Ppharmaceutical composition for prevention or treatment of anticancer-agent-resistant cancer containing cabozantinib as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of anticancer-agent-resistant cancer, containing cabozantinib as an active ingredient. The cabozantinib can function as an immune checkpoint inhibitor by blocking the binding between PD-1 and PD-L1, and thus can be effectively used in medicines for the prevention or treatment of cancer. In addition, the cabozantinib according to the present invention has an excellent anticancer effect against uterine sarcoma showing resistance to doxorubicin, and thus can be very effectively used for the prevention or treatment of resistant cancer.

Description

Pharmaceutical composition for the prevention or treatment of anticancer drug-resistant cancer containing cabozantinib as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer containing cabozantinib as an active ingredient.

[0002]

[0003] This research was supported by the Basic Research Project of the Korea Institute of Oriental Medicine (Project No. KSN1823231, KSN2411013) and the Individual Basic Research Project of the National Research Foundation of Korea (Project No. 2022R1A2C2092834).

[0004] The primary goal of cancer treatment is to cure the patient by restoring the structural and functional damage caused by cancer. If cure is impossible, the goal is to halt the progression of the cancer and alleviate symptoms, thereby extending life and improving quality of life. Cancer treatment methods are broadly divided into surgery, chemotherapy, and radiation therapy. Additionally, there are local treatment methods, hormone therapy, photodynamic therapy, laser therapy, immunotherapy, and gene therapy. Surgical treatments can be radical (for curative purposes), prophylactic (for preventative purposes), and palliative (for symptom relief). Chemotherapy is a systemic treatment using anticancer drugs that kill cancer cells, while radiation therapy kills cancer cells by shocking the tumor mass with radiation. Radiation therapy and chemotherapy are sometimes combined to reduce tumor size before surgery or to kill residual cancer cells and prevent recurrence after surgery.

[0005] Radiation therapy and chemotherapy, considered first-generation anticancer treatments, work by interfering with the unlimited proliferation and division of cancer cells, leading to their death. However, radiation therapy poses a problem: by exposing them to high-energy radiation, it not only damages the DNA of cancer cells but also induces the death of normal cells. Furthermore, chemotherapy, which administers toxic chemicals that inhibit cell division, does not specifically target cancer cells, but also inhibits the division of normal cells, leading to serious side effects such as leukopenia and hair loss.

[0006] Furthermore, anticancer drugs induce apoptosis and the cancer cells' ability to evade it creates the problem of drug resistance. The development of drug resistance poses a significant obstacle to chemotherapy-based cancer treatment. Most cancer cells have already lost the ability to suppress genetic mutations, resulting in diverse gene expression patterns within a single tumor mass. During chemotherapy, genetically resistant cells are selectively spared due to their genetic diversity. These surviving resistant cells then proliferate, ultimately leading to drug resistance in most of the tumor mass. Beyond genetic diversity or mutation-induced drug resistance, drug resistance can also arise when the patient's side effects are so severe that a sufficient dose of the drug is not administered, when oral administration results in abnormally low drug absorption, or when a physiological barrier between blood vessels and tumor tissue prevents proper drug penetration.

[0007] To overcome this resistance to anticancer drugs, the development of second-generation targeted anticancer drugs, which selectively attack cancer cells, distinguishing them from normal cells, was expected to reduce the side effects of first-generation anticancer drugs. However, targeted anticancer drugs are characterized by their ability to selectively inhibit cancer cells by acting only on cancer-causing proteins, resulting in therapeutic effects. However, the proteins that cause cancer and those that exert therapeutic effects differ depending on the type of cancer, requiring the use of anticancer drugs that are appropriate for the target protein. Furthermore, cancer cells possess mechanisms to acquire resistance to targeted anticancer drugs and possess "immune cell evasion" abilities, which involve mutations that prevent them from being targeted by targeted anticancer drugs. This can lead to cases where targeted anticancer drugs fail to recognize cancer cells. Therefore, third-generation immunotherapies are being developed to reduce the side effects and resistance associated with anticancer treatment, while also enabling immune cells to remember and continuously attack cancer cells even after treatment is discontinued.

[0008] Unlike conventional anticancer drugs that directly attack cancer cells, immunotherapy enhances the function of immune cells gathered around the patient's cancer, helping them to effectively attack cancer cells. It can be used alone or in combination with conventional anticancer drugs or targeted therapies. Immunotherapy can be categorized into immune checkpoint inhibitors, immunotherapy, therapeutic antibodies, and anticancer vaccines. Immune checkpoint inhibitors block the activity of immune checkpoint proteins involved in T cell suppression, thereby activating T cells to attack cancer cells. Antibodies that recognize CTLA-4, PD-1, or PD-L1 are commonly used. Anticancer drugs that enhance cellular immunity include NK cell therapy, T cell therapy, and CAR-T cell therapy. Therapeutic antibodies are antibody-drug conjugates that bind to cancer cells, releasing the drug and attacking them. In addition, anticancer vaccine is an immunotherapy that activates the immune system by administering to cancer patients tumor-specific antigens possessed by cancer cells or protein / peptide molecules that can enhance the overall immune response of the body, thereby activating the body's immune function and attacking cancer cells.

[0009] Accordingly, the present inventors confirmed that cabozantinib is effective in treating cancer through immune checkpoint inhibition that blocks the binding of PD-1 (Programmed cell death protein 1) and PD-L1 (Programmed death-ligand 1), and that it also has an anticancer effect on resistant cancers that show resistance to anticancer drugs.

[0010] Meanwhile, Korean Patent Registration No. 2233955 discloses a 'pharmaceutical composition for treating an immune checkpoint inhibitor containing an extract of Jerusalem artichoke aerial parts or a 3-Hydroxy-8β-tigloyloxy-1,10-dehydroariglovin compound isolated therefrom as an active ingredient', and Korean Patent Publication No. 2021-0150972 discloses a 'pharmaceutical composition for enhancing the anticancer effect of an immune checkpoint inhibitor containing a TLR5 agonist derived from flagellin as an active ingredient', but there is no disclosure regarding a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer containing the cabozantinib of the present invention as an active ingredient.

[0011] The present invention was derived from the above-mentioned needs, and the present inventors confirmed that cabozantinib has an anticancer effect by blocking the binding of PD-1 (Programmed cell death protein 1) and PD-L1 (Programmed death-ligand 1), thereby increasing the activity of T cells, which are immune cells, and reducing the size and weight of colon cancer tumors. In addition, when cabozantinib was treated on uterine sarcoma cells that showed resistance to the anticancer drug doxorubicin, it showed a cancer cell killing effect at a level similar to that of uterine sarcoma cells that did not show anticancer drug resistance, thereby confirming that it has an anticancer effect on anticancer drug-resistant cancer, thereby completing the present invention.

[0012] To solve the above problem, the present invention provides an immune checkpoint inhibitor containing cabozantinib or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] In addition, the present invention provides a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer containing the immune checkpoint inhibitor as an active ingredient.

[0014] In addition, the present invention provides an anticancer adjuvant for anticancer drug-resistant cancer comprising the above-mentioned immune checkpoint inhibitor as an active ingredient.

[0015] The present invention relates to a pharmaceutical composition for the prevention or treatment of anticancer drug-resistant cancer, containing cabozantinib as an active ingredient. Since cabozantinib can function as an immune checkpoint inhibitor by blocking the binding of PD-1 and PD-L1, it can be usefully utilized in a pharmaceutical product for the prevention or treatment of cancer. In addition, since cabozantinib according to the present invention has an excellent anticancer effect against uterine sarcoma cancer that is resistant to doxorubicin, it can be very effectively utilized in the prevention or treatment of resistant cancer.

[0016] Figure 1 shows the results of enzyme-linked immunosorbent assay (ELISA) to confirm the PD-1 / PD-L1 binding inhibition effect of cabozantinib. *** indicates that PD-1 / PD-L1 binding was statistically significantly reduced in the cabozantinib-treated group compared to the control group that was not treated with cabozantinib, and *** indicates p<0.001.

[0017] Figure 2 shows the results of confirming the cytotoxicity of cabozantinib in a Human PD-1 / NFAT Luciferase Reporter Jurkat T cell line expressing the PD-1 protein.

[0018] Figure 3 shows the results of confirming the cytotoxicity of cabozantinib in the Human PDL1 / aAPC CHO-K1 cell line expressing the PD-L1 protein.

[0019] Figure 4 shows the results of confirming the cytotoxicity of cabozantinib against colon cancer cells (MC38).

[0020] Figure 5 shows the results of confirming the PD-1 / PD-L1 binding inhibition activity of cabozantinib through a reporter assay using luciferase.

[0021] Figure 6 shows the results of confirming the cancer cell killing effect of cabozantinib after co-culture of colon cancer cells (MC38) and T cells. **, **** indicate that cell viability was statistically significantly reduced in the cabozantinib-treated group compared to the control group (no cabozantinib treatment, 0 μM). ** indicates p<0.01, and **** indicates p<0.0001.

[0022] Figure 7 shows the results of examining changes in tumor size and weight after oral administration of cabozantinib to a colon cancer animal model. *** indicates that tumor weight was statistically significantly reduced in the cabozantinib-administered group compared to the colon cancer-induced group (Vehicle) that was not administered cabozantinib, and *** indicates p<0.001.

[0023] Figure 8 shows the results of flow cytometry analysis of CD4 T cells and CD8 T cells distributed in tumors after administration of cabozantinib to a colon cancer animal model. *** and **** indicate that CD4 T cells and CD8 T cells increased statistically significantly in the cabozantinib 10 mg / kg administration group compared to the colon cancer-induced group (Vehicle). *** indicates p<0.001, **** indicates p<0.0001, and ns indicates a statistically insignificant change.

[0024] Figure 9 shows the results of flow cytometry analysis confirming CD4 T cells (A) and CD8 T cells (B) distributed in the tumor after administering an antibody against CD8 T cells to an animal model of colon cancer to remove CD8 T cells and administering cabozantinib.

[0025] Figure 10 shows the results of confirming the changes in tumor size and weight after administering antibodies against CD8 T cells to a colon cancer animal model to eliminate CD8 T cells and administering cabozantinib. ** indicates that the tumor weight was statistically significantly reduced in the cabozantinib-administered group (Isotype control-Cabozantinib) compared to the colon cancer-induced group (Isotype control-Vehicle) under conditions where antibodies against CD8 T cells were not administered, and ** is p<0.01.

[0026] Figure 11 shows the results of confirming the degree of doxorubicin resistance in MES-SA, a uterine sarcoma cell line, and MES-SA / Dx5 cell line, which is resistant to doxorubicin, a cytotoxic anticancer drug.

[0027] Figure 12 shows the results of confirming the cytotoxicity of cabozantinib in the uterine sarcoma cell line MES-SA (A) and the MES-SA / Dx5 cell line (B) resistant to the cytotoxic anticancer drug doxorubicin. **** indicates that cancer cell death was statistically significantly increased in the co-cultured T cell activation group (Vehicle) compared to MES-SA or MES-SA / Dx5 (Control), and **** is p<0.0001, and #### indicates that cancer cell death was statistically significantly increased in the cabozantinib-administered group of the T cell activation group compared to the co-cultured T cell activation group (Vehicle), and #### is p<0.0001.

[0028] In order to achieve the purpose of the present invention, the present invention provides an immune checkpoint inhibitor containing cabozantinib or a pharmaceutically acceptable salt thereof as an active ingredient.

[0029] The salts include common acid addition salts, for example, salts derived from inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, and hydrobromic acid, and salts derived from organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, benzoic acid, citric acid, maleic acid, malonic acid, malic acid, tartaric acid, gluconic acid, lactic acid, gestic acid, fumaric acid, lactobionic acid, salicylic acid, phthalic acid, embonic acid, aspartic acid, glutamic acid, and acetylsalicylic acid. The salts also include salts derived from amino acids such as glycine, alanine, valine, isoleucine, serine, cysteine, cystine, aspartic acid, glutamine, lysine, arginine, tyrosine, and proline. Additionally, the salt includes salts of sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0030] The above cabozantinib can target PD-L1 or CD80 of cancer cells, or PD-1 or CTLA-4 of T cells, and preferably target PD-L1 of cancer cells or PD-1 of T cells, but is not limited thereto.

[0031] In the present invention, the cabozantinib can function as an immune checkpoint inhibitor by blocking the interaction between PD-1 and PD-L1 and increasing the activity of T cells, which are immune cells.

[0032] The cancer cells may be derived from any one cancer selected from among colon cancer, uterine sarcoma, cervical cancer, colon cancer, liver cancer, lung cancer, breast cancer, melanoma, stomach cancer, skin cancer, bladder cancer, prostate cancer, ovarian cancer, thyroid cancer, kidney cancer, fibrosarcoma, and blood cancer, and may preferably be colon cancer cells, but are not limited thereto.

[0033] The immune checkpoint inhibitor of the present invention may further include, in addition to the above-mentioned effective ingredient, an anticancer agent; or any one antibody selected from the group consisting of an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CD80 antibody, and an anti-CTLA-4 antibody, but is not limited thereto.

[0034] The above anticancer drugs include actinomycin D, bleomycin sulfate, daunomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin, mitomycin-C, mithramycin, irinotecan, camptothecin, novobiocin, epirubicin, dactinomycin, amsacrine, teniposide, etoposide, cisplatin, carboplatin, oxaliplatin, It may be one or more selected from, but is not limited to, paclitaxel, docetaxel, gefitinib, erlotinib, and afatinib.

[0035] In addition, the present invention provides a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer, which contains the immune checkpoint inhibitor as an active ingredient.

[0036] The anticancer agent may be an anthracycline anticancer agent, preferably any one selected from doxorubicin, daunorubicin, epirubicin, idarubicin, pixantrone, mitoxantrone, sabarubicin, and valrubicin, most preferably doxorubicin, but is not limited thereto.

[0037] The above anticancer drug-resistant cancer may be any one selected from uterine sarcoma, cervical cancer, colon cancer, colorectal cancer, liver cancer, lung cancer, breast cancer, melanoma, stomach cancer, skin cancer, bladder cancer, prostate cancer, ovarian cancer, thyroid cancer, kidney cancer, fibrosarcoma, and blood cancer, and is preferably uterine sarcoma, but is not limited thereto.

[0038] The pharmaceutical composition of the present invention can be prepared in any one dosage form selected from among capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, but is not limited thereto.

[0039] The composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-mentioned effective ingredient, and may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include capsules, powders, granules, tablets, pills, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, emulsions, syrups, and aerosols. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0040] The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it is preferable to select a method of external application to the skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, epidural, or intracerebrovascular injection.

[0041] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0042] The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity. The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0043] In addition, the present invention provides an anticancer adjuvant for anticancer drug-resistant cancer comprising the above-mentioned immune checkpoint inhibitor as an active ingredient.

[0044] The above anticancer adjuvant can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers. The daily dosage of the above anticancer adjuvant is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and is preferably administered once or several times a day, but the range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The anticancer adjuvant of the present invention can be administered in various parenteral dosage forms during actual clinical administration, and when formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0045] The above anticancer adjuvant may contain one or more active ingredients exhibiting the same or similar function as the immune checkpoint inhibitor containing cabozantinib or a pharmaceutically acceptable salt thereof as an active ingredient. The above anticancer adjuvant may be administered orally or parenterally during clinical administration, and when administered parenterally, may be administered by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine epidural injection, intracerebrovascular injection, or intrathoracic injection, and may be used in the form of a general pharmaceutical formulation.

[0046]

[0047] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0048]

[0049] Example 1. Confirmation of the PD-1 / PD-L1 binding inhibition effect of cabozantinib.

[0050] A competitive ELISA assay was performed to confirm the PD-1 / PD-L1 binding inhibition effect of cabozantinib.

[0051] Specifically, a competitive ELISA assay was performed using a PD-1 / PD-L1 inhibitor ELISA screening kit according to the manufacturer's instructions. 100 μL of 1 μg / mL combined human PD-L1 dissolved in PBS was coated onto a 96-well plate overnight. The 96-well plate was washed with PBS containing 0.1% Tween 20 (PBS-T), blocked with PBS containing 2% (w / v) BSA for 1 h at room temperature, and washed again. Fifty microliters of 0.5 μg / mL biotinylated hPD-1 and various concentrations of cabozantinib were then added to the wells and incubated for 2 h at room temperature. After washing three times with PBS-T, 50 μL of 0.2 μg / mL HRP-conjugated streptavidin was added to each well and incubated for 1 h. After incubation, the plates were washed three times with 0.1% PBS-T, and relative chemiluminescence was measured using a SpectraMax L luminometer.

[0052] As a result, it was confirmed that the binding of PD-1 and PD-L1 was significantly inhibited at concentrations of 10 to 1000 nM of cabozantinib (Fig. 1).

[0053]

[0054] Example 2. Confirmation of cytotoxicity against human PD-1 / NFAT Jurkat T cells and human PD-L1 / aAPC CHO-K1 cells using cabozantinib.

[0055] In a cell experiment, PD-1 / NFAT Luciferase Reporter Jurkat T cell line, which constantly expresses PD-1 and PD-L1, and PD-L1 / aAPC CHO-K1 cell line were co-cultured and then treated with cabozantinib. As a result, it was confirmed that the survival rate of Human PD-1 / NFAT Jurkat T cells (Fig. 2) and Human PD-L1 / TCR aAPC / CHO-K1 cells (Fig. 3) decreased in a concentration-dependent manner by cabozantinib.

[0056]

[0057] Example 3. Confirmation of human PD-L1 MC38 colon cancer cytotoxicity using cabozantinib.

[0058] To determine the cytotoxicity of cabozantinib against colon cancer cells MC38, MC38 were seeded on a cell culture plate, cultured for 24 hours to allow the cells to attach to the plate, and then treated with cabozantinib. As a result, it was confirmed that the survival rate of colon cancer cells (MC38) decreased in a concentration-dependent manner of cabozantinib (Fig. 4).

[0059]

[0060] Example 4. Confirmation of the PD-1 / PD-L1 interaction inhibition effect of cabozantinib using TCR / PD-1 overexpressing T cells and TCR receptor / PD-L1 overexpressing CHO cells.

[0061] To confirm the efficacy of cabozantinib in inhibiting PD-1 / PD-L1 interaction, a cell-based PD-1 / PD-L1 blocking assay was performed. The cell-based PD-1 / PD-L1 blocking assay utilizes the principle that luciferase fluorescence decreases when TCR binds to the TCR receptor through PD-1 / PD-L1 interaction, and can be used to determine whether cabozantinib of the present invention blocks PD-1 / PD-L1 binding on the cell surface.

[0062] Specifically, TCR / PD-1 overexpressing T cells and TCR receptor / PD-L1 overexpressing CHO cells were used. 5×10 4 PD-L1 aAPC / CHO-K1 cells were seeded in 96-well plates and cultured in DMEM medium containing 10% FBS. On the day of performing the cell-based PD-1 / PD-L1 blocking assay, the medium was removed, and cabozantinib was added at various concentrations, followed by 1 × 10 5 PD-1-overexpressing T cells from dogs were added. After a certain period of time, Bio-Glo™ reagent (Promega) was mixed and luminescence was measured.

[0063] As a result, the binding of PD-1 / PD-L1 was inhibited by cabozantinib treatment, which increased the luminescence level of luciferase, and in particular, it was confirmed that the related T cell activity was increased up to 4 times through immune checkpoint blockade in the 1 μM cabozantinib treatment group (Fig. 5).

[0064]

[0065] Example 5. Confirmation of the effect of cabozantinib treatment and immune T cell co-culture on colon cancer cell death.

[0066] We confirmed the cancer cell death by co-culture of colon cancer cells (MC38) and T cells and treatment with cabozantinib. Specifically, co-culture of colon cancer cells and T cells was performed at a ratio of 1:5, and colon cancer cells treated with 10 ng / ㎖ of interferon gamma were added at a density of 5 × 10 4The cells were placed in a 96-well plate at a density of 10 cells / well and cultured for 18 hours to allow attachment to the bottom. Afterwards, 2.5 × 10 PD-1-overexpressing T cells were added. 5 Colon cancer cells were seeded into 96-well plates at a density of 1 cell / well, and cabozantinib was added, followed by co-culture for 72 hours. Subsequently, 10 μl of CCK solution was added, incubated at 37°C for 2 hours, and the absorbance at 450 nm in the microplate was measured.

[0067] As a result, it was confirmed that the survival rate of colon cancer cells was significantly reduced in the 2.5 μM or 5 μM cabozantinib treatment group compared to the control group (Fig. 6).

[0068]

[0069] Example 6. Confirmation of tumor growth inhibition by cabozantinib in an animal model of colon cancer.

[0070] MC38 colon cancer cells expressing hPD-L1 were subcutaneously administered to male C57BL / 6J mice to induce tumors, and cabozantinib (2 mg / kg or 10 mg / kg) was administered to each experimental group (6 mice per group) for approximately 3 weeks, after which the tumor size and weight were measured.

[0071] As a result, it was confirmed that the tumor size and weight were significantly reduced in the cabozantinib 2 mg / kg or 10 mg / kg administration group compared to the colon cancer-induced group (Vehicle) (Fig. 7).

[0072] Additionally, to determine the number of CD4 T cells and CD8 T cells within the tumor, tumor tissue was separated into single cells and flow cytometry analysis was performed.

[0073] As a result, the number of CD4 T cells and CD8 T cells in the experimental group administered 10 mg / kg of cabozantinib significantly increased compared to the colon cancer-induced group (Vehicle) (Fig. 8).

[0074]

[0075] Example 7. Confirmation of tumor size and weight according to T cell depletion in a colon cancer animal model.

[0076] Based on the results of Example 6 above, CD8 T cells were eliminated by intraperitoneal administration of CD8 antibodies to mice in a colon cancer-induced animal model, and then 10 mg / kg of cabozantinib was administered. Thereafter, changes in the number of intratumoral CD4 T cells and CD8 T cells following administration of the anti-CD8 antibody were confirmed through flow cytometry.

[0077] As a result, as disclosed in Figure 9(A), the number of CD4 T cells increased in the group treated with cabozantinib compared to the group not treated with cabozantinib (vehicle), similarly in both the control group (isotype control) that was not administered anti-CD8 antibody and the experimental group treated with anti-CD8 antibody.

[0078] In contrast, the number of CD8 T cells increased in the cabozantinib-treated group compared to the vehicle-treated group in the control group (isotype control) that was not administered anti-CD8 antibodies, but no CD8 T cells were identified in the anti-CD8 antibody-treated group regardless of cabozantinib treatment (Fig. 9B).

[0079] In addition, as shown in Fig. 10, the tumor size and weight were measured, and, similar to the results shown in Fig. 7, the cabozantinib-administered group showed a significant decrease compared to the colon cancer-induced group (Vehicle) in the control group (isotype control) that was not administered anti-CD8 antibodies. However, the tumor size in the group treated with anti-CD8 antibodies due to cabozantinib administration did not show a significant change compared to the colon cancer-induced group (Vehicle).

[0080] From these results, it was determined that cabozantinib of the present invention reduces the size of cancer cells through a CD8 T cell-mediated immune response.

[0081]

[0082] Example 8. Confirmation of cytotoxicity in doxorubicin-resistant cancer cells using cabozantinib.

[0083] The efficacy of cabozantinib was evaluated in the MES-SA cell line, a uterine sarcoma cell line, and the MES-SA / Dx5 cell line, which has resistance to doxorubicin.

[0084] Before evaluating the efficacy of cabozantinib, a cell viability assay was performed to determine the degree of resistance to doxorubicin.

[0085] As a result, as disclosed in Fig. 11, it was confirmed that the uterine sarcoma cell line showing resistance to doxorubicin had a 42-fold resistance to doxorubicin (resistance index 42.5).

[0086] To evaluate the efficacy of cabozantinib, MES-SA or MES-SA / Dx5 cells were seeded on cell culture plates and cultured for 24 hours to allow the cells to attach to the plates. MES-SA or MES-SA / Dx5 cells were co-cultured with T cells to induce T cell activity. Co-culture was performed in the same manner as in Example 5. Thereafter, the T cell activation group co-cultured with MES-SA or MES-SA / Dx5 cells was treated with cabozantinib to measure cytotoxicity.

[0087] As a result of measuring cytotoxicity against the MES-SA cell line, it was confirmed that cancer cell death significantly increased in the co-cultured T cell activation group (vehicle) compared to the control group (control), and that cancer cell death significantly increased in the cabozantinib-administered group of the T cell activation group compared to the T cell activation group (vehicle) (Fig. 12A).

[0088] In addition, the cytotoxicity of the MES-SA / Dx5 cell line showed almost the same results as the cytotoxicity results of the MES-SA cell line (Fig. 12B).

[0089] Through this, it was confirmed that cabozantinib of the present invention has an anticancer effect against doxorubicin-resistant cancer.

Claims

1. An immune checkpoint inhibitor containing cabozantinib or a pharmaceutically acceptable salt thereof as an active ingredient.

2. In the first paragraph, the cabozantinib is an immune checkpoint inhibitor characterized by targeting PD-L1 or CD80 of cancer cells or targeting PD-1 or CTLA-4 of T cells.

3. An immune checkpoint inhibitor according to claim 2, wherein the cancer cells are cancer cells derived from any one cancer selected from among colon cancer, uterine sarcoma, cervical cancer, colon cancer, liver cancer, lung cancer, breast cancer, melanoma, stomach cancer, skin cancer, bladder cancer, prostate cancer, ovarian cancer, thyroid cancer, kidney cancer, fibrosarcoma, and blood cancer.

4. An immune checkpoint inhibitor characterized in that, in addition to the effective ingredient in paragraph 1, it further comprises an anticancer agent; or any one antibody selected from the group consisting of an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CD80 antibody, and an anti-CTLA-4 antibody.

5. In paragraph 4, the anticancer agent is actinomycin D, bleomycin sulfate, daunomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin, mitomycin-C, mithramycin, irinotecan, camptothecin, novobiocin, epirubicin, dactinomycin, amsacrine, teniposide, etoposide, cisplatin, carboplatin, An immune checkpoint inhibitor characterized by at least one selected from oxaliplatin, paclitaxel, docetaxel, gefitinib, erlotinib, and afatinib.

6. A pharmaceutical composition for the prevention or treatment of anticancer drug-resistant cancer, containing an immune checkpoint inhibitor of any one of claims 1 to 5 as an active ingredient.

7. A pharmaceutical composition for preventing or treating cancer resistant to anticancer drugs, characterized in that the anticancer drug in claim 6 is an anthracycline anticancer drug.

8. A pharmaceutical composition for preventing or treating cancer resistant to anticancer drugs, characterized in that the anthracycline anticancer drug in paragraph 7 is any one selected from doxorubicin, daunorubicin, epirubicin, idarubicin, pixantrone, mitoxantrone, sabarubicin, and valrubicin.

9. A pharmaceutical composition for preventing or treating anticancer drug-resistant cancer, characterized in that in paragraph 6, the anticancer drug-resistant cancer is any one selected from among uterine sarcoma, cervical cancer, colon cancer, colon cancer, liver cancer, lung cancer, breast cancer, melanoma, stomach cancer, skin cancer, bladder cancer, prostate cancer, ovarian cancer, thyroid cancer, kidney cancer, fibrosarcoma, and blood cancer.

10. An anticancer adjuvant for cancer resistant to anticancer drugs, comprising an immune checkpoint inhibitor according to any one of claims 1 to 5 as an active ingredient.

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