WO2020189523A1

WO2020189523A1 - Chemokine production promoter, therapeutic agent for immune checkpoint inhibitor-resistant cancer and antitumor immunostimulator

Info

Publication number: WO2020189523A1
Application number: PCT/JP2020/010894
Authority: WO
Inventors: 啓輔日野; 惣治仁科; 恭佐々木; 山内　明; 宏太郎福田
Original assignee: 株式会社先端医療開発; 学校法人川崎学園
Priority date: 2019-03-15
Filing date: 2020-03-12
Publication date: 2020-09-24
Also published as: JP2022078362A

Abstract

A chemokine production promoter that comprises a saccharide derivative.

Description

Chemokine production promoter, immune checkpoint inhibitor, resistant cancer therapeutic agent, and antitumor immunostimulant

The present invention relates to a chemokine production promoter, an immune checkpoint inhibitor, a therapeutic agent for resistant cancer, and an antitumor immunostimulant.

Immune checkpoint inhibitors are expected as a treatment for cancer. Immune checkpoint inhibitors block the immunosuppressive mechanism mediated by immune checkpoint molecules by cancer cells, resulting in an antitumor effect by the immune system.

The immunosuppressive mechanism is enhanced in the microenvironment of advanced cancer. For example, Non-Patent Document 1 reports that anti-tumor immunity is suppressed by activation of glycolysis in cancer cells. Non-Patent Document 2 suggests that in the microenvironment of cancer, glucose metabolism in cancer cells is enhanced, so that the activity of T cells is reduced by reducing the supply of glucose to T cells. There is.

Increased chemotaxis of T cells against cancer cells is important for activation of anti-tumor immunity in the cancer microenvironment. In Non-Patent Document 1, when the expression levels of 75 types of cell chemotaxis factors (chemokines) and cytokines were examined, T cells were examined in melanoma cells having low glycolytic activity rather than melanoma cells having high glycolytic activity. It has been shown that a large amount of CXCL10, which enhances chemotaxis, is produced. In Non-Patent Document 1, although the amount of chemokine produced by melanoma cells with low glycolytic activity extracted using the rate of extracellular acidification as an index is evaluated, the production of chemokines by inhibiting the glycolytic system of cancer cells. The effect on quantity has not been investigated.

The present invention has been made in view of the above circumstances, and is a chemokine production promoter, an immune checkpoint inhibitor, a resistant cancer therapeutic agent, and an antitumor immunostimulant, which can enhance antitumor immunity in a cancer microenvironment. The purpose is to provide.

The present inventor has conducted extensive research, and in addition to enhancing chemokine production and T cell chemotaxis by cancer cells by inhibiting glycolysis in cancer cells, glucose metabolism between cancer cells and T cells We have found that interventions in balance enhance anti-tumor immunity and complete the present invention.

The chemokine production promoter according to the first aspect of the present invention is
Contains sugar derivatives.

In this case, the chemokine production promoter according to the first aspect of the present invention is
Promotes the production of CXCL9, CXCL10 and CXCL11 from cancer cells,
It may be that.

In addition, the chemokine production promoter according to the first aspect of the present invention is
Further containing biocompatible particles containing a lactic acid / glycolic acid copolymer
The sugar derivative is
Encapsulated in the biocompatible particles,
It may be that.

The immune checkpoint inhibitor-resistant cancer therapeutic agent according to the second aspect of the present invention is
Contains sugar derivatives.

The antitumor immunostimulant according to the third aspect of the present invention is
Contains sugar derivatives
Used in combination with immune checkpoint inhibitors.

Chemokine production promoter according to the first aspect of the present invention, immune checkpoint inhibitor-resistant cancer therapeutic agent according to the second aspect of the present invention, or antitumor immunostimulant according to the third aspect of the present invention. In the agent
The sugar derivative is
2-Deoxy-D-glucose,
It may be that.

According to the present invention, anti-tumor immunity in the microenvironment of cancer can be enhanced.

It is a figure which shows the influence of 2-deoxy-D-glucose (hereinafter, simply referred to as "2DG") on the amount of chemokine secreted from the liver cancer cell which concerns on Test Example 1. (A) is a figure which shows the amount of secretion of CXCL10. (B) is a figure which shows the amount of secretion of CXCL9. It is a figure which shows the enhancement of chemotaxis of T cell to the liver cancer cell by 2DG which concerns on Test Example 1. It is a figure which shows the velocity-directionality (VD) plot which concerns on Test Example 1. FIG. It is a figure which shows the migration rate of the T cell which concerns on Test Example 1. It is a figure which shows the direction of movement of the T cell which concerns on Test Example 1. It is a figure which shows the particle size distribution of 2DG nanoparticles which concerns on Test Example 2. It is a figure which shows the enhancement of the chemotaxis of T cell to the liver cancer cell by the 2DG nanoparticles which concerns on Test Example 2. It is a figure which shows the VD plot which concerns on Test Example 2. It is a figure which shows the migration rate of the T cell which concerns on Test Example 2. It is a figure which shows the direction of movement of the T cell which concerns on Test Example 2. It is a figure which shows the amount of lactic acid production from the liver cancer cell which concerns on Test Example 2. It is a figure which shows the relative expression level of IFNγ mRNA from the T cell which concerns on Test Example 2. It is a figure which shows the relative uptake amount of 2-NBDG of the liver cancer cell which concerns on Test Example 2. It is a figure which shows the relative uptake amount of 2-NBDG of the T cell which concerns on Test Example 2. It is a figure which shows the appearance of the liver tumor of the liver carcinogenic mouse which concerns on Test Example 3. It is a figure which shows the tumor maximum diameter in the liver carcinogenic mouse which concerns on Test Example 3. It is a figure which shows the total tumor volume in the liver carcinogenic mouse which concerns on Test Example 3. It is a figure which shows the mRNA expression level of CXCL9 in the tumor tissue which concerns on Test Example 3. It is a figure which shows the mRNA expression level of CXCL10 in the tumor tissue which concerns on Test Example 3. It is a figure which shows the mRNA expression level of CXCL11 in the tumor tissue which concerns on Test Example 3. It is a figure which shows the appearance of the tumor of the anti-PD-1 antibody resistant mouse which concerns on Test Example 4. It is a figure which shows the time-dependent change of the tumor volume in the anti-PD-1 antibody resistant mouse which concerns on Test Example 4. It is a figure which shows the appearance of the tumor of the anti-PD-1 antibody resistant mouse which concerns on Test Example 5. It is a figure which shows the time-dependent change of the tumor volume in the anti-PD-1 antibody resistant mouse which concerns on Test Example 5. It is a figure which shows the liver tumor tissue which was immunostained of the anti-PD-1 antibody resistance mouse which concerns on Test Example 5. It is a figure which shows the number of CD3 positive T cells in the liver tumor tissue shown in FIG. It is a figure which shows the appearance of the liver tumor of the liver carcinogenic mouse which concerns on Test Example 6. It is a figure which shows the tumor maximum diameter in the liver carcinogenic mouse which concerns on Test Example 6. It is a figure which shows the total tumor volume in the liver carcinogenic mouse which concerns on Test Example 6.

(Embodiment)
An embodiment according to the present invention will be described. The chemokine production promoter according to the present embodiment contains a sugar derivative as an active ingredient. The sugar derivative is, for example, a derivative of D-glucose that inhibits glycolysis. Preferably, the sugar derivative is a glucose derivative that does not have a hydroxyl group at the 2-position of the glucose ring. Glucose derivatives include mannose, galactose and 5-thio-glucose. The glucose derivative may have fluorine instead of hydrogen at any position on the glucose ring. Examples of the glucose derivative having fluorine include 2-fluoro-2-deoxy-D-glucose and 2-difluoro-2-D-deoxy-glucose.

The glucose derivative may have an amino group instead of a hydroxyl group at any position other than the 6-position on the glucose ring. Examples of the glucose derivative having an amino group include 2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-galactose. In addition to the above, examples of glucose derivatives include 2-F-mannose, 2-mannosamine, 2-deoxygalactose, 2-F-deoxygalactose and the like. Preferably, the sugar derivative is 2DG, which is a derivative of glucose. 2DG is taken up into cancer cells via a glucose transporter. 2DG accumulated in cancer cells inhibits glycolysis.

Preferably, the chemokine production promoter further contains biocompatible particles containing a lactic acid / glycolic acid copolymer (also referred to as a polylactide glycolide copolymer; hereinafter simply referred to as "PLGA"). Biocompatible particles are particles that are less irritating or toxic to living organisms and have the property of being decomposed and metabolized after administration. The sugar derivative is encapsulated in biocompatible particles.

PLGA is, for example, a copolymer composed of lactic acid or lactide and glycolic acid or glycolide in a ratio of 1:99 to 99: 1, preferably 75:25, more preferably 3: 1. The PLGA may be synthesized from any monomer by a known method, or a commercially available PLGA may be used. Examples of commercially available PLGA include PLGA7520 (lactic acid: glycolic acid = 75:25, average weight molecular weight 20,000, manufactured by Wako Pure Chemical Industries, Ltd.). PLGA having a content of lactic acid and glycolic acid of 25% by weight to 65% by weight is preferable in that it is amorphous and soluble in an organic solvent such as acetone.

The particle size of the biocompatible particles is less than 1000 nm, for example 2.5-900 nm, preferably 25-500 nm or 50-300 nm, more preferably 100-250 nm, still more preferably 130-200 nm, particularly preferably 150-180 nm. Is. The particle size of biocompatible particles is measured by sieving, sedimentation, microscopy, light scattering, laser diffraction / scattering, electrical resistance test, observation with a transmission electron microscope, observation with a scanning electron microscope, etc. it can. The particle size may be measured with a particle size distribution meter. The particle size can be represented by a stalk-equivalent diameter, a circle-equivalent diameter, and a sphere-equivalent diameter, depending on the measurement method. Further, the particle size may be an average particle size, a volume average particle size, an area average particle size, or the like expressed by averaging a plurality of particles as measurement targets. Further, the particle size may be an average particle size calculated from a number distribution or the like based on a measurement such as a laser diffraction / scattering method.

Specifically, when the cumulative curve is obtained with the total product of the particle population as 100%, the particle size may be 50% diameter (D ₅₀ ), which is the particle size at the point where the cumulative curve is 50%. Cumulative curve and D ₅₀ can be determined using a commercially available particle size distribution meter. Examples of the particle size distribution meter include NIKKISO Nanotrac Wave-EX150 (manufactured by Nikkiso Co., Ltd.).

The surface of the biocompatible particles may be modified with polyethylene glycol (PEG). For example, by using a PEG-modified product of PLGA in the production of the biocompatible particles, biocompatible particles whose surface is modified with PEG can be obtained. The surface is modified with PEG to improve the blood stability of biocompatible particles.

Any known production method can be adopted as a method for producing biocompatible particles in which a sugar derivative is encapsulated. For example, biocompatible particles can be produced by the underwater emulsion method. In the underwater emulsion method, two kinds of solvents, a good solvent in which PLGA dissolves and a poor solvent in which PLGA does not dissolve, are used. As a good solvent, an organic solvent in which PLGA is dissolved and miscible with a poor solvent is used. The types of the good solvent and the poor solvent are not particularly limited.

Water can be mentioned as a poor solvent. When water is used as the poor solvent, a surfactant may be added to the water. For example, polyvinyl alcohol (PVA) is preferable as the surfactant. Examples of the surfactant other than PVA include lecithin, hydroxymethyl cellulose, hydroxypropyl cellulose and the like.

Examples of good solvents include halogenated alkanes, which are organic solvents having a low boiling point and poor water solubility, acetone, methanol, ethanol, ethyl acetate, diethyl ether, cyclohexane, benzene, toluene and the like. Preferably, for example, only acetone having less adverse effect on the environment or the human body or a mixed solution of acetone and ethanol is used.

In the underwater emulsion method, first, PLGA is dissolved in a good solvent, and then a sugar derivative solution is added to the good solvent and mixed so that PLGA does not precipitate. When the mixed solution containing PLGA and the sugar derivative is added dropwise to the poor solvent under stirring, the good solvent in the mixed solution rapidly diffuses and migrates into the poor solvent. As a result, emulsification of a good solvent occurs in a poor solvent, and emulsion droplets of the good solvent are formed.

Further, due to the mutual diffusion of the good solvent and the poor solvent, the organic solvent is continuously diffused from the emulsion into the poor solvent, so that the solubility of PLGA and the sugar derivative in the emulsion droplet is lowered. Eventually, biocompatible particles of spherical crystal particles containing the sugar derivative are produced. Then, the organic solvent which is a good solvent is centrifuged or distilled under reduced pressure to obtain a powder of biocompatible particles. The obtained powder is composited as it is or, if necessary, into aggregated particles that can be redispersed by freeze-drying or the like. The content of the sugar derivative in the obtained biocompatible particles is, for example, 0.01 to 99% by weight, 0.1 to 30% by weight, 0.5 to 20% by weight, 1 to 15% by weight, and 3 to 10% by weight. % Or 5-10% by weight. The content of the sugar derivative here is the ratio of the weight of the sugar derivative to the weight of the biocompatible particles. The content rate is determined by quantifying the weight of the sugar derivative extracted from the biocompatible particles having a predetermined weight and calculating the ratio of the weight of the sugar derivative to the weight of the biocompatible particles.

A cationic polymer may be added to the poor solvent in order to increase the content of the sugar derivative in the biocompatible particles. Examples of the cationic polymer include chitosan and chitosan derivatives, cationized cellulose in which a plurality of cationic groups are bonded to cellulose, and polyamino compounds such as polyethyleneimine, polyvinylamine, and polyallylamine.

Preferably, the biocompatible particles encapsulating the sugar derivative are produced using a forced thin film microreactor. When a forced thin film type microreactor is used, first, the above-mentioned good solvent and poor solvent are introduced between the processing surfaces which are arranged so as to face each other and at least one of them rotates relative to the other. In the thin film fluid formed thereby, a good solvent and a poor solvent are mixed, and biocompatible particles in which a sugar derivative is encapsulated are precipitated in the thin film fluid. As the forced thin film type microreactor, ULREA SS-11 (manufactured by M-Technique) is preferably used.

The chemokine production promoter is produced by a known method and contains 0.000001 to 99.9% by weight, 0.00001 to 99.8% by weight, 0.0001 to 99.7% by weight, 0.001 to 9% by weight as active ingredients. 99.6% by weight, 0.01-99.5% by weight, 0.1 to 99% by weight, 0.5 to 60% by weight, 1 to 50% by weight or 1 to 20% by weight of sugar derivative or sugar derivative Includes encapsulated biocompatible particles. The chemokine production promoter may be a solid preparation or a liquid preparation.

The chemokine production promoter may contain a pharmacologically acceptable carrier or the like in addition to the sugar derivative or biocompatible particles. For example, the chemokine production promoter contains excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents and the like. May be good. In addition, additives such as preservatives, antioxidants, colorants and sweeteners may be added, if necessary.

The chemokine production promoter according to the present embodiment promotes the production of chemokine from cancer cells, particularly at least one of CXCL9, CXCL10 and CXCL11, as shown in Test Example 1 and Test Example 3 below. By binding CXCL9, CXCL10 or CXCL11 to the receptor CXCR3 expressed on the surface of CD8-positive T cells, the chemotaxis of T cells is enhanced. As a result, anti-tumor immunity is activated. Therefore, chemokine production promoters are preferably used in the treatment of cancers, especially solid cancers. Specifically, solid cancers include hepatocellular carcinoma, gastric cancer, pancreatic cancer, lung cancer, colon cancer, breast cancer, liver cancer (liver cancer), kidney cancer (kidney cancer), tongue cancer, thyroid cancer, uterine cancer, and ovarian cancer. , Prostate cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, smooth myoma and the like. Preferably, the chemokine production promoter is used in the treatment of cancer in which a polycythemia tumor is formed. A polycythemia tumor is a tumor with abundant arterial blood flow that is enhanced by contrast in a contrast examination such as contrast-enhanced computed tomography. The types of cancer in which polycythemia tumors are formed are, for example, hepatocellular carcinoma and renal cell carcinoma. In addition, solid cancers detected by positron emission tomography (PET) using fluorodeoxyglucose have enhanced glucose metabolism, so that sugar derivatives are efficiently accumulated. Therefore, the chemokine production promoter is also suitable for the treatment of solid cancer detected by PET.

The route of administration of the chemokine production promoter to humans is not particularly limited. Chemokine production promoters are administered, for example, by injection or orally. The dose of the chemokine production promoter is appropriately determined depending on the sex, age, body weight, symptoms and the like of the administration subject. The chemokine production promoter is administered so that the sugar derivative is in an effective amount. The effective amount is the amount of sugar derivative required to obtain the desired result, and is the amount required to bring about delay, inhibition, prevention, reversal or cure of the progression of the condition to be treated or treated.

The dose of the chemokine production promoter is typically 0.01 mg / kg to 1000 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, more preferably 0.2 mg / kg to 20 mg / kg. It can be administered once a day or in divided doses. When the chemokine production promoter is administered in divided portions, the chemokine production promoter is preferably administered 1 to 4 times a day. In addition, the chemokine production promoter may be administered at various administration frequencies such as daily, every other day, once a week, every other week, and once a month. Preferably, the administration frequency is easily determined by a doctor or the like. If necessary, an amount outside the above range can be used.

As described in detail above, the chemokine production promoter according to the present embodiment promotes the production of chemokines from cancer cells. Since chemokines enhance the chemotaxis of T cells to cancer cells, the chemokine production promoter can enhance anti-tumor immunity. In another embodiment, a T cell chemotaxis enhancer containing the above sugar derivative is provided.

The sugar derivative according to the present embodiment promotes the production of IFNγ from T cells, as shown in Test Example 2 below. Therefore, the chemokine production promoter according to the present embodiment can also be used as an IFNγ production promoter.

The sugar derivative inhibits glucose uptake by cancer cells, as shown in Test Example 2 below. Therefore, the chemokine production promoter according to the present embodiment can also be used as a glucose uptake inhibitor. When glucose uptake by cancer cells is suppressed, glucose uptake by T cells is relatively increased in the cancer microenvironment. Therefore, the chemokine production promoter according to the present embodiment can also be used as a glucose uptake promoter for T cells. Increased glucose uptake by T cells also enhances the chemotaxis of T cells to cancer cells.

Furthermore, the sugar derivative inhibits the production of lactic acid in cancer cells, as shown in Test Example 2 below. Therefore, the chemokine production promoter according to the present embodiment can be used as a lactic acid production inhibitor. The chemotaxis of T cells to cancer cells is also enhanced by reducing the concentration of lactic acid produced by the cancer cells.

The sugar derivative is T-celled via CXCL9-CXCR3 interaction, CXCL10-CXCR3 interaction and CXCL11-CXCR3 interaction in cancer tissue, as shown in Test Examples 3-5 below, especially when encapsulated in PLGA. It has an antitumor effect by enhancing tumor immunity. Therefore, the chemokine production promoter according to the present embodiment can be used as an immune checkpoint inhibitor-resistant cancer therapeutic agent.

The immune checkpoint inhibitor is not particularly limited as long as it is known. For example, immune checkpoint inhibitors include anti-PD-1 antibody, anti-CTLA-4 antibody and anti-PD-L1 antibody. Immune Checkpoint Inhibitor-Resistant Cancers Are Treated or Treated Even With Effective Amounts of Immune Checkpoint Inhibitors There is no tendency to delay, inhibit, reverse or cure the progression of the cancer, or immune checkpoint inhibitors It is a cancer that is less severe than an effective cancer. It should be noted that the immune checkpoint inhibitor-resistant cancer includes cases in which the tendency of delaying, inhibiting, reversing or curing the progression of cancer by the immune checkpoint inhibitor is smaller than that of other patients even in the same cancer type. Immune checkpoint inhibitor-resistant cancers include, for example, lung cancer and malignant melanoma.

In addition, the chemokine production promoter according to the present embodiment can also be used as an antitumor immunostimulant used in combination with an immune checkpoint inhibitor. The combination means that the antitumor immunostimulant and the immune checkpoint inhibitor are administered to the same patient during a predetermined period. In combination, it is preferable to administer the antitumor immunostimulant at the same time as the immune checkpoint inhibitor, but administer the other independently while the effect of one remains, for example, after a while. You may. In combination, the routes of administration of the antitumor immunostimulant and the immune checkpoint inhibitor may be the same or different. For example, in combination, the antitumor immunostimulant and immune checkpoint inhibitor are administered over a predetermined period of time according to a single regimen that specifies the dosage and usage of each of the antitumor immunostimulant and immune checkpoint inhibitor. To. In another embodiment, an immune checkpoint inhibitor used in combination with the antitumor immunostimulant is provided.

Further, the sugar derivative according to the present embodiment may be encapsulated in biocompatible particles. By encapsulating in biocompatible particles, sugar derivatives can be selectively and efficiently accumulated in the target tissue due to the EPR (Enhanced Permeability and Regeneration) effect, and a high antitumor effect can be obtained.

The anticancer agent provided in another embodiment includes the biocompatible particles in which the above-mentioned sugar derivative or sugar derivative is encapsulated, and an immune checkpoint inhibitor. That is, the anticancer agent contains both a biocompatible particle containing a sugar derivative or a sugar derivative and an immune checkpoint inhibitor in a single preparation. As a result, the sugar derivative and the immune checkpoint inhibitor can be simultaneously administered through the same route of administration. The weight ratio of the sugar derivative to the immune checkpoint inhibitor in the anticancer agent is not particularly limited, and is appropriately adjusted according to the antitumor effect and the like.

In another embodiment, a method of treating or preventing cancer or immune checkpoint inhibitor-resistant cancer of the patient is provided by administering the above sugar derivative to the patient. In addition, a method for promoting the production of chemokines in the subject by administering the above sugar derivative to the subject, a method for inhibiting glucose uptake by the target cancer cells, a method for promoting glucose uptake by the target T cells, and the subject. A method of inhibiting the production of lactic acid from cancer cells in a subject, or a method of activating anti-tumor immunity in a subject is provided.

The present invention will be described in more detail with reference to the following test examples including examples, but the present invention is not limited to the examples and test examples.

(Test Example 1: Examination of T cell chemotaxis enhancement ability through T cell chemotaxis factor secretion by suppressing glycolysis of liver cancer cells)
It was investigated whether inhibition of glycolysis by 2DG enhances the direct secretion of CXCL9 and CXCL10 from hepatocellular carcinoma cells. Huh7 (National University Corporation Tohoku University Institute of Aging Medicine Medical Cell Resource Center / Cell Bank), which is a liver cancer cell line, was seeded in 2 × 10 ⁵ cells and incubated overnight for engraftment. Huh7 was cultured at 37 ° C. in a 5% CO ₂ incubator. As the medium of Huh7, DMEM (Dulvecco's Modified Eagle's Medium) containing 10% fetal bovine serum was used.

2DG was added to the medium so as to be 0.1 mM, 1 mM and 10 mM, and incubated for 24 hours. After that, CXCL9 and CXCL10 secretion from Huh7 were stimulated by adding IFNγ 100 ng / mL and TNFα 50 ng / mL to each of the above media. The concentrations of CXCL9 and CXCL10 in the culture supernatant after 24 hours were measured by the ELISA method (Quantine ELISA Human CXCL9 / MIG Immunoassay and Quantikine ELISA Human CXCL10 / IP-10 Immunoassay, both manufactured by RISA).

In addition, CD8-positive T cells (hereinafter, simply "T cells") to liver cancer cells stimulated to secrete CXCL9 and CXCL10 by IFNγ and TNFα using a real-time cytodynamic analyzer (EZ-TAXIScan, manufactured by Effector cell Institute). It was examined whether or not the chemotaxis of () was further enhanced by 2DG. Furthermore, it was examined whether the effect of 2DG on T cell chemotaxis was canceled by a neutralizing antibody (anti-CXCR3 antibody) against CXCR3, which is a receptor for CXCL9 and CXCL10.

Peripheral blood mononuclear cells were extracted from whole human blood with Lymphoprep (manufactured by AXIS-SHIELD), and T cells were further extracted with CD8 + T cell isolation kit (manufactured by Miltenyi Biotech). In the real-time cytodynamic analyzer, a 4 μm thick chip and cover glass were used. 1 × 10 ⁵ T cells were applied to chamber A of each channel of the real-time cytodynamic analyzer. A Huh7-containing culture supernatant was applied as a sample to chamber B of each channel. Depending on the concentration of CXCL9 and CXCL10 as cell chemotaxis factors contained in chamber B, a concentration gradient of CXCL9 and CXCL10 is formed in a fine flow path interposed between chamber A and chamber B. By imaging the horizontal glass surface (terrace) arranged in the microchannel with a CCD (Charge Coupled Device) camera, the dynamics of the T cells applied to the chamber A on the glass surface can be analyzed.

The sample applied to the chamber B was prepared as follows. After seeding 5 × 10 ⁵ Huh7s and incubating overnight for engraftment, Huh7s were incubated under the following conditions. After incubation, the culture supernatant containing the exfoliated Huh7 was applied to chamber B.
(A1) Control group Incubated in a 2DG-free medium for 48 hours.
(B1) IFNγ + TNFα group After incubation in a 2DG-free medium for 24 hours, 100 ng / mL IFNγ and 50 ng / mL TNFα were added and incubated for 24 hours.
(C1) IFNγ + TNFα + 2DG group After incubation in a medium containing 10 mM 2DG for 24 hours, 100 ng / mL IFNγ and 50 ng / mL TNFα were added and incubated for 24 hours.
(D1) IFNγ + TNFα + 2DG + anti-CXCR3 antibody group After incubation in a medium containing 10 mM 2DG for 24 hours, 100 ng / mL IFNγ, 50 ng / mL TNFα and anti-CXCR3 antibody (manufactured by R & D Systems) were added and incubated for 24 hours.

The holder temperature was set to 37 ° C, and the terrace was imaged for 1.5 hours. The obtained image was analyzed using TAXIScan Analyzer 2 (manufactured by Effector cell Institute), which is dedicated software.

(result)
1 (A) and 1 (B) show the concentrations of CXCL9 and CXCL10 in the Huh7 culture supernatant, respectively. In the non-stimulated group, the concentrations of CXCL9 and CXCL10 were increased by stimulation with IFNγ and TNFα (2DG 0 mM group). The concentrations of CXCL9 and CXCL10 increased even more significantly in a 2DG concentration-dependent manner.

FIG. 2 is a diagram showing photographs of the terraces of the lanes corresponding to A1 to D1 before the start of the experiment (0 hours) and 1.5 hours after the start of the experiment. After 1.5 hours, it was observed that more T cells were migrating from chamber A to chamber B in the IFNγ + TNFα group compared to the control group. In the IFNγ + TNFα + 2DG group, it was observed that more T cells were migrating from chamber A to chamber B. The number of T cells that migrated from chamber A to chamber B in the IFNγ + TNFα + 2DG + anti-CXCR3 antibody group was lower than that in the IFNγ + TNFα + 2DG group.

FIG. 3 shows a VD plot of T cells randomly sampled for measurement on the terrace of each lane. The number of T cells to be measured is 9 in

lane

1, 20 in

lane

2, 20 in

lane

3, and 10 in lane 4. The horizontal axis of the BD plot shows the direction of movement every minute for each T cell to be measured. The direction of cell migration is 1.57 radians in the direction toward chamber B, that is, the direction toward the cell chemotaxis factor, 0 radians in the horizontal direction, and the direction opposite to the direction toward the cell chemotaxis factor. The direction is -1.57 radians. The vertical axis of the BD plot shows the movement rate per minute for each T cell to be measured.

The average values for each lane of all movement speeds and movement directions shown in FIG. 3 are shown in FIGS. 4 and 5, respectively. Compared to the control group (lane 1), T cell chemotaxis was enhanced by IFNγ and TNFα stimulation (lane 2). T cell chemotaxis was further enhanced by administration of 2DG (lane 3). T cell chemotaxis was canceled by the combined use of anti-CXCR3 antibody (lane 4). From the above results, it was proved that 2DG promotes the chemotaxis of T cells by promoting the secretion of CXCL9 and CXCL10 in liver cancer cells.

(Test Example 2: Examination of the effect of 2DG-PLGA on the function of T cells co-cultured with liver cancer cells)
Using ULREA SS-11 (manufactured by M-Technique), 2DG nanoparticles (2DG-PLGA) were prepared as follows. First, the liquid A tank was filled with the liquid A, and the tank was pressurized to 0.3 MPa. Then, the solution A was sent at a set value of 43 ° C. at 167 mL / min, and then the solution B was sent at a set value of 41 ° C. (measured value of about 29 ° C.) at 100 mL / min. Solution A is an aqueous solution containing 0.5% PVA. The solution B is a solution of PLGA: 2DG: acetone: ethanol in a weight ratio of 0.65: 0.25: 66: 33. The rotation speed was 1000 rpm, and the back pressure was 0.02 MPa. 400.5 mL of the discharged liquid was collected, and the solvent was distilled off from the discharged liquid with an evaporator for 80 minutes. As PLGA in Solution B, PLGA-7520 (lactic acid: glycolic acid = 75:25, average weight molecular weight 20,000, manufactured by Wako Pure Chemical Industries, Ltd.) was used. 224 mL of the obtained 2DG-PLGA aqueous dispersion was freeze-dried to obtain 2.16 g of 2DG-PLGA.

3.7 mg of 2DG-PLGA prepared above was weighed into a tube, and 3.7 mL of Milli-Q water was added. After stirring with a vortex mixer for 30 seconds, ultrasonic treatment was performed for 5 minutes. The particle size distribution of 2DG-PLGA was measured with NIKKISO Nanotrac Wave-EX150 (manufactured by Nikkiso Co., Ltd.) using milli-Q water as a control.

The 2DG content in 2DG-PLGA was evaluated as follows. First, a standard solution was prepared. 200 mg of 2DG was weighed in a 100 mL volumetric flask, and 50 mL of water was added to dissolve it. Next, the mixture was made up to 100 mL with acetonitrile to prepare a standard stock solution (2000 μg / mL). The standard stock solution was serially diluted with acetonitrile: water (1: 1) to a concentration of 250, 125, 62.5, 31.25 μg / mL to prepare a standard solution.

In the preparation of the sample solution, about 20 mg of 2DG-PLGA was weighed as a sample, and 10 mL of milliQ water was added. After stirring with a vortex mixer for 30 seconds, sonication was performed for 30 minutes to prepare a sample stock solution. Acetonitrile was added to 1.0 mL of the sample stock solution to make 2 mL. The solution was centrifuged at 10,000 xg for 15 minutes at 20 ° C. The supernatant after centrifugation was filtered using a 0.2 μm membrane filter, and the obtained filtrate was used as a sample solution. The sample solution was tested at n = 3. The standard solution and the sample solution were analyzed by high performance liquid chromatography (HPLC) under the following conditions, and the concentration of 2DG in the sample solution was calculated from the calibration line of the standard solution. The 2DG content was calculated based on the weight of 2DG-PLGA and the concentration of 2DG.

HPLC conditions Equipment: Shimadzu 10A series (manufactured by Shimadzu Corporation)
Detection: Shimadzu ELSD-LT
Gain: 8
Drift temperature: 40 ° C
Gas pressure: 350 kPa (actual measurement 358 kPa)
Nebulizer gas: N2
Column: Shodex Asahipak NH2P-504D (150 mm x 4.6 mm)
Column temperature: 40 ° C
Mobile phase: A: water, B: acetonitrile B. Concentration 75% → 69% (8 minutes) 69 → 75% (8 minutes → 16 minutes)
Flow rate: 1.0 mL / min Column temperature: 40 ° C
Injection volume: 20 μL
Measurement time: 16 minutes

The content of 2DG in 2DG-PLGA was 8.1 ± 0.4%. As shown in FIG. 6, the particle size distribution of 2DG-PLGA was a single peak, D ₅₀ was 166 nm ± 5.4 nm, and the span value was 1.52 ± 0.15. The 2DG-PLGA was used in the following test examples.

The cell chemotaxis of T cells co-cultured with Huh7 using Transwell (manufactured by Corning) was examined, and the effect of suppression of glucose metabolism by 2DG-PLGA on co-cultured T cells in liver cancer cells was analyzed.

A medium containing 5 μM CXCL10 was applied to the chamber B of each channel in the above-mentioned real-time cytodynamic analyzer using a 4 μm-thick tip and cover glass. After seeding 5 × 10 ⁵ Huh7s and incubating them overnight to engraft, T cells to be applied to chamber A were prepared as follows. The T cells used in the following procedure were extracted in the same manner as in Test Example 1.
(A2) PLGA + glucose-free group Huh7 medium was replaced with glucose-free medium containing 10 mM PLGA, incubated for 24 hours, and then co-cultured for 20 hours with Transwell seeded with 1 × 10 ⁵ T cells set. Was done.
(B2) A state in which the medium of the 2DG-PLGA + glucose-free group Huh7 was replaced with a glucose-free medium containing 10 mM 2DG-PLGA, incubated for 24 hours, and then Transwell in which 1 × 10 ⁵ T cells were seeded was set. Was co-cultured for 20 hours.
(C2) The medium of the group Huh7 with PLGA + glucose was replaced with a medium containing 10 mM PLGA and 900 mg / dL glucose, and after incubation for 24 hours, a Transwell in which 1 × 10 ⁵ T cells were seeded was set for 20 hours. Co-culture was performed.
(D2) The medium of the group Huh7 with 2DG-PLGA + glucose was replaced with a medium containing 10 mM 2DG-PLGA and 900 mg / dL glucose, and after incubating for 24 hours, a Transwell seeded with 1 × 10 ⁵ T cells was set. The co-culture was carried out for 20 hours in this state.

For the above A2 to D2, after co-culturing, the culture supernatant containing the exfoliated Huh7 was applied to chamber A. The holder temperature was set to 37 ° C. and the terrace was imaged for 2 hours. The obtained image was analyzed using TAXIScan Analyzer 2.

For the above A2-D2, the amount of lactic acid produced in the Huh7 culture supernatant 24 hours after the administration of PLGA or 2DG-PLGA was measured (Glycolysis Cell-Based Assay kit, manufactured by Cayman Chemical Co., Ltd.).

In addition, T cells (A3 to D2) prepared in the same manner as A2 to D2, except that the number of T cells seeded in Transwell is 1 × 10 ⁶ and the co-culture time of Huh7 and T cells is 4 hours. D3) was obtained. For A3 to D3 T cells, mRNA was extracted using RNeasy MiniKit (manufactured by Qiagen), cDNA was synthesized by SuperScript VILO (trademark), manufactured by Invitrogen), and then β was performed by real-time PCR (Polymerase Chain Reaction). -The expression levels of IFNγ mRNA corrected for actin mRNA were compared (TaqMan Gene Expression Assays, Assay ID; Hs0998291_m1 (IFNγ), Hs01060665_m1 (β-actin), Applied Biosystems).

After seeding 5 × 10 ⁴ Huh7 and incubating overnight to engraft, they were prepared in the same manner as A3 to D3 except that the number of T cells seeded in Transwell was 5 × 10 ⁵ . T cells (A4-D4) were obtained. For A4-D4 T cells, a fluorescent glucose analog 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxdiazol-4-yl) amino] -D-glucose) Glucose uptake was evaluated using. Specifically, the medium of T cells was replaced with a medium containing 2-NBDG (manufactured by Cayman Chemical) and incubated for 16 hours to separate Huh7 and T cells. Then, regarding the amount of 2-NBDG uptake per unit protein in Huh7 and the amount of 2-NBDG uptake per unit cell in T cells, 4 groups were used using a fluorescent microplate reader (FLOO star OPTIMA system, manufactured by BMG Labtechnologies). A relative comparison was made between them.

(result)
FIG. 7 is a diagram showing photographs of the terrace before the start of the experiment (0 hours) and 2 hours after the start of the experiment. In the PLGA + glucose-free group and the 2DG-PLGA + glucose-free group, almost no T cells migrated in the direction of chamber B to which CXCL10 was applied were observed. In the group with PLGA + glucose, T cells were observed to move toward chamber B. In the group with 2DG-PLGA + glucose, more T cells were migrating toward chamber B.

FIG. 8 is a diagram showing a VD plot similar to Test Example 1 above. The number of T cells to be measured is 5 in

lane

1, 5 in

lane

2, 15 in

lane

3, and 15 in lane 4. The horizontal and vertical axes of the VD plot indicate the direction of movement per minute for each T cell to be measured and the movement speed for each minute per T cell to be measured. The average values for each lane of all movement speeds and movement directions shown in FIG. 8 are shown in FIGS. 9 and 10, respectively. Under the condition with glucose, the cell chemotaxis index in both directionality and migration speed was higher in the group with 2DG-PLGA + glucose than in the group with PLGA + glucose.

FIG. 11 shows the results of the amount of lactic acid produced in the Huh7 culture supernatant 24 hours after the administration of PLGA or 2DG-PLGA in each of the above groups A2 to D2. No significant lactate production was observed with either PLGA or 2DG-PLGA administration under the condition without glucose. In the condition with glucose, the amount of lactic acid produced increased by the administration of PLGA was significantly suppressed by the administration of 2DG-PLGA.

FIG. 12 shows the relative expression level of IFNγ mRNA in T cells at 4 hours after co-culture with Huh7 in each of the above groups A3 to D3. In both cases without glucose and with glucose, the expression level of IFNγ mRNA was significantly increased when 2DG-PLGA was administered as compared with the case where PLGA was administered.

The relative uptake amount of 2-NBDG of Huh7 in the above A4 to D4 is shown in FIG. The amount of 2-NBDG uptake of Huh7 was reduced in the 2DG-PLGA-administered group as compared to the PLGA-administered group, both without glucose and with glucose. In particular, under the condition with glucose, the decrease in 2-NBDG uptake in Huh7 was significantly reduced when 2DG-PLGA was administered as compared to when PLGA was administered.

FIG. 14 shows the relative uptake of 2-NBDG of T cells in the above A4 to D4. The amount of 2-NBDG uptake of T cells, both without glucose and with glucose, was significantly increased in the 2DG-PLGA-administered group as compared to the PLGA-administered group.

From the above results, it was clarified that 2DG-PLGA enhances T cell functions such as T cell chemotaxis and IFNγ production in T cells co-cultured with liver cancer cells in a high-concentration glucose environment. In addition, as one of the mechanisms, the relative increase in glucose uptake of T cells accompanying the decrease in glucose uptake of hepatocellular carcinoma in the microenvironment of cancer, or the decrease in lactate production from hepatocellular carcinoma cells causes T cells. It was considered that the activation of the function of lactic acid was involved.

(Test Example 3: Examination of antitumor effect through enhanced expression of 2DG CXCL9, CXCL10 and CXCL11 on immune-responsive liver carcinogenic mice)
For STAM (Stelic Animal Model) mice, which are model animals for liver carcinogenesis, 200 μg of streptozotocin was subcutaneously administered to 2-day-old C57BL6 / N mouse males, and weaned 4 weeks after birth to feed on a high-fat diet. Produced by switching. 12-week-old male STM mice (manufactured by SMC Laboratories) were grouped into the following two groups.

(A1) PLGA group PLGA was administered to the tail vein once a week at 800 mg / kg / day.
(B1) 2DG-PLGA group 2DG-PLGA was administered to the tail vein once a week at 800 mg / kg / day.

2DG-PLGA of b1 was made into a suspension of 100 mg / mL with phosphate buffered saline (PBS) before each administration, and 200 μL was administered per animal. Considering that the 2DG filling rate in 2DG-PLGA is about 8%, a substantial single dose of 2DG per mouse in b1 corresponds to about 1.6 mg.

In a1 and b1, the entire liver was observed on the 21st day from the start of administration, and the maximum tumor diameter, the number of tumors, and the total tumor volume in the liver were measured. In addition, mRNA was extracted from the tumor tissue in a1 and b1 using RNeasy MiniKit (manufactured by Qiagen), cDNA was synthesized by SuperScript VILOTM (manufactured by Invitrogen), and then corrected with β-actin mRNA by real-time PCR. The expression levels of CXCL9, CXCL10 and CXCL11 mRNA were compared (TaqMan Gene Expression Assays, Assay ID; Mm00434946_m1 (CXCL9), Mm00445235_m1 (CXCL10), Mm00446262_m1 (CXCL10), Mm00444662_m1 (CXCL10)

(result)
Tumors in the liver 21 days after the start of administration are indicated by the arrows in FIG. 16 and 17 show the maximum tumor diameter and total tumor volume 21 days after the start of administration, respectively. The maximum tumor diameter and total tumor volume were significantly reduced in the 2DG-PLGA group compared to the PLGA group. Furthermore, FIGS. 18, 19 and 20 show the mRNA expression levels of CXCL9, CXCL10 and CXCL11 in the tumor tissue in each group, respectively. The mRNA expression levels of CXCL9, CXCL10 and CXCL11 were significantly increased in the 2DG-PLGA administration group as compared with the PLGA administration group.

The STAM mouse is a mouse that is not immunodeficient, including T cell immunity. Therefore, the antitumor effect mediated by the upregulation of CXCL9, CXCL10 and CXCL11 expression by 2DG-PLGA on an immune-responsive liver carcinogenesis mouse model was demonstrated.

(Test Example 4: Examination of antitumor effect of 2DG and 2DG-PLGA in anti-PD-1 antibody resistant mouse)
1 × 10 ⁷ anti-PD-1 antibody-resistant malignant melanoma cell lines B16F10 were subcutaneously transplanted into 6-week-old C57BL / 6J mice (male). In order to confirm the engraftment and proliferation of the transplanted cells, the tumor size was measured with a caliper, and when the tumor volume reached 100 to 150 mm ³ , the tumors were divided into the following 7 groups.
(A2) Control group 200 μg of isotype rat IgG was intraperitoneally administered every 3 days.
(B2) PLGA group 200 μg of isotype rat IgG was intraperitoneally administered every 3 days, and PLGA 800 mg / kg / day was administered to the tail vein once a week.
(C2) 2DG group In addition to intraperitoneal administration of 200 μg of isotype rat IgG every 3 days, 2DG was intraperitoneally administered at 100 mg / kg / day every day.
(D2) 2DG-PLGA group In addition to intraperitoneal administration of 200 μg of isotype rat IgG every 3 days, 2DG-PLGA was administered by tail vein once a week at 800 mg / kg / day.
(E2) Anti-PD-1 antibody group 200 μg of anti-PD-1 antibody (InVivoMAb anti-mouse PD-1, manufactured by BioXCell) was intraperitoneally administered every 3 days.

It should be noted that the substantial single dose of 2DG administered at 100 mg / kg / day in c2 corresponds to about 2.7 mg per mouse having an average body weight of 27 g. PLGA of b2 and 2DG-PLGA of d2 were made into a suspension of 100 mg / mL with PBS before each administration, and 200 μL was administered per animal. Considering that the 2DG filling rate in 2DG-PLGA is about 8%, the actual single dose of 2DG per mouse in the above d2 corresponds to about 1.6 mg, which is about 60% of c2. Become.

In a2 to e2, the tumor volume was measured every 3 days from the start of administration, and the transition of the tumor volume over time was evaluated. In addition, 12 days after administration, the tumor was collected, observed with the naked eye, and blood was collected.

(result)
21 and 22, respectively, show the appearance of the tumor and the time course of the tumor volume 12 days after the start of administration, respectively. Compared with the control group, the PLGA group and the anti-PD-1 antibody group, the tumor of malignant melanoma cells was smaller in the 2DG group, and the increase in tumor volume was suppressed. Furthermore, the tumors of malignant melanoma cells were clearly smaller in the 2DG-PLGA group than in the control group, PLGA group and anti-PD-1 antibody group, and the increase in tumor volume was significantly suppressed.

(Test Example 5: Examination of antitumor effect mediated by T-cell tumor immunity by 2DG-PLGA in anti-PD-1 antibody-resistant mice)
Six 1 × 10 ⁶ B16F10 mice were subcutaneously transplanted into 6-week-old C57BL / 6J mice (male). In order to confirm the engraftment and proliferation of the transplanted cells, the tumor size was measured with a caliper, and when the tumor volume reached 100 to 150 mm ³ , the tumors were divided into the following four groups.
(A3) PLGA was administered to the tail vein once a week at 800 mg / kg / day in the control group. In addition, 200 μg of isotype hamster IgG was intraperitoneally administered every 3 days after the day before the first administration of PLGA.
(B3) Anti-CXCR3 antibody group PLGA was administered to the tail vein once a week at 800 mg / kg / day. In addition, 200 μg of anti-CXCR3 antibody was intraperitoneally administered every 3 days after the day before the first administration of PLGA.
(C3) 2DG-PLGA group 2DG-PLGA was administered to the tail vein once a week at 800 mg / kg / day. In addition, 200 μg of isotype hamster IgG was intraperitoneally administered every 3 days after the day before the first administration of 2DG-PLGA.
(D3) 2DG-PLGA + anti-CXCR3 antibody group 2DG-PLGA was administered to the tail vein once a week at 800 mg / kg / day. In addition, 200 μg of anti-CXCR3 antibody was intraperitoneally administered every 3 days after the day before the first administration of 2DG-PLGA.

The PLGA of a3 and b3 and the 2DG-PLGA of c3 and d3 were made into a suspension of 100 mg / mL in PBS before each administration, and 200 μL was administered to each animal. Considering that the 2DG filling rate in 2DG-PLGA is about 8%, a substantial single dose of 2DG per mouse in c3 and d3 corresponds to about 1.6 mg.

In a3 to d3, the tumor volume was measured every 3 days from the start of administration, and the transition of the tumor volume over time was evaluated. In addition, 12 days after administration, the tumor was collected, observed with the naked eye, and blood was collected. In addition, immunostaining was performed on liver tumor tissue using a Rabbit Monoclonal CD3 antibody (manufactured by GeneTex).

(result)
23 and 24 show the appearance of the tumor and the time course of the tumor volume 12 days after the start of administration, respectively. Compared with the control group and the anti-CXCR3 antibody group, the tumor of malignant melanoma cells was clearly smaller in the 2DG-PLGA group, and the increase in tumor volume was significantly suppressed. On the other hand, in the 2DG-PLGA + anti-CXCR3 antibody group, the effect of suppressing tumor volume increase was partially reduced.

The result of immunostaining of liver tumor tissue is shown in FIG. Arrows in FIG. 25 indicate CD3-positive T cells. FIG. 26 shows the number of CD3-positive T cells per HPF (high-power field). Compared with the control group and the anti-CXCR3 antibody group, a significant increase in T cell infiltration into the tumor was observed in the 2DG-PLGA group. In addition, the enhancement of T cell infiltration into the tumor by 2DG-PLGA was suppressed by the anti-CXCR3 antibody. From the results of this test example and the result of test example 3, regarding the antitumor effect of 2DG-PLGA, T cell tumor immunity through CXCL9-CXCR3 interaction, CXCL10-CXCR3 interaction and CXCL11-CXCR3 interaction in cancer tissue Involvement was shown.

(Test Example 6: Examination of the combined effect of 2DG-PLGA and anti-PD-1 antibody on liver carcinogenic mice) 12-week-old male STM mice (manufactured by SMC Laboratories) were grouped into the following four groups.
(A4) Control group 200 μg of isotype rat IgG was intraperitoneally administered every 3 days.
(B4) Anti-PD-1 antibody group 200 μg of anti-PD-1 antibody was intraperitoneally administered every 3 days.
(C4) Anti-PD-1 antibody + 2DG group In addition to intraperitoneal administration of 200 μg of anti-PD-1 antibody every 3 days, 2DG was intraperitoneally administered at 100 mg / kg / day every day.
(D4) Anti-PD-1 antibody + 2DG-PLGA group In addition to intraperitoneal administration of 200 μg of anti-PD-1 antibody every 3 days, 2DG-PLGA was administered by tail vein once a week at 800 mg / kg / day.

It should be noted that the substantially single dose of 2DG administered at 100 mg / kg / day administered with c4 corresponds to about 2.7 mg as in the case of c2 above. 2DG-PLGA of d4 was made into a suspension of 100 mg / mL in PBS before each administration, and 200 μL was administered to each animal. Considering that the 2DG filling rate in 2DG-PLGA is about 8%, the actual single dose of 2DG per mouse in d4 corresponds to about 1.6 mg, which is about 60% of c4. ..

In a4 to d4, the entire liver was observed on the 21st day from the start of administration, the maximum tumor diameter, the number of tumors, and the total tumor volume in the liver were measured, and blood was collected.

(result)
Tumors in the liver 21 days after the start of administration are indicated by the arrows in FIG. 28 and 29 show the maximum tumor diameter and total tumor volume 21 days after the start of administration, respectively. The combined use of anti-PD-1 antibody and 2DG reduced the maximum tumor diameter and total tumor volume compared to the anti-PD-1 antibody group. Furthermore, the combined use of anti-PD-1 antibody and 2DG-PLGA was found to significantly suppress the total tumor volume.

Table 1 shows serological findings and liver weight on the 21st day after the start of administration. No significant difference was found between all groups. From the above, the combined effect of 2DG-PLGA and anti-PD-1 antibody on a mouse model of liver carcinogenesis was shown.

The present invention enables various embodiments and modifications without departing from the broad spirit and scope of the present invention. Moreover, the above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the scope of claims, not by the embodiment. Then, various modifications made within the scope of the claims and the equivalent meaning of the invention are considered to be within the scope of the present invention.

This application is based on Japanese Patent Application No. 2019-48743, filed on March 15, 2019. The specification of Japanese Patent Application No. 2019-48743, the scope of claims, and the entire drawing shall be incorporated into this specification as a reference.

The present invention is suitable for pharmaceuticals.

Claims

Contains sugar derivatives,
Chemokine production promoter.
Promotes the production of CXCL9, CXCL10 and CXCL11 from cancer cells,
The chemokine production promoter according to claim 1.
Further containing biocompatible particles containing a lactic acid / glycolic acid copolymer
The sugar derivative is
Encapsulated in the biocompatible particles,
The chemokine production promoter according to claim 1 or 2.
Contains sugar derivatives,
Immune checkpoint inhibitor A drug for the treatment of resistant cancer.
Contains sugar derivatives
Used in combination with immune checkpoint inhibitors,
Anti-tumor immunostimulant.
The sugar derivative is
2-Deoxy-D-glucose,
The chemokine production promoter according to any one of claims 1 to 3, the immune checkpoint inhibitor-resistant cancer therapeutic agent according to claim 4, or the antitumor immunostimulant according to claim 5.