CN117794579A - Cancer therapeutic agent - Google Patents

Abstract

The present invention provides a therapeutic agent for cancer against a cancer tissue containing CD 209-positive cells. The therapeutic agent for cancer against a cancer tissue containing CD 209-positive cells contains gel particles containing a modified polysaccharide containing a hydrophobic group.

Description

Cancer therapeutic agent

Technical Field

The present invention relates to a cancer therapeutic agent and the like.

Background

As factors affecting the malignancy of cancer, the importance of immune cells, particularly macrophages (tumor-associated macrophages, abbreviated TAMs), present in cancer tissues is pointed out. Agents have been developed that exert therapeutic effects on cancer by inhibiting or modulating the function of TAMs. However, there is a limitation in selectivity, and there is no practical agent so far in terms of effectiveness or safety. Accordingly, it is desirable to seek techniques that enable selective delivery of agents to TAMs.

CD209 (alias: DC-SIGN) is known to be a type of immune polysaccharide binding receptor protein (lectin), and is selectively expressed in a part of TAM, and has good correlation with the stage and prognosis of cancer. CD209 is considered a good target for drug delivery to TAMs.

It has been reported that hydrophobized polysaccharides such as cholesterol pullulan can self-organize to form a nanogel, and have high biocompatibility, functions as a protein carrier, and capabilities of decomposing and inhibiting aggregation of an entrapped protein, and therefore can be used as an antigen carrier by complexing with a protein (non-patent document 1).

Prior art literature

Non-patent literature

Non-patent document 1: muraoka, D.et al, ACS Nano,8 (9), 9209-9218,2014

Disclosure of Invention

Technical problem to be solved by the invention

The technical problem of the present invention is to provide a therapeutic agent for cancer against a cancer tissue containing CD 209-positive cells.

Technical solution for solving technical problems

The present inventors have conducted intensive studies in view of the above-described technical problems, and as a result, found that: gel particles containing modified polysaccharides containing hydrophobic groups are selectively incorporated into CD209 positive cells. The inventors of the present invention have further studied based on this finding, and as a result, completed the present invention. Namely, the present invention includes the following.

Item 1. A cancer therapeutic agent for a cancer tissue containing CD 209-positive cells, which contains gel particles containing a modified polysaccharide containing a hydrophobic group.

The therapeutic agent for cancer according to item 1, wherein the gel particles are nanogel particles.

The therapeutic agent for cancer according to item 1 or 2, wherein the structural polysaccharide of the modified polysaccharide comprises at least 1 selected from the group consisting of pullulan, mannan and glucan.

The therapeutic agent for cancer according to any one of items 1 to 3, wherein the structural polysaccharide of the modified polysaccharide comprises pullulan.

The therapeutic agent for cancer according to any one of items 1 to 4, wherein the hydrophobic group includes a hydrophobic group having a sterol skeleton.

The therapeutic agent for cancer according to any one of items 1 to 5, wherein the modified polysaccharide has a weight average molecular weight of 5000 ～ 2,000,000.

The therapeutic agent for cancer according to any one of items 1 to 6, wherein the gel particles have a weight average particle diameter of 20 to 200nm.

The therapeutic agent for cancer according to any one of items 1 to 7, wherein the gel particles contain a drug.

The therapeutic agent for cancer according to item 8, wherein the agent contains at least 1 selected from the group consisting of an adjuvant, a cancer antigen, an anticancer agent and a nucleic acid drug.

The therapeutic agent for cancer according to any one of items 1 to 9, wherein the CD 209-positive cell is a macrophage.

The cancer therapeutic agent according to any one of items 1 to 10, which is used for administration to a patient having a cancer tissue containing CD 209-positive cells selected from patients having a cancer tissue.

The cancer therapeutic agent according to any one of items 1 to 11, for use in combination with a pharmaceutical agent.

Item 13. A delivery vehicle to CD 209-positive cells comprising gel particles comprising a modified polysaccharide comprising a hydrophobic group.

The delivery vehicle of item 13, wherein the CD209 positive cells are present in the cancerous tissue.

Item 15. A detection agent for tissue comprising CD 209-positive cells comprising the delivery vehicle of item 13 or 14 and a contrast agent.

Item 15 is a diagnostic drug for cancer therapeutic agent according to any one of items 1 to 12, which contains a CD209 binding molecule.

Item 1A. A method of treating cancer comprising: a step of administering gel particles containing a modified polysaccharide containing a hydrophobic group to a patient having a cancerous tissue containing CD 209-positive cells.

Item 1B the method of item 1A, comprising: a step of selecting a patient having a cancer tissue containing CD 209-positive cells from patients having a cancer tissue; and a step of administering gel particles containing a modified polysaccharide containing a hydrophobic group to the selected patient.

The gel particles containing a modified polysaccharide having a hydrophobic group or the preparation containing the gel particles, which are useful as a therapeutic agent for cancer against a cancer tissue containing CD 209-positive cells.

Use of a gel particle containing a modified polysaccharide containing a hydrophobic group or a preparation containing the gel particle in the manufacture of a cancer therapeutic agent for a cancer tissue containing CD 209-positive cells.

The use of a gel particle containing a modified polysaccharide containing a hydrophobic group or a preparation containing the above gel particle as a cancer therapeutic agent against a cancer tissue containing CD 209-positive cells in item 1E.

The method for delivering a drug or a contrast agent to a CD 209-positive cell, comprising the step of applying the drug or the contrast agent to a gel particle containing a modified polysaccharide having a hydrophobic group.

The delivery method of item 2B, wherein the CD209 positive cells are present in the cancerous tissue.

The gel particles containing a modified polysaccharide having a hydrophobic group or the preparation containing the gel particles, which are used as a carrier for delivering to CD 209-positive cells.

Use of a gel particle comprising a modified polysaccharide comprising a hydrophobic group or a formulation comprising the gel particle for the manufacture of a carrier for delivery to CD209 positive cells.

Use of a gel particle comprising a modified polysaccharide comprising a hydrophobic group or a formulation comprising the gel particle as a carrier for delivery to CD209 positive cells.

Item 3A. A method of detecting a tissue containing CD 209-positive cells, comprising: a step of administering the delivery vehicle of item 13 or 14 and a contrast agent or a preparation containing them to a subject having a tissue containing CD 209-positive cells.

Item 3B. The delivery vehicle and contrast agent of item 13 or 14, or a formulation containing the same, for use as a detection agent for tissue containing CD209 positive cells.

Use of the delivery vehicle of item 13 or 14 and a contrast agent or a formulation containing them in the manufacture of a detector for a tissue containing CD209 positive cells.

The use of the delivery vehicle of item 13 or 14 and a contrast agent or a formulation containing the same as a detector of a tissue containing CD209 positive cells.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, a therapeutic agent for cancer against a cancer tissue containing CD 209-positive cells can be provided.

Drawings

FIG. 1 shows the results of measuring the uptake rate of rhodamine-labeled pullulan nanogel in each cell by a flow cytometer in test example 1.

FIG. 2 shows the results of measuring the uptake rate of rhodamine-labeled pullulan nanogel in each cell by a flow cytometer in test example 2.

FIG. 3 shows the results of observation of uptake (red) of rhodamine-labeled pullulan nanogels in each cell with a fluorescence microscope in test example 3.

FIG. 4 shows the results of measuring the uptake intensity (fluorescence intensity) of rhodamine-labeled pullulan nanogel in each cell by a flow cytometer in test example 4.

FIG. 5 shows the results of measuring the uptake intensity (fluorescence intensity) of rhodamine-labeled pullulan nanogel in each cell by a flow cytometer in test example 5.

FIG. 6 shows the results of observation with a fluorescence microscope and evaluation of uptake of rhodamine-labeled pullulan nanogels in each cell with a flow cytometer in test example 6.

FIG. 7 shows the results of evaluating uptake of rhodamine-labeled pullulan nanogels in the indicated cell mass by flow cytometry in test example 7.

FIG. 8 shows the results of evaluation of uptake of rhodamine-labeled pullulan nanogels by fluorescent microscopy in test example 8.

FIG. 9 shows the results of evaluating uptake of rhodamine-labeled pullulan nanogels in the indicated cell mass by flow cytometry in test example 9.

FIG. 10 shows the results of evaluating the cell activity using the cell count kit-8 (Cell Counting Kit-8) in test example 10. The vertical axis represents the relative proportion of viable cell count, and the horizontal axis represents the applied concentration of pullulan nanogel PE complex or PE. In the legend, 5a indicates the case of using CMS5a cells, and 209b indicates the case of using cells expressing mouse SIGNR1/CD209b in CMS5a cells.

FIG. 11 shows the results of measuring tumor sizes with time in test example 11. The vertical axis represents tumor size (unit: mm) ³ ) The horizontal axis represents the number of days elapsed from the start of administration.

Detailed Description

In this specification, the expression "containing" and "including" includes the concepts of "containing" and "including", "consisting essentially of … …" and "consisting of … … only".

In one embodiment, the present invention relates to a cancer therapeutic agent (also referred to as "cancer therapeutic agent of the present invention" in the present specification) for a cancer tissue containing CD 209-positive cells, which comprises gel particles containing a modified polysaccharide containing a hydrophobic group. This will be described below.

The modified polysaccharide is not particularly limited as long as it is a compound modified with a polysaccharide and contains a hydrophobic group as a modification group.

The polysaccharide constituting the modified polysaccharide (i.e., the polysaccharide before modification) is not particularly limited as long as it is a polymer in which sugar residues are bonded by glycosidic bonds. As the sugar residue constituting the polysaccharide, for example, a residue of a sugar such as a monosaccharide, a disaccharide, or an oligosaccharide derived from glucose, mannose, galactose, or fucose can be used. The sugar residues may be bound in 1,2-, 1,3-, 1, 4-or 1, 6-glycosidic linkages, which may be any of the alpha-or beta-type linkages. The polysaccharide may be any of linear or branched polysaccharides. The sugar residue is preferably a glucose residue, and as the polysaccharide, for example, pullulan, mannan, glucan, amylose, amylopectin, etc. of natural or synthetic origin can be used, and pullulan, mannan, glucan, etc. are preferably used, pullulan, mannan, etc. are more preferably used, and pullulan, etc. are particularly preferably used.

The weight average molecular weight of the polysaccharide is not particularly limited as long as the modified polysaccharide can be formed into gel particles, and is, for example, 5,000 ～ 2,000,000. The weight average molecular weight is preferably 10,000 ～ 1,000,000, more preferably 20,000 ～ 500,000, further preferably 40,000 ～ 250,000, further preferably 80,000 ～ 125,000.

As the polysaccharide, a commercially available product or a product obtained by a known production method can be used.

The hydrophobic group is not particularly limited as long as it is a group having hydrophobicity and can make the modified polysaccharide constitute gel particles. The hydrophobic group is preferably a hydrophobic group having a sterol skeleton, a hydrocarbon group, or the like, and particularly preferably a hydrophobic group having a sterol skeleton.

The sterol skeleton is an alcohol having a hydroxyl group bonded to a cyclopentahydrophenanthrene ring represented by formula (I). The symbols A to D of the formula (I) represent the rings constituting the cyclopentahydrophenanthrene ring.

In the sterol skeleton, a double bond may be present on the cyclopenthydrophenanthrene ring, and the bonding position of the hydroxyl group is not particularly limited. Preferably a solid alcohol having a hydroxyl group bonded to the C-3 position and a double bond in the B ring; or a stanol comprising a saturated ring, wherein a hydroxyl group is bonded to the C-3 position. Examples of the hydrophobic group having a sterol skeleton include a group derived from a compound in which a sterol skeleton is modified, for example, a hydrocarbon group (for example, a linear or branched alkyl group having 1 to 20 carbon atoms) is substituted in a ring-constituting carbon. The term "group derived from … …" as used herein means a group obtained by removing a functional group such as a hydrogen atom or a hydroxyl group from a certain compound.

Examples of the hydrophobic group having a sterol skeleton include a cholesterol-derived group, a cholestanol-derived group, a lanosterol-derived group, an ergosterol-derived group, a β -sitosterol-derived group, a campesterol-derived group, a stigmasterol-derived group, and a brassicasterol-derived group. Among these, cholesterol-derived groups, cholestanol-derived groups, lanosterol-derived groups, ergosterol-derived groups and the like are preferable, and cholesterol-derived groups are more preferable.

The hydrocarbon group as the hydrophobic group is not particularly limited, and examples thereof include a chain (preferably straight-chain) hydrocarbon group (preferably an alkyl group) having 8 to 50 carbon atoms (preferably 10 to 30, more preferably 12 to 20).

The weight average molecular weight of the modified polysaccharide is not particularly limited as long as the modified polysaccharide can be formed into gel particles, and is, for example, 5,000 ～ 2,000,000. The weight average molecular weight is preferably 10,000 ～ 1,000,000, more preferably 20,000 ～ 500,000, further preferably 40,000 ～ 250,000, further preferably 80,000 ～ 125,000.

The number of the hydrophobic groups contained in the modified polysaccharide is not particularly limited as long as the modified polysaccharide can be formed into gel particles, and the number of the hydrophobic groups is, for example, 1 to 10, preferably 1 to 5 per 100 saccharide residues constituting the polysaccharide.

The hydrophobic group may be attached to the polysaccharide directly or indirectly (e.g., via a linker).

As the modified polysaccharide, for example, it is preferable that the primary hydroxyl groups of, for example, 1 to 10 (preferably 1 to 5) sugar units per 100 sugar residues constituting the polysaccharide are represented by the formula (II): -O- (CH) ₂ ) _m CONH(CH ₂ ) _n NH-CO-O-R (II) (wherein R represents a hydrophobic group having a sterol skeleton or a hydrocarbon group; m represents 0 or 1; and n represents an arbitrary positive integer). n is preferably 1 to 8.

The modified polysaccharides can be synthesized according to or based on a known method (for example, international publication No. WO 00/12564). As an example, the following method can be mentioned. First, a hydrocarbon or sterol having a hydroxyl group and having 12 to 50 carbon atoms is reacted with OCN-R ¹ NCO (wherein R is ¹ Is a hydrocarbon group having 1 to 50 carbon atoms. ) The diisocyanate compound shown is reacted to produce an isocyanate group-containing hydrophobic compound obtained by reacting 1 molecule of a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms. Next, the obtained isocyanate group-containing hydrophobic compound is further reacted with a polysaccharide to produce a polymer containing a carbon atom as a hydrophobic groupA polysaccharide containing a hydrophobic group, which is a hydrocarbon group or a sterol group having a number of 12 to 50. The obtained reaction product is purified with a ketone solvent, whereby a high-purity polysaccharide containing a hydrophobic group can be produced.

The modified polysaccharides may be 1 kind alone or 2 or more kinds in combination.

The gel particles contain modified polysaccharides. The term "gel particles" refers to polymer gel particles having a hydrogel structure. Hydrogels are those obtained by swelling a three-dimensional network structure formed by crosslinking hydrophilic polymers with water. In the gel particles, the modified polysaccharides are self-organized via a physical crosslinking moiety formed based on hydrophobic interactions of hydrophobic groups to form a three-dimensional network structure.

The shape of the gel particles is not particularly limited, and is generally spherical.

The gel particles are preferably nano-sized (i.e., nanogel particles) and have a weight average particle diameter of, for example, 200nm or less, preferably 10 to 200nm, more preferably 15 to 200nm, and still more preferably 20 to 200nm. The upper limit of the weight average particle diameter is preferably 100nm, more preferably 70nm, and further preferably 50nm. The particle size can be measured by dynamic light scattering.

The gel particles may contain other substances than the modified polysaccharides. Examples of the other substance include proteins, peptides, nucleic acids, saccharides, low-molecular compounds, high-molecular compounds, and inorganic substances, and further include a complex thereof. More specifically, examples of the other substance include an adjuvant, a cancer antigen, an anticancer agent, a drug such as a nucleic acid drug, a contrast agent, and the like.

The adjuvant may be selected from inactivated bacterial cells, bacterial cell extracts, lipopolysaccharides, lipopeptides, synthetic low-molecular compounds, etc., and preferably imidazoquinoline (e.g., R848 or imiquimod, etc.), saponin (e.g., quilA or QS21, etc.), STING agonists (e.g., cyclic di-GMP, etc.), monophosphoryl lipids, lipopeptides, etc. are used. Examples of the adjuvants other than the above include taxane drugs, anthracycline drugs, JAK/STAT inhibitors, indole Dioxygenase (IDO) inhibitors, tryptophan Dioxygenase (TDO) inhibitors, and the like. Among these inhibitory substances, neutralizing antibodies to the factor are included in addition to compounds having antagonism to the factor.

The cancer antigen may be exemplified by an antigen polypeptide, preferably an antigen polypeptide. An antigenic polypeptide is an antigen or a partial peptide thereof that is expressed in large amounts in cancer cells, and in some cases, is expressed only by cancer cells. The antigenic polypeptide can be expressed within a cancer cell, or on the surface of a cancer cell.

The antigen polypeptide is not limited, can be selected from ERK1, ERK2, MART-1/Melan-A, gp, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin B (Cyclophilin B), colorectal associated antigen (CRC) -C017-1A/GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate Specific Antigen (PSA), PSA-1, PSA-2, PSA-3, prostate Specific Membrane Antigen (PSMA) T cell receptor/CD 3-zeta chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5 GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8 and GAGE-9, BAGE, RAGE, LAGE-1, NAG, gnT-V, MUM-1, CDK4, tyrosinase, P53, MUC family, HER2/neu, P21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, P120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), cytoliner protein, connexin 37, ig idiotype, P15, gp75, GM2 ganglioside, GD2 ganglioside, human papilloma virus protein, smafamily of tumor antigens, smaP-1, P1A, EBV encoded Nuclear Antigen (NA) -1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, c-erbB-2, their partial peptides.

Examples of the anticancer agent include alkylating agents, metabolic antagonists, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum agents, molecular target agents, hormonal agents, biological agents, and the like.

Examples of alkylating agents include cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ramustine.

Examples of the metabolic antagonist include elcitabine, carmofur, capecitabine, tegafur-uracil, tegafur-gimeracil-octreotide-potassium octreotide, gemcitabine, cytarabine octadecyl phosphate, nelarabine, fluorouracil, fludarabine, pemetrexed, pentastatin, methotrexate, cladribine, deoxyfluorouridine, hydroxyurea, mercaptopurine, and the like.

Examples of microtubule inhibitors include alkaloid anticancer agents such as vincristine, and taxane anticancer agents such as docetaxel and paclitaxel.

Examples of antibiotic anticancer agents include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, aclacinomycin, idarubicin, pirarubicin, pelomycin, mitoxantrone, amrubicin, and imipramine Ding Sizhi.

Examples of the topoisomerase inhibitor include CPT-11 having a topoisomerase I inhibitory activity, irinotecan, nogetecan (Nogitean), etoposide having a topoisomerase II inhibitory activity, and Sobrozocine.

Examples of the platinum preparation include cisplatin, nedaplatin, oxaliplatin, and carboplatin.

Examples of hormonal agents include dexamethasone, finasteride, tamoxifen, anastrozole, exemestane, ethinyl estradiol, chlordydrogesterone, goserelin, bicalutamide, flutamide, prednisolone, leuprorelin, letrozole, estramustine, toremifene, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, and melandrane.

Examples of biological agents include interferon α, β, and γ, interleukin 2, ubenimex (Ubenimex), dry BCG, and extracellular toxins. Specific examples of the extracellular toxins include those produced by bacteria such as pseudomonas exotoxin (Pseudomonas Exotoxin, PE), which has an enzymatic activity of nad+ -diphtheria amide-ADP-ribosyl transferase (nad+ -diphtheria amide-ADP-ribosyl transferase), and which inhibit protein synthesis and exert strong toxicity. Up to now, conjugates of PE and antibodies such as CD22 antibodies have been approved as anticancer agents.

Examples of the molecular target include rituximab, alemtuzumab, trastuzumab, cetuximab, panitumumab, imatinib, dasatinib, nilotinib, gefitinib, erlotinib, temsirolimus (Temsirolimus), bevacizumab, VEGF trap, sunitinib, sorafenib, tolizumab, bortezomib, gemtuzumab-ozumicin (Gemtuzumab-ozigamicin), temozogamicin-ozuzumab (iblitumomab-ozigamicin), temozolomab (Ibritumomab Tiuxetan), tamibarotene, and retinoic acid. In addition to the specific molecular target drugs, the present invention may also include various kinds of tyrosine kinase inhibitors such as human epidermal growth factor receptor 2 inhibitors, epidermal growth factor receptor inhibitors, bcr-Abl tyrosine kinase inhibitors, epidermal growth factor tyrosine kinase inhibitors, mTOR inhibitors, vascular endothelial growth factor receptor 2 inhibitors (α -VEGFR-2 antibodies) and the like, angiogenesis inhibitors such as MAP kinase inhibitors and the like, cytokine inhibitors, proteasome inhibitors, antibody-anticancer agent complexes and the like. Antibodies are also included in these inhibitors.

The nucleic acid drug is not particularly limited as long as it is a pharmaceutically active ingredient of nucleic acid.

The contrast medium is not particularly limited as long as it can visualize the site where the contrast medium exists. Examples of the contrast agent include X-ray contrast agents such as barium sulfate, bismuth subcarbonate, bismuth oxide, zirconium oxide, ytterbium fluoride, iodoform, barium apatite, barium titanate, lanthanum glass, barium glass, and strontium glass; a contrast medium for computed tomography (Computed Tomography, CT) such as an iodine contrast medium; a contrast agent for MRI such as gadolinium preparation and superparamagnetic iron oxide preparation (Super Paramagnetic Iron Oxide, SPIO); single photon emission computed tomography (Single photon emission computed tomography, SPECT) of technetium 99m (99 mTc), molybdenum 99 (99 Mo), and the like uses a radioisotope, other various radioisotopes, or substances containing the same, and the like.

The content of the other substance is, for example, 0 to 10000 parts by mass, 0 to 1000 parts by mass, 0 to 500 parts by mass, 0 to 100 parts by mass, 0 to 50 parts by mass, or 0 to 10 parts by mass, relative to 100 parts by mass of the content of the modified polysaccharide.

The gel particles can be produced according to a known method. For example, the modified polysaccharide-containing solution can be produced by subjecting the solution to ultrasonic treatment, or adding a modifying agent such as urea, DMSO, or a surfactant to the solution and then removing the modifying agent by dialysis. The former method is more preferable from the viewpoint of simplicity. The latter method can be carried out according to or based on known methods.

The modified polysaccharide-containing solution can be prepared by dissolving the modified polysaccharide in a solvent. As the solvent, for example, an organic solvent such as water or DMSO can be used. The modified polysaccharide solutions can generally be prepared using water as a solvent. In this case, a buffer such as PBS is preferably used instead of water.

The concentration of the modified polysaccharide in the modified polysaccharide-containing solution is not particularly limited, and is, for example, 5 to 100. Mu.M, preferably 5 to 80. Mu.M, more preferably 8 to 50. Mu.M, from the viewpoint of the formation efficiency of gel particles, etc.

For example, a plastic tube containing a modified polysaccharide-containing solution is fixed to water in a bath-type ultrasonic tank, and ultrasonic treatment is performed by irradiating the water with ultrasonic waves. The conditions of the ultrasonic treatment are not particularly limited, and are, for example, 10 to 40 ℃ (preferably 20 to 35 ℃), 10 to 50kHz (preferably 20 to 40 kHz), 30 to 200W (preferably 70 to 150W), and 2 to 30 minutes (preferably 5 to 15 minutes).

In one embodiment, the gel particles are used as a cancer therapeutic against a cancer tissue containing CD209 positive cells. The gel particles target CD209, bind to CD209 expressing cells, and ingest the gel particles. Thus, the components contained in the gel particles can be selectively delivered to the cancerous tissue containing CD 209-positive cells. Specifically, for example, a drug (adjuvant, cancer antigen, anticancer agent, nucleic acid drug, etc.) contained in the gel particles is selectively delivered to a cancer tissue containing CD 209-positive cells, and an immune activation effect, an anticancer effect, etc. are exerted, whereby cancer can be treated. Specifically, for example, cancer can be treated by taking in CD 209-positive cells (preferably macrophages and tumor-associated macrophages) with a cancer antigen contained in gel particles, presenting the antigen to the cells, and activating lymphocytes such as T cells.

Since the gel particles are targeted at CD209 and bind to cells expressing CD209 on the cell membrane, they can be used as a carrier for delivery to CD 209-positive cells (the carrier for delivery of the present invention). CD209 positive cells are preferably present in cancerous tissue. The delivery vehicle of the present invention can deliver other substances (preferably, cancer tissues containing CD 209-positive cells) to CD 209-positive cells by administering (preferably, by carrying (e.g., binding, holding) the other substances on the gel particles) together with the substances to be delivered (other substances that the gel particles may contain (e.g., adjuvants, cancer antigens, anticancer agents, agents such as nucleic acid drugs, contrast agents, etc.)). As one embodiment of the carrier for delivery, for example, a drug delivery carrier can be cited.

In one embodiment, the present invention relates to a detection agent for a tissue containing CD 209-positive cells, which contains the delivery vehicle of the present invention and a contrast agent, and can be used as a detection agent for a tissue containing CD 209-positive cells (preferably, a cancer tissue containing CD 209-positive cells), for example, as a detection agent for MRI, PET, CT or the like, depending on the type of the contrast agent.

In the case of humans, CD209 is a lectin receptor of the C-type, denoted DC-SIGN, expressed on the cell surface. CD209 of other animals may be determined based on sequence identity to human CD 209/DC-SIGN. For example, the functional homolog of mouse CD209 is CD209b, which is sometimes also denoted as sign 1.

The cancer tissue containing CD 209-positive cells can be determined, for example, by screening a specific cell population (preferably macrophages) in the cancer tissue to determine the degree to which CD 209-positive cells are contained in the cancer tissue. The screening and determination can be performed according to a known method. For example, an immune tissue staining method can be cited. In this method, the CD 209-positive cells in the cancer tissue are stained with an anti-human CD209 antibody (fluorescence method or chromogenic method), and positive or negative is determined based on the amount of pigment (for example, the amount of fluorescence or visible light) emitted from the stained cells. The staining can typically be performed using CD209 binding molecules. Accordingly, in one embodiment thereof, the present invention relates to a concomitant diagnostic agent (diagnostic agent of the present invention) for the cancer therapeutic agent of the present invention, which contains a CD 209-binding molecule. The effectiveness of the cancer therapeutic agent of the present invention can be predicted by a method comprising a step of bringing a CD 209-binding molecule into contact with cells in a cancer tissue of a subject.

The CD209 binding molecule is not particularly limited as long as it is a molecule capable of binding to CD209 (preferably, capable of specifically binding). Examples of the CD209 binding molecule include antibodies, more specifically, immunoglobulins, fab and F (ab') ₂ Minibodies (minibodies), scFv-Fc, fv, scFv, bispecific antibodies (diabodies), trispecific antibodies (triabodies), tetraspecific antibodies (tetrabodies), monomeric antibodies (monobodies), and the like.

The series of operations of the above-described screening and determination can be performed, for example, by observation with a microscope. The positive or negative determination can be performed according to a known method based on the amount of pigment emitted from the stained cells. This determination can be performed by a known method. For example, a dye amount emitted from a negative control cell (specifically, for example, a cell stained with an isotype control antibody) is used as a dye amount of the background, a cell having a dye amount lower than the background is determined as a negative cell, and a cell having a fluorescence amount higher than the background is determined as a positive cell. As a simpler method, a method of performing reverse transcription quantitative PCR of CD209 on mRNA extracted from cancer tissue and determining the amount of CD209 mRNA can be considered.

Preferably, a patient having a cancer tissue containing CD 209-positive cells is selected from patients having a cancer tissue, and the cancer therapeutic agent of the present invention or the delivery vehicle of the present invention is administered to the selected patient. Therefore, the cancer therapeutic agent of the present invention or the delivery vehicle of the present invention is preferably used in a manner to be administered to a patient having a cancer tissue containing CD 209-positive cells selected from patients having a cancer tissue.

The cancer therapeutic agent of the present invention or the delivery vehicle of the present invention is not particularly limited as long as it contains gel particles, and may contain other components as needed. The other component is not particularly limited as long as it is a pharmaceutically acceptable component. As other components, additives are contained in addition to the components having pharmacological actions. Examples of the additives include a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a humectant, a coloring material, a perfume, and a chelating agent.

The cancer therapeutic agent of the present invention or the delivery vehicle of the present invention may contain, in addition to the gel particles, other substances (for example, an adjuvant, a cancer antigen, an anticancer agent, a drug such as a nucleic acid drug, a contrast agent, and the like) that the gel particles can contain. The cancer therapeutic agent of the present invention or the delivery vehicle of the present invention may be used in combination with other substances (for example, an adjuvant, a cancer antigen, an anticancer agent, a drug such as a nucleic acid drug, a contrast agent, etc.) which the gel particles can contain.

The target to which the cancer therapeutic agent of the present invention or the delivery vehicle of the present invention is applied is not particularly limited, and examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, deer, and the like. Examples of the cells include animal cells. The type of the cells is not particularly limited, and examples thereof include blood cells, hematopoietic stem cells, precursor cells, gametes (sperm, ovum), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, liver cells, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells, and the like.

The cancer therapeutic agent of the present invention or the cancer to be treated with the delivery vehicle of the present invention is not particularly limited, and examples thereof include leukemia (including chronic lymphocytic leukemia and acute lymphocytic leukemia), lymphoma (including non-hodgkin lymphoma, T-cell lymphoma, B-cell lymphoma, burkitt lymphoma, malignant lymphoma, diffuse lymphoma, follicular lymphoma), myeloma (including multiple myeloma), breast cancer, large intestine cancer, kidney cancer, stomach cancer, ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer, esophageal cancer, liver cancer, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal carcinoma, pleural carcinoma, male blastoma, endometrial hyperplasia, endometriosis, embryonal carcinoma, fibrosarcoma, kaposi's sarcoma, hemangioma, spongiform hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, myeloblastoma, glioma, neuroblastoma, myoma, and the like.

The cancer therapeutic agent of the present invention or the delivery vehicle of the present invention may be in any form, for example, in the form of tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, buccal tablets, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), oral preparations such as jelly, preparations for injection (for example, intravenous injection) intramuscular injection, subcutaneous injection, intradermal injection), external preparations (for example, ointments, external application, lotions), suppositories, inhalants, eye drops, eye ointments, nasal drops, ear drops, liposome, etc. non-oral preparations.

The route of administration of the cancer therapeutic agent of the present invention or the delivery vehicle of the present invention is not particularly limited as long as a desired effect can be obtained, and examples thereof include enteral administration such as oral administration, tube feeding, and enema administration; intravenous administration, intra-arterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and other non-oral administration.

The content of the active ingredient in the cancer therapeutic agent of the present invention or the delivery vehicle of the present invention is not limited, and may be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight, depending on the mode of use, the object to be applied, the state of the object to be applied, and the like.

The amount of the cancer therapeutic agent of the present invention or the delivery vehicle of the present invention to be administered is not particularly limited as long as it is an effective amount capable of exhibiting a drug effect, and is usually 0.1 to 1000mg/kg body weight per day, preferably 0.5 to 500mg/kg body weight per day in the case of oral administration, and 0.01 to 100mg/kg body weight, preferably 0.05 to 50mg/kg body weight per day in the case of non-oral administration, based on the weight of the effective ingredient. The amount of the above-mentioned drug may be appropriately increased or decreased depending on the age, condition, symptom, etc.

Examples

The present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Preparation example 1 preparation of pullulan nanogel

Cholesterol modified pullulan (CHP) was prepared by introducing 1.2 cholesterol per 100 monosaccharides of pullulan having a weight average molecular weight of 100,000 according to the conventional report (Macromolecules 1993,23,3062-3068).

CHP was added to PBS to 1mg/mL, and the mixture was stirred at 25℃for 15 hours to dissolve the CHP, and then intermittently irradiated with ultrasonic waves for 6 minutes using a probe type ultrasonic irradiator. After the solution was subjected to centrifugation at 20000g for 30 minutes at 25 ℃, the supernatant was subjected to filtration sterilization through a 0.22um filter to obtain pullulan nanogel (nanogel weight average molecular weight 450,000).

The average particle diameter of the obtained pullulan nanogel was measured by analysis using a Dynamic Light Scattering (DLS) accumulation method. As a result, the average particle diameter of the pullulan nanogel was 32.2.+ -. 2.1nm and the polydispersity index was 0.26.+ -. 0.01.

Test example 1 binding of pullulan nanogel to mouse CD209

Expression vectors encoding cDNA of mouse SIGNR1/CD209b or mouse DC-SIGN/CD209a were transiently introduced into HEK293 cells by lipofection. As a control, an expression Vector containing no cDNA was used, and the same was done (Vector control). After 48 hours, rhodamine-labeled pullulan nanogels were applied to each cell at the concentrations shown in fig. 1, and incubated at 37 ℃ for 3 hours. Cells were washed with cold PBS, exfoliated with EDTA-containing cold PBS, and suspended as single cells. The uptake rate of rhodamine-labeled pullulan nanogels in each cell was measured using a flow cytometer.

The results are shown in FIG. 1. In the case of mouse SIGNR1/CD209 b-expressing cells, uptake of rhodamine-labeled pullulan nanogel was confirmed in 48.9% (at 2. Mu.g pullulan nanogel/mL) or 67.8% (at 10. Mu.g pullulan nanogel/mL) cells, whereas in the case of mouse DC-SIGN/CD209 a-expressing cells, a low value of 5.5% (at 2. Mu.g pullulan nanogel/mL) or 16.6% (at 10. Mu.g pullulan nanogel/mL) was confirmed. Mouse SIGNR1/CD209b functions similarly to human DC-SIGN/CD 209. In addition, the introduction efficiency was about 40% as evaluated by transient expression of an expression vector encoding a green fluorescent protein (Green fluorescent protein, GFP) cDNA.

Test example 2 binding of pullulan nanogel to human CD209

Expression vectors encoding cDNA of mouse SIGNR1/CD209b, mouse DC-SIGN/CD209a, or human DC-SIGN/CD209 were transiently introduced into HEK293 cells by lipofection in the same manner as in test example 1. As a control, the same procedure was carried out using an expression Vector containing no cDNA (Vector control). After 48 hours, rhodamine-labeled pullulan nanogels were applied to each cell at the concentrations shown in fig. 2, and incubated at 37 ℃ for 2 hours. Cells were washed with cold PBS, exfoliated with EDTA-containing cold PBS, and suspended as single cells. The uptake rate of rhodamine-labeled pullulan nanogels in each cell was measured using a flow cytometer.

The results are shown in FIG. 2. The uptake of rhodamine-labeled pullulan nanogels was confirmed in 3.1%, 22.3% and 31.4% of the cells expressing mouse DC-SIGN/CD209a, mouse SIGNR1/CD209b and human DC-SIGN/CD 209. At this time, the introduction efficiency was evaluated to be about 29% by transient expression with an expression vector encoding Green Fluorescent Protein (GFP) cDNA.

Test example 3 specific binding of pullulan nanogel to CD209

The expression vector encoding the cDNA of the indicated C-type lectin was transiently introduced into COS7 cells by lipofection. As a control, the same procedure was carried out using an expression Vector containing no cDNA (Vector control). After 48 hours, rhodamine-labeled pullulan nanogels were applied to each cell at a concentration of 2 μg/mL and incubated for 2 hours at 37 ℃. Cells were washed with cold PBS and exfoliated with TryPLE (Thermo Scientific) to become single-cell suspension. Uptake (red) of rhodamine-labeled pullulan nanogels in each cell was observed with a fluorescence microscope.

The results of the human C-type lectin-expressing cells are shown in FIG. 3-1, and the results of the mouse C-type lectin-expressing cells are shown in FIG. 3-2. Uptake of rhodamine-labeled pullulan nanogels was only observed in cells expressing human DC-SIGN/CD209, human L-SIGN, mouse SIGNR1/CD209b, and not confirmed in other cells expressing C-type lectin.

Test example 4 polysaccharide nanogels and binding of Linear polysaccharide to CD209

Rhodamine-labeled Pullulan nanogel (CHP in fig. 4) or rhodamine-labeled Pullulan (linear, monomeric) (pullulon 400K in fig. 4) was administered to mouse RAW264.7 cells stably expressing the mouse SIGNR1/CD209b gene (RAW SR-1 in fig. 4) at the concentrations indicated in fig. 4, and incubated at 37 ℃ for 3 hours. Cells were scraped off with a spatula and suspended as single cells. The uptake intensity (fluorescence intensity) of rhodamine-labeled pullulan nanogels in each cell was measured with a flow cytometer. Further, the same experiment was performed using mannans (linear, monomeric) and mannan nanogels.

The results are shown in FIG. 4. Both the pullulan nanogel and the pullulan are ingested by RAW SR-1 cells in a concentration-dependent manner. The pullulan nanogel (nanoparticulate) has higher uptake than pullulan (monomeric, linear).

Test example 5 SIGNR1 dependence of pullulan nanogels on RAW SR-1 cell binding

The same experiment as in test example 4 was performed. But cells were treated with sign r1 specific antibodies (clone 22D1 (IgG) or clone ERTR9 (IgM)) for 1 hour before adding rhodamine-labeled pullulan nanogels.

The results are shown in FIG. 5. In cells treated with 22D1 antibody (IgG), a significant reduction in uptake of pullulan nanogels was confirmed. It is considered that since 22D1 binds to the sugar chain recognition site of SIGNR1/CD209b, the binding of SIGNR1/CD209b to pullulan nanogel is hindered by 22D1, and thus the uptake of pullulan nanogel is suppressed. It is considered that the inhibition of the uptake of pullulan nanogel does not occur because ERTR9 does not bind to the sugar chain recognition site of sign r1/CD209 b. According to the result, the specific binding between the pullulan nanogel and SIGNR1 is supported.

Test example 6 specific binding of pullulan nanogel to CD209

The expression vector encoding the cDNA of mouse SIGNR1/CD209b was transiently introduced into CMS5a cells of the mouse fibrosarcoma cell line by electroporation. The following day, rhodamine-labeled pullulan nanogel was administered to each cell at a concentration of 2 μg/mL, and anti-SIGNR 1 antibody (22D 1) was administered at the concentration shown in fig. 6, and incubated at 37 ℃ for 2 hours. Cells were exfoliated with trypsin and suspended in a manner to become single cells. Uptake of rhodamine-labeled pullulan nanogels in each cell was assessed by fluorescence microscopy and flow cytometry.

The results are shown in FIG. 6. Uptake of rhodamine-labeled pullulan nanogels was clearly confirmed in SIGNR1/CD209b expressing cells (Control in FIG. 6). In contrast, the uptake of rhodamine-labeled pullulan nanogels decreased in a concentration-dependent manner by the addition of the 22D1 antibody. Similar to FIG. 5, the specific binding of pullulan nanogel to SIGNR1/CD209b is supported.

Test example 7 Selective uptake of pullulan nanogels in tumor-associated macrophages (TAM)

The CT26 cell line of the colorectal cancer cell line of the mice was transplanted under the skin of normal BALB/c mice (7 weeks old, female), tumors were recovered after 8 days, and after being broken into cell suspensions by GentleMACS (Miltenyi), the cell suspensions were cultured on a culture plate with RPMI1640 medium containing 10% fetal bovine serum. Rhodamine-labeled pullulan nanogels were applied thereto at a concentration of 2 μg/mL and incubated at 37 ℃ for 2 hours. Cells were then trypsinized off and suspended as single cells. The cells were stained with anti-CD 45 antibody, CD11b antibody, gr1 antibody, SIGNR1/CD209b antibody, MHC class II antibody, and uptake of rhodamine-labeled pullulan nanogels in the indicated cell mass was evaluated by flow cytometry.

The results are shown in FIG. 7. Among the myeloid cells, high uptake of rhodamine-labeled pullulan nanogels was clearly confirmed in cd11b+gr1-cd209b+ cells (CD 209b positive TAM) (right in fig. 7).

Test example 8 Selective uptake of pullulan nanogels in tumor-associated macrophages (TAM)

The CT26 cell line of the colorectal cancer cell line of the mice was transplanted under the skin of normal BALB/c mice (7 weeks old, female), tumors were recovered after 8 days, and after being broken into cell suspensions by GentleMACS (Miltenyi), the cell suspensions were cultured on a culture plate with RPMI1640 medium containing 10% fetal bovine serum. Rhodamine-labeled pullulan nanogels were applied thereto at a concentration of 2 μg/mL and incubated at 37 ℃ for 2 hours. At this time, the sign r1 antibody (22D 1) was added simultaneously at the concentration shown in fig. 8. Then, uptake of rhodamine-labeled pullulan nanogels was evaluated by fluorescent microscopy observation. The number of cells that strongly emit light in the field of view of x 400 was taken as the number of cells taken up.

The results are shown in FIG. 8. In the presence of the 22D1 antibody, uptake of rhodamine-labeled pullulan nanogels was significantly inhibited.

Test example 9 Selective uptake of pullulan nanogels in tumor-associated macrophages (TAM)

The mouse colorectal cancer cell line CT26 cells were transplanted subcutaneously into normal BALB/c mice (7 weeks old, female), and after 8 days rhodamine-labeled pullulan nanogel (600 μg) was administered intravenously. The SIGNR1 antibody (22D 1) was administered simultaneously at the indicated concentrations. After 6 hours, tumors were harvested, disrupted by GentleMACS (Miltenyi) to prepare cell suspensions, stained with anti-CD 45 antibody, CD11b antibody, gr1 antibody, CD209b antibody, CD206 antibody, MHC class II antibody, and the uptake of rhodamine-labeled pullulan nanogels in the indicated cell mass was evaluated by flow cytometry.

The results are shown in FIG. 9. The high uptake of rhodamine-labeled pullulan nanogels was clearly confirmed in CD11b+Gr1-CD209b+ cells (CD 209 positive TAM) (FIG. 9 left). In contrast, the concentration of the sign r1 antibody dependently inhibited the uptake of pullulan nanogels in the above cells.

Test example 10. Pseudomonas aeruginosa exotoxin (Pseudomonas Exotoxin), PE) complex induced cytostatic

The expression vector encoding the cDNA of mouse SIGNR1/CD209b was transiently introduced into CMS5a cells of the mouse fibrosarcoma cell line by electroporation. The next day, pullulan nanogel PE complex or PE was administered to each cell at a concentration of 0.3, 1, 3, 9, 10, 30, 90 μg/mL and incubated for 1 hour at 37 ℃. After 1 hour, the cells were rinsed with 10% FCS RPMI, cultured with 10% FCS RPMI for 24 hours, and then evaluated for cell activity with Cell Counting Kit-8.

The results are shown in FIG. 10. In the case of PE alone, the cell inhibitory activity was observed independently of the expression of SIGNR1/CD209b and in the case of the pullulan nanogel PE complex, the excellent cell inhibitory activity was confirmed in the cells expressing SIGNR1/CD209 b.

Test example 11 cell inhibition by pullulan nanogel PE complex

Mice colorectal cancer cell line CMS5a cells were transplanted subcutaneously into normal BALB/c mice (8 weeks old, female), and after 8, 10, 12 days, pullulan nanogel PE complex and PE (0.1 μg) were administered intravenously together with CpG (50 ug). Tumor size was measured over time.

The results are shown in FIG. 11. Compared with the group administered with CpG alone and the group administered with pullulan nanogel PE alone, it was observed that a more excellent tumor proliferation inhibition effect was obtained by the combined administration of the pullulan nanogel PE complex and CpG. When mice were administered PE, they died within 24 hours of administration.

Claims (16)

1. A cancer therapeutic agent for a cancer tissue containing CD 209-positive cells, characterized by:

comprises gel particles comprising modified polysaccharides comprising hydrophobic groups.

2. The cancer therapeutic agent according to claim 1, wherein:

the gel particles are nano gel particles.

3. The cancer therapeutic agent according to claim 1 or 2, wherein:

the structural polysaccharide of the modified polysaccharide comprises at least 1 selected from pullulan, mannan, and glucan.

4. The cancer therapeutic agent according to any one of claims 1 to 3, wherein:

the structural polysaccharide of the modified polysaccharide comprises pullulan.

5. The cancer therapeutic agent according to any one of claims 1 to 4, wherein:

the hydrophobic group includes a hydrophobic group having a sterol backbone.

6. The cancer therapeutic agent according to any one of claims 1 to 5, wherein:

the modified polysaccharide has a weight average molecular weight of 5000 ～ 2,000,000.

7. The cancer therapeutic agent according to any one of claims 1 to 6, wherein:

the weight average particle diameter of the gel particles is 20-200 nm.

8. The cancer therapeutic agent according to any one of claims 1 to 7, wherein:

the gel particles contain a pharmaceutical agent.

9. The cancer therapeutic agent according to claim 8, wherein:

the agent comprises at least 1 selected from the group consisting of an adjuvant, a cancer antigen, an anticancer agent, and a nucleic acid drug.

10. The cancer therapeutic agent according to any one of claims 1 to 9, wherein:

the CD 209-positive cells are macrophages.

11. The cancer therapeutic agent according to any one of claims 1 to 10, wherein:

for administration to a patient selected from patients having cancerous tissue, having cancerous tissue containing CD 209-positive cells.

12. The cancer therapeutic agent according to any one of claims 1 to 11, wherein:

for use in combination with a medicament.

13. A vector for delivery to CD 209-positive cells, characterized in that:

comprises gel particles comprising modified polysaccharides comprising hydrophobic groups.

14. The delivery vehicle of claim 13, wherein:

CD209 positive cells are present in cancerous tissue.

15. A test agent for a tissue comprising CD 209-positive cells, comprising:

a delivery vehicle according to claim 13 or 14 and a contrast agent.

16. A concomitant diagnostic drug for the cancer therapeutic agent according to any one of claims 1 to 12, characterized in that:

containing a CD209 binding molecule.