Type 2 innate lymphoid cells with cytotoxic activity, method for producing same, and use thereof

Abstract

The present invention relates to: a cancer immunotherapeutic composition comprising type 2 innate lymphoid cells with cytotoxic activity; a pharmaceutical composition for preventing or treating solid tumors, comprising same as an active ingredient; and a method for culturing type 2 innate lymphoid cells. By using the type 2 innate lymphoid cells of the present invention, which are immune cells that secrete cytotoxic substances by treatment with cytokines, cellular metabolite agonists, or fatty acid metabolites, anticancer effects can be obtained by improving low specificity to cancer cells, damage to normal tissues, and side effects on actively dividing and proliferating tissues, which result from anticancer therapies through existing hormone regulation, chemical drug-induced cancer cell death, and physical removal. In addition, the present invention can be applied to solid tumors where existing cancer immunotherapeutic agents have been difficult to apply.

Description

Type 2 innate lymphocyte cells with cytotoxicity, method for producing the same, and use thereof

[0001] The present invention relates to type 2 innate lymphocyte cells having cytotoxicity, a method for producing the same, and a use thereof.

[0002] Cancer is a leading chronic disease, accounting for 26% of deaths in Korea. Various anticancer drugs are being studied for its treatment. However, existing cancer treatments rely on chemical and physical methods to destroy and eliminate cancer cells, causing significant damage to normal tissues and lacking specificity for cancer cells. To address this issue, research is underway into developing immunotherapy drugs that target cancer cells by modulating the activity of existing immune cells.

[0003] When cancer develops, various immune cells infiltrate the tumor microenvironment (TME) surrounding the cancer cells and work to eliminate them. Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) are known to secrete cytotoxic substances such as Granzyme B (GMZB) and promote cancer cell death. However, as cancer progresses, immune cells infiltrating the TME become less active and exhausted due to persistent immune responses, making them ineffective at eliminating cancer cells. Therefore, immunotherapy approaches that reactivate these impaired immune cells are gaining attention, with research focusing on NK cells and T lymphocytes. While T lymphocyte-based immunotherapy has shown efficacy in some specific cancer types, such as hematological malignancies, it remains difficult to apply to diverse cancer types, such as solid tumors. Therefore, there is a need to develop cancer treatment technologies that can act on various types of cancer by developing immune anticancer drugs that utilize immune cells other than T lymphocytes.

[0004] Accordingly, the present inventors developed a technology for producing type 2 innate lymphoid cells (ILC2s) that secrete GZMB, and confirmed the therapeutic efficacy of lung cancer, a type of solid cancer, through ILC2s that secrete GZMB, thereby confirming that it can be applied to solid cancers to which existing immunotherapy agents were difficult to apply, thereby completing the present invention.

[0005] An object of the present invention is to provide an immuno-oncology composition comprising type 2 innate lymphoid cells that secrete cytotoxic substances induced by cytokines, cell metabolite agonists or fatty acid metabolites.

[0006] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating solid cancer, which comprises innate lymphocytes as an active ingredient.

[0007] Another object of the present invention is to provide a method for culturing type 2 innate lymphocytes with increased ability to secrete cytotoxic substances, comprising the steps of: (a) treating type 2 innate lymphocytes with a cytokine, a cell metabolite agonist, or a fatty acid metabolite to induce secretion of a cytotoxic substance; and (b) culturing the type 2 innate lymphocytes.

[0008] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0009] The present invention relates to an immuno-oncology composition comprising type 2 innate lymphoid cells that secrete cytotoxic substances induced by cytokines, cell metabolite agonists or fatty acid metabolites.

[0010] In the present invention, cytokines refer to proteins (~5-20 kDa) that play a role in cell signaling. Cytokines include interleukins, chemokines, interferons, lymphokines, tumor necrosis factors, monokines, and colony-stimulating factors, and these cytokines can be produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells and monocytes, endothelial cells, fibroblasts, and stromal cells.

[0011] In the present invention, the cytokine may be at least one selected from the group consisting of IL (interleukin)-2, IL-7, and IL-33. In addition, the innate lymphoid cells may be induced to secrete cytotoxic substances by IL-33.

[0012] In the present invention, the cell metabolite agonist refers to a substance capable of regulating cellular respiration and promoting cellular metabolism. According to one embodiment of the present invention, the cell metabolite may be a peroxisome proliferator-activated receptor (PPAR). In addition, it was confirmed that the optimal concentration of the PPAR agonist treated to increase the secretion of GZMB as the cytotoxic substance is 10 to 50 uM.

[0013] In the present invention, the fatty acid may be at least one selected from the group consisting of palmitic acid and oleic acid. In addition, according to one embodiment of the present invention, it was confirmed that the optimal concentration of the fatty acid treated to increase the secretion of GZMB as a cytotoxic substance is a concentration of 100 to 200 uM. In the present invention, immune cells refer to cells that regulate immunity as a defense mechanism against pathogens, foreign substances, viruses, etc. that invade from the outside or directly attack pathogens, etc.

[0014] In the present invention, lymphocytes are a type of white blood cell created by differentiation and maturation of hematopoietic stem cells into lymphoid progenitor cells during the process of blood cell differentiation from stem cells in bone marrow.

[0015] In the present invention, innate lymphoid cells are a type of immune cell and refer to a group of lymphocyte lineage cells that have innate immune characteristics without expressing antigen-specific receptors, which are characteristic of adaptive immune cells. The innate lymphoid cells are developed by differentiation from common lymphoid progenitors (CLPs). In addition, the innate lymphoid cells are known to be classified into type I innate lymphoid cells (ILC1), type II innate lymphoid cells (ILC2), and type III innate lymphoid cells (ILC3) according to the cytokines they secrete and the transcription factors that regulate the development and function of these cells.

[0016] In the present invention, the type 2 innate lymphoid cells (ILC2) may have cytotoxicity.

[0017] Meanwhile, in the past, the anticancer effect was shown through a mechanism via activated dendritic cells and T cells through cytokines and chemokines secreted from activated type 2 innate lymphocytes. However, in the case of the type 2 innate lymphocytes of the present invention, it was confirmed that the anticancer effect was shown alone without other immune cells by being induced into type 2 innate lymphocytes that secrete cytotoxic substances by IL-33.

[0018] In the present invention, the cytotoxic substance may be Granzyme B.

[0019] The present inventors developed an in vitro culture technique for isolating ILC2 cells and acquiring cytotoxic capacity. First, using a culture medium containing 1) various cytokine combinations, 2) a PPAR agonist that can promote cell metabolism, and 3) fatty acid metabolites, differentiation and culture of type 2 innate lymphoid cells (cytotoxic ILC2s) with anticancer effects were achieved. By culturing ILC2 cells for an optimal culture time, cells with cytotoxic capacity could be produced. While ILC2 cells generally do not exhibit cytotoxicity, the in vitro culture method and conditions of the present invention enable the production of ILC2 cells with cytotoxic capacity (differentiation into GZMB+ ILC2s). Meanwhile, innate lymphoid cells express various cytokine receptors and have the characteristic of being activated by cytokine stimulation. Furthermore, it was confirmed that the properties and activity of immune cells can be modulated by promoting cell respiration and metabolism or by altering fatty acid metabolites contained in the culture medium. In the present invention, we confirmed the conditions under which ILC2 cells differentiate into cells with cytotoxic capabilities such as GZMB secretion in an in vitro culture situation by 1) using a culture medium containing a combination of IL-2, IL-7, and IL-33 cytokines, 2) adding a PPAR agonist capable of promoting cell metabolism, and 3) using a culture medium containing fatty acids, thereby suggesting the possibility of using ILC2 cells as a cancer treatment agent.

[0020] In the present invention, immunotherapy means killing cancer cells by activating the immune cells of the human body, and it means showing a therapeutic effect on cancer by directly activating the patient's immune cells.

[0021] In addition, the present invention relates to a pharmaceutical composition for preventing or treating solid cancer, which comprises the above-mentioned immuno-oncology composition as an active ingredient.

[0022] In the present invention, an active ingredient means a component that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself.

[0023] In the present invention, prevention refers to any act of inhibiting or delaying the occurrence of cancer by administering a composition according to the present invention.

[0024] In the present invention, treatment refers to or includes alleviating, inhibiting the progression or preventing a disease, disorder or condition, or one or more symptoms thereof, and “active ingredient” or “pharmaceutically effective amount” may mean any amount of the composition used in the process of practicing the invention provided herein that is sufficient to alleviate, inhibit the progression or prevent a disease, disorder or condition, or one or more symptoms thereof.

[0025] In the present invention, the method of administration of the pharmaceutical composition is not particularly limited, but may be administered parenterally, such as intravenously, subcutaneously, intraperitoneally, by inhalation, or topically, or may be administered orally, depending on the intended method. The dosage may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity. The daily dosage refers to the amount of a therapeutic substance according to a daily pattern that is sufficient to treat the alleviated disease state by administering it to an individual in need of treatment. The effective amount of a therapeutic substance varies depending on the specific compound, the disease state and its severity, and the individual in need of treatment, and can be routinely determined by those skilled in the art.

[0026] In the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier and / or additive. For example, it may include sterile water, physiological saline, a conventional buffer (such as phosphoric acid, citric acid, or other organic acids), a stabilizer, a salt, an antioxidant (such as ascorbic acid), a surfactant, a suspending agent, an isotonic agent, or a preservative. For topical administration, it may also include a combination with an organic substance such as a biopolymer, an inorganic substance such as hydroxyapatite, specifically, a collagen matrix, a polylactic acid polymer or copolymer, a polyethylene glycol polymer or copolymer, and chemical derivatives thereof. When the pharmaceutical composition according to one embodiment is prepared in a dosage form suitable for injection, the immune cells or a substance that increases their activity may be dissolved in a pharmaceutically acceptable carrier or may be frozen in a dissolved solution state.

[0027] In the present invention, the pharmaceutical composition may appropriately contain, if necessary, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc. depending on the administration method or formulation. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, 19th ed., 1995]. The pharmaceutical composition may be manufactured in a unit dose form or may be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets, or capsules.

[0028] In the present invention, the solid cancer may be at least one selected from the group consisting of lung cancer, brain tumor, head and neck cancer, breast cancer, thymoma, squamous cell carcinoma, esophageal cancer, pancreatic cancer, colon cancer, liver cancer, stomach cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, colon cancer, lymphoma, multiple myeloma, sarcoma, malignant melanoma, and skin cancer.

[0029] In addition, the present invention relates to a method for culturing type 2 innate lymphocytes with increased ability to secrete cytotoxic substances, comprising the steps of: (a) treating type 2 innate lymphocytes with a cytokine, a cell metabolite agonist, or a fatty acid metabolite to induce secretion of a cytotoxic substance; and (b) culturing the type 2 innate lymphocytes.

[0030] In the present invention, when culturing the innate lymphocyte cells, at least one cytokine selected from the group consisting of IL-2, IL-7, and IL-33 may be added.

[0031] In the present invention, the cytokine can be treated to type 2 innate lymphoid cells at a concentration of 10 to 50 ng / mL, and preferably at a concentration of 30 ng / mL.

[0032] In the present invention, the cell metabolites may be treated to type 2 innate lymphoid cells at a concentration of 10 to 50 uM.

[0033] In the present invention, the fatty acid may be treated to type 2 innate lymphocyte cells at a concentration of 100 to 200 uM.

[0034] In addition, the cytokine, cell metabolite agonist or fatty acid metabolite can be treated 1 to 5 times to type 2 innate lymphoid cells, and preferably 3 times. Meanwhile, since the corresponding features in the above details can be replaced by the above-described part, their description is omitted.

[0035] According to the method for culturing innate lymphocytes according to the present invention, type 2 innate lymphocytes, which are immune cells that secrete cytotoxic substances, can be produced by treating cytokines, cell metabolite agonists, or fatty acid metabolites. In addition, an immuno-oncology composition comprising type 2 innate lymphocytes having cytotoxic activity according to the present invention as an active ingredient can be used as a pharmaceutical composition for preventing or treating solid cancer. An immuno-oncology composition using type 2 innate lymphocytes having cytotoxic activity can be applied as an alternative to the low specificity for cancer cells, damage to normal tissues, and side effects on tissues with active division and proliferation of anticancer treatments through the induction of apoptosis and physical removal of cancer cells through existing hormone regulation and chemical drugs, and has the advantage of being usable in solid cancers to which the application of existing immuno-oncology agents was difficult.

[0036] Figure 1 illustrates an overview of a cancer treatment technology using cytotoxic ILC2 of the present invention.

[0037] Figure 2 shows the culture conditions according to treatment with cytokines, cell metabolite agonists, or fatty acid metabolites for cytotoxic ILC2 differentiation.

[0038] Figure 3 illustrates various cytotoxic gene expression in ILC2s. Figure 3A illustrates various cytotoxic gene expression in ILC2s activated by IL-33. Figure 3B illustrates that the gene sets expressed in ILC2s activated by IL-33 are similar to gene sets mediating cytotoxicity.

[0039] Figure 4 illustrates the secretion of cytotoxic substances in response to various stimuli. Figure 4A shows the secretion of three types of cytotoxic substances under various cytokine stimulation conditions. Figure 4B shows the production of the cytotoxic substances Granzyme B (GZMB) and ECP, confirmed using an immunofluorescence staining technique.

[0040] Figure 5 shows the results of GZMB+ ILC2 culture according to cytokine combination treatment. Figure 5A shows the distribution of GZMB+ ILC2 according to various cytokine combinations. Figure 5B shows the proportion of GZMB+ ILC2 changed by various cytokine combinations.

[0041] Figure 6 shows the results of GZMB+ ILC2 culture according to treatment with PPAR agonist as a cell metabolite agonist.

[0042] Figure 7 shows the results of GZMB+ ILC2 culture according to palmitic acid and oleic acid treatment as fatty acid metabolites.

[0043] Figure 8 shows the viability of GZMB+ ILC2 cells according to the concentration of fatty acid treatment.

[0044] Figure 9 shows the results of cancer cell apoptosis mediated by GZMB+ ILC2s. Figure 9A shows changes in cancer cell apoptosis induced by ILC2s using immunofluorescence staining. Figure 9B shows the rate of cancer cell apoptosis induced by ILC2s using flow cytometry. Figure 9C shows ILC2-mediated cancer cell apoptosis over time using real-time immunofluorescence staining. Figure 9D compares the changes in cancer cell apoptosis capacity over time depending on the number of ILC2s.

[0045] Figure 10 illustrates the GZMB-specific apoptotic capacity of ILC2s. Figure 10A illustrates the experimental conditions used in Figure 10. Figure 10B confirms the reduced apoptotic capacity of ILC2s by treatment with a GZMB inhibitor. Figure 10C compares the apoptotic rate of cancer cells according to the amount of GZMB secreted.

[0046] Figure 11 shows the difference in GZMB secretion ability depending on whether PD-1 is expressed.

[0047] Figure 12 shows the results of the cancer treatment efficacy verification through GZMB+ ILC2. Figure 12A shows a description of the experimental conditions performed in Figure 12. Figure 12B shows the functional changes of ILC2 depending on the presence or absence of PD-1 expression. Figure 12C compares the IL-5 / IL-13 secretion rates depending on the presence or absence of PD-1 expression. Figure 12D compares the degree of cancer progression depending on the presence or absence of PD-1 expression in ILC2. Figure 12E shows the results of measuring the degree of cancer progression depending on the presence or absence of PD-1 expression in ILC2. Figure 12F compares the degree of cancer progression by measuring the change in lung weight depending on the presence or absence of PD-1 expression in ILC2.

[0048] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0049]

[0050] Example 1. Culture for cytotoxic ILC2 differentiation

[0051]

[0052] Mice were injected intratracheally with 30 μL of PBS (phosphate-buffered saline) containing 250 ng of recombinant IL-33 for 3 consecutive days (days 0, 1, and 2). On day 7, the lungs of the mice were finely dissociated, and immune cells were isolated using digestion media containing collagenase IV and DNase I. Afterwards, ILC2s were isolated using fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) with fluorescently labeled monoclonal antibodies. Then, IL-2 (10 ng / mL), IL-7 (10 ng / mL), and IL-33 (30 ng / mL) were added to 10% FBS + RBMI1640 medium and cultured in vitro. In vitro cultures were performed at a density of 5 × 10 6 10 mL of culture medium was used per ILC2, and T75 flasks or 100 mm × 20 mm cell culture vessels were used. The culture medium was replaced once every 3–4 days, and after 7 days of in vitro culture, ILC2 secreting the cytotoxic substance GZMB were produced (Fig. 2).

[0053]

[0054] Example 2. Confirmation of the cytotoxic substance secretion ability of activated ILC2s.

[0055]

[0056] When the expression of various cytotoxic substance genes in ILC2 present in the lungs of mice was compared 7 days after three injections of 250 ng of Interleukin (IL)-33, a cytokine that can activate type 2 innate lymphoid cells (ILC2), it was confirmed that the expression of granzyme B (GZMB) was increased by IL-33 (Fig. 3).

[0057]

[0058] Example 3. Confirmation of culture conditions for ILC2s secreting GZMB.

[0059]

[0060] 3.1. Culture using cytokines

[0061]

[0062] To determine the culture conditions that can increase the secretion of GZMB, an in vitro culture experiment was conducted using ILC2 isolated from the lungs of mice 7 days after three injections of 250 ng of IL-33.

[0063] In vitro culture conditions were used in which various cytokines capable of activating ILC2s, IL-25, IL-33, and TSLP, were added at a concentration of 30 ng / mL each to RPMI1640 medium containing 10% FBS. When cultured for 7 days using each medium, ILC2s differentiated under conditions in which IL-33 was added actively secreted Eosinophil cationic protein (ECP), granzyme A (GZMA), and granzyme B (GZMB), as confirmed by enzyme-linked immunosorbent assay (ELISA) analysis and immunofluorescence (IF) imaging technique (Fig. 4).

[0064]

[0065] Meanwhile, to confirm the efficacy of IL-15 and IL-18, cytokines known to be able to increase the secretion of GMZMB, the efficacy of combinations of various cytokines including IL-33 was verified. IL-2 and IL-7 were added at 10 ng / mL to RPMI1640 culture medium containing 10% FBS, and IL-15, IL-18, and IL-33 were added at a concentration of 30 ng / mL and cultured for 7 days. It was confirmed that all culture mediums containing IL-33 significantly increased the secretion ability of GZMB compared to the basic culture medium containing only IL-2 and IL-7. On the other hand, when a culture medium containing IL-15 or IL-18 was used, the secretion of GZMB in ILC2 did not increase, and when treated together with IL-33, it showed a slight decrease in the secretion of GZMB. Therefore, it was confirmed that the secretion of GZMB was effectively increased when 30 ng / mL of IL-33 was treated with 10 ng / mL of IL-2 and IL-7 in RPMI1640 culture medium containing 10% FBS (Fig. 5).

[0066]

[0067] 3.2. Culturing with cell metabolite agonists

[0068]

[0069] To determine the culture conditions that can increase the secretion of GZMB, an in vitro culture experiment was conducted using ILC2 isolated from the lungs of mice 7 days after three injections of 250 ng of IL-33.

[0070] In vitro culture was performed by adding an agonist that can promote the function of PPAR (Peroxisome Proliferator-Activated Receptor), a substance that mediates the activity of cell metabolism, to RPMI1640 culture medium containing 10% FBS. PPAR agonists 1) TDZ; thiazolidinedione (TOCRIS, England, 50uM), 2) GW0724 (Sigma-Aldrich, USA, 5uM), 3) GW501516 (Sigma-Aldrich, USA, 5uM) were added to the culture medium at the above concentrations. When cultured for 24 hours using each culture medium, it was confirmed that the GZMB secretion ability was significantly increased under the condition in which the PPAR agonist was added. Meanwhile, when various concentrations of PPAR agonist were treated, it was confirmed that the GZMB secretion ability was reduced when a high concentration of PPAR agonist was used, and it was confirmed that the optimal concentration of PPAR agonist for increasing GZMB secretion was TDZ: 10-50uM, GW0742: 5uM, GW501516: 5uM (Fig. 6).

[0071]

[0072] 3.3. Cultivation with fatty acid metabolites

[0073]

[0074] To determine the culture conditions that can increase the secretion of GZMB, an in vitro culture experiment was conducted using ILC2 isolated from the lungs of mice 7 days after three injections of 250 ng of IL-33.

[0075] Since the cytotoxicity of ILC2 is related to fatty acid metabolism, 1) Palmitate (Palmitic acid) and 2) Oleate (Oleic acid) were added to RPMI1640 medium containing 10% FBS for in vitro culture. Palmitate and oleate were used in BSA (Bovine Serum Albumin)-conjugated form for easy uptake into cells, and the ratio of palmitate / oleate and BSA was 6:1, pH 7.4. When cultured for 24 hours using a culture medium containing 0-200 μM palmitate or oleate, ILC2 differentiated in the culture condition with palmitate and oleate showed a significant increase in GZMB secretion. In addition, this GZMB secretion was shown to increase as the concentration of palmitate and oleate in the culture medium increased (Fig. 7).

[0076] Meanwhile, it was confirmed that the Viability of ILC was significantly reduced when the concentration of fatty acids in the culture medium was 200 uM or higher, so it was confirmed that using a concentration of fatty acids of 100-200 uM was the optimal condition for increasing the Viability of cultured cells and GZMB secretion (Fig. 8).

[0077]

[0078] Example 4. Confirmation of the anticancer efficacy of ILC2s secreting GZMB.

[0079]

[0080] GZMB produced through in vitro culture + To confirm whether ILC2s actually exhibit anticancer efficacy, we examined their ability to eliminate mouse lung cancer cell lines.

[0081] GZMB prepared by treating 30 ng / mL of IL-33 with 10 ng / mL of IL-2 and IL-7 in RPMI1640 medium containing 10% FBS+ ILC2 was placed in a test tube containing a lung cancer cell line (TC-1, derived from primary lung epithelial cells of C57BL / 6 mice), and the degree of apoptosis (measured by checking the expression of annexin V) of the lung cancer cell line was examined for 3 hours (Fig. 9A, Fig. 9B). When compared with the efficacy of eosinophils (Eos), which were previously known to be able to induce apoptosis of cancer cells, it was confirmed that ILC2 effectively eliminated the lung cancer cell line, and real-time imaging revealed that ILC2 directly induced apoptosis of the lung cancer cell line (Fig. 9C, Fig. 9D).

[0082]

[0083] Meanwhile, when GZMB inhibitor (GZMBi) that can suppress the secretion of GZMB in ILC2 was treated together, the ability to remove lung cancer cell lines was confirmed to disappear, and when the secretion of other types of substances (IL-5, IL-13) was inhibited (aIL-5 / 13), the removal ability was confirmed to increase (Fig. 10A, Fig. 10B). In addition, the concentration of GZMB and the ratio of removed cancer cell lines (TC-1) showed a positive correlation (Fig. 10C). Therefore, GZMB produced through in vitro culture + ILC2 has anticancer properties that eliminate lung cancer, and it was found that the anticancer properties vary depending on the secretion ability of GZMB.

[0084]

[0085] Example 5. Confirmation of the efficacy of anticancer treatment using ILC2.

[0086]

[0087] In general, drugs that inhibit the action of immune checkpoint receptors such as PD-1 are prescribed together in the clinical treatment of lung cancer patients, and it is known that PD-1 is also expressed in ILC2. Therefore, we investigated whether inhibition of PD-1 could increase anticancer efficacy in ILC2. Wild-type (WT) and PD-1-deficient ILC2 (Pdcd1 - / - When ILC2 secreting GZMB was produced using Pdcd1 - / - We confirmed that secretion of GZMB was significantly increased in ILC2. Therefore, Pdcd1 was more abundant than WT ILC2. - / - It was found that there was a possibility that the anticancer efficacy would be greater when ILC2 was used (Fig. 11).

[0088]

[0089] Next, to confirm whether the cancer treatment mechanism using GZMB+ ILC2 produced through in vitro culture can be applied in the preclinical stage, its efficacy was confirmed in a mouse lung cancer model.

[0090] Lung cancer was induced in mice by injection of TC-1 lung cancer cell line, and then WT ILC2 and Pdcd1 secreting GZMB - / - The anticancer efficacy of ILC2s was verified by injecting ILC2s individually. Injection of WT ILC2s showed some recovery of ILC2 function (increased IL-5 and IL-13 secretion), but did not effectively eliminate lung cancer. However, Pdcd1 - / - In the case of ILC2, we were able to confirm that lung cancer was significantly reduced along with the functional recovery of ILC2 (Fig. 12). Therefore, GZMB together with immunotherapy + It is expected that the method using ILC2 can be applied as a new treatment for lung cancer.

[0091]

[0092] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An immuno-oncology composition comprising type 2 innate lymphoid cells that secrete cytotoxic substances induced by cytokines, cell metabolite agonists or fatty acid metabolites.

2. In paragraph 1, An immuno-anticancer composition, wherein the cytokine is at least one selected from the group consisting of IL-2, IL-7, and IL-33.

3. In paragraph 1, An immuno-anticancer composition wherein the above cell metabolite is PPAR (Peroxisome Proliferator-Activated Receptor).

4. In paragraph 1, An immuno-anticancer composition, wherein the fatty acid is at least one selected from the group consisting of palmitic acid and oleic acid.

5. In paragraph 1, The above type 2 innate lymphocyte cell is an innate lymphocyte cell having cytotoxicity.

6. In paragraph 1, Innate lymphoid cells, wherein the cytotoxic substance is Granzyme B.

7. A pharmaceutical composition for preventing or treating solid cancer, comprising the immunotherapy composition of any one of claims 1 to 6 as an active ingredient.

8. In paragraph 7, A pharmaceutical composition, wherein the solid cancer is at least one selected from the group consisting of lung cancer, brain tumor, head and neck cancer, breast cancer, thymoma, squamous cell carcinoma, esophageal cancer, pancreatic cancer, colon cancer, liver cancer, stomach cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, colon cancer, lymphoma, multiple myeloma, sarcoma, malignant melanoma, and skin cancer. 9.(a) a step of inducing secretion of cytotoxic substances by treating type 2 innate lymphoid cells with cytokines, cell metabolite agonists, or fatty acid metabolites; and (b) A method for culturing type 2 innate lymphocytes having increased secretion capacity of cytotoxic substances, comprising the step of culturing type 2 innate lymphocytes.

10. In paragraph 9, A method for culturing type 2 innate lymphocyte cells, wherein the cytokine is treated to type 2 innate lymphocyte cells at a concentration of 10 to 50 ng / mL.

11. In paragraph 9, A method for culturing type 2 innate lymphocyte cells, wherein the above cell metabolites are treated to type 2 innate lymphocyte cells at a concentration of 10 to 50 uM.

12. In paragraph 9, A method for culturing type 2 innate lymphocyte cells, wherein the fatty acid is treated to type 2 innate lymphocyte cells at a concentration of 100 to 200 uM.

Images