KR20180007726A - Anti-cancer adjuvant - Google Patents

Abstract

The present invention relates to a supplement capable of being used in cancer treatment by using a cell therapy agent, and a method using the same. According to the present invention, since it is possible to prevent reduction in activities of effector cells injected for treatment due to immunosuppression shown in tumor microenvironment in target objects suffering from cancer, therapeutic effects for cancer using cell therapies remarkably increase.

Description

ANTI-CANCER ADJUVANT

The present invention relates to adjuvants which can be used in the treatment of cancer diseases using cell therapy agents.

Adoptive immune cell therapy is a method of injecting cells into a patient by activating the cells in vitro, and a cell therapy agent including dendritic cells, T cells, and NK cells is being developed for clinical application. Among them, NK cells are one of the innate immune cells, which are capable of killing various kinds of cancer cells and recognizing cancer cells regardless of the presence or absence of the antigen.

In vitro expanded NK cells have excellent in vitro killing activity against solid cancer cell lines in vitro, but they do not show clear cure effects in actual clinical trials. The main reason for the lack of clear effects of cell therapy in clinical trials of patients with solid tumors is the serious immune suppression in the tumor microenvironment.

The microenvironment of the tumor has the following characteristics. 1) Collagen or junction protein constitutes an extracellular matrix that physically interferes with the invasion of immune cells. 2) Immunosuppressive cells such as regulatory T cells and regulatory B cells And 3) secretion of immunosuppressive cytokines such as IL-10 and TGF-beta is known to suppress the function of T cells and NK cells acting as actual effect cells.

Therefore, in order to overcome these limitations, there is a need for continuous research on a method for increasing the therapeutic effect of a cell therapy agent by controlling the environment of the body.

In order to solve the above problems, the present invention provides a method for increasing the ability of killing natural killer cells to kill cancer cells, that is, the therapeutic effect, in treatment of cancer diseases using natural killer cells or cell therapy using NK cell- A kit for the treatment of cancer diseases, and a method of administration for the treatment of cancer diseases.

The inventors of the present invention have been studying a method of increasing the therapeutic ability of an effector cell in cell therapy. In the case of administering a TGF-beta inhibitor in combination with a cell treatment using NK cells, The present inventors have completed the present invention by experimentally confirming that the problem of deterioration of anticancer activity can be solved and the treatment effect is remarkably higher than that of single administration.

Accordingly, in this aspect, the present invention provides an anticancer adjuvant for use in the treatment of cancer diseases using a cell therapy agent comprising TGF-beta (Transforming growth factor beta) inhibitor and a natural killer cell (NK cell) .

In addition, the present invention provides TGF-beta inhibitors and natural killer cells, and provides a pharmaceutical combination for individual, sequential or simultaneous use in the treatment of cancer diseases.

The present invention also provides a kit for the treatment of cancer diseases for individual, sequential or simultaneous use in the treatment of cancer diseases, comprising TGF-beta inhibitor and natural killer cells:

The present invention also provides a cell therapy adjuvant for use in cell therapy using natural killer cells (NK cells), which comprises a TGF-beta inhibitor.

The present invention also relates to a method for separately, sequentially or simultaneously administering a cell therapy agent and a TGF-beta inhibitor containing NK cells to a subject using the adjuvant, the pharmaceutical combination drug or the cancer disease treatment kit of the present invention, A method for treating a disease.

By using the present invention, it is possible to prevent the activity of the effector cells administered for treatment by the immunosuppression phenomenon appearing in the tumor microenvironment of a tumor-bearing subject, from deteriorating, Can be improved.

FIG. 1 is a schematic diagram showing an administration schedule of an NK cell therapeutic agent and a TGF-beta inhibitor EW-7197 according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of inhibiting tumor growth according to the treatment of NK cell therapy with TGF-beta inhibitor according to an embodiment of the present invention.
FIGS. 3A and 3B are graphs showing the effect of EW-7197 on the proliferation of A375 cell line and PANC-1 cell line. FIG.

Hereinafter, the present invention will be described in detail.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, unless further departing from the scope of the invention as defined by the following claims. It is not. It is to be understood that the scope of the present invention includes all modifications, equivalents, and alternatives falling within the scope and spirit of the present invention.

And a cell therapy adjuvant for use in the treatment of cancer diseases using a cell therapy agent comprising a TGF-beta (Transforming growth factor beta) inhibitor and a natural killer cell (NK cell) .

The adjuvant including the TGF-beta (Transforming growth factor beta) inhibitor may be a chemotherapeutic assistant or a cell therapy adjuvant for use in the treatment of cancer diseases using a cell therapy agent containing a natural killer cell (NK cell) have.

In the present invention, "adjuvant" serves to increase the effects of therapeutic agents and substances, and the anticancer adjuvant may be used as an anticancer agent, And the cell therapy adjuvant means a preparation for enhancing the therapeutic effect of an effective cell by allowing the cell therapy agent administered to the subject to be increased or decreased in the activity of the effect cell.

Transforming growth factor beta (TGF-beta) is a protein called beta-transforming growth factor-beta, which induces apoptosis in the body and plays a role in lymphocyte function and immune cell function.

In the present invention, an inhibitor means a substance that interferes with the function, expression or secretion of a specific substance and acts to suppress the reaction. The TGF-beta inhibitor inhibits the activity or secretion of TGF- Of the substance.

The TGF-beta inhibitor may be selected from the group including, for example, EW-7197, LY2157299, GC1008, PF-03446962, IMC-TR1, LY2382770, SD-208, LY3022859 and ACE- It is not.

According to one embodiment of the present invention, the TGF-? Inhibitor is selected from the group consisting of N - ((4 - ([1,2,4] triazolo [1,5-a] pyridin- -2-yl) -1H-imidazol-2-yl) methyl) -2-fluoroaniline or a derivative thereof.

The TGF-beta inhibitor may be included in the present invention without being limited to the method of administration such as oral administration, intravenous administration, subcutaneous administration and the like. In the case of a preparation for intravenous administration, it can be administered simultaneously or sequentially with NK cells. In the case of an oral administration agent, it can be administered separately from NK cells, which can be determined by therapeutic judgment depending on the subject to be treated.

The inventors of the present invention have found that when the TGF-beta inhibitor is coadministered in cell therapy, the immune cells administered to the subject can be inhibited from being degraded by the tumor microenvironment and thus the anticancer activity can be remarkably improved. Therefore, when the TGF-beta inhibitor of the present invention is used as an anti-cancer adjuvant or a cell therapy adjuvant, the therapeutic effect can be improved.

In the present invention, "cell therapy" refers to a method of preparing cells having normal functions and injecting them into a target body to treat the cells. Specifically, cells that are expected to have immunity against tumors are treated with a tumor May be an immunotherapy that anticancer effects are expected.

In one embodiment of the present invention, when the in vitro cultured NK cells are injected into a subject to treat cancer, when the TGF-beta inhibitor is administered to a subject, deterioration of cancer cell killing ability of NK cells can be prevented, It was experimentally confirmed that the anticancer activity was remarkably higher than that of the single treatment.

The adjuvants of the present invention can be prepared by incorporating pharmaceutically suitable and physiologically acceptable ingredients, and examples thereof include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants, Of a solubilizing agent may be used, but is not limited thereto.

The adjuvants of the present invention may be formulated into adjuvants for administration by including at least one pharmaceutically acceptable carrier in addition to the active ingredient. Examples of the carrier, excipient or diluent which can be included in the adjuvant of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate But are not limited to, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The adjuvants of the present invention may be formulations for oral or parenteral administration. For example, the oral preparation may be a capsule, a tablet, a coated tablet, a lozenge, a granule, a powder, a syrup, a suspension, an emulsion, a juice, an aerosol, a left parenteral, Emulsions, and freeze-dried preparations. In the case of parenteral formulations, it can be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, rectal, or intradermal routes.

In one embodiment of the present invention, it has been confirmed that NK cells can be administered intravenously, and adjuvant can be administered by intramuscular injection to improve anticancer activity.

In the case of preparations for oral administration, acceptable pharmaceutical carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof. The carrier may also comprise all components of the coating composition which may include plasticizers, pigments, coloring agents, stabilizers and fluidizing agents. Examples of suitable coating materials include cerulose, such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate and hydroxypropylmethylcellulose acetate succinate, Os polymer; But are not limited to, polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins, zein, shellac, and polysaccharides. In addition, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, fluidizers, stabilizers, porosifiers and surfactants. Any pharmaceutically acceptable excipient includes, but is not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants.

The diluent is generally required to increase the volume of the solid dosage form, thereby providing a particle size for compression of the tablet or formation of beads and granules. Suitable diluents are selected from the group consisting of calcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dried starch, hydrolyzed starch, But are not limited to, silicon, titanium oxide, magnesium aluminum silicate, and powdered sugar. Binders are used to impart adhesion properties to solid dosage forms and to ensure that tablets or beads or granules remain intact after being formulated into the dosage form. Suitable binding materials include natural and synthetic materials such as starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycols, waxes, acacia, tragacanth, sodium alginate Cellulose derivatives such as cellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate But are not limited to, synthetic polymers such as copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid, and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc and mineral oil.

Disintegrators are used to facilitate the collapse or breakage of the dosage form after administration and generally include starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropylcellulose, But are not limited to, cross-linked polymers such as starch, clay, cerulose, arginine, gum or cross-linked PVP.

Stabilizers are used, for example, to inhibit or retard drug degradation reactions, including oxidation reactions. Suitable stabilizers include antioxidants, butylated hydroxytoluene (BHT), ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; Sulfites such as sodium metabisulfite; Cysteine and its derivatives; Citric acid; But are not limited to, propyl gallate, and butylated hydroxyanisole (BHA).

Oral dosage forms such as capsules, tablets, solutions and suspensions may be formulated to have controlled release. For example, one or more compounds and optionally one or more additional active ingredients may be formulated as nanoparticles, microparticles, and combinations thereof, encapsulated in a soft or hard gelatin or non-gelatin capsule, or dispersed in a dispersion medium to form an oral suspension or syrup Can be formed. The particles may be formed into a drug and a controlled release polymer or matrix. Alternatively, the drug particles may be coated with one or more controlled release coatings before incorporation into the finished dosage form.

Formulations for parenteral administration may be prepared in an aqueous composition using techniques known to those skilled in the art. In general, such compositions include injectable formulations, for example, solutions or suspensions; Solid forms suitable for use in preparing solutions or suspensions upon addition of reconstitution medium prior to injection, such as micro- or nanoparticles; Emulsions such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions, liposomes or emulsions thereof.

The carrier may be, for example, water, ethanol, one or more polyols such as glycerol, propylene glycol, and liquid polyethylene glycol, oils such as vegetable oils such as peanut oil, corn oil, sesame oil, But are not limited thereto. Proper fluidity can be maintained by using coatings such as lecithin or by maintaining the required particle size in the case of dispersions, or by using surfactants. It may also include, but is not limited to, isotonic agents of sugars or salts such as sodium chloride.

Solutions or dispersions of the active compounds as the free acid or free base or a pharmaceutically acceptable salt thereof may be prepared in water or other solvent or dispersion medium suitably mixed with one or more pharmaceutically acceptable excipients. Excipients include, but are not limited to, for example, surfactants, dispersants, emulsifiers, pH adjusting agents, and combinations thereof.

Suitable surfactants can be anionic, cationic, amphoteric or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylates, sulfonates and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates such as sodium dodecylbenzenesulfonate and alkyl aryl sulfonates; Dialkyl sodium sulfosuccinates such as sodium dodecylbenzenesulfonate; Dialkyl sodium sulfosuccinates such as sodium bis- (2-ethylthioxyl) -sulfosuccinate; And alkyl sulphates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, brominated seturonium, stearyldimethylbenzylammonium chloride, polyoxyethylene and coconut amines. Examples of nonionic surfactants are ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, poloxamer 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include, but are not limited to, sodium N-dodecyl-alanine, sodium N-lauryl-iminodipropionate, myristole amphoacetate, lauryl betaine and lauryl sulfobetaine It is not.

In addition, the preparation may contain preservatives that inhibit the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid and thimerosal. The formulation may also contain, but is not limited to, an antioxidant capable of preventing the degradation of the active ingredient (s).

In one embodiment of the invention, the adjuvants of the invention may be administered separately, sequentially or co-administering or administering a cell therapy agent or immunocyte. The above-mentioned administration includes both oral administration and parenteral administration such as tablets, capsules and the like.

In one embodiment of the invention, the cancer disease is selected from the group consisting of breast cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, , Ovarian cancer, endometrial cancer, cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer and bone cancer.

According to still another aspect of the present invention, there is provided a pharmaceutical combination comprising a TGF-beta inhibitor and a natural killer cell.

The above pharmaceutical combination agent is for administering TGF-beta inhibitor and natural killer cells to a subject separately, sequentially or concurrently, and may have different administration characteristics depending on the type of the TGF-beta inhibitor used concurrently.

In one embodiment of the present invention, natural killer cells are administered intravenously and EW-7197, which is a TGF-beta inhibitor, is orally administered separately, and when the above-mentioned coadminister is administered to a subject, it can have a remarkable anticancer effect Respectively.

In one embodiment of the present invention, EW-7197 and NK cells that are co-administered may be a 0.1mg / kg to ^{10mg / kg, 1x10 8 cells /} kg to about 5x10 ⁸ cells / kg, respectively. The specific dose may be determined in consideration of the patient's condition, age, height, size of the lesion, condition, and the like.

Other anticancer agents, anticancer activities, constituents and components to be added for the preparation are the same as those described above, and thus are not described in duplicate.

According to still another aspect of the present invention, there is provided a kit for the treatment of cancer diseases comprising a TGF-beta inhibitor and natural killer cells.

The kit may comprise a TGF-beta inhibitor in a first compartment and a natural killer cell in a second compartment and may be administered to the subject separately, sequentially or concurrently.

The compartment may be a container capable of accommodating the pharmaceutical preparation according to the present invention, and may be, for example, a vial, but is not limited thereto.

The anticancer agent, the anticancer activity, the constituents and the constituents added for the preparation in the kit are the same as those described above, and thus are not described in duplicate.

According to still another aspect of the present invention, there is provided a method of treating cancer, comprising administering to a subject a cell treatment agent containing NK cells and a TGF-beta inhibitor separately, sequentially or concurrently, using the adjuvant, the pharmaceutical adjuvant, And a method for treating cancer diseases using the same.

The anticancer agent, the anticancer activity, the constituents and the constituents added for the preparation in the above method are the same as those described above, and thus are not described in duplicate.

Hereinafter, the present invention will be described in detail with reference to Production Examples and Experimental Examples. The following Examples and Experiments are illustrative of the present invention and are not intended to limit the scope of the present invention.

[ Test Example  1] Concomitant administration Anticancer effect  Confirm

NK cells were collected from human blood and centrifuged at 2500 rpm for 30 minutes using Ficoll (Ficoll-paque ™ PLUS, GE healthcare), and peripheral blood mononuclear cells were isolated from the buffy coat. Thereafter, hRPMI medium containing 10% FBS and 1% penicillin / streptomycin in RPMI1640 medium at a ratio of 1: 0.5: 0.5 in the presence of IL-2 500 U / ml using Jurkat cell line and EBV-LCL cell line irradiated with 100 Gy And cultured with hRPMI medium supplemented with 500 U / ml of IL-2 once every 4 days. The natural killer cells cultured for about 2 weeks are placed in a solution containing 90% FBS and 10% DMSO, placed in an isopropanol jar, frozen at -80 ° C and stored. The cells were dissolved in a 37 ° C water bath before use in vivo and counted using a hematocytometer to prepare ⁵ × 10 ⁷ cells / ml.

EW-7197 (medipacto) was prepared as a TGF-β inhibitor and dissolved in artificial gastric fluid (2 g / L NaCl, 3.2 g / L Pepsin, 0.06 M HCl) and used at 2.5 mg / kg.

Tumors were induced by injecting melanoma cell line A375, subcutaneously, at 2.5 x 10 ⁶ cells at 7 to 8 weeks of age in immunodeficient mice (NSG, 25-30 g). After 7 days of tumor formation, 2.5 mg / kg of TGF-beta inhibitor, EW-7197, was orally administered five times per week and NK cells were intravenously injected once every 7 days with 1 x 10 ⁷ cells (Fig. 1). Total 25 days, and tumor size was measured once every 3 days. After 28 days, the mice were killed and the tumors were weighed. The results are shown in Fig.

As shown in Fig. 2, the NK cells alone or in combination with the TGF-beta inhibitor alone and the NK cells and the TGF-beta inhibitor in the NK cell or TGF-beta inhibitor alone Although the antitumor effect was observed, it was confirmed that the tumor volume of the combined administration group was 50% or more lower than that of the single administration group. Therefore, it was confirmed that the combination of NK cell and TGF-beta inhibitor can significantly improve the anticancer activity.

[ Test Example  2] TGF -β inhibitor on tumor cells

Experiments were performed to determine whether the EW-7197 formulation directly affects tumor cell proliferation.

1 × 10 ⁴ cells were seeded in a 96-well culture plate, and EW-7197 was treated with 10, 100, and 1000 nM for 48 hours, followed by CCK-8 And the cell viability of A375 (FIG. 3 a) and PANC-1 (FIG. 3 b) cell lines was shown in a graph.

As shown in FIGS. 3A and 3B, when the treatment concentration of EW-7197 was increased in both cancer cell lines, cell viability was decreased, but there was no significant difference. Thus, the combined effect of EW-7197 .

Claims (11)

TGF-beta (Transforming growth factor beta) inhibitors,
Anticancer adjuvant for use in the treatment of cancer diseases using cellular therapeutic agents including NK cells. The method according to claim 1,
Wherein the TGF-beta inhibitor is at least one selected from the group consisting of EW-7197, LY2157299, GC1008, PF-03446962, IMC-TR1, LY2382770, SD-208, LY3022859 and ACE-041. The method according to claim 1,
The TGF- [beta] -inhibitor is an N - ((4 - ([1,2,4] triazolo [1,5-a] pyridin-6-yl) -5- (6- methylpyridin- 2-yl) methyl) -2-fluoroaniline or derivatives thereof. The method according to claim 1,
The cancer diseases include cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, colon cancer, fallopian tube cancer, endometrial cancer Wherein the cancer is selected from the group consisting of cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer and bone cancer. The method according to claim 1,
Wherein the anticancer adjuvant is for individual, sequential or simultaneous use with a cell therapy agent. TGF-beta < / RTI > inhibitors and natural killer cells,
A pharmaceutical combination for individual, sequential or simultaneous use in the treatment of cancer diseases. The method according to claim 6,
The TGF- [beta] -inhibitor is an N - ((4 - ([1,2,4] triazolo [1,5-a] pyridin-6-yl) -5- (6- methylpyridin- 2-yl) methyl) -2-fluoroaniline or a derivative thereof. TGF-beta < / RTI > inhibitors and natural killer cells,
Kits for the treatment of cancer diseases for individual, sequential or simultaneous use in the treatment of cancer diseases. 9. The method of claim 8,
A TGF-beta inhibitor is included in the first compartment, and
Wherein the natural killer cells are contained in the second compartment and can be administered to the subject individually, sequentially or concurrently. 9. The method of claim 8,
The TGF- [beta] -inhibitor is an N - ((4 - ([1,2,4] triazolo [1,5-a] pyridin-6-yl) -5- (6- methylpyridin- 2-yl) methyl) -2-fluoroaniline or a derivative thereof. TGF-beta (Transforming growth factor beta) inhibitors,
Cell therapy adjuvant for use in cell therapy using natural killer cells (NK cells).

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