestinal tract (Fig. 3b). That is, it was clarified that the glycan-presenting particles presenting the high-mannose type glycan of glycan pattern A (oligosaccharide with only mannose at the terminal) have an excretion mechanism to the outside of the body and show type 1 in vivo kinetics.

[0114] (iii) GNS- MDA-MB-231 As a sugar chain component, it has a sugar chain pattern (Figures 2a and 2b) containing 18% of five types of terminal sialic acid-type sugar chains with two or three strands and 82% of five types of high-mannose-type sugar chains. When presenting the sugar chain pattern of MDA-MB-231, a metastatic breast cancer cell, it was completely different from the in vivo behavior of GNS-MCF7. At 3 hours after intravenous administration, accumulation in the axillary and supraclavicular lymph nodes was particularly prominent (indicated by arrows), showing type 3 in vivo behavior. Also, until about 1 to 2 hours after administration, the distribution of nanoparticles could be confirmed in the lungs, heart, liver, spleen, kidneys, etc., but most of them were excreted outside 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, at the terminal determine the metastatic and organotropism of breast cancer cells.

[0115] (iv) GNS-A549 As a sugar chain component, it has a sugar chain pattern (Figures 2a and 2b) containing 19% of five types of terminal sialic acid-type sugar chains with two or three strands, 11% of sugar chains with a galactose terminus, 8% of sugar chains with N-acetylglucosamine at the terminal, and further 62% of seven types of high-mannose-type sugar chains. When presenting the sugar chain pattern of A549, it was widely distributed throughout the body even at 3 hours after intravenous administration. In particular, accumulation in the lungs, liver, and spleen was very prominent, and distribution could also be confirmed in the brain and kidneys, showing type 4 in vivo behavior.

[0116] (v) GNS-HepG2 In the case of nanoparticles presenting the glycan pattern of HepG2, which has a glycan pattern containing 6% of two types of terminal sialic acid-type glycans, either double-stranded or triple-stranded, 9% of glycans with a galactose terminus, 13% of glycans with N-acetylglucosamine at the terminus, and further 72% of seven types of high-mannose-type glycans (Figs. 2a and 2b), it showed a type 4 in vivo behavior very similar to GNS-A549. However, it was also revealed that GNS-A549, in which the proportion of Neu5Acα2,6Gal or Neu5Acα2,3Gal units in the total glycans is 19%, has clearly higher accumulation amounts in the lung, liver, and spleen than 6% GNS-HepG2. However, since the organ tropism (accumulation property) 3 hours after administration of these nanoparticles presenting two types of cancer cell-derived glycans is significantly different between the case of GNS-HepG2 and the case of GNS-MDA-MB-231, it was considered that these differences are due to the binding mode of terminal sialic acid and galactose (the difference between α2,6 linkage and α2,3 linkage) affecting the organ tropism of the nanoparticles after intravenous administration.

[0117]

Table 5

[0118] Example 2 (1) Glycan presentation particles having artificially prepared glycan patterns with different binding modes (α2,6 bond and α2,3 bond) of terminal sialic acid and galactose MDA-MB-231 cells, A549 cells, and HepG2 cells are all human cancer cells that significantly express two types of terminal sialic acid-type glycans, either double-stranded or triple-stranded, as glycan components. However, in the results of Example 1, the in vivo behavior and organ tropism were different for MDA-MB-231 cells, A549 cells, and HepG2 cells. From this, it was predicted that the binding mode of sialic acid and galactose (the difference between α2,6 linkage and α2,3 linkage) determines the difference in the in vivo behavior of these glycan pattern-presenting nanoparticles after intravenous administration. Therefore, in this example, nanoparticles presenting only one of the individual terminal sialic acid-type glycans artificially linked by α2,6 linkage and α2,3 linkage were prepared, and their respective in vivo behavior and organ tropism were examined to confirm the difference in in vivo behavior due to the difference in terminal sialic acid-type glycans.

[0119] Since the main sugar chain in the egg white of the ruddy duck has a multi-branched sugar chain structure with galactose at the non-reducing end, sialic acid was added to the terminal galactose and modified by two types of sialyltransferases with known substrate specificities, α2,3-(N)-sialyltransferase derived from Pasteurella multocida and human α2,6-(N)-sialyltransferase, to induce a sugar chain pattern containing only either Neu5Acα2,3Gal or Neu5Acα2,6Gal (Figure 4a).

[0120] (1-1) Method for modifying the sialic acid transferase of the ovalbumin glycan profile of Ruddy duck : To Ruddy duck egg white (17 mg / 50 μL in Milli Q, equivalent to approximately 1 mM LacNAc unit), add 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 further add milliQ (10 μL) to make the total volume 100 μL, and react at 37 °C for 20 hours. Similarly, to Ruddy duck egg white (17 mg / 50 μL in Milli Q, equivalent to approximately 1 mM LacNAc unit), add 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, pH 6.5), and further add milliQ (10 μL) to make the total volume 100 μL, and react at 37 °C for 20 hours.

[0121] (1-2) Glycan profiles of Button Quail, Chicken, Ruddy duck ovalbumin and sialic acid-modified Ruddy duck ovalbumin : According to the above step (A), 500 μg equivalent of the total protein of five types of egg white and sialic acid-modified egg white is reacted with 1,4-dithithreiol (DTT, 20 μL, 120 mM / MilliQ) at 60 °C for 30 minutes, and then with iodoacetamide (IAA, 40 μL, 123 mM / MilliQ) in the cold dark place at room temperature for 1 hour. 400 U of trypsin (Sigma Aldrich) is added to the protein mixture, and after treating at 37 °C overnight, hydrolysis is stopped by heating at 90 °C for 10 minutes. 2 U of PNGaseF (Roche Applied Science) is added to this reaction solution, reacted at 37 °C overnight, and the solvent is distilled off using a centrifugal concentrator under reduced pressure (SpeedVac) to dryness to prepare a sample for sugar chain profiling (Figs. 4b and 4c).

[0122] 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 as sugar chain components, 40% are sugar chains with N-acetylglucosamine at the end, and only a small amount of galactose at the end is observed, and no sialic acid is present at all. Regarding Chicken egg white, as sugar chain components, sialic acid was not detected, about 40% were multi-branched sugar chains with 3 to 5 branches having N-acetylglucosamine at the end, 30% had galactose at the end, and the rest were high-mannose type sugar chains. In the egg white of Ruddy duck, as sugar chain components, no sialic acid was observed at all, and multi-branched sugar chains with 3 to 5 branches having galactose residues at the non-reducing end accounted for about 65% of the whole, and the rest were confirmed to have about 25% high-mannose type sugar chains and about 10% sugar chains with N-acetylglucosamine at the end. Also, it was clarified that no fucose residues were present in the sugar chains of these three types of avian egg white.

[0123] According to the scheme of Figure 4a, the ovalbumin of Ruddy duck was treated with α2,3-(N)-sialyltransferase (recombinant Pasteurella multocida) and α2,6-(N)-sialyltransferase (recombinant human) in the presence of CMP-sialic acid, and 2,3S-Ruddy duck and 2,6S-Ruddy duck with multiple sialic acid additions were induced.

[0124] Furthermore, as a result of the sugar chain profile by the glycotyping method (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537), 2,3S-Ruddy duck and 2,6S-Ruddy duck were also very similar to the sugar chain profiles of human cancer cells A549 and HepG2 (Figures 2b and 4c).

[0125] (2) Organ tropism of glycan presentation particles presenting artificially prepared glycan patterns Ovalbumin sugar chains of Button Quail, Chicken, and Ruddy duck prepared by the method of Example 2(1), two types of sugar chain samples with modified sugar chain patterns of Ruddy duck ovalbumin sugar chains (both prepared from an equivalent amount of 500 μg in terms of total protein), and further glycopeptides of chicken egg yolk (500 μg, 17 nmol; sialylglycopeptide, SGP, Tokyo Chemical Industry, with α2,6-linked sialic acid and galactose bound at the sugar chain site) were PNGase-treated according to the steps of Example 1(2-1) to prepare a crude product. 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 steps of Example 1(2-3) and then purified to produce sugar chain pattern-presenting nanoparticles (GNSs). The average particle diameter of the obtained sugar chain pattern-presenting nanoparticles was 14.4 - 24.4 nm as a result of measurement with a fiber optic dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Electronics). Nanoparticles presenting these sugar chains were intravenously administered to mice by the method described in Example 1(2-4), and their in vivo kinetics and organ tropism were observed with a near-infrared fluorescence microscope (exposure time 1 sec, excitation wavelength 710 nm, detection wavelength 820 nm) (Figs. 5a - 5c).

[0126] As shown in Fig. 5, it can be seen that the sugar chain structure and its profile are clear, and by presenting the sugar chain patterns obtained by modifying the sugar chains of quail, chicken, and duck egg whites and chicken egg yolk, which are safe and inexpensive biomaterials, on nanoparticles, the sugar chain-presenting particles of the present invention can be manufactured.

[0127] Specifically, (i) The retention of GNS-Button Quail prepared from Button Quail egg white, in which high mannose-type sugar chains account for about 50% of the whole, is slightly prolonged in the mouse body (Fig. 5b), but the in vivo kinetics 3 hours after administration (Fig. 5c) is very similar to that of GNS-MCF7 (Fig. 3c), and most of it is excreted outside the body (type 1 in vivo kinetics).

[0128] (ii) GNS-Chicken and GNS-Ruddy duck, which presented the sugar chain patterns of Chicken and Ruddy duck, showed extremely high accumulation in the liver and spleen (Figs. 5b and 5c) (type 2 biodistribution). This is thought to largely depend on the specific interaction between the highly branched major sugar chains with galactose or N-acetylglucosamine without sialic acid at the termini of GNS-Chicken and GNS-Ruddy duck and asialoglycoprotein receptors typified by mammalian hepatic lectin highly expressed in these organs and cells (e.g., Y. C. Lee et al., Acc. Chem. Res. 1997, 28, 321-327, etc.).

[0129] (iii) From the analysis of the biodistribution and organ tropism by two kinds of nanoparticles (GNS-2,3S-Ruddy duck and GNS-2,6S-Ruddy duck) presenting the sugar chain pattern derived from Ruddy duck egg white and GNS-2,6S-A2 prepared from chicken yolk glycopeptide, it was revealed that the major terminal sialic acid-type sugar chains of MDA-MB-231 cells are double-stranded or triple-stranded complex-type sugar chains containing Neu5Acα2,6Gal units at the termini (Figs. 5b and 5c). Furthermore, from the differences in the biodistribution and organ tropism of GNS-MCF7 (Figs. 3b and 3c) and GNS-Button Quail (Figs. 5b and 5c), it became clear that the ratio (distribution density) of the sugar chains containing Neu5Acα2,6Gal units at the termini (or high mannose-type sugar chains) to the total sugar chains on the exosome membrane surface determines the metastatic ability and organ tropism of breast cancer cells (as shown in Fig. 5b, accumulation in the axillary and supraclavicular lymph nodes can be confirmed in GNS-2,6S-Ruddy duck and GNS-2,6S-A2) (type 3 biodistribution).

[0130] On the one hand, (iv) since the in vivo dynamics and organ tropism of GNS-2,3S-Ruddy duck (Figs. 5b and 5c) are very similar to those of GNS-A549 and GNS-HepG2 (type 4 in vivo dynamics), the main terminal sialic acid-type sugar chain structures of A549 cells and HepG2 cells are double-stranded or triple-stranded complex sugar chains containing the Neu5Acα2,3Gal unit at the end. Furthermore, from the difference in organ tropism between these two cancer cell-derived exosome models (Fig. 3c), it was suggested that the content rate of double-stranded or triple-stranded complex sugar chains containing the Neu5Acα2,3Gal unit at the end strongly affects not only the organ tropism but also the retention (blood concentration and half-life) of nanoparticles after intravenous administration.

[0131]

Table 6

[0132] Example 3 In Example 2, it was revealed that the ratio of the terminal sialic acid-type sugar chain component and the high-mannose-type sugar chain component to the total sugar chain, whose binding position to galactose was controlled by enzymatic modification of the egg white sugar chain of Ruddy duck, had a great influence on the in vivo dynamics and organ tropism. Therefore, a sugar chain with a higher content rate of high-mannose-type sugar chain (about 70%) than that of Button Quail egg white (50%) and a simpler sugar chain pattern, derived from Japanese Quail egg white (S.-I. Nishimura et al., J. Agri. Food Chem. 2018, in press), and the 2,6S-A2 sugar chain derived from chicken egg yolk glycopeptide were mixed at an arbitrary ratio and presented on nanoparticles to prepare the sugar chain-presenting particles of the present invention. The average particle diameter of the obtained cancer sugar chain pattern-presenting nanoparticles was 27.3 nm as a result of measurement with a fiber optic dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Electronics Co., Ltd.).

[0133] (3-1) To three types of sugar chain samples prepared by adding 0.6 nmol, 1.2 nmol, and 2.4 nmol of 2,6S-A2 sugar chain sample (17 nmol; prepared from 500 μg of SGP) to a sugar chain sample derived from Japanese Quail egg white (about 6 nmol; equivalent to 100 μg in terms of total protein), respectively. The results of analyzing the profiles of these sugar chains by the glycoblotting method (the % in the figure indicates the ratio of the 2,6S-A2 sugar chain to the whole calculated from each peak area) are shown in Fig. 6a and Table 7. These sugar chain patterns were presented on nanoparticles (100 μL, 1 μM / MilliQ: PC-SH / AOHEG-SH = 40 / 1) according to the steps of Example 1 (2-3) to prepare a total of five types of sugar chain pattern-presenting nanoparticles (GNSs). Nanoparticles presenting sugar chain patterns artificially prepared by mixing two types of sugar chains (patterns) were prepared.

[0134]

Table 7

Industrial Applicability

[0135] The present invention can construct sugar chain-presenting particles derived from cancer cells, and these particles are useful for cancer prevention and treatment.

Claims

1. A sugar chain-presenting particle which is a nanoparticle having a sugar chain on its surface, wherein (1) the average particle diameter of the sugar chain-presenting particle is in the range of 10 to 100 nm, (2) at least a part of the surface of the nanoparticle is coated with a phospholipid, (3) the sugar chain on the nanoparticle surface is a sugar chain pattern determined based on a sugar chain pattern derived from cancer cells or a profile of this sugar chain (hereinafter referred to as a cancer sugar chain pattern), and the cancer sugar chain pattern is (1) a cancer sugar chain pattern A in which 50% to 95% of the sugar chain is a terminal high-mannose type sugar chain and the terminal sialic acid type sugar chain is 0% or more and less than 2%, (2) a cancer sugar chain pattern B in which the terminal high-mannose type sugar chain of the sugar chain is less than 45%, the terminal sialic acid type sugar chain is 0% or more and less than 2%, and further includes a terminal galactose type sugar chain and a terminal N-acetylglucosamine type sugar chain, (3) a cancer sugar chain pattern C in which 5 to 60% of the sugar chain is a terminal sialic acid type sugar chain and the content of the terminal α2,6-sialic acid type sugar chain is more than the content of the terminal α2,3-sialic acid type sugar chain, (4) a sugar chain pattern selected from the group consisting of a cancer sugar chain pattern D in which 5 to 60% of the sugar chain is a terminal sialic acid type sugar chain and the content of the terminal α2,3-sialic acid type sugar chain is more than the content of the terminal α2,6-sialic acid type sugar chain. The sugar chain-presenting particle.

2. The cancer sugar chain pattern A is a sugar chain pattern whose in vivo behavior mainly depends on the terminal high-mannose type sugar chain, the cancer sugar chain pattern B is a sugar chain pattern whose in vivo behavior mainly depends on the terminal galactose type sugar chain or the terminal N-acetylglucosamine type sugar chain, the cancer sugar chain pattern C is a sugar chain pattern whose in vivo behavior mainly depends on the terminal α2,6-sialic acid type sugar chain, and the cancer sugar chain pattern D is a sugar chain pattern whose in vivo behavior mainly depends on the terminal α2,3-sialic acid type sugar chain. The sugar chain-presenting particle according to claim 1.

3. The phospholipid coating at least a part of the nanoparticle surface is a sulfide conjugate of an alkanethiol having a phosphorylcholine group, the sugar chain on the nanoparticle surface is a sulfide conjugate of an alkanethiol immobilized with a sugar chain, and the nanoparticle surface is coated with a monolayer of a sulfide conjugate of an alkanethiol having a phosphorylcholine group and a sulfide conjugate of an alkanethiol immobilized with a sugar chain. The sugar chain-presenting particle according to any one of claims 1 to 2.

4. The sulfide conjugate of the alkanethiol having a phosphorylcholine group is represented by the following general formula (A): 【Chemical 1】 (In the general formula (A), n3 is an integer in the range of 2 to 30, and the -S- terminal is a supporting site that forms a sulfide bond with the nanoparticle.) The sugar chain-presenting particle according to claim 3, wherein the sulfide conjugate of the alkanethiol immobilized with the sugar chain is represented by the following general formula (B). [Chemical Formula 2] (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 supporting site that sulfide-bonds to the nanoparticle, and R 10 is a sugar chain-containing site.)

5. The sugar chain-presenting particle according to any one of claims 1 to 4, further having a drug site.

6. The sugar chain-presenting particle according to claim 3 or 4, wherein the monolayer further contains a sulfide conjugate of an alkanethiol having a drug site, which is represented by the following general formula (C). [Chemical Formula 3] In the general formula (C), n1 is an integer from 2 to 30, n2 is an integer from 2 to 30, the -S- end is a supporting site that sulfide-bonds to the nanoparticle, and R 20 is a drug-containing site.

7. A sugar chain-presenting particle kit containing two or more types of sugar chain-presenting particles having different cancer sugar chain patterns, wherein the sugar chain-presenting particles are the sugar chain-presenting particles according to any one of claims 1 to 6.

8. The sugar chain-presenting particle kit according to claim 7, wherein the different cancer sugar chain patterns are any two or more sugar chain patterns among the sugar chain patterns A to D according to claim 1.

9. The sugar chain-presenting particle kit according to claim 7, wherein the sugar chain-presenting particle is a sugar chain-presenting particle further having the drug site according to claim 5 or 6.

10. A cancer metastasis preventive drug containing the sugar chain-presenting particle according to claim 5 or 6 as an active ingredient.

11. A cancer therapeutic drug containing the sugar chain-presenting particle according to claim 5 or 6 as an active ingredient.

12. Comprising profiling the sugar chains of cancer cells collected from a subject and determining a sugar chain pattern based on the profiled sugar chains, The determination of the sugar chain pattern is such that the profiled sugar chains are (1) Cancer sugar chain pattern A, in which 50% to 95% of the sugar chains are terminal high-mannose type sugar chains, and the terminal sialic acid type sugar chains are 0% or more and less than 2%. (2) Cancer sugar chain pattern B, in which the terminal high-mannose type sugar chains of the sugar chains are less than 45%, and the terminal sialic acid type sugar chains are 0% or more and less than 2%, and further contain terminal galactose type sugar chains and terminal N-acetylglucosamine type sugar chains. (3) Cancer sugar chain pattern C, in which 5 to 60% 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 more than the content of the terminal α2,3 sialic acid type sugar chains. Determining whether it is a sugar chain pattern selected from the group consisting of cancer sugar chain pattern D, wherein 5 to 60% of the sugar chain is a terminal sialic acid type sugar chain and the content of the terminal α2,3 sialic acid type sugar chain is greater than the content of the terminal α2,6 sialic acid type sugar chain. Method for determining the sugar chain pattern of cancer cells.

13. Sugar chain pattern A is a sugar chain pattern whose in vivo behavior mainly depends on the terminal high mannose type sugar chain. Sugar chain pattern B is a sugar chain pattern whose in vivo behavior mainly depends on the terminal galactose type sugar chain or the terminal N-acetylglucosamine type sugar chain. Sugar chain pattern C is a sugar chain pattern whose in vivo behavior mainly depends on the terminal α2,6 sialic acid type sugar chain. The determination method according to claim 12, wherein sugar chain pattern D is a sugar chain pattern whose in vivo behavior mainly depends on the terminal α2,3 sialic acid type sugar chain.

14. When the sugar chain profile of cancer cells is sugar chain pattern A, the cancer cells possessed by the subject show type 1 in vivo behavior in which the excretion of the particles to the outside of the body is promoted compared to the sugar chain-presenting particles having no sugar chains, suggesting that the cancer cells have a low tendency to metastasize. When the sugar chain profile is sugar chain pattern B, the cancer cells possessed by the subject show type 2 in vivo behavior in which they accumulate in the liver and spleen compared to the sugar chain-presenting particles having no sugar chains, suggesting that the cancer cells have a tendency to metastasize to the liver and spleen. When the sugar chain profile is sugar chain pattern C, the cancer cells possessed by the subject show type 3 in vivo behavior in which they accumulate in the axilla and supraclavicular lymph nodes, suggesting that the cancer cells have a tendency to metastasize to the axilla and supraclavicular lymph nodes. The determination method according to claim 12 or 13, wherein when the sugar chain profile is sugar chain pattern D, the cancer cells possessed by the subject show type 4 in vivo behavior in which they are distributed to organs such as the lung, liver, spleen, brain, and kidney compared to the sugar chain-presenting particles having no sugar chains, suggesting that the cancer cells have a tendency to metastasize to the lung, liver, spleen, brain, and kidney.

15. The method for producing a sugar chain-presenting particle according to any one of claims 1 to 6, comprising presenting a cancer sugar chain pattern on the surface of a nanoparticle whose at least a part of the surface is coated with a phospholipid.

16. The cancer sugar chain pattern to be presented is a cancer sugar chain pattern obtained by excising the sugar chains of cancer cells collected from a subject, or a sugar chain pattern determined based on the profile obtained by profiling the sugar chains of cancer cells collected from a subject. The production method according to claim 15.

17. The method for producing according to claim 15 or 16, wherein the nanoparticles having at least a part of the surface coated with a phospholipid can be carried out by mixing a crosslinking precursor X represented by the following general formula (D), a phospholipid precursor represented by the following general formula (E), and colloidal nanoparticles to obtain surface-modified nanoparticles having the crosslinking precursor X and the phospholipid supported on the surface of the nanoparticles. 【Chemical Formula 4】 (In the general formula (D), n1 is an integer of 2 to 30, and n2 is an integer of 2 to 30.) 【Chemical Formula 5】 (In the general formula (E), n3 is an integer in the range of 2 to 30.)

18. The presentation of the cancer glycan pattern is carried out by reacting the aminooxy group of the crosslinking precursor X represented by the general formula (D) introduced into the surface-modified nanoparticles with the reduced end of the glycan contained in the cancer glycan pattern obtained by cleaving the glycan of cancer cells collected from a subject, or the glycan pattern determined based on the profile obtained by profiling the glycan of cancer cells collected from a subject, by the glycoblotting method. The manufacturing method according to claim 17.

19. The glycan pattern determined based on the profile is a glycan pattern in which the types and contents of the glycan components are the same as those of the profile, or a glycan pattern in which some of the types and contents of the glycan components are the same as those of the profile. The manufacturing method according to any one of claims 16 to 18.

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