Combination preparation for interleukin-2 immune anticancer agent

Abstract

The present invention relates to a combination preparation comprising: an interleukin-2 (IL-2) immune anticancer drug; and a compound represented by chemical formula 1, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof, whereby the combination preparation can exhibit the effects of enhancing the efficacy of an interleukin-2 immune anticancer drug and alleviating vascular leakage syndrome, which is a side effect of the interleukin-2 immune anticancer drug. [Chemical formula 1]

Description

Combination agents for interleukin-2 immunotherapy

[0001] The present invention relates to a combination preparation for immunotherapy against cancer.

[0002] Cancer is a disease characterized by localized, abnormal cell growth with the potential to spread throughout the body. There are numerous types of cancer, including lung, bladder, prostate, pancreatic, cervical, brain, stomach, colorectal, and melanoma. In the past, the most common methods of treating cancer (oncology) were surgery, radiation therapy, or chemotherapy. However, cancer immunotherapy has recently emerged as a promising treatment option for oncology.

[0003] Cancer immunotherapy is a branch of oncology that uses the immune system to treat cancer, in stark contrast to conventional treatments that involve direct tumor removal or treatment. This therapeutic concept is based on the identification of numerous proteins on the surface of T cells that act to suppress the immune function of these cells.

[0004] The most fundamental problem in tumor immunity is how to activate the immune system to recognize and eliminate these antigens. In this regard, a novel approach that genetically engineered tumor cells to secrete specific cytokines has revolutionized tumor immunity.

[0005] The theoretical basis for genetically modified tumor vaccines based on these immunotherapies is that the host possesses antigens that enable it to recognize the tumor as an external agent. While human T and B lymphocytes have developed the ability to distinguish a nearly infinite number of antigens through antigen receptors, the success of tumor immunity requires two criteria: first, tumor cells must express novel antigens (peptides) not expressed by normal cells; and second, immune cells must recognize these antigens and be appropriately activated.

[0006] In particular, combination therapy for cancer treatment is becoming increasingly popular as the benefits of attacking the disease through multiple modalities are recognized. Combination therapy is not only useful in cases where resistance to anticancer drugs has developed, but also has the advantage of enhancing the efficacy of anticancer drugs when they are effective, allowing for a reduction in the dose administered. This allows for maximizing anticancer efficacy while minimizing the toxicity and side effects of anticancer drugs on various organs. Although numerous effective combination therapies have been identified over the past several decades, the continued high annual cancer death toll necessitates the identification of effective therapies for use in cancer treatment.

[0007] Interleukins are proteins secreted primarily by cells, including white blood cells, and are a type of cytokine, a signaling molecule. They play a crucial role in the immune response, inflammatory response, and the development, interaction, and regulation of cells in the blood system. Interleukins can be broadly divided into four groups based on their structural characteristics. Among them, interleukin-2 (IL-2) is the first reproducible cancer immunotherapy with ambivalent effects on the immune system. Interleukin-2, also known as a T-cell growth factor, regulates immune responses by dually activating effector T cells and regulatory T cells. Interleukin-2 binds to the interleukin-2 receptor (IL-2R) present on the cell membrane of T helper cells, thereby determining the direction of T cell differentiation. The IL-2 receptor consists of three polypeptide chains: α, β, and γ. Each immune cell expresses a different receptor chain. For example, regulatory T cells have high expression of the α chain. Interleukin-2 binds to all three receptors simultaneously on cells that have a third receptor, IL-2α. In other white blood cells, this can weaken the body's immune response, but in white blood cells residing in blood vessels, it can cause a fatal disease that increases vascular leakiness.

[0008] IL-2 therapy has been commonly used to treat metastatic renal cell carcinoma and metastatic melanoma. The role of interleukin-2 in lymphocyte regulation suggests new directions for cancer immunotherapy. Furthermore, increased tumor-infiltrating lymphocytes induced by interleukin-2 administration have demonstrated significant anticancer effects in melanoma patients. In particular, high-dose interleukin-2 immunotherapy is known to exhibit remarkable anticancer effects against metastatic renal cell carcinoma and melanoma.

[0009] However, interleukin-2, despite its remarkable anticancer properties, has significant limitations, including a short half-life and serious side effects on endothelial cells, limiting its use in patients. In particular, serious side effects, such as vascular leak syndrome and capillary leak syndrome, associated with high-dose doses limit its use despite its promising efficacy. Interleukin-2 drug toxicity is known to occur in most organ systems, including the heart, lungs, kidneys, and central nervous system.

[0010] Vascular leak syndrome (VLS) is characterized by fluid accumulation, peripheral edema, ascites, pleural effusion, and pulmonary edema. While the cause of this condition remains unclear, it is believed to be caused by increased circulating TNF-α levels, which, due to various factors, activates neutrophils and increases vascular permeability. This condition most commonly occurs in patients receiving high doses of cytokines such as interleukin-2 (IL-2) or in bone marrow transplant recipients.

[0011] Despite the side effects, IL-2 immunotherapy remains an essential tool for patients who lack specific receptors and ligands and thus cannot use next-generation immunotherapy. The present inventors discovered that co-administration of CU06-1004, a compound known to block endothelial dysfunction, with IL-2 immunotherapy reduced the side effect of vascular leak syndrome, leading to the completion of the present invention.

[0012] The purpose of the present invention is to provide a combination preparation that can alleviate the side effect of vascular leak syndrome that occurs during interleukin-2 (IL-2) immunotherapy and increase the anticancer effect of interleukin-2.

[0013] Another object of the present invention is to provide an anticancer adjuvant for an interleukin-2 immunotherapy.

[0014] Another object of the present invention is to provide a pharmaceutical composition that enhances the efficacy of an interleukin-2 immunotherapy agent.

[0015] Another object of the present invention is to provide a pharmaceutical composition that alleviates or reduces side effects of an interleukin-2 immunotherapy agent.

[0016] To achieve the above purpose,

[0017] One aspect of the present invention is an interleukin-2 (IL-2) immunotherapy; and

[0018] A combination immunotherapy agent for cancer is provided, comprising a compound represented by the following chemical formula 1, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof.

[0019] [Chemical Formula 1]

[0020]

[0021] Another aspect of the present invention provides an immunotherapy adjuvant for an interleukin-2 immunotherapy agent comprising a compound represented by the above chemical formula 1, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof.

[0022] Another aspect of the present invention provides a pharmaceutical composition for enhancing the efficacy of an interleukin-2 immunotherapy agent, comprising the compound, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0023] The combination immunotherapy agent according to the present invention can exhibit the effect of enhancing the efficacy of an interleukin-2 immunotherapy agent and the effect of alleviating vascular leak syndrome, a side effect of an interleukin-2 immunotherapy agent.

[0024] Figure 1a is a graph of an MTT assay after treatment of HUVEC with an interleukin-2 immunotherapy agent.

[0025] Figure 1b is a photograph showing the results of immunofluorescence staining of F-actin and VE-cadherin.

[0026] Figure 1c is a graph measuring TEER for a HUVEC monolayer.

[0027] Figure 1d is a graph measuring FITC-dextran for HUVEC monolayers.

[0028] Figure 2a is a schematic diagram showing the procedure from preparation to sacrifice of a tumor-free experimental animal model.

[0029] Figures 2b to 2f are photographs confirming the results of Evans blue staining of the mouth corners of tumor-free mice in each drug administration group and of organs removed from the mice.

[0030] Figures 2g to 2j are graphs showing the detection values ​​of Evans blue staining for each organ extracted from tumor-free mice in each drug administration group.

[0031] Figure 3a is a photograph of the spleen removed from tumor-free mice in each drug administration group.

[0032] Figure 3b is a graph showing the spleen weight of tumor-free mice in each drug administration group.

[0033] Figure 4a is a schematic diagram showing the procedure from preparation to sacrifice of a tumor-bearing experimental animal model.

[0034] Figures 4b and 4c are photographs measuring the size of tumors before and after excision in tumor-bearing mice of each drug administration group.

[0035] Figure 4d is a graph measuring the tumor size in tumor-bearing mice in each drug administration group.

[0036] Figure 4e is a graph measuring the tumor weight in tumor-bearing mice of each drug administration group.

[0037] Figures 5a to 5d are photographs confirming the results of Evans blue staining of the mouth corners of tumor-bearing mice in each drug administration group and of organs removed from the mice.

[0038] Figures 5e to 5h are graphs showing the detection values ​​of Evans blue staining for each organ extracted from tumor-bearing mice in each drug administration group.

[0039] Figure 6a is a photograph showing the results of immunofluorescence staining of CD8+ T cells in each drug administration group.

[0040] Figure 6b is a graph showing the number of CD8+ T cells per unit area in each drug administration group.

[0041] Figure 6c is a graph showing the measurement of TNFα expression in each drug administration group.

[0042] Figure 7a is a schematic diagram showing the procedure from preparation to sacrifice of an experimental animal model bearing a lung tumor.

[0043] Figure 7b is a graph showing the detection values ​​of Evans blue staining confirmed in lungs extracted from mice with lung tumors in each drug administration group.

[0044] Figure 7c is a graph measuring the weight of lungs removed from mice with lung tumors in each drug administration group.

[0045] Figure 8 is a photograph confirming vascular leakage near the tumor in tumor-bearing mice of each drug administration group.

[0046] Hereinafter, the present invention will be described in detail.

[0047] The embodiments of the present invention may be modified in various different forms, and the scope of the present invention is not limited to the embodiments described below. Furthermore, the embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Furthermore, throughout the specification, the term "including" a certain component does not exclude other components, but rather implies the inclusion of other components, unless specifically stated otherwise.

[0048]

[0049] One aspect of the present invention provides a combination preparation for immunotherapy against cancer.

[0050] Specifically, an immuno-oncology combination preparation is provided, comprising an interleukin-2 (IL-2) immuno-oncology agent; and a compound represented by the following chemical formula 1, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof.

[0051] Interleukin-2 (IL-2) is an immune-boosting protein that modulates the immune response by dually activating both effector and regulatory T cells. As a signaling molecule called a cytokine, it simultaneously binds to T cell receptors called IL-2β and IL-2γ, thereby increasing T cell activity.

[0052] The term "interleukin-2" as used herein is used interchangeably with "IL-2." Interleukin-2 can be any wild-type interleukin-2 obtained from any vertebrate source, including mammals, for example, primates (e.g., humans) and rodents (e.g., mice and rats). The interleukin-2 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing interleukin-2. Furthermore, the interleukin-2 can be wild-type interleukin-2 or a variant thereof.

[0053] In this specification, interleukin-2 or its variants are collectively referred to, and interleukin-2 can also be referred to as interleukin-2 protein or interleukin-2 polypeptide. Interleukin-2, interleukin-2 protein, interleukin-2 polypeptide, and interleukin-2 variants specifically bind to, for example, the interleukin-2 receptor. This specific binding can be confirmed by methods known to those skilled in the art.

[0054] The above interleukin-2 may be a mature form. Specifically, the mature interleukin-2 may not contain a signal sequence, and in this case, the interleukin-2 may be used as a concept that includes a truncated fragment of the N-terminus or C-terminus of wild-type interleukin-2.

[0055] Additionally, the fragment of interleukin-2 may be in a form in which 1 to 25 amino acids are deleted consecutively from the N-terminus of the protein having the amino acid sequence. Additionally, the fragment of interleukin-2 may be in a form in which 1 to 25 amino acids are deleted consecutively from the C-terminus of the protein.

[0056] Interleukin-2 variant refers to a form in which a portion of an amino acid of full-length interleukin-2 or a fragment of the above-mentioned interleukin-2 is substituted. That is, the interleukin-2 variant may have an amino acid sequence different from that of wild-type interleukin-2 or a fragment thereof. However, the interleukin-2 variant may have an activity equivalent to or similar to that of wild-type interleukin-2. Here, interleukin-2 activity may mean, for example, specific binding to an interleukin-2 receptor, and this specific binding can be measured by a method known to those skilled in the art.

[0057] At this time, the “other amino acid” introduced by the substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0058] In addition, the interleukin-2 variant may be characterized by low toxicity in vivo. In this case, the low toxicity in vivo may be a side effect caused by the binding of interleukin-2 to the alpha chain of the interleukin-2 receptor (IL-2Rα). Various interleukin-2 variants have been developed to improve the side effects caused by the binding of interleukin-2 and IL-2Rα, and such interleukin-2 variants may use those disclosed in U.S. Patent No. 5,229,109 and Republic of Korea Patent No. 1,667,096.

[0059] The compound of formula 1 that can be used in combination with interleukin-2 is known as CU06-1004.

[0060] [Chemical Formula 1]

[0061]

[0062] The above compound can be prepared according to a known method, and as a specific example, International Publication No. WO 2011 / 053048 discloses the above compound as a novel vascular leak blocker, International Publication No. WO 2021 / 118003 discloses a high-yield preparation method of the above compound, and International Publication No. WO 2021 / 225233 discloses a crystalline form of the above compound.

[0063] The above compound can be separated into alpha-isomers and beta-isomers, as described in International Publication No. WO 2021 / 225233, and it is known that the alpha-isomer can exist in various crystalline forms.

[0064] In the present invention, the compound according to chemical formula 1 includes alpha-isomers, beta-isomers, mixtures thereof, and even various crystal forms.

[0065] The compound represented by the above chemical formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt, and as a salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The acid addition salt is obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc.; non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc.; organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, Includes phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, etc.

[0066] The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the derivative of chemical formula 1 in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., adding an organic acid or inorganic acid, filtering and drying the resulting precipitate, or by distilling the solvent and an excess acid under reduced pressure, drying, and crystallizing in the presence of an organic solvent.

[0067] Additionally, it is also possible to prepare and use addition salts here with inorganic bases. Pharmaceutically acceptable metal salts can be prepared using bases. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are pharmaceutically suitable for preparing metal salts. In addition, the corresponding salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable anion salt (e.g., silver nitrate).

[0068] In the immuno-oncology combination formulation according to the present invention, the compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof may be administered in various oral and parenteral formulations during clinical administration, and more preferably, the parenteral formulation may be used. Parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

[0069] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0070] In order to formulate a dosage form for parenteral administration, the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof is mixed with a stabilizer or buffer in water to prepare a solution or suspension, which can be prepared in an ampoule or vial unit dosage form. The composition may be sterilized and / or contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying promoters, salts for osmotic pressure control, and / or buffers, and other therapeutically useful substances, and may be formulated according to conventional mixing, granulation, or coating methods.

[0071] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms 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 dosage forms for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, and emulsions. Non-aqueous solvents and suspending agents that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0072] Interleukin-2 is typically provided in an injectable form, but is not limited thereto. As a specific example of a combination formulation, the CU06-1004 compound may be provided dissolved in a liquid interleukin-2 injectable formulation, or a formulation of the CU06-1004 compound may be provided separately from the liquid interleukin-2 injectable formulation.

[0073] They may be provided in the form of a single or mixed composition, and each may be administered separately or in the form of one composition.

[0074] The immunotherapy agent and the CU06-1004 compound can be administered simultaneously, sequentially, or with a time interval between them. When administered sequentially, the immunotherapy agent can be administered before the CU06-1004 compound, or the immunotherapy agent can be administered after the CU06-1004 compound. However, the above administration method is merely an example, and the administration method can be modified to enhance the immunotherapy effect and achieve the desired therapeutic effect.

[0075] Immunotherapy combination agents can activate one or more immune factors selected from the group consisting of helper T cells, cytotoxic T cells, natural killer (NK) cells, and cytokines. By activating these immune factors, they exhibit the effect of enhancing the anticancer effect of immunotherapy.

[0076] At this time, the above-mentioned immuno-oncology combination agent can be administered in combination with other immuno-oncology agents to prevent or treat cancer.

[0077] The above cancers are pseudomyxoma, intrahepatic cholangiocarcinoma, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, It may be at least one selected from the group consisting of duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoid tumor, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, triple-negative breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid tumor, vaginal cancer, spinal cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, and thymic cancer.

[0078] The CU06-1004 compound according to the present invention can function as an anticancer adjuvant agent that enhances the anticancer efficacy of an interleukin-2 immunotherapy agent during anticancer therapy using the interleukin-2 immunotherapy agent.

[0079] The CU06-1004 compound according to the present invention can function as an anticancer adjuvant that alleviates or reduces the side effects of vascular leak syndrome of an interleukin-2 immunotherapy agent during anticancer therapy through administration of an interleukin-2 immunotherapy agent.

[0080] The specific description of the above anticancer adjuvant and combination is the same as the specific description of the above immuno-oncology combination agent.

[0081]

[0082] *Another aspect of the present invention provides a pharmaceutical composition for enhancing the efficacy of an immunotherapy-based anticancer agent. The specific description of the pharmaceutical composition for enhancing the efficacy of an immunotherapy-based anticancer agent is identical to the specific description of the immunotherapy-based anticancer combination agent.

[0083]

[0084] Hereinafter, the present invention will be described in detail through examples and experimental examples.

[0085] However, the examples and experimental examples described below are only specific examples of one aspect of the present invention, and the present invention is not limited thereto.

[0086] Preparation: In vitro cell culture

[0087] To culture HUVEC (Human Umbilical Vein Endothelial Cells), EBM (Endothelial cell basal medium) supplemented with 5% FBS (fetal bovine serum) and 1% penicillin / streptomycin was used, and DMEM (Dulbecco's modified Eagle's media) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin was used to culture B16F10 melanoma cells.

[0088]

[0089] Example 1: Measurement of cell viability using the MTT assay

[0090] To determine whether the cytotoxicity of interleukin-2 (IL-2) immunotherapy at different concentrations was inhibited by the drug CU06-1004, an MTT assay was performed to measure cell viability.

[0091] Human umbilical vein endothelial cells (HUVECs) were seeded in a 24-well plate (4 × 10^4 cells / well). After treatment with CU06-1004 and interleukin-2, the cells were maintained in medium containing 0.2% FBS for 12 h. 0.5 mg / mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well, and the cells were incubated at 37°C for 3 h. The supernatant was removed, and 200 μL of dimethyl sulfoxide (DMSO) + isopropyl alcohol was added to dissolve the formazan product. The absorbance, which is proportional to the number and proliferation rate of living cells, was measured at a wavelength of 540 nm using a microplate reader.

[0092] Figure 1a is an MTT assay graph of a single-administration group without CU06-1004 and a combination-administration group injected with CU06-1004 after treatment with 50 kU / ml and 100 kU / ml of interleukin-2 immunotherapy. As shown in the Figure 1a graph, in the single-administration group, cell viability decreased when treated with 50 kU / ml and 100 kU / ml of interleukin-2 immunotherapy, but in the combination-administration group, cell viability was maintained even when treated with 50 kU / ml and 100 kU / ml of interleukin-2 immunotherapy.

[0093] The above results demonstrate that the cytotoxicity of interleukin-2 immunotherapy is inhibited by the drug CU06-1004.

[0094]

[0095]

[0096] *Example 2: F-actin and VE-cadherin immunofluorescence staining experiment

[0097] Changes in AJ (adherent junction) proteins were confirmed through F-actin and VE-cadherin immunofluorescence staining.

[0098] To perform immunofluorescence staining, mice were first anesthetized by injection of 2.5% tribromoethanol and then perfused with 50 ml of PBS or saline via the left ventricle of the heart. The obtained tissues were then fixed with 4% paraformaldehyde (PFA) for 16 hours, dehydrated in a 15% sucrose solution, and then dehydrated in a 30% sucrose solution until the tumors sank to the bottom of the container. Mouse tumor tissues were sectioned into 20-30 μm thick sections using a cryostat. Approximately 7-10 slices were obtained from each tissue, and the tissues were stored at -80°C. To check for vascular leakage, tumor tissues were treated with 0.5% PBS Triton X-100 (PBST) for 5 minutes, incubated with blocking solution for 1 hour at room temperature, and then treated with primary antibody for double staining and incubated O / N.

[0099] The proteins CD31 / PECAM1 (R&D Systems; AF3628; 1:200, Santa Cruz, SC-1506; 1:200), VE-cadherin (Vascular endothelial cadherin) (Santa Cruz; SC-9989; 1:200), and collagen IV (Millipore; 1982483; 1:200) were treated for 16 h at 4°C, followed by secondary antibody treatment for 1 h at room temperature. Subsequently, the slides were treated with DAPI (1:1000) for 20 min before mounting for nuclear staining. Immunofluorescence images were captured using a confocal microscope (Carl Zeiss 880, Germany).

[0100] Figure 1b is a photograph showing the results of F-actin and VE-cadherin immunofluorescence staining in the control group, the interleukin-2 monotherapy group, and the interleukin-2 and CU06-1004 combination therapy group.

[0101] Immunofluorescence staining images of the control group confirmed that in the normal state, thin F-actin fibers rarely appeared and formed a ring shape. In addition, AJ proteins in normal HUVECs formed a linear pattern at the cell border.

[0102] In the interleukin-2 monotherapy group, F-actin formed thicker and more prominent F-actin fiber bundles, failing to form a ring shape. Furthermore, the linear pattern of AJ proteins was disrupted, as evidenced by the presence of gaps between VE-cadherin.

[0103] In contrast, the group administered with interleukin-2 and CU06-1004 in combination showed ring formation, similar to normal F-actin. Furthermore, unlike the results of the monotherapy group, a linear pattern of AJ protein was observed.

[0104]

[0105] *That is, interleukin-2 interferes with the interaction of AJ proteins induced by VE-cadherin phosphorylation, altering cell morphology and creating gaps between adjacent cells. These results demonstrate that co-administration of CU06-1004 with interleukin-2 alleviates these side effects.

[0106]

[0107] Example 3: TEER and FITC-dextran measurement experiments on HUVEC monolayers

[0108] Trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-dextran) were measured for HUVEC monolayers to determine endothelial barrier integrity and permeability.

[0109] Figure 1c is a graph showing TEER measurements in the control group, the interleukin-2 monotherapy group, and the interleukin-2 and CU06-1004 combination therapy group.

[0110] Compared to the control group, the TEER value was reduced in the interleukin-2 monotherapy group, confirming damage to the endothelial barrier caused by interleukin-2. On the other hand, the TEER value was not reduced in the interleukin-2 and CU06-1004 combination therapy group, indicating that combination therapy with CU06-1004 prevents damage to the endothelial barrier caused by interleukin-2.

[0111] Figure 1d is a graph showing the measurement of FITC-dextran in the control group, the interleukin-2 monotherapy group, and the interleukin-2 and CU06-1004 combination therapy group. Fluorescein intensity is expressed as a % relative to the control group.

[0112] In the interleukin-2 monotherapy group, fluorescein intensity was confirmed to be over 120%. This confirmed that the interleukin-2 monotherapy group lost circulatory normality and exhibited abnormally high permeability.

[0113] In contrast, in the group receiving combination therapy with interleukin-2 and CU06-1004, fluorescein intensity did not exceed 100%. This confirmed that permeability did not increase in the combination therapy group and that normal circulation was maintained.

[0114] The above experimental results demonstrated that CU06-1004 could suppress the side effects caused by interleukin-2 by protecting the viability of endothelial cells and reducing their permeability.

[0115]

[0116] Example 4: Experiment to confirm vascular leak syndrome using Evans blue staining in tumor-free mice.

[0117] To maintain the efficacy of high-dose interleukin-2 drugs while suppressing side effects, interleukin-2 and CU06-1004 were injected and the results were observed in various institutions.

[0118] Figure 2a is a schematic diagram showing the preparation and sacrifice procedures of a tumor-free experimental animal model. The specific administration method is as follows. The drug was administered to each mouse in each drug administration group three times a day for three days, and then once a day on the fourth day. The control group was administered vehicle (10 mpk), the interleukin-2 monotherapy group was administered interleukin-2 (75,000 U), and the interleukin-2 and CU06-1004 combination administration group was administered interleukin-2 (75,000 U) and CU06-1004 (10 mpk).

[0119] Interleukin-2 was injected intraperitoneally into mice at a concentration of 75,000 U / mouse (20 g). 1 mg of interleukin-2 was dissolved in 1 ml of autoclaved DW containing 0.6% acetic acid and 0.1% BSA to make a 1 mg / ml solution. 50 μl aliquots were used and stored in a deep freezer.

[0120] Evans blue staining was performed to confirm vascular leak syndrome in C57BL / 6 mice. Mice were intravenously injected with Evans blue reagent before tissue collection after drug administration. Two hours after the final drug administration, the mice were sacrificed and their organs were removed.

[0121] Figure 2b is a photograph confirming the results of Evans blue staining confirmed around the mouth of each mouse in the vehicle-administered control group, the interleukin-2-only administration group, and the interleukin-2 and CU06-1004 co-administration group.

[0122] Figures 2c to 2f are photographs confirming the results of Evans blue staining confirmed in the lungs, liver, hands, and feet of each mouse in the vehicle-administered control group, the interleukin-2-only administration group, and the interleukin-2 and CU06-1004 co-administration group.

[0123] Figures 2g to 2j are graphs showing the results of Evans blue staining confirmed in the lungs, liver, hands, and feet of each mouse in the vehicle-administered control group, the interleukin-2-only administration group, and the interleukin-2 and CU06-1004 co-administration group.

[0124] As a result, Evans blue staining was observed with the naked eye in the organs and body parts extracted from the interleukin-2 monotherapy group mice, and compared to the vehicle-administered control group, the Evans blue reagent was detected at a dose of 1.5 to 2.5 times that of the vehicle-administered control group, confirming serious side effects. However, in the interleukin-2 and CU06-1004 co-administration group mice, both the visual observation and the detection values ​​of the Evans blue reagent were confirmed to be similar to those of the vehicle-administered control group, proving that serious side effects were significantly alleviated.

[0125]

[0126] Example 5: Comparison of spleen size and weight in tumor-free mice

[0127] Since the spleen is an important immune organ composed of lymphocytes and various immune cells, it is possible to determine whether side effects are induced in vivo by comparing the size and weight of the spleen removed from each drug-administered group of mice that do not have tumors.

[0128] After preparing a tumor-free experimental animal model as described in Example 4 above, the spleen was removed from the sacrificed mouse 2 hours after the last drug administration.

[0129] Figure 3a is a photograph of the spleen removed from tumor-free mice in each drug administration group.

[0130] Figure 3b is a graph showing the spleen weight of tumor-free mice in each drug administration group.

[0131] Through the photo in Fig. 3a and the graph in Fig. 3b, it was confirmed that the size and weight of the spleen in the body were increased by the interleukin-2 drug, and it was confirmed that the increase in the size and weight of the spleen was maintained in the group administered with the combination of interleukin-2 and CU06-1004.

[0132] The above results demonstrate that the substance is safe and does not cause side effects in vivo, even when administered alone or in combination with an anticancer agent.

[0133]

[0134] Example 6: Confirmation of the efficacy enhancement effect of anticancer combination agents in tumor-bearing mice.

[0135] Figure 4a is a schematic diagram illustrating the preparation and sacrifice procedures for a tumor-bearing experimental animal model. The specific administration method is as follows.

[0136] 5x10^5 (cells) of B16F10 melanoma cells were injected subcutaneously into the right side of 7-week-old C57BL / 6 male mice. The mice were then divided into three groups: a vehicle-administered control group, an interleukin-2 monotherapy group, and an interleukin-2 and CU06-1004 combination therapy group.

[0137] Seven days after melanoma cell administration, the drug was administered to each mouse in each drug administration group three times a day for three days, and once a day on the 11th day. At this time, the control group was administered vehicle (10 mpk), the interleukin-2 monotherapy group was administered interleukin-2 (75,000 U), and the interleukin-2 and CU06-1004 combination administration group was administered interleukin-2 (75,000 U) and CU06-1004 (10 mpk).

[0138] Two hours after the last drug administration, mice were sacrificed and tumors were removed.

[0139] Figures 4b and 4c show photographs of tumor sizes measured before and after excision in tumor-bearing mice from each drug-administered group. The tumors in the control group were significantly larger than those in the interleukin-2 monotherapy group and the interleukin-2 plus CU06-1004 combination therapy group, confirming the anticancer effect of the interleukin-2 immunotherapy.

[0140] Figure 4d is a graph measuring the tumor size in tumor-bearing mice from each drug-administered group. On average, tumors in the control group were approximately 450 mm 3 The size of the tumor was confirmed to be about 160 mm, while the tumor in the interleukin-2 monotherapy group was about 160 mm. 3 The size is about 130mm, and the tumor in the group receiving combination therapy with interleukin-2 and CU06-1004 is about 130mm. 3 Measured by size, it was confirmed that the anticancer effect was further enhanced when interleukin-2 and CU06-1004 were administered together.

[0141] Figure 4e is a graph measuring the tumor weights of tumor-bearing mice in each drug administration group. On average, tumors in the control group weighed approximately 0.37 g, while tumors in the interleukin-2 monotherapy group weighed approximately 0.12 g, and tumors in the interleukin-2 and CU06-1004 combination group weighed approximately 0.07 g, confirming that the anticancer effect was further enhanced when interleukin-2 and CU06-1004 were administered together.

[0142] As a result, it was demonstrated that the co-administration of interleukin-2 and CU06-1004 had an effect of enhancing the efficacy of interleukin-2 immunotherapy.

[0143]

[0144] Example 7: Confirmation of the alleviation of vascular leakage syndrome by anticancer combination agents in tumor-bearing mice.

[0145] After preparing a tumor-bearing experimental animal model as described in Example 6 above, Evans blue staining was performed in the same manner as in Example 4, and each organ was removed from the sacrificed mouse 2 hours after the last drug administration.

[0146] Figures 5a to 5d are photographs confirming the results of Evans blue staining confirmed in the lungs, liver, hands, and feet of each mouse in the vehicle-administered control group, the interleukin-2-only administration group, and the interleukin-2 and CU06-1004 co-administration group.

[0147] Figures 5e to 5h are graphs showing the results of Evans blue staining confirmed in the lungs, liver, hands, and feet of each mouse in the vehicle-administered control group, the interleukin-2-only administration group, and the interleukin-2 and CU06-1004 co-administration group.

[0148] As a result, Evans blue staining was observed with the naked eye in the organs and body parts extracted from the interleukin-2 monotherapy group mice, and compared to the vehicle-administered control group, the Evans blue reagent was detected at a dose of 1.5 to 2.5 times that of the vehicle-administered control group, confirming serious side effects. However, in the interleukin-2 and CU06-1004 co-administration group mice, both the visual observation and the detection values ​​of the Evans blue reagent were confirmed to be similar to those of the vehicle-administered control group, proving that serious side effects were significantly alleviated.

[0149] As a result, it was verified that the combined administration of interleukin-2 and CU06-1004 had a significant effect in alleviating vascular leak syndrome even in tumor-bearing cells.

[0150]

[0151] Example 8: Experiment to confirm changes in immune cells due to interleukin-2 immunotherapy

[0152] 1. Immunofluorescence staining experiment

[0153] To confirm changes in immune cells due to interleukin-2 immunotherapy, staining was performed using a specific marker, the CD8 marker, known as a cytotoxic T cell marker.

[0154] To perform immunofluorescence staining, mice were first anesthetized by injection of 2.5% tribromoethanol and then perfused with 50 ml of PBS or saline via the left ventricle of the heart. The obtained tissues were then fixed with 4% paraformaldehyde (PFA) for 16 hours, dehydrated in a 15% sucrose solution, and then dehydrated in a 30% sucrose solution until the tumors sank to the bottom of the container. Mouse tumor tissues were sectioned into 20-30 μm thick sections using a cryostat. Approximately 7-10 slices were obtained from each tissue, and the tissues were stored at -80°C. To check for vascular leakage, tumor tissues were treated with 0.5% PBS Triton X-100 (PBST) for 5 minutes, incubated with blocking solution for 1 hour at room temperature, and then treated with primary antibody for double staining and incubated O / N.

[0155] To investigate the increased T cells in tumors, CD8 (Abcam; ab22378; 1:200) was incubated for 16 h at 4°C, followed by secondary antibody incubation for 1 h at room temperature. Immunofluorescence was imaged using a confocal microscope.

[0156] The photograph in Fig. 6a and the graph in Fig. 6b confirm that the number of CD8+ T cells significantly increased in both groups injected with interleukin-2. In particular, the number of CD8+ T cells in the group administered with combination of interleukin-2 and CU06-1004 was confirmed to be higher than that in the group administered with interleukin-2 alone, verifying that the efficacy of immune anticancer treatment was enhanced through combination of interleukin-2 and CU06-1004.

[0157] 2. Experiment to measure TNFα expression using ELISA

[0158] To confirm the increased T cell function, serum was isolated from mice and an enzyme-linked immunosorbent assay (ELISA) was used. To analyze cytokine levels between drug treatment groups, TNFα (Mouse TNF-alpha) expression in each mouse serum was measured.

[0159] As a result of analyzing the TNF alpha level through the graph in Figure 6c, it was confirmed that TNFα was significantly increased in the group injected with interleukin-2.

[0160]

[0161] Example 9: Confirmation of the effectiveness of combination immunotherapy in lung tumor-bearing mice.

[0162] Figure 7a is a schematic diagram illustrating the preparation and sacrifice procedures for an experimental animal model bearing lung tumors. The specific administration method is as follows.

[0163] Seven-week-old male C57BL / 6 mice were injected with 7x10^5 B16F10 melanoma cells via the tail vein. This induced tumor implantation in lung tissue. The mice were then divided into three groups: a vehicle-administered control group, an interleukin-2 monotherapy group, and an interleukin-2 and CU06-1004 combination therapy group.

[0164] Seven days after melanoma cell administration, the drug was administered to each mouse in each drug administration group three times a day for three days, and once a day on the 11th day. At this time, the control group was administered vehicle (10 mpk), the interleukin-2 monotherapy group was administered interleukin-2 (75,000 U), and the interleukin-2 and CU06-1004 combination administration group was administered interleukin-2 (75,000 U) and CU06-1004 (10 mpk).

[0165] 1. Confirm the Evans staining results of the lungs of each drug administration group.

[0166] Evans blue staining was performed to confirm vascular leak syndrome in C57BL / 6 mice. After drug administration, the mice were intravenously injected with Evans blue reagent. Two hours after the final drug administration, the mice were sacrificed and their lungs were removed.

[0167] Figure 7b is a graph showing the detection values ​​of Evans blue staining confirmed in lungs extracted from mice with lung tumors in each drug administration group.

[0168] Compared to the control group administered vehicle, the interleukin-2 monotherapy group detected approximately twice as much Evans blue reagent, confirming serious side effects. However, in the group of mice administered interleukin-2 and CU06-1004 in combination, the detection value of Evans blue reagent was confirmed to be similar to that of the control group administered vehicle, proving that vascular leak syndrome was significantly alleviated.

[0169]

[0170] *2. Comparison of lung weights in each drug administration group

[0171] Since tumor implantation is induced in lung tissue, a decrease in lung weight directly corresponds to a decrease in tumor weight.

[0172] Figure 7c is a graph measuring the weight of lungs removed from mice with lung tumors in each drug administration group.

[0173] It was confirmed that the lung weight of the interleukin-2 monotherapy group decreased more than that of the control group, and that the lung weight of the interleukin-2 and CU06-1004 combination therapy group decreased even more, confirming that the anticancer effect was further enhanced when interleukin-2 and CU06-1004 were combined.

[0174] As a result, it was demonstrated that the co-administration of interleukin-2 and CU06-1004 had an effect of enhancing the efficacy of interleukin-2 immunotherapy.

[0175]

[0176] Example 10: Confirmation of the effectiveness of combination immunotherapy in tumor-bearing mice.

[0177] Figure 8 is a photograph confirming the vascular leakage near the tumor in tumor-bearing mice of each drug administration group. Figure 8 shows the results of comparing the appearance of the blood vessels near the tumor in the mouse models of each group. When the skin of the area with the tumor was peeled off and the blood vessels were compared, in the group injected with interleukin-2, the tumor size was smaller, but the degree of vascular leakage seemed quite serious. However, in the combination administration group, the degree of vascular leakage was reduced in proportion to the smaller tumor size.

[0178] As a result, it was demonstrated that the combined administration of interleukin-2 and CU06-1004 enhanced the efficacy of interleukin-2 immunotherapy and alleviated vascular leak syndrome.

[0179]

[0180] Through the above examples, it was confirmed that an immuno-oncology combination preparation comprising an interleukin-2 (IL-2) immuno-oncology agent according to the present invention; and a compound represented by the above chemical formula 1, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof can be administered.

[0181] Based on the above results, it is expected that the efficacy of interleukin-2 immunotherapy can be enhanced by using the above immunotherapy combination agent, and that it is expected to have an effect of alleviating vascular leak syndrome.

Claims

1. Interleukin-2 (IL-2) anticancer agent; and An immunotherapy combination preparation comprising a compound represented by the following chemical formula 1, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 1] 2. In paragraph 1, The above immuno-oncology combination agent is an immuno-oncology combination agent that exhibits an effect of enhancing the efficacy of interleukin-2 immuno-oncology agents.

3. In paragraph 1, The above immunotherapy combination agent is an immunotherapy combination agent that alleviates or reduces vascular leak syndrome.

4. In paragraph 1, The compound of the above immuno-oncology combination agent is an immuno-oncology combination agent that can be administered simultaneously, sequentially, or alternately with an interleukin-2 immuno-oncology agent.

5. In paragraph 1, The above-mentioned immuno-oncology combination agent is an immuno-oncology combination agent that activates one or more immune factors selected from the group consisting of helper T cells, cytotoxic T cells, natural killer (NK) cells, and cytokines.

6. In paragraph 1, The above combination immunotherapy agent is used for the treatment of pseudomyxoma, intrahepatic biliary tract cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, One or more selected from the group consisting of kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, stomach cancer, gastric carcinoid tumor, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, triple-negative breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid tumor, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsillar cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, and thymic cancer Combination immunotherapy agents for preventing or treating cancer.

7. An anticancer adjuvant for interleukin-2 immunotherapy comprising a compound represented by the following chemical formula 1, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] 8. A pharmaceutical composition for enhancing the efficacy of an interleukin-2 immunotherapy agent, comprising a compound represented by the following chemical formula 1, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] 9. A pharmaceutical composition for reducing side effects of an interleukin-2 immunotherapy agent, comprising a compound represented by the following chemical formula 1, a stereoisomer thereof, a crystalline form thereof, or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1]

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