JP7703811B2 - Glycan-presenting particles and method for producing same - Google Patents

Description

The present invention relates to a glycan-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 glycan-presenting particle that presents a cancer glycan pattern on the particle surface, and a method for producing the same.
CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Japanese Patent Application No. 2019-171135, filed on September 20, 2019, the entire disclosure of which is expressly incorporated herein by reference.

In order to enhance the therapeutic effect of a drug, it is important to have a technology (drug delivery system, hereafter abbreviated as DDS) that selectively delivers the drug to a specific organ or tissue where the target molecule that causes the disease or is an aggravating factor of the pathology is highly expressed. Currently, anticancer nanomedicines using PEG-modified liposomes have been approved and marketed. The efficacy of these nanomedicines has been said to depend essentially on the enhanced permeability and retention (EPR) effect, which is prominent in cancer tissues, but it has become clear that the EPR effect is greatly influenced by many factors, including the type, site, and progression of cancer, as well as various adhesion molecules that constitute the cancer cells and the surrounding interstitium (extracellular matrix) (Non-Patent Documents 1, 2, etc.). Research and development of DDS using various artificial extracellular particles coated with polyethylene glycol (PEG)-modified compounds and inorganic/metal nanoparticles (cored with gold nanoparticles, quantum dots, silica-based particles, etc.) that are favorable for the EPR effect and allow for more advanced molecular design has been actively promoted.

However, recent research results have shown that most nanomedicines, including liposomes and metal nanoparticles, do not actually achieve the expected pharmacokinetics (e.g., uptake in cancer tissue) in the living body of experimental animals to which they are administered (Non-Patent Document 3). Surprisingly, a survey of 232 papers published in the past 10 years showed that only about 0.7% (average) of the administered dose of nanomedicine reached the targeted cancer tissue. Most nanoparticle-type drugs administered intravascularly are said to rapidly accumulate in the liver and are taken up by phagocytes such as macrophages, where they are decomposed and excreted, but this is said to be largely dependent on the properties of the "protein corona" on the surface of the nanoparticles, which is formed by 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 even organ tropism (metastasis destination) of individual cancer cells are determined by exosomes (extracellular nanoparticles) derived from cancer cells, research on cancer cell-derived exosomes has been actively conducted, and there are also cases aiming at the development of new pharmaceuticals that utilize the functions of exosomes and the various microRNAs contained therein (e.g., Non-Patent Document 5).

Exosomes are membrane vesicles formed intracellularly and released to the outside of the cell, and function as a tool for intercellular information transmission. However, due to the heterogeneity of exosome size and the molecules they carry, as well as the difficulty of purifying exosomes derived from cancer cells and tissues, the specific molecular mechanism of organ tropism remains largely unknown.

Patent Document 1: WO2017/131242A1/US2020138973(A1)

Non-patent document 1: Prabhakar et al., Cancer Res. 2013, 73, 2412-2417
Non-patent document 2: Danhier, J. Control. Release 2016, 244, 108-121
Non-patent document 3: WCW Chan et al., Nat. Mater. 2016, 1, 1-12
Non-patent document 4: KA Dawson et al., Nat. Biotech. 2012, 7, 779-786
Non-patent document 5: R. Kalluri, J. Clin. Invest. 2016, 126, 1208-1215
Non-patent document 6: S.-I. Nishimura et al., J. Am. Chem. Soc. 2011, 133, 12507-12517
Non-patent document 7: S.-I. Nishimura et al., ACS Chem. Biol. 2015, 10, 2073-2086;
Non-patent document 8: M. Colombo, et al., Annu. Rev. Cell Dev. Biol. 2014, 30, 255-289
Non-patent document 9: S.-I. Nishimura et al., Angew. Chem. Int. Engl. Ed. 2005, 44, 91-96
The entire disclosures of Patent Document 1 and Non-Patent Documents 1 to 9 are expressly incorporated herein by reference.

Exosomes released from many cells are present in the blood, but for example, in cancer patients, it is difficult to purify only exosomes derived from cancer cells and tissues, so the specific organ tropism mechanism at the molecular level remains largely unknown.

The objective of the present invention is to artificially construct particles that mimic exosomes, which are a tool for intercellular information transmission, and to provide a new technology that utilizes the in vivo dynamics of exosomes released from the sender of information.

More specifically, the present invention aims to provide a method for predicting the dynamics and organ tropism after in vivo administration of glycan-presenting particles carrying various molecules that are extremely useful as models of cancer cell-derived exosomes and are the subject of metastasis function analysis, and to provide technology for realizing the development of new nanoparticle DDS for preventing and treating cancer metastasis that will enable clinical use in humans. Another aim of the present invention is to provide a method for determining glycan patterns that can suggest the metastatic potential of cancer cells.

The present invention focuses on the fact that the surface of exosomes presents glycans in a pattern similar to that of the glycans of the cells that release them. That is, for example, in the case of cancer cell metastasis, exosomes released from cancer cells are thought to play a leading role in guiding the in vivo dynamics of cancer cells. Various cells and organs that take up exosomes are likely to first encounter the bulky glycocalyx on the exosome membrane surface. In the present invention, focusing on this fact, it was found that the in vivo dynamics of cancer cell-derived exosomes, particularly their movement and excretion within the body, and their distribution or accumulation in organs and tissues (organ tropism), are led and determined by the glycan pattern on the exosome membrane surface, and the present invention was completed.

Specifically, human cancer cell-derived exosome-mimicking microparticles could be produced by presenting all N-glycans of cultured human cancer cells on the nanosome membrane surface using the glycoblotting method, and these were directly administered intravenously to mice to observe their pharmacokinetics in real time, revealing that the pharmacokinetics and organ tropism of human cancer cell-derived exosomes could be easily determined. Furthermore, based on the results, artificially created particles mimicking exosomes that presented cancer-specific glycan patterns reproduced the intended pharmacokinetics after administration, demonstrating that this technology enables drug discovery based on the new concept of molecular design of a tailor-made DDS that is adapted to the organ tropism of individual cancer metastases, thus completing the present invention.

The present invention is as follows.
[1]
A glycan-presenting particle which is a nanoparticle having a glycan on the surface thereof,
(1) the average particle size of the glycan-presenting particle is in the range of 10 to 100 nm;
(2) At least a portion of the surface of the nanoparticle is covered with a phospholipid;
(3) The glycans on the nanoparticle surface have a glycan pattern specific to cancer cells or a glycan pattern determined based on the glycan profile (hereinafter referred to as a cancer glycan pattern).
Glycan-displaying particles.
[2]
Cancer glycan patterns are
(1) Glycosylation pattern A, whose pharmacokinetics are mainly dependent on terminal high mannose type glycans;
(2) Glycosylation pattern B, whose pharmacokinetics are mainly dependent on terminal galactose-type or terminal N-acetylglucosamine-type glycans;
(3) Glycosylation pattern C, whose pharmacokinetics are mainly dependent on terminal α2,6 sialic acid type glycans;
(4) The glycan-presenting particle according to [1], which has a glycan pattern selected from the group consisting of glycan pattern D, the pharmacokinetics of which mainly depends on terminal α2,3 sialic acid type glycans.
[3]
Cancer glycan pattern A is characterized in that 45 mol % or more of the glycans are terminal high mannose type glycans, and 0% or more and less than 2% are terminal sialic acid type glycans;
Cancer glycan pattern B is characterized in that terminal high mannose type glycans are less than 45% and terminal sialic acid type glycans are 0% or more and less than 2%;
Cancer glycan pattern C is characterized in that 2-100% 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;
The glycan-presenting particle according to [2], wherein cancer glycan pattern D is a glycan in which 2-100% of the glycans are terminal sialic acid glycans and the content of α2,3 sialic acid glycans is higher than the content of α2,6 sialic acid glycans.
[4]
The glycan-presenting particle according to any one of [1] to [3], wherein the phospholipid covering at least a part of the nanoparticle surface is a sulfide bond of an alkanethiol having a phosphorylcholine group, the glycan on the nanoparticle surface is a sulfide bond of an alkanethiol having a glycan immobilized thereon, and the nanoparticle surface is a nanoparticle covered with a monomolecular film of the sulfide bond of an alkanethiol having a phosphorylcholine group and the sulfide bond of an alkanethiol having a glycan immobilized thereon.
[5]
The sulfide bond of an alkanethiol having a phosphorylcholine group is represented by the following general formula (A):
(In general formula (A), n3 is an integer ranging from 2 to 30, and the -S- end is a supporting site that is sulfide-bonded to the nanoparticle.)
The sugar chain-presenting particle according to [4], wherein the sulfide bond of alkanethiol to which the sugar chain is immobilized is represented by the following general formula (B):
(In general formula (B), n1 is an integer from 2 to 30, n2 is an integer from 2 to 30, the -S- end is a supporting moiety that is sulfide-bonded to the nanoparticle, and R ¹⁰ is a sugar chain-containing moiety.)
[6]
The sugar chain-presenting particle according to any one of [1] to [5], further comprising a drug moiety.
[7]
The sugar chain-presenting particle according to [4] or [5], wherein the monolayer further contains a sulfide bond of an alkanethiol having a drug moiety, represented by the following general formula (C):
In general formula (C), n1 is an integer from 2 to 30, n2 is an integer from 2 to 30, the -S- terminus is a loading moiety that is sulfide-bonded to the nanoparticle, and R ²⁰ is a drug-containing moiety.
[8]
A glycan-presenting particle kit comprising two or more types of glycan-presenting particles having different cancer glycan patterns, the glycan-presenting particles being the glycan-presenting particles described in any one of [1] to [7].
[9]
The glycan-presenting particle kit according to [8], wherein the different cancer glycan patterns are any two or more of the glycan patterns A to D according to [2].
[10]
The glycan-presenting particle kit according to [8], wherein the glycan-presenting particle is a glycan-presenting particle further having the drug moiety according to [6] or [7].
[11]
A drug for preventing cancer metastasis, comprising the glycan-presenting particle according to [6] or [7] as an active ingredient.
[12]
A cancer therapeutic agent comprising the glycan-presenting particle according to [6] or [7] as an active ingredient.
[13]
A method for determining a glycan pattern of cancer cells, comprising profiling glycans of cancer cells collected from a subject, and determining the glycan pattern based on the profiled glycans.
[14]
The determination of the glycan pattern is carried out by determining whether the profiled glycans are
(1) Glycosylation pattern A, whose pharmacokinetics are mainly dependent on terminal high mannose type glycans;
(2) Glycosylation pattern B, whose pharmacokinetics are mainly dependent on terminal galactose-type or terminal N-acetylglucosamine-type glycans;
The method of determining the glycan pattern described in [13], which is carried out by identifying which glycan pattern is selected from the group consisting of (3) glycan pattern C, whose pharmacokinetics are mainly dependent on terminal α2,6 sialic acid type glycans, and (4) glycan pattern D, whose pharmacokinetics are mainly dependent on terminal α2,3 sialic acid type glycans.
[15]
Cancer glycan pattern A is characterized in that 45 mol % or more of the glycans are terminal high mannose type glycans, and 0% or more and less than 2% are terminal sialic acid type glycans;
Cancer glycan pattern B is characterized in that terminal high mannose type glycans are less than 45% and terminal sialic acid type glycans are 0% or more and less than 2%;
Cancer glycan pattern C is characterized in that 2-100% 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;
The method for determining cancer glycan pattern D according to [14], wherein 2-100% of glycans are terminal sialic acid type glycans and the content of α2,3 sialic acid type glycans is higher than the content of α2,6 sialic acid type glycans.
[16]
When the glycan profile of the cancer cells is glycan pattern A, the cancer cells of the subject exhibit type 1 pharmacokinetics, suggesting that the cancer cells have a low tendency to metastasize;
When the glycan profile is glycan pattern B, the cancer cells of the subject exhibit type 2 pharmacokinetics, 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 of the subject exhibit type 3 pharmacokinetics, suggesting that the cancer cells have a tendency to metastasize to the axillary and supraclavicular lymph nodes;
The method of determining according to [14] or [15], wherein when the glycan profile is glycan pattern D, the cancer cells carried by the subject exhibit type 4 pharmacokinetics, suggesting that the cancer cells have a tendency to metastasize to the lungs, liver, spleen, brain, and kidneys.
[17]
A method for producing a glycan-presenting particle according to any one of [1] to [7], comprising presenting a cancer glycan pattern on the surface of a nanoparticle at least a portion of which is coated with a phospholipid.
[18]
The manufacturing method described in [17], wherein the presented cancer glycan pattern is a cancer glycan pattern obtained by excising glycans from 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.
[19]
The nanoparticles having at least a part of the surface coated with a phospholipid can be produced by mixing a crosslinked precursor X represented by the following general formula (D) and a phospholipid precursor represented by the following general formula (E) with a colloidal nanoparticle to obtain a surface-modified nanoparticle carrying the crosslinked precursor X and the phospholipid on the surface of the nanoparticle, according to the production method described in [17] or [18].
(In general formula (D), n1 is an integer from 2 to 30, and n2 is an integer from 2 to 30.)
(In general formula (E), n3 is an integer ranging from 2 to 30.)
[20]
The cancer glycan pattern is presented by reacting, by glycoblotting, an aminooxy group of the crosslinked precursor X represented by general formula (D) introduced into the surface-modified nanoparticle with a cancer glycan pattern obtained by cutting out the glycan of a cancer cell collected from a subject, or with a reducing end of a glycan contained in a glycan pattern determined based on a profile obtained by profiling the glycans of the cancer cell collected from the subject, in the manufacturing method described in [19].
[21]
The method for producing a glycan pattern according to any one of [18] to [20], wherein the glycan pattern determined based on the profile is a glycan pattern in which the types and amounts of glycan components are identical to those of the profile, or a glycan pattern in which the types and amounts of glycan components are partially identical to those of the profile.

According to the present invention, it is possible to provide glycan-presenting particles having a cancer glycan pattern that exhibits specific pharmacokinetics and can predict the metastatic destination of cancer cells from a primary focus. By intravenously administering these glycan-presenting particles to mice, for example, it is possible to visualize the process of cancer metastasis led by the cancer glycan pattern in real time. Furthermore, according to the present invention, it is also possible to provide a DDS technology that is effective in preventing 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 potential of cancer cells.

Figure 1 shows the method for producing glycan-displaying particles that display the glycan pattern of cancer cells on their surface and the results of in vivo imaging of their dynamics in the body. Figure 2 shows the N-glycan profile of cultured human cancer cells. (a) Glycan structure analysis by MALDI-TOFMS, IS = internal standard, (b) structures of major glycan components of each cancer cell (top 10 structures in expression amount), and the pie chart shows the results of glycotyping analysis. Figure 3 shows the experimental results of the in vivo dynamics of glycan-presenting particles, which are nanoparticles that present the glycan patterns of cancer cells. (a) In vivo dynamics (numbers indicate zeta potential) of glycan-presenting particles presenting glycans of four types of human cancer cells and a control up to 3 hours after intravenous administration, (b) distribution of glycan-presenting particles in major organs dissected after 3 hours. Figure 3 shows the experimental results of the in vivo dynamics of glycan-presenting particles, which are nanoparticles that present the glycan pattern of cancer cells. (c) Organ targeting of glycan-presenting particles: The relative fluorescence intensity in each organ is shown when the fluorescence intensity in the brain of control particles without glycans is set to 1.0. However, axillary and supraclavicular lymph node tissues were not removed by dissection. Figure 4 shows the results of an experiment to modify the glycan pattern of ruddy duck egg white to that of human cancer cells. (a) The structure of the hyperbranched glycan found in ruddy duck egg white and the modification method using sialyltransferase, (b) the hierarchical structure of the glycans of button quail, chicken, and ruddy duck egg white, and two types of glycan patterns modified from ruddy duck egg white (2,3S-ruddy duck and 2,6S-ruddy duck). Figure 4 shows the results of an experiment to modify the glycan pattern of human cancer cells from Ruddy duck egg white glycans. (c) The proportion of major glycan components (top 10 components in expression) among the glycans presented on glycan-presenting particles. The pie graph shows the results of glycotyping analysis. Figure 5 shows the results of investigating the pharmacokinetics and organ targeting of nanoparticles presenting egg white and egg yolk glycans, modified glycan patterns, and α2,6-sialic acid glycans. (a) Pharmacokinetics (numbers indicate zeta potential) of glycan-presenting particles and control, including nanoparticles presenting egg white glycans of button quail, chicken, and ruddy duck, two types of glycans that were modified from ruddy duck egg white glycans and sialylated, and nanoparticles presenting glycans derived from chicken egg yolk glycopeptides (GNS-2,6S-A2), up to 3 hours after intravenous administration. (b) Distribution of glycan-presenting particles (GNS-Button Quail, GNS-Chicken, GNS-Ruddy duck, GNS-2,3S-Ruddy duck, GNS-2,6S-Ruddy duck, GNS-2,6S-A2) in the major organs of mice dissected after 3 hours. Figure 5 shows the results of investigating the pharmacokinetics and organ targeting of nanoparticles that display the glycan patterns of egg white and egg yolk, modified glycan patterns, and α2,6-sialic acid glycan patterns. (c) Organ targeting of nanosomes: The relative fluorescence intensity in each organ is shown when the near-infrared fluorescence intensity in the brain of the control nanoparticles without glycans is set to 1.0. However, the axillary and supraclavicular lymph node tissues were not removed by dissection. Figure 6 shows the experimental results of the in vivo dynamics of glycan-presenting particles prepared by mixing Japanese Quail egg white glycans and hen egg yolk glycopeptide-derived glycans (2,6S-A2). (a) The glycan profile of nanoparticles that displayed glycans (approximately 6 nmol) derived from Japanese Quail egg white was added with 0.6 nmol, 1.2 nmol, and 2.4 nmol of 2,6S-A2-derived glycans, respectively, and mixed.

<Glycan-displaying particles>
The present invention relates to a sugar chain-presenting particle, which is a nanoparticle having a sugar chain on the surface thereof. The sugar chain-presenting particle comprises:
(1) the average particle size of the glycan-presenting particle is in the range of 10 to 100 nm;
(2) At least a portion of the surface of the nanoparticle is covered with a phospholipid;
(3) The glycans on the nanoparticle surface have a glycan pattern derived from cancer cells, or a pattern that mimics a glycan pattern determined based on the profile of this glycan (cancer glycan pattern).

The glycan-presenting particles of the present invention have a phospholipid coating and a cancer glycan pattern on the surface of the nanoparticles. In this specification, the glycan pattern means a pattern specified by the presence or absence of a specific type of glycan or the amount of a specific type of glycan present in a glycan population consisting of two or more different glycans based on the glycan profile of the surface. The glycan pattern is specified by the type of glycan component and its content.

Therefore, in the present invention, a glycan-presenting particle that captures and presents the glycan of a cancer cell as it is is a glycan-presenting particle having a glycan pattern derived from a cancer cell. In addition, a glycan-presenting particle that has been profiled for the glycan of a cancer cell and has a glycan pattern determined based on the profile and has this determined glycan pattern is also a glycan-presenting particle in the present invention. The glycan pattern determined based on the profile may be either a glycan pattern in which the type and content of glycan components are the same as those in the profile, or a glycan pattern in which the type and content of glycan components are partly the same as those in the profile. When the type and content of glycan components are the same as those in the profile, some 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, the glycan pattern can be a glycan pattern in which glycan components belonging to one or more types with a high content are included and glycan components belonging to one or more types with a low content are reduced or deleted. 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, the glycan pattern determined from the profile can be a glycan pattern in which the terminal N-acetylglucosamine type glycans with a low content are reduced or deleted. Alternatively, when one type contains multiple glycan components, the glycan pattern determined from the profile can be a glycan pattern in which the content ratio of glycan components belonging to the same type is changed. Since the pharmacokinetics of glycan-presenting particles change depending on the type of glycan, the glycan pattern can also be determined by appropriately changing the profile in consideration of the change in pharmacokinetics.

The sugar chain patterns of the present invention can be classified into four cancer sugar chain patterns, A to D, based on the pharmacokinetics of the sugar chain-presenting particles of the present invention.
(1) Glycosylation pattern A, whose pharmacokinetics are mainly dependent on terminal high mannose type glycans;
(2) Glycosylation pattern B, whose pharmacokinetics are mainly dependent on terminal galactose-type glycans or terminal N-acetylglucosamine-type glycans;
(3) Glycosylation pattern C, whose pharmacokinetics are mainly dependent on terminal α2,6 sialic acid type glycans;
(4) Glycosylation pattern D, whose pharmacokinetics depend mainly on terminal α2,3 sialic acid type glycans.

Pharmacokinetics refers to the movement of glycan-presenting particles over time through the circulatory system and the like within the body of an animal to which the glycan-presenting particles have been administered, and their distribution over time to each organ and tissue. In this specification, the property of distribution or accumulation over time in each organ or tissue in pharmacokinetics may be specifically referred to as organ tropism.

The cancer cells targeted in the present invention are those that release exosomes, which are targets for the glycan-presenting particles of the present invention to mimic, and include cancer cells collected from a living body and cancer cells cultured after collection. The cancer cells may be single cells cultured in pure culture, or a mixture of multiple cancer cells with different malignancies, etc.

In addition, the glycan patterns of existing cancer cells that are commonly used can also be used as glycan patterns in the glycan-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 (gastric cancer), MIAPaCa-2 (pancreatic cancer), A431 (skin cancer), and SKOV (ovarian cancer).

Glycosylation particles are particles that have a function (intercellular signaling-like function) that mimics the function of exosomes, which are particles used in intercellular signaling. Exosomes contain nucleic acids, proteins, etc., and are transmitted to recipient cells via exosomes secreted from cells. Exosomes function as a communication tool between cells. The glycan-presenting particles produced in the present invention, like original exosomes, have the function of transmitting information to recipient cells and tissues and organs in which they are assembled.

The nanoparticles can be metal nanoparticles or semiconductor nanoparticles. There are no particular limitations on the material of the metal nanoparticles, but they can be gold, platinum, silver, or iron magnetic material, and the metal nanoparticles can be gold nanoparticles, platinum nanoparticles, silver nanoparticles, or iron magnetic material nanoparticles. In particular, gold nanoparticles, platinum nanoparticles, and silver nanoparticles are preferred metal nanoparticles from the viewpoint of safety to living organisms.

There are no particular limitations on the material of the semiconductor nanoparticles. The semiconductor nanoparticles can also be quantum dots. Quantum dots are small clusters of hundreds to thousands of semiconductor atoms, measuring just over 10 nm, and are fluorescent nanoparticles. The wavelength (color) of the emitted fluorescence varies depending on the particle size. Commercially available products can be used, and when quantum dots are used as the nanoparticles of the complex of the present invention, a fluorescent complex can be produced, making it possible to monitor dynamics in the body.

The nanoparticles may have a particle diameter 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, even more preferably in the range of 5 to 30 nm, and even more preferably in the range of 10 to 30 nm. The glycan-presenting particles having a phospholipid coating and a cancer glycan pattern on the surface of the nanoparticles have an average particle diameter 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, even 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 diameter of the nanoparticles and the glycan-presenting particles can be measured by a dynamic light scattering method. As a measuring device, a fiber optic dynamic light scattering photometer (for measuring particle size distribution) can be used. More specifically, a fiber optic dynamic light scattering photometer FDLS-3000 (manufactured by Otsuka Electronics) can be used.

The glycan-presenting particles of the present invention have nanoparticle surfaces coated with phospholipids. Metal nanoparticles completely coated with a phospholipid alkanethiol mixed monolayer with an average particle size of about 20 nm are stable nanoparticles that do not form a protein corona due to nonspecific adsorption even in blood, and when administered intravenously to mice, they do not accumulate in specific organs and remain uniformly throughout the body even up to 3 hours after administration (Non-Patent Documents 6-7, Patent Document 1).

The sulfide bond of an alkanethiol having a phosphorylcholine group can be a phospholipid mimetic represented by the general formula (A).
In general formula (A), n3 is an integer ranging from 2 to 30, and the -S- end is a supporting site that is sulfide-bonded to the nanoparticle.

The phospholipid mimetic modifies the surface of the nanoparticle and provides the function of preventing nonspecific adsorption of the protein corona to the surface of the glycan-presenting particle of the present invention. From this perspective, n3 is an integer in the range of 2 to 30, preferably 5 to 20, and more preferably 7 to 15.

It is preferable that the amount of phospholipid or phospholipid pseudo-substance loaded on each nanoparticle is 80% or more of the metal elements (reaction points) on the surface of the nanoparticle.

The glycan-presenting particle of the present invention has a cancer glycan pattern on the surface of the nanoparticle. This cancer glycan pattern is a glycan pattern that presents glycans derived from cancer cells that release exosomes as an information sender, or a cancer glycan pattern determined by profiling the glycans of cancer cells. It is preferable that the glycan pattern derived from cancer cells is a glycan pattern of glycans derived from cancer cells collected from a specific cancer treatment patient in order to improve the predictability of the pharmacokinetics of exosomes released from the cells. Existing glycan patterns derived from cancer cells that are used as standard can also be used for prediction. The glycan pattern derived from cancer cells can be presented by excising the glycans of the cancer cells and capturing them on the surface of the nanoparticle. In the excision of glycans from cells and capture on nanoparticles, it is preferable that the glycans of the cells are presented as they are on the surface of the nanoparticle, but it does not matter if the glycan components vary within the range of the cancer glycan pattern determined in the cells.

The above-mentioned glycan profile derived from cancer cells can be identified according to a general protocol of the glycoblotting method (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537). The relationship between the expression levels of major structural motifs and the major structural motifs can be clarified by glycotyping analysis using an internal standard compound as an index. In addition, glycans can be supported on nanoparticles by existing methods (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). For details, see Example 1 (A).

As a result of analyzing the in vivo dynamics of glycan-presenting particles having the glycan patterns of cancer cell glycans, it was found that the glycan patterns of the cancer cells examined were at least (1) glycan pattern A, the pharmacokinetics of which is mainly dependent on terminal high mannose type glycans;
(2) Glycosylation pattern B, whose pharmacokinetics are mainly dependent on terminal galactose-type or terminal N-acetylglucosamine-type glycans;
(3) Glycosylation pattern C, whose pharmacokinetics are mainly dependent on terminal α2,6 sialic acid type glycans;
(4) It was revealed that the pharmacokinetics of a glycan-presenting particle having a cancer glycan pattern is determined depending on the cancer glycan pattern, and that the pharmacokinetics of the glycan-presenting particle having a cancer glycan pattern is determined depending on the cancer glycan pattern (see Examples 1, 2, and 3).

The pharmacokinetics of glycan-presenting particles having cancer glycan patterns A to D are as follows.
Glycan-presenting particles having cancer glycosylation pattern A exhibit type 1 pharmacokinetics in which excretion of particles from the body is promoted, as compared with glycosylation particles not having a glycosylation chain.
Glycan-presenting particles having cancer glycosylation pattern B exhibit type 2 pharmacokinetics, accumulating in the liver and spleen, as compared with glycosylation particles not having a glycosylation chain.
Glycan-presenting particles having cancer glycosylation pattern C exhibit type 3 pharmacokinetics, accumulating in the axillary and supraclavicular lymph nodes, compared to glycan-presenting particles not having a glycosylation chain.
Glycan-presenting particles having cancer glycoprotein pattern D exhibit type 4 pharmacokinetics, which are distributed (dispersed and accumulated) in organs such as the lungs, liver, spleen, brain, and kidneys, as compared with glycoproteins not having glycoproteins.

The terminal high mannose type glycan constituting the above glycan pattern means a glycan in which the terminal of the glycan is branched at two or more branches, and all of the sugars in the branched parts are mannose. Examples of terminal high mannose type glycans are shown below.

The term "terminal galactose-type glycan" constituting the above glycan pattern means a glycan in which one or more terminal sugars of one glycan are galactose. Examples of terminal galactose-type glycans are shown below.

The term "terminal N-acetylglucosamine-type glycan" constituting the above glycan pattern means a glycan in which one or more terminal sugars of one glycan are N-acetylglucosamine. Examples of terminal N-acetylglucosamine-type glycans are shown below.

The terminal sialic acid type glycan constituting the above glycan pattern means a glycan in which one or more terminal sugars of one glycan are sialic acid. Terminal sialic acid type glycans include complex type glycans containing a Neu5Acα2,6Gal unit at the end and complex type glycans containing a Neu5Acα2,3Gal unit at the end. In this specification, a complex type glycan containing a Neu5Acα2,6Gal unit at the end is referred to as a terminal α2,6 sialic acid type glycan, and a complex type glycan containing a Neu5Acα2,3Gal unit at the end is referred to as a terminal α2,3 sialic acid type glycan. Examples of terminal sialic acid type glycans are shown below.

In the present invention, four types of cultured human cancer cells (MCF7, MDA-MB-231, A549, HepG2) for which information on metastatic potential and organs where metastatic recurrence occurs (organ tropism) were used to (1) profile the state of post-translational glycan modification (N-glycan) of all proteins present in the cancer cells and on the membrane surface. Next, (2) the glycans of those cancer cells were directly captured in nanosomes (fluorescent nanoparticles prepared from quantum dots: Non-Patent Documents 6-7, Patent Document 1) by glycoblotting (Non-Patent Document 9) to produce glycan-presenting particles presenting glycans derived from each cancer cell. Furthermore, the pharmacokinetics of these particles when administered intravenously to mice was observed in real time by near-infrared fluorescence spectroscopy using glycan-presenting particles that did not present glycans as a control (see Example 1).

Based on these results, (3) we artificially determined the glycan pattern based on the glycan profile of the above-mentioned cultured human cancer cells, and created artificial glycan-presenting particles that present glycans within the range of the determined glycan pattern. These were administered intravenously to mice using a similar method, and their pharmacokinetics were observed. As a result, it was demonstrated that the glycan pattern of exosomes derived from cancer cells leads the pharmacokinetics of exosomes and ultimately determines the organ tropism of cancer cells (see Example 2).

(1) Cancer glycan pattern A
In the cancer glycan pattern A, 45 mol % or more of the glycans contained in the glycan are terminal high mannose type glycans. Hereinafter, unless otherwise specified, % of glycan means mol %. The content of terminal high mannose type glycans (HM) in the glycan-presenting particles of the present invention having the cancer glycan pattern A shown in the examples is as follows. The terminal sialic acid type glycan is 0% in all cases.

In addition to terminal high mannose type glycans, cancer glycan pattern A can contain terminal galactose type glycans, terminal N-acetylglucosamine type glycans, and terminal sialic acid type glycans as glycan components, but the terminal sialic acid type glycans are 0% or more and less than 2%. When the terminal sialic acid type glycans are 2% or more, it can be determined to be glycan pattern C or glycan pattern D, and the effect of the terminal sialic acid type glycans becomes preferential (described later). If the terminal sialic acid type glycans are less than 2% and the terminal high mannose type glycans are 45% or more, the glycan-presenting particles will exhibit type 1 pharmacokinetics and will not exhibit distribution to specific organs (organ tropism), regardless of the type and amount of the other glycan components, terminal galactose type glycans and terminal N-acetylglucosamine type glycans. The terminal high mannose type glycans can be in the range of 50% to 95%, for example.

As shown in Figure 3a, in the case of NS (control), which is a glycan-presenting particle having no glycan, fluorescence from the particles was observed throughout almost the entire body even 180 minutes after intravenous administration, whereas in the case of GNS-MCF-7 of Example 1 having cancer glycan pattern A, most of the particles were excreted from the body 180 minutes after intravenous administration. This phenomenon was also observed in the case of GNS-Button Quail of Example 2 (see Figure 5) and 0/6/32 of Example 3. It can be seen that the glycan-presenting particles having cancer glycan pattern A show type 1 pharmacokinetics in which excretion of particles from the body is promoted, compared to glycan-presenting particles having no glycan.

(2) Cancer glycosylation pattern B
Cancer glycan pattern B is a cancer glycan pattern in which terminal high mannose type glycans are less than 45% and terminal sialic acid type glycans are 0% or more and less than 2%. Glycan-presenting particles having a glycan pattern in which terminal high mannose type glycans are less than 45% and terminal sialic acid type glycans are 0% or more and less than 2% show type 2 pharmacokinetics. Although terminal galactose type glycans and terminal N-acetylglucosamine type glycans can be included as glycan components other than terminal high mannose type glycans and terminal sialic acid type glycans, as long as terminal high mannose type glycans are less than 45% and terminal sialic acid type glycans are 0% or more and less than 2%, they can be determined to be glycan pattern B even if the quantitative ratio of terminal galactose type glycans and terminal N-acetylglucosamine type glycans changes. Glycan-presenting particles of cancer glycan pattern B show type 2 pharmacokinetics.

The contents of terminal high mannose type glycan (HM), terminal galactose type glycan (Gal) and terminal N-acetylglucosamine type glycan (NAc-G) in the glycan-presenting particles 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 all cases.

As shown in Figures 5b and c, for example, the example of GNS-Chicken in Example 2 having cancer glycan pattern B accumulated in the liver and spleen 180 minutes after intravenous administration. This phenomenon was also observed in the example of GNS-Ruddy Duck in Example 2 (see Figures 5b and c). In other words, it can be seen that glycan-presenting particles having cancer glycan pattern B exhibit type 2 pharmacokinetics, which accumulate in the liver and spleen, compared to control particles without glycans.

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

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

The contents of α2,3 sialic acid type glycan (α2,3), α2,6 sialic acid type glycan (α2,6) and terminal high mannose type glycan (HM) in 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. Terminal galactose type glycan (Gal) and terminal N-acetylglucosamine type glycan (NAc-G) are not shown.

As shown in Figures 3b and c, NS (control), which is a particle without a glycan, showed fluorescence from the particles throughout almost the entire body even 180 minutes after intravenous administration. In contrast, in the case of 2,6-Ruddy Duck of Example 2, which has a cancer glycan pattern of pattern C (see Figures 5b and c), the particles accumulated in the axilla and supraclavicular lymph nodes, for example, 180 minutes after intravenous administration, compared to NS. This shows that glycan-presenting particles with cancer glycan pattern C exhibit type 3 pharmacokinetics, which accumulate in the axilla and supraclavicular lymph nodes, compared to glycan-presenting particles without a glycan.

In the case of 2,3-Ruddy Duck of Example 3 having a cancer glycan pattern of pattern D (see Figures 5b and c), 180 minutes after intravenous administration, the particles were distributed (dispersed and accumulated) in organs such as the lungs, liver, spleen, brain, and kidneys. This shows that glycan-presenting particles having cancer glycan pattern D show type 4 pharmacokinetics, which are distributed (dispersed and accumulated) in organs such as the lungs, liver, spleen, brain, and kidneys, compared to glycan-presenting particles without glycans.

Next, the identification of α2,6 sialic acid type glycans and α2,3 sialic acid type glycans will be described. In the case of GNS-MDA-MB-231 in Example 1, 180 minutes after intravenous administration, the particles accumulated in the axilla and supraclavicular lymph nodes (particularly, Figures 3b and c). This phenomenon was similar to that of 2,6-Ruddy Duck in Example 2, which has cancer glycan pattern C. In Example 1, it was not specified whether the terminal sialic acid type glycan was an α2,3 sialic acid type glycan or an α2,6 sialic acid type glycan, but based on the results of this pharmacokinetics, GNS-MDA-MB-231 in Example 1 was determined to be glycan pattern C and is shown in Table 4.

In the case of GNS-A549 in Example 1, 180 minutes after intravenous administration, the particles were distributed (dispersed and accumulated) in organs such as the lungs, liver, spleen, brain, and kidneys (particularly Figures 3b and c). This phenomenon was similar to that of 2,3-Ruddy Duck in Example 2, which has cancer glycan pattern D. In Example 1, it was not specified whether the terminal sialic acid type glycan was an α2,3 sialic acid type glycan or an α2,6 sialic acid type glycan, but from the results of this pharmacokinetics, GNS-A549 and GNS-HepG2 in Example 1 were determined to be glycan pattern D and are shown in Table 4. In this specification, the specification of whether the glycan is an α2,6 sialic acid type glycan or an α2,3 sialic acid type glycan is determined by its function, but it can also be specified by analytical methods such as chemical and enzymatic decomposition properties.

The cancer glycan pattern can be the glycans themselves of cancer cells that are commercially available and commonly used for research purposes as described above, or the glycans of cancer cells of a specific cancer treatment patient. Furthermore, the cancer glycan pattern can be a glycan pattern determined based on the profile obtained by profiling the glycans of cancer cells. The glycan pattern determined based on the profile can be either a glycan pattern in which the type and content of glycan components are the same as those of the profile, or a glycan pattern in which the type and content of glycan components are partly the same as those of the profile. When the type and content of glycan components are the same as those of the profile, some 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, the glycan pattern can be a glycan pattern in which glycan components belonging to one or more types with a high content are included and glycan components belonging to one or more types with a low content are reduced or deleted. A glycan pattern determined based on the obtained profile by profiling the glycans of cancer cells is a glycan pattern that has different types of glycan components and different amounts of each glycan component from the above-mentioned cancer glycan pattern derived from cancer cells, but shows the same pharmacokinetics as the cancer glycan pattern derived from cancer cells. When profiling the glycans of cancer cells, if the glycan components contained in the profile are classified into several types, a glycan pattern that shows the same pharmacokinetics as the cancer glycan pattern derived from the cancer cells from which the glycans were profiled can be obtained by including glycan components belonging to one or more types with a high content as they are and reducing or deleting 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 a certain glycan pattern has a type of glycan component and a content of each glycan component that are different from the cancer glycan pattern derived from cancer cells, but has pharmacokinetics that are mainly dependent on terminal high mannose type glycans, it shows type 1 pharmacokinetics.
Similarly, when a certain glycan pattern has a type of glycan component and a content of each glycan component that are different from the cancer glycan pattern derived from cancer cells, but has glycan pattern B whose pharmacokinetics are mainly dependent on terminal galactose-type glycans or terminal N-acetylglucosamine-type glycans, it shows type 2 pharmacokinetics.
When a certain glycan pattern has a type of glycan component and a content of each glycan component that are different from the cancer glycan pattern derived from cancer cells, but has pharmacokinetics that are mainly dependent on terminal α2,6 sialic acid type glycans, it shows type 3 pharmacokinetics.
When a certain glycan pattern has a type of glycan component and a content of each glycan component that are different from the cancer glycan pattern derived from cancer cells, but has pharmacokinetics that are mainly dependent on terminal α2,3 sialic acid type glycans, it shows type 4 pharmacokinetics.

For example, in the prevention or treatment of cancer cells at a primary focus metastatic site, the glycans of the cancer cells are profiled, and based on the obtained profile, it is determined whether the cancer has cancer glycan patterns A to D. The glycan-presenting particles of the present invention can be prepared and provided using known methods, with the glycan-presenting particles having the selected glycan pattern.

The selected glycan pattern can be artificially modified by, for example, treating an existing glycan known to contain a specific glycan with an enzyme. Examples of existing glycans include glycans contained in egg whites and egg yolks of quail, chicken, duck, etc., as shown in Example 2. Glycans of existing materials can also be used as a specific glycan pattern, either as is or after appropriate processing. For example, as shown in Example 2, glycans can be treated with an enzyme to be used as a material that provides a desired cancer glycan pattern. Alternatively, materials containing multiple glycans with known compositions can be mixed in an appropriate ratio to be used as a material that provides a desired cancer glycan pattern (see Example 2).

The glycans from egg whites of quails, chickens, and ducks, and hen's egg yolks used in Example 2 are safe and inexpensive biological materials with clear glycan structures and expression profiles, so by presenting these glycans as they are or after modifying them as raw materials on nanoparticles to form glycan-presenting particles, it is possible to reproduce the same in vivo dynamics as exosomes derived from cancer cells. The glycan pattern can be not only of biological origin, but also a mixture of artificially synthesized glycan components. The artificial synthesis of glycan components can be carried out by known methods.

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

The sugar chain on the surface of the nanoparticle can be a sulfide bond of an alkanethiol presenting a sugar chain represented by the following general formula (B).
In general formula (B), n1 is an integer from 2 to 30, n2 is an integer from 2 to 30, the -S- terminus is a supporting moiety that is sulfide-bonded to the nanoparticle, and R ¹⁰ is a glycan-containing moiety. The glycan of the glycan-containing moiety R ¹⁰ is a cancer glycan pattern, and has glycan components and their composition ratios according to glycan patterns A to D determined based on the glycan of cancer cells and on the glycan profile.

The sugar chain-presenting particle of the present invention can be represented, for example, diagrammatically by the following general formula (10).

The residues supported on the nanoparticle (NP) of general formula (10) are a sugar chain-containing group represented by general formula (B) having R ¹⁰ as a sugar chain-containing moiety and a phospholipid group represented by general formula (A). Both are supported on the nanoparticle (NP) via the sulfide (-S-) at the end of each group. In an actual complex, one or more phospholipid groups represented by general formula (A) are supported on the nanoparticle NP. Furthermore, R ¹⁰ includes a plurality of sugar chain component-containing groups consisting of sugar chain components that constitute the sugar chain pattern.

The glycan-presenting particles of the present invention have pharmacokinetics that correspond to each pattern of glycan, and therefore these properties can be utilized to accumulate in specific tissues or organs. Therefore, by selecting the pattern of glycans, it is possible to obtain glycan-presenting particles that can accumulate in specific tissues or organs, and these properties can also be utilized to provide DDS.

The glycan-presenting particle of the present invention can further have a drug moiety. There are no particular limitations on the drug in the drug moiety, but it can be, for example, an anticancer drug or an anti-inflammatory drug.

In the sugar chain-presenting particle of the present invention, for example, a drug-containing group represented by general formula (C) can be supported on the surface of the nanoparticle as a drug moiety.
In general formula (C), n1 is an integer from 2 to 30, n2 is an integer from 2 to 30, the -S- terminus is a loading moiety that is sulfide-bonded to the nanoparticle, and R ²⁰ is a drug-containing moiety.

The drug-containing group represented by general formula (C) can maintain the distance between the drug-containing group and the nanoparticle within a desired range by selecting n1 and n2 in the formula. n1 is an integer in the range of 2 to 30, preferably 5 to 16, and more preferably 4 to 15. n2 is an integer in the range of 2 to 30, preferably 5 to 20, and more preferably 7 to 15.

The sugar chain-presenting particle of the present invention having a drug moiety can be represented, for example, as a schematic diagram by the following general formula (11).

The residues supported on the nanoparticle (NP) of general formula (11) are, from the top, a glycan-containing group represented by general formula (B), a phospholipid group represented by general formula (A), and a drug-containing group represented by general formula (C). In the actual complex, one or more of the glycan-containing group represented by general formula (B), the phospholipid group represented by general formula (A), and the drug-containing group represented by general formula (C) are supported on the nanoparticle NP as sulfide bonds.

When the glycan-presenting particle of the present invention has a drug moiety, it encompasses cancer preventive drugs and therapeutic drugs that contain this glycan-presenting particle as an active ingredient. The cancer preventive drug and therapeutic drug of the present invention preferably have a cancer glycan pattern in which the glycan-presenting particle captures and presents glycans derived from cancer cells as is, but may also be a glycan-presenting particle having cancer glycan pattern B, C, or D determined based on the profile obtained by profiling this glycan. The glycan-presenting particle exhibits pharmacokinetics of types 2 to 4 according to each glycan pattern, and is useful for the prevention and treatment of specific metastatic cancers.

In the case of a glycan-presenting particle having cancer glycan pattern B, it is useful for preventing or treating metastatic cancer to the liver or spleen,
In the case of glycan-presenting particles having cancer glycan pattern C, they are useful for preventing and treating metastatic cancer to the axillary and supraclavicular lymph nodes,
Glycan-presenting particles having cancer glycoconjugate pattern D are useful for preventing and treating metastatic cancer to organs such as the lungs, liver, spleen, brain, and kidneys.

(Glycan-displaying 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 particle contained in the kit is any one of the above-mentioned glycan-presenting particles of the present invention. The different cancer glycan patterns in the kit are any two or more of the above-mentioned glycan patterns A to D. Glycan patterns A to D are selected, for example, by determining the glycan pattern by profiling the glycans of cancer cells collected from a cancer patient.

In the glycan-presenting particle kit of the present invention, glycan-presenting particles having a determined glycan pattern are selected and used, but depending on the type of cancer, multiple glycan patterns can also be used in combination. For example, two types of glycan patterns B and C can 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).

In the glycan-presenting particle kit of the present invention, the glycan-presenting particle may further have a drug moiety. Examples of drugs include preventive and therapeutic drugs for canker sores.

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, they can be used parenterally in the form of a sterile solution with water or other pharma- ceutically acceptable liquid, or in the form of an injection of a suspension. For example, they can be formulated by appropriately combining with 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 dose form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is such that an appropriate dose within the indicated range can be obtained.

Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be used in combination with suitable solubilizing agents, such as alcohol, specifically ethanol, polyalcohols, such as propylene glycol and polyethylene glycol, and nonionic surfactants, such as polysorbate 80 (TM) and HCO-60.

Oily liquids include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Buffers such as phosphate buffers and sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants may also be used. The prepared injection solution is usually filled into a suitable ampule. Liposomes can also be used to encapsulate the drug for cellular delivery.

Depending on the purpose of use, the glycan-presenting particles and glycan-presenting particle kits of the present invention can be administered to cancer patients or patients suspected of having cancer via an appropriate administration route for the prevention or treatment of metastatic cancer to organs.

Administration may be oral or parenteral, preferably parenteral, and specific examples include injections, nasal administration, pulmonary administration, transdermal administration, etc. Examples of injections include intravenous, intramuscular, intraperitoneal, and subcutaneous injections, which can be used for systemic or local administration.

The dose and administration method of the glycan-presenting particle and glycan-presenting particle kit of the present invention can be appropriately selected depending on the age, weight, sex, and the nature or severity of the symptoms to be treated of the patient. The dose of the pharmaceutical composition containing the glycan-presenting particle and glycan-presenting particle kit of the present invention can be selected, for example, from the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dose can be selected from the range of 0.01 to 100,000 mg/body per patient, but is not necessarily limited to these numerical values. The dose and administration method vary depending on the age, weight, sex, symptoms, etc. of the patient, but can be appropriately selected by the person concerned.

<Method of producing sugar chain-presenting particles>
The present invention includes the above-mentioned method for producing the sugar chain-presenting particle of the present invention.
The sugar chain-presenting particle of the present invention can be prepared by presenting a desired cancer sugar chain pattern on the surface of a nanoparticle at least partially coated with a phospholipid. The method for preparing the nanoparticle at least partially coated with a phospholipid will be described later.

As described above, the desired cancer glycan pattern may be a cancer glycan pattern in which the glycans of cancer cells collected from a subject are cut out and used as is, or the glycans of existing materials are used as is or appropriately modified to a specific glycan pattern. A specific glycan pattern is a glycan pattern determined based on the profile obtained by profiling the glycans of cancer cells collected from a subject. A glycan pattern determined based on a profile may be either a glycan pattern in which the type and content of glycan components are the same as the profile, or a glycan pattern in which the type and part of the content of glycan components are the same as the profile. When the type and part of the content of glycan components are the same as the profile, some 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, the glycan pattern may include glycan components belonging to one or more types with a high content, and reduce or delete glycan components belonging to one or more types with a low content. When profiling the glycans of cancer cells, if the glycan components contained in the profile are classified into several types, a glycan pattern that shows the same pharmacokinetics as the cancer glycan pattern derived from the cancer cells whose glycans were profiled can be obtained by containing glycan components belonging to one or more types with a high content as they are and reducing or deleting 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.

A conceptual diagram of the method for producing glycan-presenting particles of the present invention (a method for producing an exosome model presenting the glycan pattern of cancer cells on its surface and in vivo imaging of its pharmacokinetics and organ targeting) is shown in Figure 1. (a) shows the basic structure of glycan-presenting particles: anti-aggregation nanoparticles whose surface is completely covered with a self-assembled mixed monolayer of an alkanethiol having a phosphorylcholine group at its head and an appropriate amount of an aminooxy linker. (b) shows the basic principle of the glycoblotting method, in which the reducing end of the glycan (a compound containing an aldehyde group or a ketone group) reacts specifically with the aminooxy group on the nanoparticle surface to form an oxime bond. (c) shows a method for presenting a glycan pattern derived from cancer cells on the nanoparticle surface and in vivo imaging. The glycans are captured and displayed on the surface of nanoparticles using the glycoblotting method, which utilizes a chemical reaction specific to glycans with reducing ends (equivalent to aldehyde groups) from a mixture containing glycans prepared from collected or cultured cancer cells.

Nanoparticles having at least a portion of their surface coated with phospholipids can be produced by mixing a crosslinked precursor X represented by the following general formula (D) and a phospholipid precursor represented by the following general formula (E) with colloidal nanoparticles to obtain surface-modified nanoparticles carrying the crosslinked precursor X and phospholipid on the nanoparticle surface. This method is described in Patent Document 1, the entire disclosure of which is expressly incorporated herein by reference.

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

(In general formula (E), n3 is an integer ranging from 2 to 30.)

In the general formula (D), n1 is an integer ranging from 2 to 30, preferably from 5 to 20, and more preferably from 7 to 15. n2 is an integer ranging from 2 to 30, preferably from 5 to 20, and more preferably from 7 to 15. The general formula (E) is the same as the phospholipid mimetic group represented by the general formula (A) except that the terminal is an SH group. In the general formula (E), n3 is an integer ranging from 2 to 30, preferably from 5 to 20, and more preferably from 7 to 15. Both the crosslinked precursor X represented by the general formula (D) and the phospholipid mimetic precursor represented by the general formula (E) are commercially available products, and can also be prepared by the method described in the reference literature. (Reference literature: T. Ohyanagi, et. al., J. Am. Chem. Soc. 2011, 133, 12507-12517)

The mixing ratio of the crosslinked precursor X represented by general formula (D), the phospholipid pseudo-substance precursor represented by general formula (E), and the nanoparticles can be appropriately determined taking into consideration the desired loading amount of the crosslinked precursor X represented by general formula (D) and the phospholipid pseudo-substance precursor represented by general formula (E) on the nanoparticles.

In the crosslinked precursor X shown by general formula (D) in the scheme below, n1 is 6, n2 is 9, and n3 of the phospholipid mimetic precursor shown 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 glycan structure-containing group shown by general formula (B) to the phospholipid mimetic group shown by general formula (A) can be, for example, in the molar ratio range of 1:100 to 100:1, and preferably in the range of 1:10 to 10:1, and can be a ratio equivalent thereto.

The nanoparticles are similar to those described for the glycan-presenting particles. In the above scheme, colloidal quantum dots (QDs (QD: Quantum Dot)) were used as the raw material for the metal nanoparticles. Colloidal quantum dots have protective groups on the surface of luminescent semiconductor nanoparticles with diameters 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. When the nanoparticles are metal nanoparticles, colloidal metal nanoparticles can also be used as the raw material. Colloidal quantum dots and colloidal metal nanoparticles are commercially available.

The desired cancer glycan pattern is presented on the surface of the obtained surface-modified nanoparticles. The presentation of glycans can be performed by the glycoblotting method, in which the aminooxy group of the crosslinked precursor X represented by general formula (D) introduced into the surface-modified nanoparticles obtained above reacts specifically with glycans having a reducing end (equivalent to an aldehyde group).

The glycan-presenting particles of the present invention having a drug moiety present or carry a desired cancer glycan pattern and a drug-containing moiety on the surface of the surface-modified nanoparticle. The presentation or carrying of the glycan and the drug-containing moiety can be performed sequentially or simultaneously. For example, the drug-containing moiety can be carried after the glycan is presented by the glycoblotting method as described above. The drug-containing moiety can be obtained by mixing the surface-modified nanoparticle presenting the glycan with a drug-containing precursor Z represented by the following general formula (G) and linking it to the crosslinked precursor X on the surface-modified nanoparticle to further form a drug-containing group represented by general formula (C).

(In general formula (G), ^R20 is a drug-containing moiety.)

The glycan-containing portion and the drug-containing portion in the manufacturing method of the present invention are synonymous with the glycan-containing portion and the drug-containing portion in the particles of the present invention. The drug-containing precursor Z represented by general formula (G) can be synthesized, for example, according to the method described in Patent Document 1 (WO2017/131242A1/US2020138973(A1)).

(Method for determining glycan patterns of cancer cells)
The present invention involves profiling glycans present on the surface of cancer cells collected from a subject, and determining a glycan pattern based on the profiled glycans.
The profile of glycans on the surface of cancer cells can be determined by known analytical methods, as described above.

The glycan pattern obtained is determined by determining whether the profiled glycans are:
(1) Glycosylation pattern A, whose pharmacokinetics are mainly dependent on terminal high mannose type glycans;
(2) Glycosylation pattern B, whose pharmacokinetics are mainly dependent on terminal galactose-type or terminal N-acetylglucosamine-type glycans;
This can be achieved by identifying which glycan pattern is selected from the group consisting of (3) glycan pattern C, whose pharmacokinetics are mainly dependent on terminal α2,6 sialic acid type glycans, and (4) glycan pattern D, whose pharmacokinetics are mainly dependent on terminal α2,3 sialic acid type glycans.

More specifically, the sugar chain pattern is, for example,
Cancer glycan pattern A is characterized in that 45 mol % or more of the glycans are terminal high mannose type glycans, and 0% or more and less than 2% are terminal sialic acid type glycans;
Cancer glycan pattern B is characterized in that terminal high mannose type glycans are less than 45% and terminal sialic acid type glycans are 0% or more and less than 2%;
Cancer glycan pattern C is characterized in that 2-100% of the glycans are terminal sialic acid glycans, and the content of terminal α2,6 sialic acid glycans is greater than the content of terminal α2,3 sialic acid glycans;
In cancer sugar chain pattern D, 2-100% of the sugar chains are terminal sialic acid type sugar chains, and the content of terminal α2,3 sialic acid type sugar chains is higher than the content of terminal α2,6 sialic acid type sugar chains.

In the determination method of the present invention, when the determined glycan pattern is glycan pattern A, it shows type 1 pharmacokinetics, suggesting that the cancer cells have a low tendency to metastasize. When the determined glycan pattern is glycan pattern B, it shows type 2 pharmacokinetics, suggesting that the cancer cells have a tendency to metastasize to the liver and spleen. When the determined glycan pattern is glycan pattern C, it shows type 3 pharmacokinetics, suggesting that the cancer cells have a tendency to metastasize to the axillary and supraclavicular lymph nodes. When the determined glycan pattern is glycan pattern D, it shows type 4 pharmacokinetics, suggesting that the cancer cells have a tendency to metastasize to the lungs, liver, spleen, brain and kidneys.

Based on the glycan pattern of cancer cells determined by the method of the present invention, medical professionals can diagnose the metastatic potential of the target cancer cells by considering whether the glycan pattern of the cancer cells possessed by the subject indicates any of types 1 to 4 of endothelial dynamics.

The present invention will be described in more detail below with reference to examples. However, the examples are merely illustrative of the present invention, and the present invention is not intended to be limited to the examples.

Example 1
(1) Profile the post-translational glycan modification status (glycan expression patterns and their expression levels) of all proteins present in cancer cells and on the cell membrane surface

Glycoprofiles of human cancer cells :
Using approximately 5 x ¹⁰⁵ cells (approximately 100 μg of total protein) of four types of human cultured cancer cells (MCF7, MDA-MB-231, A549, HepG2), the total glycan profiles of these cancer cells were analyzed in detail according to a general protocol for cell glycoblotting (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537) (Fig. 2a). Next, the relationship between the expression levels of major structural motifs and their glycan structures was clarified by glycotyping analysis using internal standard compounds as indicators (Fig. 2b).

(2) Method for producing nanoparticles that display cancer glycan patterns (glycan-displaying particles) and analysis of their in vivo dynamics by real-time imaging after intravenous administration to mice (see Figure 1 for the principle and protocol)

(2-1) Preparation of cancer cell-derived glycans :
Human cultured cancer cells MCF7 (ATCC), A549 (JCBR), and HepG2 (RIKEN) were cultured at 1 x ¹⁰⁶ cells each in D-MEM High-glucose medium (Wako) containing 10% fetal bovine serum (FBS) at 37℃ and 5% _CO2 for 48 hours. For MDA-MB-231 (RIKEN), 1 x ¹⁰⁶ cells were cultured in Leibovitz's L-15 medium (Wako) containing 10% fetal bovine serum (FBS) at 37℃ for 48 hours to obtain 2-3 x ¹⁰⁶ cells (equivalent to approximately 500μg of total protein). The cells were detached with 1 mL of ice-cold 0.2 M phosphate buffer (pH 7.4), suspended in 1 mL of phosphate buffer (pH 7.4) containing 10 mM EDTA, and centrifuged at 10,000 g for 10 minutes at 4℃ to remove the supernatant. The residue was solubilized by adding 100 μL of a solution consisting of 0.1% SDS, 1% Triton-X100, and 100 mM ammonium bicarbonate, and the total protein was quantified by the BCA (bicinchoninic acid) method. An amount equivalent to 500 μg of protein was 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) at room temperature for 1 hour in a cool, dark place. 400 U of trypsin (Sigma Aldrich) was added to the protein mixture, which was then treated overnight at 37°C and heated at 90°C for 10 minutes to stop hydrolysis. 2 U of PNGaseF (Roche Applied Science) was added to the reaction solution, which was then reacted overnight at 37°C, and the solvent was removed using a centrifugal concentrator (SpeedVac) under reduced pressure to dry the mixture, preparing a sample for analysis.

(2-2) Method for preparing nanoparticles for displaying sugar chains :
According to the standard 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), quantum dots (1 μM TOPO-coated QD800 in decane, Thermo Fischer) were coated with 11-mercaptoundecylphosphorylcholine (hereafter abbreviated as PC-SH) and 11,11'-dithio bis[undec-11-yl 12-(aminooxyacetyl)amino We prepared nanoparticles (PC-SH/AOHEG-SH=80/1) completely coated with a mixed monolayer consisting of two types of alkanethiol derivatives of hexa(ethyleneglycol) (hereafter abbreviated as AOHEG-SH). Specifically, quantum dots (200μL, 1μM TOPO-coated QD800 in decane) were added to a mixed solvent of MeOH (200μL) and i-PrOH (400μL), and centrifuged at 15,000 g at room temperature for 5 minutes to insolubilize and precipitate the TOPO-coated QD800, and the supernatant was removed. The residue was added with n-hexane (400μL) to solubilize the TOPO-coated QD800, and PC-SH (32μL, 100 mM/MeOH, Medicinal Chemistry Pharmaceuticals), AOHEG-SH (2μL, 10 mM/MilliQ, Medicinal Chemistry Pharmaceuticals), NaBH ₄ (1μL, 12 wt% in 14 N NaOH) and Milli Q (200μL) were added to this solution and vigorously stirred at room temperature for 30 minutes using a vortex mixer. After leaving it 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 (500μL) (100μL, 2μM/MilliQ was used immediately for glycan blotting of cancer cells). At the same time, nanoparticles without sugar chains, coated with a monolayer of PC-SH only (PC-SH/AOHEG-SH=100/0), were prepared as a control.

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

(2-4) Pharmacokinetics and organ targeting of cancer glycan pattern-displaying nanoparticles (glycan-displaying particles) :
Cancer glycan 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 spectroscopy 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 major organs were removed and their fluorescence intensities were observed. In the following examples, the in vivo dynamics of the cancer glycan pattern-presenting nanoparticles (glycan-presenting particles) is described as the movement and excretion of the particles in the body after administration, and the distribution and accumulation of the particles in each organ or tissue is particularly described as organ tropism.

Figure 3 shows the pharmacokinetics of nanoparticles (GNS-MCF7, GNS-MDA-MB-231, GNS-A549, GNS-HepG2) presenting glycans derived from four types of human cultured cancer cells (MCF7, MDA-MB-231, A549, HepG2) and the control nanoparticles within 3 hours after intravenous administration to mice, as well as their distribution in each organ when dissected 3 hours later.

(i) Control (nanoparticles without glycans)
These experiments confirmed that the control, which was coated with a monolayer using only PC-SH, did not show any 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 (whole body) except the pancreas (S.-I. Nishimura et al., J. Am. Chem. Soc. 2011, 133, 12507-12517).

(ii) GNS-MCF7
As shown in Figures 2a and 2b, when the glycan pattern of non-metastatic breast cancer cells MCF7, which has a glycan pattern containing seven types of high mannose glycans and two types of galactose-terminated glycans that account for approximately 94% of the total glycans, was presented, most of the glycans were rapidly excreted from the body within approximately 15 minutes after intravenous administration without accumulating in any specific organs (Figure 3a). Even in mice dissected 3 hours later, almost no accumulation in any organs was observed, and some of the glycans were mixed into the feces in the gastrointestinal tract (Figure 3b). In other words, it was revealed that glycan-presenting particles presenting high mannose glycans of glycan pattern A (oligosaccharides with only mannose at the end) have an excretion mechanism and exhibit type 1 pharmacokinetics.

(iii) GNS- MDA-MB-231
The glycan pattern included five types of terminal sialic acid-type glycans, either di- or tri-antennary, which accounted for 18% of the total glycans, and five types of high mannose-type glycans, which accounted for 82% (Fig. 2a and 2b). When the glycan pattern of metastatic breast cancer cells, MDA-MB-231, was presented, the pharmacokinetics was completely different from that of GNS-MCF7, and accumulation in the axillary and supraclavicular lymph nodes was particularly prominent 3 hours after intravenous administration (indicated by arrows), indicating type 3 pharmacokinetics. In addition, the distribution of nanoparticles was confirmed in the lungs, heart, liver, spleen, kidneys, etc. up to 1 to 2 hours after administration, but most of them were excreted from the body by 3 hours. These results suggest that glycans containing Neu5Acα2,6Gal or Neu5Acα2,3Gal units at the terminus, which are glycan components on the surface of nanoparticles, determine the metastatic potential and organ tropism of breast cancer cells.

(iv) GNS-A549
The glycan pattern contained five types of terminal sialic acid-type glycans with two or three chains (19% of the total glycans), galactose-terminated glycans (11%), N-acetylglucosamine-terminated glycans (8%), and seven types of high mannose-type glycans (62%) (Fig. 2a and 2b). When the glycan pattern of A549 was presented, it was widely distributed throughout the body even 3 hours after intravenous administration, with particularly significant accumulation in the lungs, liver, and spleen, and distribution in the brain and kidneys, indicating type 4 pharmacokinetics.

(v) GNS-HepG2
Nanoparticles presenting the glycan pattern of HepG2, which contains two types of terminal sialic acid glycans (di- or tri-antennary) at the terminal (6%), galactose-terminated glycans (9%), N-acetylglucosamine-terminated glycans (13%), and seven types of high mannose-type glycans (72%) (Fig. 2a and 2b), showed type 4 pharmacokinetics very similar to that of GNS-A549. However, it was also revealed that GNS-A549, which contains 19% of Neu5Acα2,6Gal or Neu5Acα2,3Gal units in its total glycans, accumulated in the lungs, liver, and spleen at a significantly higher level than GNS-HepG2, which contains 6% of Neu5Acα2,3Gal units in its total glycans. However, the organ targeting (accumulation) of nanoparticles presenting glycans derived from these two types of cancer cells 3 hours after administration was significantly different in the case of GNS-HepG2 from that of GNS-MDA-MB-231. These differences were thought to be due to the binding mode of terminal sialic acid and galactose (α2,6 linkage vs. α2,3 linkage) influencing the organ targeting of nanoparticles after intravenous administration.

Example 2
(1) Glycan-presenting particles with artificially engineered glycan patterns that have different linkages between terminal sialic acid and galactose (α2,6 linkage and α2,3 linkage)
MDA-MB-231 cells, A549 cells, and HepG2 cells are all human cancer cells that significantly express diantennary or triantennary terminal sialic acid glycans as glycan components. However, in the results of Example 1, the pharmacokinetics and organ tropism were different between MDA-MB-231 cells, A549 cells, and HepG2 cells. From this, it was predicted that the binding mode of sialic acid and galactose (α2,6 bond or α2,3 bond) determines the difference in the pharmacokinetics of these glycan pattern-presenting nanoparticles after intravenous administration. Therefore, in this Example, nanoparticles that artificially display only one of the individual terminal sialic acid glycans linked by α2,6 bond or α2,3 bond were prepared in advance, and the pharmacokinetics and organ tropism of each were examined, and the difference in the pharmacokinetics due to the difference in the terminal sialic acid glycan was confirmed.

In ruddy duck egg white, the major glycan at the non-reducing end is a multibranched galactose glycan structure. Two sialyltransferases with known substrate specificities, Pasteurella multocida-derived α2,3-(N)-sialyltransferase and human α2,6-(N)-sialyltransferase, were used to modify the terminal galactose by adding sialic acid to induce a glycan pattern containing only Neu5Acα2,3Gal or Neu5Acα2,6Gal (Fig. 4a).

(1-1) Method for modifying ruddy duck egg white glycan profile using sialyltransferase :
Ruddy duck egg white (17 mg/50 μL in MilliQ, equivalent to approximately 1 mM LacNAc unit) was mixed with CMP-Neu5Ac (10 μL, 200 mM/milliQ, Yamasa), α2,3-(N)-sialyltransferase (5 μL, 1000 mU/mL, Sigma Aldrich), HEPES-NaOH buffer (10 μL, 1 M/milliQ, pH 8.0), and milliQ (10 μL) to make a total volume of 100 μL, and the mixture was incubated at 37°C for 20 hours. Similarly, 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 milliQ (10 μL) were added to ruddy duck egg white (17 mg/50 μL, equivalent to approximately 1 mM LacNAc unit) to make a total volume of 100 μL, and the mixture was reacted at 37°C for 20 hours.

(1-2) Glycosylation profiles of button quail, chicken, and ruddy duck egg white proteins and sialic acid-modified ruddy duck egg white :
Following the above process (A), five types of egg whites and sialic acid-modified egg whites were 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) at room temperature for 1 hour in a cool, dark place. 400 U of trypsin (Sigma Aldrich) was added to the protein mixture, which was then incubated overnight at 37°C and heated at 90°C for 10 minutes to stop hydrolysis. 2 U of PNGaseF (Roche Applied Science) was added to the reaction solution, which was then incubated overnight at 37°C. The solvent was removed under reduced pressure using a centrifugal concentrator (SpeedVac) to dry the mixture, and samples for glycan profiling were prepared (Figures 4b and 4c).

As is clear from Figures 4b and 4c, the glycans of Button Quail egg white are approximately 50% high mannose type glycans, 40% N-acetylglucosamine-terminated glycans, and a small amount of galactose termini, with no sialic acid at all. In chicken egg white, no sialic acid was detected as a glycan component, and approximately 40% were N-acetylglucosamine-terminated 3- to 5-branched glycans, 30% were galactose termini, and the remainder were high mannose type glycans. In Ruddy duck egg white, no sialic acid was detected as a glycan component, and approximately 65% were 3- to 5-branched glycans with galactose residues at the non-reducing end, with the remaining 25% being high mannose type glycans and approximately 10% being N-acetylglucosamine-terminated glycans. It was also confirmed that the glycans of these three bird egg whites are completely free of fucose residues.

According to the scheme in Figure 4a, ruddy duck egg white was treated with α2,3-(N)-sialyltransferase (recombinant Pasteurella multocida) and α2,6-(N)-sialyltransferase (recombinant human) in the presence of CMP-sialic acid to induce 2,3S-ruddy duck and 2,6S-ruddy duck with a large number of sialic acids added.

In addition, the glycan profiles of 2,3S-Ruddy duck and 2,6S-Ruddy duck were found to be very similar to those of human cancer cells A549 and HepG2 (Figs. 2b and 4c) by glycotyping (S.-I. Nishimura et al., Mol. Cell. Proteomics 2010, 9, 523-537).

(2) Organ targeting of glycan-presenting particles displaying artificially produced glycan patterns The egg white glycans of button quail, chicken, and ruddy duck prepared by the method of Example 2(1), glycan samples of two types of glycan patterns obtained by modifying the egg white glycan of ruddy duck (each prepared from an amount equivalent to 500 μg of total protein), and glycopeptide from chicken egg yolk (500 μg, 17 nmol; sialylglycopeptide, SGP, Tokyo Chemical Industry, sialic acid and galactose are bound in an α2,6-linked manner at the glycan site) were treated with PNGase according to the procedure of Example 1(2-1) to prepare crude products. The mixtures containing these glycans were mixed and reacted with a nanoparticle solution (100 μL, 2 μM/MilliQ: PC-SH/AOHEG-SH=80/1) according to the procedure of Example 1(2-3), and then purified to prepare glycan-pattern-presenting nanoparticles (GNSs). The average particle size of the obtained glycan pattern-presenting nanoparticles was 14.4-24.4 nm as measured by a fiber optic dynamic light scattering spectrometer FDLS-3000 (Otsuka Electronics Co., Ltd.). These glycan-presenting nanoparticles were intravenously administered to mice as described in Example 1 (2-4), and their pharmacokinetics and organ tropism were observed by near-infrared fluorescence microscopy (exposure time 1 sec, excitation wavelength 710 nm, detection wavelength 820 nm) (Fig. 5a-5c).

As shown in Figure 5, the glycan structure and its profile are clear, and it can be seen that the glycan-presenting particles of the present invention can be produced by presenting the glycan pattern obtained by modifying the glycans of quail, chicken, and duck egg white and chicken egg yolk, which are safe and inexpensive biological materials, on nanoparticles.

Specifically, (i) the retention time of GNS-Button Quail, which was prepared from Button Quail egg white, in which high mannose-type glycans account for approximately 50% of the total, in the mouse body was somewhat extended (Fig. 5b), but the pharmacokinetics 3 hours after administration (Fig. 5c) were very similar to those of GNS-MCF7 (Fig. 3c), with most of it being excreted from the body (type 1 pharmacokinetics).

(ii) GNS-Chicken and GNS-Ruddy duck, which show the glycan patterns of chicken and ruddy duck, show remarkably high accumulation in the liver and spleen (Fig. 5b and 5c) (type 2 pharmacokinetics). This is thought to be largely due to the specific interaction between the major highly branched glycans of GNS-Chicken and GNS-Ruddy duck, which have no sialic acid and terminate with galactose or N-acetylglucosamine, and asialoglycoprotein receptors, such as mammalian hepatic lectin, which are highly expressed in these organs and cells (e.g., Y. C. Lee et al., Acc. Chem. Res. 1997, 28, 321-327, etc.).

(iii) Analysis of the in vivo kinetics and organ tropism of two types of nanoparticles (GNS-2,3S-Ruddy duck and GNS-2,6S-Ruddy duck) that display glycan patterns derived from Ruddy duck egg white and GNS-2,6S-A2 prepared from chicken egg yolk glycopeptides revealed that the major terminal sialic acid-type glycans in MDA-MB-231 cells are di- or tri-antennary complex glycans containing Neu5Acα2,6Gal units at their termini (Figs. 5b and 5c). Furthermore, the difference in the in vivo kinetics and organ tropism of GNS-MCF7 (Figs. 3b and 3c) and GNS-Button Quail (Figs. 5b and 5c) revealed that the ratio (distribution density) of glycans containing Neu5Acα2,6Gal units at their termini to the total glycans (or high mannose-type glycans) on the exosome membrane surface determines the metastatic potential and organ tropism of breast cancer cells (as shown in Fig. 5b, GNS-2,6S-Ruddy duck nanoparticles). Accumulation in the axillary and supraclavicular lymph nodes was confirmed in duck, GNS-2, and 6S-A2 (type 3 pharmacokinetics).

On the other hand, (iv) the pharmacokinetics 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 pharmacokinetics), suggesting that the major terminal sialic acid type glycan structures in A549 and HepG2 cells are di- or tri-antennary complex glycans containing Neu5Acα2,3Gal units at their termini, and the difference in organ tropism between these two cancer cell-derived exosome models (Fig. 3c) suggests that the content of di- or tri-antennary complex glycans containing Neu5Acα2,3Gal units at their termini strongly influences not only the organ tropism of nanoparticles after intravenous administration but also their retention (blood concentration and half-life).

Example 3
In Example 2, it was revealed that the ratio of terminal sialic acid-type glycan components, in which the binding site with galactose was controlled by enzymatic modification of Ruddy duck egg white glycans, and high mannose-type glycan components to the total glycans has a significant effect on pharmacokinetics and organ tropism. Therefore, glycans derived from Japanese quail egg white (S.-I. Nishimura et al., J. Agri. Food Chem. 2018, in press), which have a higher content of high mannose-type glycans (about 70%) than button quail egg white (50%) and a simpler glycan pattern, and 2,6S-A2 glycans derived from chicken egg yolk glycopeptides were mixed in any ratio and presented on nanoparticles to produce the glycan-presenting particles of the present invention. The average particle diameter of the obtained cancer glycan pattern-presenting nanoparticles was 27.3 nm as measured by a fiber optic dynamic light scattering spectrometer FDLS-3000 (Otsuka Electronics).

(3-1)
Three types of glycans were prepared by adding 0.6 nmol, 1.2 nmol, and 2.4 nmol of 2,6S-A2 glycan sample (17 nmol; prepared from 500 μg of SGP) to a glycan sample derived from Japanese Quail egg white (approximately 6 nmol; prepared from 100 μg of total protein). The profiles of these glycans were analyzed by glycoblotting (the percentages in the figure indicate the proportion of the total 2,6S-A2 glycans calculated from each peak area) and are shown in Figure 6a and Table 7. These glycan 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), and a total of five types of glycan pattern-presenting nanoparticles (GNSs) were prepared. Nanoparticles presenting artificial glycan patterns were prepared by mixing two types of glycans (patterns).

The present invention makes it possible to construct glycan-presenting particles derived from cancer cells, which are useful for the prevention and treatment of cancer.

Claims (19)

A glycan-presenting particle which is a nanoparticle having a glycan on the surface thereof,
(1) the average particle size of the glycan-presenting particle is in the range of 10 to 100 nm;
(2) At least a portion of the surface of the nanoparticle is covered with a phospholipid;
(3) the glycan on the nanoparticle surface is a glycan pattern derived from a cancer cell or a glycan pattern determined based on the profile of this glycan (hereinafter referred to as a cancer glycan pattern);
Cancer glycan patterns are
(1) Cancer glycan pattern A, in which 50% to 95% of the glycans are terminal high mannose type glycans and terminal sialic acid type glycans are 0% or more and less than 2%;
(2) Cancer glycan pattern B, in which terminal high mannose-type glycans are less than 45%, terminal sialic acid-type glycans are 0% or more and less than 2%, and further contains terminal galactose-type glycans and terminal N-acetylglucosamine-type glycans;
(3) Cancer glycan pattern C, in which 5-60% of the glycans are terminal sialic acid glycans and the content of terminal α2,6 sialic acid glycans is higher than the content of terminal α2,3 sialic acid glycans;
(4) A glycan-presenting particle having a glycan pattern selected from the group consisting of cancer glycan pattern D, in which 5-60% of the glycans are terminal sialic acid type glycans and the content of terminal α2,3 sialic acid type glycans is higher than the content of terminal α2,6 sialic acid type glycans. Cancer glycan pattern A is a glycan pattern whose pharmacokinetics mainly depends on terminal high mannose type glycans,
Cancer glycan pattern B is a glycan pattern whose pharmacokinetics are mainly dependent on terminal galactose-type glycans or terminal N-acetylglucosamine-type glycans.
Cancer glycan pattern C is a glycan pattern whose pharmacokinetics are mainly dependent on terminal α2,6 sialic acid type glycans;
The sugar chain-presenting particle according to claim 1, wherein the cancer sugar chain pattern D is a sugar chain pattern whose pharmacokinetics mainly depends on terminal α2,3 sialic acid type sugar chains. The glycan-presenting particle according to any one of claims 1 to 2, wherein the phospholipid that covers at least a part of the nanoparticle surface is a sulfide bond of an alkanethiol having a phosphorylcholine group, the glycan on the nanoparticle surface is a sulfide bond of an alkanethiol having a glycan immobilized thereon, and the nanoparticle surface is covered with a monolayer of the sulfide bond of an alkanethiol having a phosphorylcholine group and the sulfide bond of an alkanethiol having a glycan immobilized thereon. The sulfide bond of an alkanethiol having a phosphorylcholine group is represented by the following general formula (A):
(In general formula (A), n3 is an integer ranging from 2 to 30, and the -S- end is a supporting site that is sulfide-bonded to the nanoparticle.)
The sugar chain-presenting particle according to claim 3 , wherein the sulfide bond of alkanethiol to which the sugar chain is immobilized is represented by the following general formula (B):
(In general formula (B), n1 is an integer from 2 to 30, n2 is an integer from 2 to 30, the -S- end is a supporting moiety that is sulfide-bonded to the nanoparticle, and R ¹⁰ is a sugar chain-containing moiety.) The glycan-presenting particle according to any one of claims 1 to 4, further comprising a drug moiety. 5. The sugar chain-presenting particle according to claim 3, wherein the monolayer further comprises a sulfide bond of an alkanethiol having a drug moiety, represented by the following general formula (C):
In general formula (C), n1 is an integer from 2 to 30, n2 is an integer from 2 to 30, the -S- terminus is a loading moiety that is sulfide-bonded to the nanoparticle, and R ²⁰ is a drug-containing moiety. A glycan-presenting particle kit comprising two or more types of glycan-presenting particles having different cancer glycan patterns, the glycan-presenting particles being the glycan-presenting particles described in any one of claims 1 to 6. The glycan-presenting particle kit according to claim 7, wherein the different cancer glycan patterns are any two or more of the glycan patterns A to D according to claim 1. The glycan-presenting particle kit according to claim 7, wherein the glycan-presenting particle further has a drug moiety according to claim 5 or 6. A drug for preventing cancer metastasis, comprising the glycan-presenting particle according to claim 5 or 6 as an active ingredient. A cancer treatment drug containing the glycan-presenting particle according to claim 5 or 6 as an active ingredient. The method includes profiling glycans of cancer cells collected from a subject, and determining a glycan pattern based on the profiled glycans;
The determination of the glycan pattern is carried out by determining whether the profiled glycans are
(1) Cancer glycan pattern A, in which 50% to 95% of the glycans are terminal high mannose type glycans and terminal sialic acid type glycans are 0% or more and less than 2%;
(2) Cancer glycan pattern B, in which terminal high mannose-type glycans are less than 45% and terminal sialic acid-type glycans are 0% or more and less than 2%, and further contains terminal galactose-type glycans and terminal N-acetylglucosamine-type glycans;
(3) Cancer glycan pattern C, in which 5-60% of the glycans are terminal sialic acid glycans and the content of terminal α2,6 sialic acid glycans is higher than the content of terminal α2,3 sialic acid glycans;
(4) by identifying whether the glycan pattern is selected from the group consisting of cancer glycan pattern D, in which 5-60% of the glycans are terminal sialic acid type glycans and the content of terminal α2,3 sialic acid type glycans is higher than the content of terminal α2,6 sialic acid type glycans;
A method for determining the glycan pattern of cancer cells. Glycosylation pattern A is a glycan pattern whose pharmacokinetics mainly depends on terminal high mannose type glycans;
Glycosylation pattern B is a glycan pattern whose pharmacokinetics mainly depends on terminal galactose-type glycans or terminal N-acetylglucosamine-type glycans;
Glycosylation pattern C is a glycan pattern whose pharmacokinetics mainly depends on terminal α2,6 sialic acid type glycans;
The method for determining sugar chain pattern D according to claim 12, wherein the pharmacokinetics of sugar chain pattern D is mainly dependent on terminal α2,3 sialic acid type sugar chains. When the glycan profile of cancer cells is glycan pattern A, the cancer cells of the subject exhibit type 1 pharmacokinetics in which excretion of particles from the body is promoted compared to glycan-presenting particles that do not have glycans, suggesting that the cancer cells have a low tendency to metastasize;
When the glycan profile is glycan pattern B, the cancer cells present in the subject exhibit type 2 pharmacokinetics, which accumulates in the liver and spleen, compared with glycan-presenting particles not having glycans, 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 of the subject show type 3 pharmacokinetics, which are accumulated in the axillary and supraclavicular lymph nodes, suggesting that the cancer cells have a tendency to metastasize to the axillary and supraclavicular lymph nodes;
The method of claim 12 or 13, wherein when the glycan profile is glycan pattern D, the cancer cells present in the subject exhibit type 4 pharmacokinetics distributed to the organs of the lungs, liver, spleen, brain, and kidneys compared to glycan-presenting particles not having glycans, suggesting that the cancer cells have a tendency to metastasize to the lungs, liver, spleen, brain, and kidneys. A method for producing a glycan-presenting particle according to any one of claims 1 to 6, comprising presenting a cancer glycan pattern on the surface of a nanoparticle at least part of which is coated with a phospholipid. The method of claim 15, wherein the presented cancer glycan pattern is a cancer glycan pattern obtained by excising glycans from cancer cells collected from a subject, or a glycan pattern determined based on a profile obtained by profiling the glycans of cancer cells collected from a subject. The nanoparticles having at least a part of the surface coated with a phospholipid can be produced by mixing a crosslinked precursor X represented by the following general formula (D) and a phospholipid precursor represented by the following general formula (E) with a colloidal nanoparticle to obtain a surface-modified nanoparticle carrying the crosslinked precursor X and the phospholipid on the surface of the nanoparticle. The production method according to claim 15 or 16.
(In general formula (D), n1 is an integer from 2 to 30, and n2 is an integer from 2 to 30.)
(In general formula (E), n3 is an integer ranging from 2 to 30.) The method of claim 17, wherein the cancer glycan pattern is presented by reacting, by glycoblotting, an aminooxy group possessed by the crosslinked precursor X represented by general formula (D) introduced into the surface-modified nanoparticle with a cancer glycan pattern obtained by cutting out the glycan of a cancer cell collected from a subject, or with a reducing end possessed by a glycan contained in a glycan pattern determined based on a profile obtained by profiling the glycan of a cancer cell collected from a subject. The method of any one of claims 16 to 18, wherein the glycan pattern determined based on the profile is a glycan pattern in which the type and content of glycan components are the same as those in the profile, or a glycan pattern in which part of the type and content of glycan components are the same as those in the profile.

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