Interleukin complex and use thereof

Abstract

Provided is a use of an interleukin (IL) complex in preparation of drugs for treating lung cancer, liver cancer, sarcoma, colorectal cancer, pancreatic cancer, prostate cancer, and melanoma in a subject in need thereof. Compared with wild-type IL-2, the provided IL complex can more strongly activate tumor-killing immune cells such as CD8+ T cells and NK cells and can also be less likely to activate the growth of Treg cells for inhibiting an immune response, has more excellent drug administration convenience, can achieve the effect of better inhibiting the growth and reproduction of tumor cells in an acceptable toxic dose of organisms, and can inhibit the growth of tumor cells of more types of tumors.

Description

Interleukin complex and use thereof Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an interleukin complex and uses thereof. Background Art

[0002] Cancer is a major threat to human health and a cause of death. Traditional treatments include surgery, radiotherapy, chemotherapy, and targeted drug therapy. However, local treatments like surgery and radiotherapy are only suitable for larger lesions and cannot achieve the same systemic therapeutic effects, making tumors prone to metastasis and recurrence. Systemic treatments like chemotherapy and targeted drugs can treat smaller, less readily detected tumor lesions. However, due to the genetic heterogeneity of tumor cells, even after specific chemotherapy or targeted drugs are used to kill tumor cells, drug-insensitive tumor cells will continue to grow and multiply, leading to drug resistance. Activating the human immune system and amplifying the body's own tumor-killing immune cells, such as CD8-positive T cells (CD8+ T cells) and NK cells, is a new approach to inhibiting tumor cell growth and reproduction. It allows the immune system to recognize different tumor cells, comprehensively killing them and reducing the rate of tumor metastasis and recurrence.

[0003] Interleukin-2 (IL-2), discovered in 1976 and then known as T-cell growth factor (TCGF), is a globular glycoprotein that plays an important role in maintaining the normal function of T lymphocytes and NK cells. Natural IL-2 is a polypeptide composed of 133 amino acid residues with a molecular weight of approximately 15 kDa. IL-2 is primarily produced by activated T cells. It promotes T cell proliferation and differentiation, maintaining T cell activity; stimulates the generation, proliferation, and activation of natural killer (NK) cells; induces the generation, proliferation, and activation of cytotoxic T lymphocytes (CTLs); and induces and activates lymphokine-activated killer (LAK) cells and tumor-infiltrating lymphocytes. It also promotes the expression of cytokines and cytolytic molecules by T cells and promotes the proliferation of B cells (Waldmann et al., Nat Rev Immunol 6, 595-601 (2009)). These cells have the potential to directly or indirectly kill cells infected with exogenous microorganisms and cancerous cells. Therefore, IL-2 has excellent antiviral and anticancer effects and has broad clinical application potential.

[0004] The use of interleukin-2 (IL-2) to activate the human immune system to inhibit tumor growth was first reported in the 1990s. The wild-type IL-2 drug, called aldesleukin, was approved by the U.S. Food and Drug Administration in 1992 and 1998 for the treatment of metastatic renal cell carcinoma and metastatic melanoma, respectively, demonstrating promising efficacy. However, this approach has serious limitations, particularly due to the drug's high toxicity and the low dosage required, below 37 μg (drug weight) / kg (body weight). Higher doses of IL-2 can cause severe toxicities, including vascular leak syndrome, pulmonary edema, hypotension, and cardiotoxicity. At clinical doses, aldesleukin's ability to inhibit tumor growth is limited, resulting in a relatively low overall tumor inhibition rate. Furthermore, aldesleukin's approved indications are relatively narrow, limited to melanoma and renal cancer. At the same time, while wild-type IL-2 activates tumor-killing immune cells such as CD8+ T cells, it also activates the growth of regulatory T cells (Treg) that suppress immune responses, thereby limiting its effectiveness in inhibiting the growth and reproduction of tumor cells.

[0005] Current methods for suppressing tumors, such as the use of toxic chemicals and radiation, can easily lead to drug resistance and recurrence if they fail to completely kill all tumor cells. Furthermore, there is a lack of a safe and effective immune-stimulating method to stimulate the body's own immune system to suppress tumor growth, thereby achieving long-term survival benefits. Currently, there is a need for an immune-stimulating method that can activate tumor-killing immune cells, such as CD8+ T cells, while minimizing the activation of regulatory T cells that suppress immune responses. At the same time, this immune-stimulating method is sufficiently low in toxicity that it can be tolerated by animals (including humans), thereby utilizing the stimulation of the animal's own immune system to inhibit the growth of a variety of different tumors.

[0006] Summary of the Invention

[0007] In order to improve the above technical problems, the present invention provides the use of an interleukin complex, which uses an appropriate dosage to maximize the activation of tumor-killing immune cells such as CD8+T cells and minimize the growth of regulatory T cells that activate and suppress immune responses. Combined with the low toxicity of the complex, it can achieve the activation of tumor-killing T cells such as CD8+T cells and NK cells and other immune cells within an acceptable toxic dose for animals (including humans), thereby inhibiting the growth and reproduction of various tumor cells.

[0008] The key to this invention lies in the use of a novel interleukin complex within a specific dosage range to inhibit tumor growth in various animal models of tumors. The dosage range used in animals can be converted to dosages for other species based on the affinity of the interleukin complex for receptors in different species.

[0009] In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, comprising administering to the subject an interleukin complex, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1 and the sushi domain of the IL15Rα receptor as shown in SEQ ID NO.2.

[0010] In one embodiment of the present invention, the aforementioned subject is a mammal, including humans, non-human primates, cows, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice.

[0011] In one embodiment of the present invention, the aforementioned non-human primate is a monkey or a chimpanzee.

[0012] In one embodiment of the present invention, the solid tumor is selected from lung cancer, sarcoma, prostate cancer, solid tumors of the digestive tract and melanoma;

[0013] Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

[0014] In one embodiment of the present invention, the aforementioned method for treating colorectal cancer comprises administering the aforementioned interleukin complex to a subject, with each administration dose being 0.3-10 mg / kg;

[0015] Preferably, the dose administered to the subject each time is 1.5-5 mg / kg;

[0016] Preferably, the drug is administered to the subject once or twice a week;

[0017] Preferably, the drug is administered to the subject a total of three, five or six times.

[0018] In one embodiment of the present invention, the aforementioned method for treating liver cancer comprises administering the aforementioned interleukin complex to a subject, with each administration dose being 1.5-5 mg / kg;

[0019] Preferably, the dose administered to the subject each time is 3-5 mg / kg / w;

[0020] Preferably, the drug is administered to the subject once a week for a total of three times.

[0021] In one embodiment of the present invention, the aforementioned method for treating pancreatic cancer comprises administering the aforementioned interleukin complex to a subject, with each administration dose being 1.5-5 mg / kg;

[0022] Preferably, the dose administered to the subject each time is 3-5 mg / kg / w;

[0023] Preferably, the drug is administered to the subject once a week for a total of three times.

[0024] In one embodiment of the present invention, the aforementioned method for treating sarcoma comprises administering the aforementioned interleukin complex to a subject, with each administration dose being 0.03-10 mg / kg / w;

[0025] Preferably, the dose administered to the subject each time is 0.3-2.1 mg / kg / w;

[0026] Preferably, the drug is administered to the subject once or twice a week;

[0027] Preferably, the drug is administered to the subject three, four or six times in total.

[0028] In one embodiment of the present invention, the aforementioned method for treating prostate cancer comprises administering the aforementioned interleukin complex to a subject, with each administration dose being 0.03-10 mg / kg / w;

[0029] Preferably, the dose administered to the subject each time is 0.3-2.1 mg / kg / w;

[0030] Preferably, the drug is administered to the subject once or twice a week;

[0031] Preferably, the drug is administered to the subject three, four or six times in total.

[0032] In one embodiment of the present invention, the aforementioned method for treating lung cancer comprises administering the aforementioned interleukin complex to a subject, with each administration dose being 0.03-10 mg / kg;

[0033] Preferably, the dose administered to the subject each time is 0.3-2.1 mg / kg;

[0034] Preferably, the drug is administered to the subject once or twice a week;

[0035] Preferably, the drug is administered to the subject three, four or six times in total.

[0036] In one embodiment of the present invention, the aforementioned method for treating melanoma comprises administering the aforementioned interleukin complex to a subject, with each administration dose being 0.3-1.9 mg / kg or 2.1-10 mg / kg;

[0037] Preferably, the dose administered to the subject each time is 0.3-1.9 mg / kg;

[0038] Preferably, the drug is administered to the subject once or twice a week for a total of five times.

[0039] In one embodiment of the present invention, the aforementioned method for treating breast cancer comprises administering the aforementioned interleukin complex to a subject, with each administration dose being 0.03-2.1 mg / kg;

[0040] Preferably, the dose administered to the subject each time is 1-2.1 mg / kg;

[0041] Preferably, the drug is administered to the subject once a week for a total of three times.

[0042] In another aspect, the present disclosure provides a use of an interleukin complex in the preparation of a medicament for treating solid tumors in a subject in need thereof, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1 and the sushi domain of the IL15Rα receptor as shown in SEQ ID NO.2.

[0043] In one embodiment of the present invention, the aforementioned subject is a mammal, including humans, non-human primates, cows, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice.

[0044] In one embodiment of the present invention, the aforementioned non-human primate is a monkey or a chimpanzee.

[0045] In one embodiment of the present invention, the solid tumor is selected from lung cancer, sarcoma, prostate cancer, solid tumors of the digestive tract and melanoma;

[0046] Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

[0047] In one embodiment of the present invention, the aforementioned drug for treating colorectal cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 15-500 mg;

[0048] Preferably, the content of the interleukin complex is 75-250 mg.

[0049] In one embodiment of the present invention, the aforementioned drug for treating liver cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 75-250 mg;

[0050] Preferably, the content of the interleukin complex is 150-250 mg.

[0051] In one embodiment of the present invention, the aforementioned drug for treating pancreatic cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 75-250 mg;

[0052] Preferably, the content of the interleukin complex is 150-250 mg.

[0053] In one embodiment of the present invention, the aforementioned drug for treating sarcoma is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 1.5-500 mg;

[0054] Preferably, the content of the interleukin complex is 50-105 mg.

[0055] In one embodiment of the present invention, the aforementioned drug for treating prostate cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 1.5-500 mg;

[0056] Preferably, the content of the interleukin complex is 50-105 mg.

[0057] In one embodiment of the present invention, the aforementioned drug for treating lung cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 1.5-500 mg;

[0058] Preferably, the content of the interleukin complex is 50-105 mg.

[0059] In one embodiment of the present invention, the aforementioned drug for treating melanoma is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 15-95 mg or 105-500 mg;

[0060] Preferably, the content of the interleukin complex is 15-95 mg.

[0061] In one embodiment of the present invention, the aforementioned drug for treating breast cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 1.5-105 mg;

[0062] Preferably, the content of the interleukin complex is 50-105 mg.

[0063] On the other hand, the present disclosure provides a method for treating lung cancer, liver cancer, sarcoma, colorectal cancer, pancreatic cancer, prostate cancer, and melanoma in a subject in need thereof using an interleukin complex, comprising determining the dosage to be administered to the subject using the difference in equilibrium dissociation constant (KD) of the interleukin complex for the CD122 receptor of subjects belonging to different species, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1 and the sushi domain of the IL15Rα receptor as shown in SEQ ID NO.2.

[0064] In some embodiments, the present disclosure provides KD values ​​for the binding of the aforementioned interleukin complex to CD122 of subjects belonging to different species, and the dose conversion ratio of the aforementioned interleukin complex in different species is estimated by the ratio between the KD values ​​between different species. In some embodiments, the dosage of the aforementioned interleukin complex provided by the present disclosure for humans is 0.035 times the dosage for mice.

[0065] In another aspect, the present disclosure provides a method for treating a solid tumor in a human in need thereof, comprising administering to the human an interleukin complex, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1 and the sushi domain of the IL15Rα receptor as shown in SEQ ID NO.2.

[0066] In one embodiment of the present invention, the solid tumor is selected from lung cancer, sarcoma, prostate cancer, solid tumors of the digestive tract and melanoma;

[0067] Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

[0068] In one embodiment of the present invention, the aforementioned method for treating colorectal cancer comprises administering the aforementioned interleukin complex to a human, with each administration dose being 0.01-0.35 mg / kg;

[0069] Preferably, the dosage for each administration to humans is 0.0525-0.175 mg / kg;

[0070] Preferably, the drug is administered to humans once or twice a week;

[0071] Preferably, the drug is administered to a human three, five or six times in total.

[0072] In one embodiment of the present invention, the aforementioned method for treating liver cancer comprises administering the aforementioned interleukin complex to a human, with each administration dose being 0.0525-0.175 mg / kg;

[0073] Preferably, the dosage for each administration to humans is 0.105-0.175 mg / kg;

[0074] Preferably, the drug is administered to humans once a week for a total of three times.

[0075] In one embodiment of the present invention, the aforementioned method for treating pancreatic cancer comprises administering the aforementioned interleukin complex to a human, with each administration dose being 0.0525-0.175 mg / kg;

[0076] Preferably, the dosage for each administration to humans is 0.105-0.175 mg / kg;

[0077] Preferably, the drug is administered to humans once a week for a total of three times.

[0078] In one embodiment of the present invention, the aforementioned method for treating sarcoma comprises administering the aforementioned interleukin complex to a human, with each administration dose being 0.001-0.35 mg / kg;

[0079] Preferably, the dosage for each administration to humans is 0.01-0.0735 mg / kg;

[0080] Preferably, the drug is administered to humans once or twice a week;

[0081] Preferably, the drug is administered to a human three, four or six times in total.

[0082] In one embodiment of the present invention, the aforementioned method for treating prostate cancer comprises administering the aforementioned interleukin complex to a human, with each administration dose being 0.001-0.35 mg / kg;

[0083] Preferably, the dosage for each administration to humans is 0.01-0.0735 mg / kg;

[0084] Preferably, the drug is administered to humans once or twice a week;

[0085] Preferably, the drug is administered to a human three, four or six times in total.

[0086] In one embodiment of the present invention, the aforementioned method for treating lung cancer comprises administering the aforementioned interleukin complex to a human, with each administration dose being 0.001-0.35 mg / kg;

[0087] Preferably, the dosage for each administration to humans is 0.01-0.0735 mg / kg;

[0088] Preferably, the drug is administered to humans once or twice a week;

[0089] Preferably, the drug is administered to a human three, four or six times in total.

[0090] In one embodiment of the present invention, the aforementioned method for treating melanoma comprises administering the aforementioned interleukin complex to a human, with each administration dose being 0.01-0.0665 mg / kg or 0.0735-0.35 mg / kg;

[0091] Preferably, the dosage for each administration to humans is 0.01-0.0665 mg / kg;

[0092] Preferably, the drug is administered to humans once or twice a week for a total of five times.

[0093] In one embodiment of the present invention, the aforementioned method for treating breast cancer comprises administering the aforementioned interleukin complex to a human, with each administration dose being 0.001-0.0735 mg / kg;

[0094] Preferably, the dosage for each administration to humans is 0.035-0.0735 mg / kg;

[0095] Preferably, the drug is administered to humans once a week for a total of three times.

[0096] On the other hand, the present invention provides a use of an interleukin complex in the preparation of a medicament for treating solid tumors in a human in need thereof, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1 and the sushi domain of the IL15Rα receptor as shown in SEQ ID NO.2.

[0097] In one embodiment of the present invention, the solid tumor is selected from lung cancer, sarcoma, prostate cancer, solid tumors of the digestive tract and melanoma;

[0098] Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

[0099] In one embodiment of the present invention, the aforementioned drug for treating colorectal cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 0.525-17.5 mg;

[0100] Preferably, the content of the interleukin complex is 2.625-8.75 mg.

[0101] In one embodiment of the present invention, the aforementioned drug for treating liver cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 2.625-8.75 mg;

[0102] Preferably, the content of the interleukin complex is 5.25-8.75 mg.

[0103] In one embodiment of the present invention, the aforementioned drug for treating pancreatic cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 2.625-8.75 mg;

[0104] Preferably, the content of the interleukin complex is 5.25-8.75 mg.

[0105] In one embodiment of the present invention, the aforementioned drug for treating sarcoma is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 0.0525-17.5 mg;

[0106] Preferably, the content of the interleukin complex is 1.75-3.675 mg.

[0107] In one embodiment of the present invention, the aforementioned drug for treating prostate cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 0.0525-17.5 mg;

[0108] Preferably, the content of the interleukin complex is 1.75-3.675 mg.

[0109] In one embodiment of the present invention, the aforementioned drug for treating lung cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 0.0525-17.5 mg;

[0110] Preferably, the content of the interleukin complex is 1.75-3.675 mg.

[0111] In one embodiment of the present invention, the aforementioned drug for treating melanoma is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 0.525-3.325 mg or 3.675-17.5 mg;

[0112] Preferably, the content of the interleukin complex is 0.525-3.325 mg.

[0113] In one embodiment of the present invention, the aforementioned drug for treating breast cancer is a unit dose preparation, wherein the content of the aforementioned interleukin complex is 0.0525-3.675 mg;

[0114] Preferably, the content of the interleukin complex is 1.75-3.675 mg.

[0115] In another aspect, the present invention provides a method for administering the aforementioned drug, which includes intravenous, intradermal, subcutaneous, oral inhalation, transdermal, transmucosal administration, and enteral administration such as rectal administration. Beneficial effects

[0116] The interleukin complex provided by the present invention can more strongly activate tumor-killing immune cells such as CD8+ T cells and NK cells than wild-type IL-2, while at the same time less effectively activating the growth of Treg cells that suppress immune responses. It is more convenient to administer and can achieve a better effect of inhibiting the growth and reproduction of tumor cells within a toxic dose acceptable to the organism, and can inhibit the growth of tumor cells of more types of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 shows the anti-tumor effect of the modified interleukin complex injected intraperitoneally at doses of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) in a CT26 colon cancer subcutaneous transplantation model in mice. The figure shows the tumor size at different days after administration.

[0118] FIG2 shows the anti-tumor effect of the modified interleukin complex injected intraperitoneally at doses of 1.5, 3.0, and 5.0 mg (drug weight) / kg (body weight) in a CT26 colon cancer subcutaneous transplantation model in mice. The figure shows the tumor size at different days after administration.

[0119] Figure 3: Antitumor effects of the engineered interleukin complex injected intraperitoneally at doses of 0.03, 0.3, 1, 1.9, 2.1, and 10 mg (drug weight) / kg (body weight) in a subcutaneous transplant model of MC38 colon cancer in mice. The figure shows the tumor size at different days after administration.

[0120] Figure 4 shows the anti-tumor effect of the modified interleukin complex injected intraperitoneally at doses of 1.5, 3.0, and 5.0 mg (drug weight) / kg (body weight) in a Hepa 1-6 liver cancer subcutaneous transplantation model in mice. The figure shows the tumor size at different days after administration.

[0121] FIG5 shows the anti-tumor effect of the modified interleukin complex injected intraperitoneally at doses of 1.5, 3.0, and 5.0 mg (drug weight) / kg (body weight) in a Pan02 pancreatic cancer subcutaneous transplantation model in mice. The figure shows the tumor size at different days after administration.

[0122] Figure 6 shows the anti-tumor effect of the modified interleukin complex injected intraperitoneally at doses of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) in a subcutaneous transplantation model of S180 sarcoma in mice. The figure shows the tumor size at different days after administration.

[0123] Figure 7 shows the anti-tumor effect of the engineered interleukin complex injected intraperitoneally at doses of 0.03, 0.3, 1, 1.9, 2.1, and 10 mg (drug weight) / kg (body weight) in a B16F10 melanoma subcutaneous transplantation model in mice. The figure shows the tumor size at different days after administration.

[0124] Figure 8 shows the anti-tumor effect of the modified interleukin complex injected intraperitoneally at doses of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) in the RM-1 prostate cancer subcutaneous transplantation model in mice. The figure shows the tumor size at different days after administration.

[0125] Figure 9 shows the anti-tumor effect of the modified interleukin complex injected intraperitoneally at doses of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) in the NCI-H460 lung cancer subcutaneous transplantation model in mice. The figure shows the tumor size at different days after administration.

[0126] Figure 10 shows the anti-tumor effect of the modified interleukin complex injected intraperitoneally at doses of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) in the EMT6 breast cancer mouse subcutaneous transplantation model. The figure shows the tumor size at different days after administration. DETAILED DESCRIPTION

[0127] definition

[0128] It will be understood that the interleukin complexes described above will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. A large number of suitable formulations can be found in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), in particular Chapter 87 of Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TMSM102, DOPE, cholesterol, and PEG 1000-DMG), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be suitable for use in the treatment or therapy according to the present application, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated for the route of administration.

[0129] As used herein, a "subject" is an animal, such as a mammal, including primates (e.g., humans, non-human primates such as monkeys and chimpanzees), non-primates (e.g., cows, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice). In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

[0130] As used herein, a "unit dose formulation" can be a packaged formulation, such as a tablet, capsule, or powder packaged in a bottle, bag, box, or ampoule. A unit dose formulation can be a capsule, tablet, or any dosage form in an appropriate number in packaged form. A unit dose refers to a unit suitable for a single dose for a human or other mammal.

[0131] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with the administration of the drug. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference work in this field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as immobilized oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0132] Preparations to be used for clinical in vivo administration must be sterile. This can be easily achieved by filtration through sterile filtration membranes.

[0133] The aforementioned drugs are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent for injection, such as water, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol, methyl paraben, phenol, or m-cresol; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate, and an agent for regulating osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The parenteral formulation can be packaged in ampoules, vials, disposable syringes, glass or plastic multiple-dose vials, or injection pens. There are two main types of injection pens. One is a disposable pre-filled pen that contains medication and does not require a replacement cartridge. It can be thrown away after use. The other is a more commonly used durable injection pen, which consists of an injection pen and a drug cartridge. After use, the cartridge can be replaced and the pen can be used again.

[0134] Suitable medicines for injection purposes include sterile aqueous solutions (water-soluble in this case) or dispersions and sterile powders for the immediate preparation of sterile injections or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluidity should reach a degree that is easy to inject. It must be stable under manufacturing and storage conditions and must be able to prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and a suitable mixture thereof. For example, by utilizing a coating such as lecithin, maintaining the desired particle size in the case of dispersions, and utilizing a surfactant, suitable fluidity can be maintained. The prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, metacresol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0135] As required, sterile injection solution can be prepared by the medicine of the present invention being mixed with a kind of or combination (as required) suitable solvent with above-listed composition with the required amount, then filter sterilization.Generally speaking, dispersion is prepared by the medicine of the present invention being mixed with the sterile carrier containing dispersion medium and those required other components listed above.With regard to the sterile powder for the preparation of sterile injection solution, preparation method is to obtain vacuum drying and freeze drying of powder, and this powder comprises active ingredient and any other desired component, and they are from the aseptic filtered solution of aforementioned these components.

[0136] For administration by inhalation, the drug is delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

[0137] The drug can also be administered systemically through mucosal or transdermal administration. For transmucosal or transdermal administration, a penetrant suitable for the permeation barrier is used in the formulation. Such penetrants are generally known in the art and include detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the drug of the present invention can be formulated into an ointment, an unguent, a gel, or a cream as generally known in the art.

[0138] The drug may also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.

[0139] In one embodiment, the medicament of the present invention can be prepared with a carrier that prevents it from being rapidly eliminated by the body, such as a sustained / controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.

[0140] For example, the active ingredients can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions.

[0141] Sustained-release formulations can be prepared. Examples of suitable sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the drug of the present invention, in the form of shaped articles such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methylpropionate) or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for shorter periods of time. Polylactic acid (PLA) and polylactic-glycolic acid copolymers (PLGA) have been the focus of recent research. In addition, there are albumin microspheres, chitosan microspheres, gelatin microspheres, etc.

[0142] Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0143] It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically separable units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of the agent of the invention calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the described embodiments are dictated by and directly dependent on the unique characteristics of the agent of the invention and the specific therapeutic effect to be achieved, and the limitations inherent in the art of formulating such agents of the invention for treating individuals.

[0144] The medication can be included in a container, pack, or dispenser together with instructions for administration.

[0145] As used herein, an "effective amount" refers to an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic amount is an amount that achieves the desired therapeutic effect. This amount may be the same as or different from a prophylactic effective amount, which is the amount necessary to prevent the onset of a disease or disease symptoms. An effective amount can be administered in one or more administrations, applications, or dosages. A "therapeutically effective amount" (i.e., an effective dose) of a therapeutic drug depends on the therapeutic drug selected. The administration of the composition may range from one or more times per day to one or more times per week; including every other day. Those skilled in the art will appreciate that certain factors may affect the dosage and time required to effectively treat a subject, including but not limited to the severity of the subject's disease or condition, previous treatment, overall health and / or age, and other diseases present. In addition, treating a subject with a therapeutically effective amount of a therapeutic drug described herein may include a single treatment or a series of treatments.

[0146] As used herein, the term "treat" generally refers to administering or administering a therapeutic agent to a patient, or administering or administering a therapeutic agent to a tissue or cell line isolated from a patient, who has a disease, disease symptoms, or disease predisposition, with the purpose of treating, curing, alleviating, relieving, altering, remedying, improving, enhancing, or affecting the disease, disease symptoms, or disease predisposition. This can include ameliorating the disease state, eliminating the lesion, or improving the prognosis.

[0147] As used herein, the terms "tumor" and "cancer" generally refer to cells that exhibit at least partial loss of proliferation control during normal growth and / or development. For example, common tumors or cancer cells have generally lost contact inhibition and may be invasive and / or have the ability to metastasize. In some embodiments, the interleukin complexes disclosed herein can inhibit the growth of solid tumors, especially solid tumors of the digestive tract. In some embodiments, the interleukin complexes disclosed herein can inhibit the growth of lung cancer, liver cancer, sarcoma, colorectal cancer, pancreatic cancer, prostate cancer, and melanoma. In some embodiments, the interleukin complexes disclosed herein are administered to a subject at a dose of 0.3-10 mg (drug weight) / kg (subject body weight) to inhibit tumor growth.

[0148] As used herein, "solid tumor" refers to all neoplastic cell growth and proliferation, except hematological cancers (e.g., lymphoma, leukemia, and multiple myeloma), and all precancerous and cancerous cells and tissues. Examples of solid tumors include, but are not limited to, soft tissue sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovialoma, mesothelioma, Ewing's tumor, and sarcoma. tumor) and other bone tumors (e.g., osteosarcoma, malignant fibrous histiocytoma), leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor , cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial cancers, brain / CNS tumors (e.g., astrocytoma, glioma, glioblastoma, childhood tumors such as atypical teratoma / rhabdoid tumor, germ cell tumor, embryonal tumor, ependymoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Some of the most common solid tumors for which the compositions and methods of the present disclosure will be useful include: head and neck cancer, rectal adenocarcinoma, glioma, medulloblastoma, urothelial carcinoma, pancreatic cancer, uterine cancer (e.g., endometrial cancer, fallopian tube cancer), ovarian cancer, cervical cancer, prostate cancer, non-small cell lung cancer (squamous and adenocarcinoma), small cell lung cancer, melanoma, breast cancer, bladder cancer, ductal carcinoma in situ, renal cell carcinoma and hepatocellular carcinoma, adrenal tumors (e.g., adrenocortical carcinoma), esophageal cancer, eye cancer (e.g., melanoma, retinoblastoma), gallbladder cancer, gastrointestinal cancer, Wilms' tumor, heart cancer, head and neck cancer, laryngeal and hypopharyngeal cancer, oral cancer (e.g., lip, mouth, salivary gland), nasopharyngeal cancer, neuroblastoma, peritoneal cancer, pituitary cancer, Kaposi's sarcoma, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, parathyroid cancer, vaginal tumors, and metastases of any of the foregoing.

[0149] As used herein, examples of "solid tumors of the digestive tract" include, but are not limited to, liver cancer, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer.

[0150] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0151] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0152] Example 1: Preparation of interleukin complexes engineered from mammalian cells

[0153] 1.1 Synthesis and construction of expression plasmids

[0154] Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the target gene fragments of the modified interleukin (SEQ ID NO.1) and sushi-hIgG4Fc (SEQ ID NO.2) proteins and clone them between the EcoRI and HindIII sites of the pZD vector to obtain two expression plasmids. The synthesized or constructed plasmids were then transformed into DH10B, sequenced and maintained in bacteria.

[0155] 1.2 Plasmid extraction and HEK293 cell preparation

[0156] 1.2.1 Plasmid extraction

[0157] The above plasmid preparation was performed according to the operating procedures mentioned in the "Qiagen Mini-prep Kit" and "Qiagen Endofree Maxi-prep Kit".

[0158] 1.2.2 HEK293 cell preparation

[0159] Use a density of 1-1.2 × 10 6 HEK293 cells (National Research Council, Canada) at 400 μg / mL were used for transient expression.

[0160] 1.3 HEK293 transient expression

[0161] 1.3.1 Reagent Preparation

[0162] A) G418 solution: Weigh 250 mg of Geneticin™ and dissolve in 4.5 mL of ultrapure water. Dose to 5 mL with ultrapure water, filter through a 0.22 μm filter, and store at -20°C.

[0163] B) PEI solution: Dissolve 50 mg of PEI in 45 mL of ultrapure water. Adjust the pH to 7.0 with 1 M NaOH, dilute to 50 mL with ultrapure water, filter through a 0.22 μm filter, and store at -20°C.

[0164] C) Culture medium: Add 10 mL of 10% Pluronicd™ F-68 and 500 μL of G418 to 1 liter of FreeStyle™ 293 Expression Medium;

[0165] D) The plasmid was prepared in advance in a 2 mL endotoxin-free centrifuge tube;

[0166] E) Prepare fresh passages to 1-1.2×10 cells / mL according to the volume required for transfection. 6 cells / mL of cell suspension.

[0167] 1.3.2 Preparation of transfection reagent-plasmid complex

[0168] Solution A: Plasmid 1μg / mL + Opti-MEM™ 33.3μL / mL

[0169] Solution B: PEI 2μg / mL+Opti-MEMTM 33.3μL / mL

[0170] Pour 1 mL of solution B into 1 mL of solution A, mix well, incubate for 10 minutes, and then add the cell suspension.

[0171] The modified interleukin and sushi-hIgG4Fc were mixed at a mass ratio of 1:1 (total amount of 1 μg / mL) and then transfected.

[0172] 1.3.3 Fluid exchange

[0173] After culturing for 4 small trials at 115 rpm, 36.8°C, and 5% CO2, the culture medium was centrifuged at 800 g for 5 minutes and replaced with FreeStyle™ 293 Expression medium without Pluronicd™ F-68 and G418.

[0174] 1.3.4 Expression culture and harvest

[0175] After culturing at 115 rpm, 36.8°C, 5% CO2 for 5 days, the cells were centrifuged at 8500 rpm for 15 minutes, and the cell supernatant was collected for purification.

[0176] 1.4 Purification and preparation

[0177] The engineered interleukin was co-expressed with sushi-hIgG4Fc, forming a heterodimeric complex, designated the engineered interleukin complex, which carries an Fc tag. Affinity purification was performed using Mabselect Sure (Protein A, GE Healthcare) according to the purification medium instructions. Further purification was performed using gel filtration chromatography (Superdex 200 pg, GE Healthcare) to obtain a highly pure protein and remove aggregates. Purity analysis was performed using a high-performance liquid chromatography-molecular sieve column (SEC-HPLC). A single peak on the purified protein by SEC indicated successful complex formation.

[0178] Example 2: Modified interleukin complex activates immune cell signaling pathway

[0179] Based on modern immunology research, tumor-killing T cells, such as CD8+ T cells, play a role in suppressing tumor growth and are beneficial to tumor growth. Treg cells negatively regulate the immune system, inhibiting immune cell growth and promoting tumor growth. To achieve the goal of suppressing tumors through interleukin-activated immune system activation in animals, a cytokine is required that maximizes the activation of cytotoxic T cells, such as CD8+ T cells, while minimizing the activation of Treg cells.

[0180] STAT5 is a member of the STAT family of transcription factors. In response to cytokines and growth factors, STAT family members are phosphorylated by receptor-associated kinases, then form homodimers or heterodimers that translocate to the nucleus, where they act as transcriptional activators. STAT5 is activated and mediates responses to numerous cellular ligands, such as IL-2, IL-3, IL-7, GM-CSF, erythropoietin, thrombopoietin, and various growth hormones. Assaying the phosphorylation level of STAT5 at tryptophan 694 is a commonly used quantitative assay for cytokine receptor activation.

[0181] CD8+ T cells and Treg cells were resuspended in 20 mL of complete culture medium (RPMI-1640 (Gibco, Catalog No.: 72400-047) and 10% FBS (Biowest, Catalog No.: S1580-500), transferred to a T25 culture flask (NEST, Catalog No.: 707003), and cultured in a 37°C, 5% CO2 incubator overnight.

[0182] On the second day, transfer the overnight cultured CD8+ T cells and Treg cells into 50 mL centrifuge tubes (NEST, Cat. No. 602052), centrifuge at 300 g for 5 minutes, and discard the supernatant. Resuspend the cells in complete medium and adjust the number of CD8+ T cells and Treg cells to 2.4 × 10 6 Cells / mL, 50 μL / well were added to a 96-well plate (NEST, Catalog No. 701101). Wild-type IL-2 was diluted to a starting concentration of 10 nM. Wild-type IL-2, IL-15, and the modified interleukin complex were diluted to a starting concentration of 100 nM. Five-fold serial dilutions were then performed to a total of 10 concentrations. 50 μL / well was added to the plated CD8+ T cells and Treg cells, respectively. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 15 minutes. After incubation, 100 μL of Fix Buffer I (BD, Catalog No. 557870) was immediately added to each well and the cells were fixed in a 37°C, 5% CO2 incubator for 10 minutes. 100 μL of Stain Buffer (BD, Catalog No. 554656) was added to each well and the cells were centrifuged at 300 g for 5 minutes. The supernatant was discarded.

[0183] Antibodies targeting the surface of CD8+ T cells (tube 1) and Treg cells (tube 2) were prepared as follows: Tube 1: APC-H7 mouse anti-human CD3 (BD, cat. no.: 560176) and BV605 mouse anti-human CD8 (BD, cat. no.: 564116); Tube 2: APC-H7 mouse anti-human CD3 (BD, cat. no.: 560176), BV650 mouse anti-human CD4 (BD, cat. no.: 563875), PE mouse anti-human CD25 (BD, cat. no.: 555432); After mixing, 100 μL / well was added to the corresponding cells, incubated in the dark at 4°C for 30 minutes, 100 μL stain buffer was added to each well, and the cells were centrifuged at 300 g for 5 minutes, and the supernatant was discarded. Add 100 μL of Perm Buffer III (BD, Cat. No. 558050) to each well and incubate on ice for 30 minutes. After incubation, add 100 μL of stain buffer to each well, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0184] Antibodies targeting CD8+ T cells (tube 3) and Treg cells (tube 4) were prepared as follows: Tube 3: AF647 Mouse Anti-Stat5 (pY694) (BD, Catalog No. 562076); Tube 4: AF647 Mouse Anti-Stat5 (pY694) (BD, Catalog No. 562076) and V450 Mouse Anti-Human Foxp3 (BD, Catalog No. 561182). Mix well and add 100 μL per well to the corresponding cells. Incubate overnight at 4°C in the dark. The next day, add 100 μL of stain buffer to each well, centrifuge at 300 g for 5 minutes, and discard the supernatant. Resuspend the cells in 80 μL of stain buffer per well. After setting compensation, analyze the cells using a flow cytometer. Data were analyzed using FlowJo [v10.8] and graphed using Graphpad Prism [v9.0].

[0185] Table 1 EC50 of interleukin-activated START5 signaling pathway in immune cells

[0186] The lower the EC50, the lower the dose at which the START5 signaling pathway of immune cells can be activated, thereby further stimulating cell proliferation. To maximize the activation of CD8+ T cells and minimize the activation of Treg cells, the lower the CD8+T EC50 / Treg EC50 ratio, the better. At this point, the cytokine can activate the proliferation of CD8+ T cells at a lower concentration, while the activity of stimulating Treg cell proliferation at this concentration is relatively small. The data in Table 1 show that the modified interleukin complex can preferentially stimulate the cell proliferation signaling pathway of CD8+ T cells compared to IL-2 and IL-15. In combination, by selectively stimulating the proliferation of immune cells, it can achieve the effect of inhibiting tumor cell growth.

[0187] Example 3: Modified interleukin complex activates immune cell proliferation

[0188] As described in Example 2, to achieve the goal of suppressing tumors by activating the animal's immune system through interleukin, it is necessary to use a cytokine that can maximize the activation of cytotoxic T cells, such as CD8+ T cells, and minimize the activation of Treg cells. In addition to quantitatively detecting immune cell signaling pathways, the activation and proliferation of different immune cells by interleukins can also be determined by quantitatively detecting the proliferation of activated immune cells.

[0189] To recover CD8+ T cells and Treg cells, add 10 mL of complete culture medium (RPMI-1640 (Gibco, Catalog No.: 72400-047) and 10% FBS (Biowest, Catalog No.: S1580-500) to resuspend the cells and count them to adjust the cell number to 4 × 10 5 Cells / mL, 50 μL / well were added to 96-well white-bottom plates (NEST, Catalog No. 701301), with two replicates for each sample. Wild-type IL-2 and IL-15 and the modified interleukin complex were diluted to a starting concentration of 1000 nM. Five-fold serial dilutions were then performed to a total of 10 concentrations, and 50 μL / well was added to the plated CD8+ T cells and Treg cells, respectively. After mixing, the 96-well plates were incubated in a 37°C, 5% CO2 incubator for 4 days. After incubation, 50 μL of CELLTiter-Glo-2.0 Assay (Promega, Catalog No. G9243) was added to each well, mixed, and shaken at 550 rpm on a horizontal shaker for 5 minutes. After standing at room temperature for 10 minutes, the chemiluminescence (RLU) was measured using a microplate reader. The detection principle is described in the CELLTiter-Glo-2.0 Assay user manual. Chemiluminescence can be used to quantify cell viability.

[0190] Table 2 EC50 of interleukins activating immune cell proliferation

[0191] The lower the EC50, the lower the dose at which immune cell proliferation can be activated. To maximize CD8+ T cell activation and minimize Treg cell activation, the CD8+T EC50 / Treg EC50 ratio should be as low as possible. At this point, the cytokine can activate CD8+ T cell proliferation at a lower concentration, while the activity in stimulating Treg cell proliferation at this concentration is relatively low. The data in Table 2 show that the modified interleukin complex can preferentially activate CD8+ T cells compared to IL-2 and IL-15. This, combined with the selective stimulation of immune cell proliferation, achieves the effect of inhibiting tumor cell growth.

[0192] Example 4: Antitumor effect in a subcutaneous colorectal cancer transplantation model

[0193] Subcutaneous tumor transplantation animal models play a crucial role in basic research on tumor development, progression, and metastasis, as well as in drug development and treatment. Cancer research relies on the establishment of animal models and the evaluation of drug efficacy. Tumor cell lines are transplanted subcutaneously into the same or xenogeneic animals to form tumor masses. These are typically used to screen and test drugs that inhibit tumor cell proliferation and have gained widespread recognition and use in the pharmaceutical industry.

[0194] In this example, colon cancer CT26 cells in the exponential growth phase were resuspended in PBS solution to 4.0×10 6 BALB / c mice were subcutaneously inoculated with 4.0×10 5 CT26 cells were inoculated. On the 4th day after inoculation, 56 mice were selected and divided into 7 groups, with 8 mice in each group. They were the modified interleukin complex monotherapy group at a dose of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) and the solvent control group, and the intraperitoneal injection was administered at 4 μL / g (body weight). The first dose was given on the day of grouping, which was counted as day 0 (D0) after administration. The drugs were administered on D0, D3, D7, D10 and D14. The subcutaneous transplanted tumor model of colorectal cancer was studied, and the tumor volume was observed, measured and recorded. The efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0195] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2 Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0196] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment and control groups at a specific time point. T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0197] On day 17 after the start of drug administration, the average tumor volume of mice in the solvent control group (Group 1) was 3257 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table.

[0198] Table 3 Average tumor volume and TGI% of modified interleukin complexes at different doses in the CT26 colon cancer subcutaneous transplantation model on day 17 after administration

[0199] As shown in Figure 1 and Table 3, differences in mean tumor volume between each dose group and the vehicle control group were evident around days 6-10 after dosing. With the exception of the lowest dose, 0.03 mg (drug weight) / kg (body weight), which exhibited faster tumor growth compared to the control group, all other dose groups demonstrated tumor inhibition. By day 17, the tumor inhibition effect was even more pronounced in all dose groups, except the 0.03 mg (drug weight) / kg (body weight) group. The 0.3 mg (drug weight) / kg (body weight) group demonstrated a TGI% of 14%, demonstrating a tumor inhibitory effect. The 10 mg (drug weight) / kg (body weight) group achieved a TGI% of 76%, demonstrating a highly significant tumor inhibitory effect. It is clearly evident that the tumor inhibition effect gradually increased with increasing dose, forming a clear dose-dependent inhibitory trend. It is reasonable to speculate that this interleukin complex can also inhibit tumor growth at doses greater than 10 mg (drug weight) / kg (body weight) that are well tolerated by mice.

[0200] Example 5: Antitumor effect in a subcutaneous colorectal cancer transplantation model

[0201] In this example, colon cancer CT26 cells in the exponential growth phase were resuspended in PBS solution to 5.0×10 6 BALB / c mice were subcutaneously inoculated with 5.0×10 5 Seven days after inoculation of CT26 cells, 40 mice were divided into four groups of 10 mice each. The modified interleukin complex was administered intraperitoneally at doses of 1.5 mg (drug weight) / kg (body weight), 3 mg (drug weight) / kg (body weight), and 5 mg (drug weight) / kg (body weight) respectively, as well as a solvent control group. The mice were injected intraperitoneally with 4 μL / g (body weight) of body weight. Day 0 (D0) was defined as post-dose administration. Dosing was performed once weekly for a total of three weeks (three times) to study the CT26 colorectal cancer model in mice. Efficacy was evaluated based on relative tumor inhibition (TGI).

[0202] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2 Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0203] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment and control groups at a specific time point. T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0204] 24 days after the start of drug administration, the average tumor volume of the solvent control group (Group 1) was 2499 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table. Table 4 Average tumor volume and TGI% of the modified interleukin complex at different doses on day 24 in the CT26 colon cancer subcutaneous transplant model in mice

[0205] As can be seen from the information in Figure 2 and Table 4, around 5-7 days after administration, the difference in average tumor volume between each dose group and the solvent control group was very obvious. By the 24th day, the tumor inhibition effect of each dose group was more obvious. A TGI% of 36% was observed in the 1.5 mg (drug weight) / kg (body weight) group, showing a tumor inhibition effect. The tumor inhibition effects of the 3.0 and 5.0 mg (drug weight) / kg (body weight) groups were similar, with TGI% exceeding 50%, showing a very outstanding tumor inhibition effect. Statistical analysis showed that the relative tumor volumes of the three dose groups showed significant differences compared with the solvent control group (p<0.01). During the experiment, the mice in the group receiving the modified interleukin complex at the set dose tolerated it well throughout the experiment, with no mouse deaths or significant weight loss.

[0206] Example 6: Antitumor Effect in a Subcutaneous Colorectal Cancer Transplantation Model

[0207] In this example, MC38 colon cancer cells in the exponential growth phase were resuspended in PBS solution to 8.0×10 6 8.0×10 cells / mL were inoculated subcutaneously on the right front back of C57BL / 6J mice. 5MC38 cells were used to establish a subcutaneous transplant tumor model of colorectal cancer. On the 4th day after inoculation, 56 mice were selected and divided into 7 groups, with 8 mice in each group. They were the modified interleukin complex single-drug administration group at a dose of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) and the solvent control group, and the intraperitoneal injection was administered at 4 μL / g (body weight). The first administration on the day of grouping was counted as day 0 (D0) after administration, and the drugs were administered on D0, D3, D7, D10, D14, and D17. The small sarcoma subcutaneous transplant tumor model was studied, the tumor volume was observed, measured, and recorded, and the efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0208] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2 Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0209] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment and control groups at a specific time point. T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0210] On day 18 after the start of drug administration, the average tumor volume of mice in the solvent control group (Group 1) was 2480 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table.

[0211] Table 5 Average tumor volume and TGI% of different doses of modified interleukin complexes in the MC38 colon cancer subcutaneous transplantation model on day 18 after administration

[0212] As can be seen from Figure 3 and Table 5, differences in average tumor volume between each dose group and the solvent control group were already evident around days 8-11 after administration. With the exception of the lowest dose, the 0.03 mg (drug weight) / kg (body weight) group, where tumor growth was faster than in the control group, all other dose groups demonstrated a tumor inhibitory effect. By day 18, with the exception of the 0.03 mg (drug weight) / kg (body weight) group, the tumor inhibitory effect was even more pronounced in all dose groups. The 0.3 mg (drug weight) / kg (body weight) group demonstrated a TGI% of 15%, demonstrating a tumor inhibitory effect. In the 10 mg (drug weight) / kg (body weight) group, a trend of tumor shrinkage was observed, with a TGI% reaching 96%, demonstrating a very significant tumor inhibitory effect. It can be clearly seen that the effect of inhibiting tumor growth gradually increased with increasing dose, forming a clear dose-dependent trend of inhibiting tumor growth. It is reasonable to speculate that the interleukin complex can also exert the effect of inhibiting tumor growth at a higher dose of greater than 10 mg (drug weight) / kg (body weight) that mice can tolerate.

[0213] Example 7: Antitumor Effect in a Subcutaneous Hepatocellular Carcinoma Model

[0214] In this example, Hepa 1-6 liver cancer cells in the exponential growth phase were resuspended in PBS solution to a concentration of 3.0×10 6 3×10 cells / mL were subcutaneously inoculated on the right back of C57BL / 6 mice. 5 Hepa 1-6 cells were added, and tumor growth was observed regularly. When the tumor grew to an average volume of 60-80 mm 3 Mice were randomly divided into 40 groups (10 mice / group) based on tumor size and body weight. The modified interleukin complex was administered at doses of 1.5 mg / kg (body weight), 3 mg / kg (body weight), and 5 mg / kg (body weight) alone, or as a solvent control. Animals were dosed once weekly for three consecutive weeks, for a total of three doses. The Hepa 1-6 mouse liver cancer model was studied by intraperitoneal injection of 4 μL / g (body weight), starting on day 0 (D0). Dosing was continued weekly for three weeks (three doses). Efficacy was evaluated based on relative tumor inhibition (TGI).

[0215] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0216] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment group and the control group at a specific time point. T and C are the relative tumor volume (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0217] 58 days after the start of drug administration, the average tumor volume of the solvent control group (Group 1) was 3515 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table. Table 6 Average tumor volume and TGI% of the modified interleukin complex at various doses in the Hepa 1-6 liver cancer subcutaneous transplant model on day 58 after administration

[0218] As shown in Figure 4 and Table 6, within 7 days after dosing, significant differences in mean tumor volume were observed between each dose group and the solvent control group. By day 58, tumor inhibition was evident in all dose groups. The 1.5 mg (drug weight) / kg (body weight) group achieved a TGI% of 90%, demonstrating excellent tumor inhibition. The 3.0 and 5.0 mg (drug weight) / kg (body weight) groups showed similar tumor inhibition, with the TGI% reaching 100%, achieving complete remission and demonstrating a highly significant tumor inhibition effect. Statistical analysis revealed that the relative tumor volumes of all three dose groups were significantly different from those of the solvent control group (p < 0.05). It is clear that the tumor growth inhibition effect gradually increased with increasing dose, forming a clear dose-dependent trend, with complete remission achieved at doses greater than 3.0 mg (drug weight) / kg (body weight). It is reasonable to speculate that the interleukin complex can also inhibit tumor growth at higher doses, greater than 5.0 mg (drug weight) / kg (body weight), which are well tolerated by mice. During the experiments described in this example, the experimental mice tolerated the treatment well, with no mortality. During the experiments, mice receiving the modified interleukin complex at the prescribed doses tolerated the treatment well throughout the experiment, with no mortality or significant weight loss.

[0219] Example 8: Antitumor Effect in a Subcutaneous Pancreatic Cancer Transplantation Model

[0220] In this example, pancreatic cancer Pan02 cells in the exponential growth phase were resuspended in PBS solution to 5.0×10 7 5.0×10 cells / mL were inoculated subcutaneously on the right front back of C57BL / 6 mice. 6 Pan02 cells were used to establish a pancreatic cancer subcutaneous transplantation tumor model. On the 8th day after inoculation, 40 mice (average tumor volume 102.55 mm 3 Four groups of 10 mice were divided into two groups: one receiving the engineered interleukin complex alone at doses of 1.5, 3, and 5 mg / kg (body weight) and the other receiving the solvent. The drug was administered intraperitoneally at a dose of 4 μL / g (body weight) once weekly for three doses in a mouse Pan02 pancreatic cancer model. Efficacy was evaluated based on relative tumor inhibition (TGI).

[0221] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2 Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0222] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment and control groups at a specific time point. T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0223] On day 28 after the start of drug administration, the average tumor volume of mice in the solvent control group (Group 1) was 1548 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table.

[0224] Table 7 Average tumor volume and TGI% of different doses of modified interleukin complexes in the Pan02 pancreatic cancer mouse subcutaneous transplantation model on day 28 after administration

[0225] As can be seen from the information in Figure 5 and Table 7, the difference in average tumor volume between each dose group and the solvent control group was evident on days 7-14 after administration. By day 28, the tumor inhibition effect of each dose group was very obvious. A TGI% of 16% was observed in the 1.5 mg (drug weight) / kg (body weight) group, showing a significant tumor inhibition effect. The tumor inhibition effects of the 3.0 and 5.0 mg (drug weight) / kg (body weight) groups were similar, with TGI% reaching 27% and 30%, respectively, showing an outstanding tumor inhibition effect. Statistical analysis showed that the relative tumor volumes of the three dose groups showed significant differences compared with the solvent control group (p<0.05). During the experiment, the mice in the group receiving the modified interleukin complex at the set dose were well tolerated throughout the experiment, with no mouse deaths or significant weight loss.

[0226] Example 9: Antitumor Effect in a Subcutaneous Sarcoma Transplantation Model

[0227] In this example, sarcoma S180 cells in the exponential growth phase were resuspended in PBS solution to 1.6×10 7 1.6×10 cells / mL were inoculated subcutaneously on the right front back of BALB / c mice. 6 S180 cells were used to establish a subcutaneous sarcoma transplant tumor model. On the third day after inoculation, 56 mice were selected and divided into 7 groups, with 8 mice in each group. They were the modified interleukin complex monotherapy group at doses of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) and the solvent control group, and the intraperitoneal injection was administered at 4 μL / g (body weight). The first dose was given on the day of grouping, which was counted as day 0 (D0) after administration, and the drugs were administered on D0, D3, D7, D10, D14, and D17. The small sarcoma subcutaneous transplant tumor model was studied, the tumor volume was observed, measured, and recorded, and the efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0228] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2 Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0229] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment and control groups at a specific time point. T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0230] On day 19 after the start of drug administration, the average tumor volume of the solvent control group mice (Group 1) was 2084 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table. Table 8 Average tumor volume and TGI% of the modified interleukin complex at different doses in the S180 sarcoma subcutaneous transplantation model on day 19 after administration

[0231] As can be seen from Figure 6 and Table 8, differences in mean tumor volume between each dose group and the solvent control group were evident around days 5-9 after dosing. By day 19, tumor inhibition was evident in all dose groups. Even at the lowest dose, the 0.03 mg (drug weight) / kg (body weight) group, a TGI% of 2% was observed, demonstrating a tumor inhibitory effect. In the 10 mg (drug weight) / kg (body weight) group, a trend of tumor shrinkage was observed, with a TGI% reaching 93%, demonstrating a very significant tumor inhibitory effect. It is clearly evident that the tumor growth inhibitory effect gradually increased with increasing dose, forming a clear dose-dependent trend in tumor growth inhibition. It is reasonable to speculate that this interleukin complex can also exert a tumor growth inhibitory effect at higher doses, greater than 10 mg (drug weight) / kg (body weight), which are tolerated by mice.

[0232] Example 10: Anti-tumor effect in a subcutaneous melanoma transplantation model

[0233] In this example, melanoma B16F10 cells in exponential growth phase were resuspended in PBS solution to 2.0×10 6 2.0×10 cells / mL were inoculated subcutaneously on the right front back of C57BL / 6J mice. 5B16F10 cells were used to establish a subcutaneous melanoma transplant tumor model. On the fourth day after inoculation, 56 mice were selected and divided into 7 groups, with 8 mice in each group. They were the modified interleukin complex monotherapy group at doses of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) and the solvent control group, and the intraperitoneal injection was administered at 4 μL / g (body weight). The first dose was given on the day of grouping, which was counted as day 0 (D0) after administration. The drugs were administered on D0, D3, D7, D10 and D14. The small melanoma subcutaneous transplant tumor model was studied, and the tumor volume was observed, measured and recorded. The efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0234] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2 Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0235] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment and control groups at a specific time point. T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0236] On day 14 after the start of drug administration, the average tumor volume of the solvent control group mice (Group 1) was 3761 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table.

[0237] Table 9 Average tumor volume and TGI% of modified interleukin complexes at different doses in the B16F10 melanoma subcutaneous transplantation model on day 14 after administration

[0238] As shown in Figure 7 and Table 9, differences in mean tumor volume between each dose group and the solvent control group were evident around days 7-10 after dosing. With the exception of the lowest dose, the 0.03 mg (drug weight) / kg (body weight) group, where tumor growth was faster than in the control group, all other dose groups demonstrated a tumor-suppressing effect. By day 14, the tumor-suppressing effect was even more pronounced in all dose groups, except the 0.03 mg (drug weight) / kg (body weight) group. The 0.3 mg (drug weight) / kg (body weight) group demonstrated a TGI% of 35%, demonstrating a tumor-suppressing effect. The 10 mg (drug weight) / kg (body weight) group achieved a TGI% of 69%, demonstrating a highly significant tumor-suppressing effect. It is clear that the tumor-suppressing effect gradually increased with increasing dose, forming a clear dose-dependent inhibitory trend. It is reasonable to speculate that this interleukin complex can also inhibit tumor growth at doses greater than 10 mg (drug weight) / kg (body weight) that are well tolerated by mice.

[0239] Example 11: Antitumor Effect in a Subcutaneous Prostate Cancer Model

[0240] In this example, prostate cancer RM-1 cells in the exponential growth phase were resuspended in PBS solution to 8.0×10 6 8.0×10 cells / mL were inoculated subcutaneously on the right front back of C57BL / 6J mice. 5 RM-1 cells were used to establish a subcutaneous transplant tumor model of prostate cancer. On the 5th day after inoculation, 56 mice were selected and divided into 7 groups, with 8 mice in each group. They were the modified interleukin complex single-drug administration group at a dose of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) and the solvent control group, and the drug was administered by intraperitoneal injection at 4 μL / g (body weight). The first dose was given on the day of grouping, which was counted as day 0 (D0) after administration, and the drugs were administered on D0, D3, D7 and D10. The subcutaneous transplant tumor model of mouse prostate cancer was studied, and the tumor volume was observed, measured and recorded, and the efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0241] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2 Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0242] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment and control groups at a specific time point. T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0243] On day 13 after the start of drug administration, the average tumor volume of mice in the solvent control group (Group 1) was 2307 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table.

[0244] Table 10 Average tumor volume and TGI% of different doses of modified interleukin complexes in the RM-1 prostate cancer subcutaneous transplantation model on day 13 after administration

[0245] As can be seen from the information in Figure 8 and Table 10, around 6-10 days after administration, differences in average tumor volume between each dose group and the solvent control group were already evident. On day 13 after administration, a TGI% of 7% was observed in the 0.03 mg (drug weight) / kg (body weight) group, demonstrating a tumor inhibitory effect. In the 10 mg (drug weight) / kg (body weight) group, the TGI% reached 81%, demonstrating a very significant tumor inhibitory effect. It can be clearly seen that as the dose increases, the effect of inhibiting tumor growth also gradually increases, forming a clear dose-dependent trend of inhibiting tumor growth. It is reasonable to infer that at higher doses greater than 10 mg (drug weight) / kg (body weight) that mice can tolerate, the interleukin complex can also exert an inhibitory effect on tumor growth.

[0246] Example 12: Antitumor Effect in a Subcutaneous Lung Cancer Transplantation Model

[0247] In this example, lung cancer NCI-H460 cells in the exponential growth phase were resuspended in PBS solution to 1.0×10 8 cells / mL, and 1.0×10 7NCI-H460 cells were used to establish a subcutaneous transplantation tumor model of lung cancer. On the 4th day after inoculation, 56 mice were selected and divided into 7 groups, with 8 mice in each group. They were the modified interleukin complex monotherapy group at doses of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) and the solvent control group, and the drug was administered by intraperitoneal injection at 4 μL / g (body weight). The first dose was given on the day of grouping, which was counted as day 0 (D0) after administration, and the drugs were given on D0, D7 and D14. The small sarcoma subcutaneous transplantation tumor model was studied, the tumor volume was observed, measured and recorded, and the efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0248] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2 Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0249] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment and control groups at a specific time point. T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0250] On the 15th day after the start of drug administration, the average tumor volume of the solvent control group mice (Group 1) was 1926 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table.

[0251] Table 11 Average tumor volume and TGI% of different doses of modified interleukin complexes in the NCI-H460 lung cancer mouse subcutaneous transplantation model on day 19 after administration

[0252] As can be seen from Figure 9 and Table 11, differences in average tumor volume between each dose group and the solvent control group were already evident around days 4-8 after administration. By day 15, tumor inhibition was evident in all dose groups. Even at the lowest dose, the 0.03 mg (drug weight) / kg (body weight) group, a TGI% of 6% was observed, demonstrating a tumor inhibitory effect. The 10 mg (drug weight) / kg (body weight) group achieved a TGI% of 32%, demonstrating a very significant tumor inhibitory effect. It can be clearly seen that the tumor growth inhibition effect gradually increased with increasing dose, forming a clear dose-dependent trend in tumor growth inhibition. It is reasonable to speculate that this interleukin complex can also exert a tumor growth inhibitory effect at higher doses, greater than 10 mg (drug weight) / kg (body weight), which are tolerated by mice.

[0253] Example 13: Antitumor Effect in a Subcutaneous Breast Cancer Model

[0254] In this example, breast cancer EMT6 cells in the exponential growth phase were resuspended in PBS solution to 4.0×10 6 4.0×10 cells / mL were inoculated subcutaneously on the right front back of BALB / c mice. 5 EMT6 cells were used to establish a subcutaneous transplantation tumor model of breast cancer. On the 4th day after inoculation, 56 mice were selected and divided into 7 groups, with 8 mice in each group. They were the modified interleukin complex single-drug administration group at a dose of 0.03, 0.3, 1, 1.9, 2.1 and 10 mg (drug weight) / kg (body weight) and the solvent control group, and the intraperitoneal injection was administered at 4 μL / g (body weight). The first dose was given on the day of grouping, which was counted as day 0 (D0) after administration, and the drugs were administered on D0, D7, and D14. The mouse breast cancer subcutaneous transplantation tumor model was studied, the tumor volume was observed, measured, and recorded, and the efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0255] After tumor inoculation, the animals were checked daily for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. Tumor volume was measured using a bidirectional measurement method. First, the long and short diameters of the tumor were measured using a vernier caliper, and then the formula T = 0.5 × a × b was used. 2 Tumor volume was calculated. a and b represent the long and short diameters of the tumor, respectively.

[0256] Relative tumor inhibition rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of tumor volume or weight of the treatment and control groups at a specific time point. T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV x 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt - V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0257] On the 20th day after the start of administration, the average tumor volume of the mice in the solvent control group (Group 1) was 2529 mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table.

[0258] Table 12 Average tumor volume and TGI% of modified interleukin complexes at different doses in the EMT6 breast cancer mouse subcutaneous transplantation model on day 20 after administration

[0259] As can be seen from the information in Figure 10 and Table 12, around 9-13 days after administration, differences in average tumor volume between each dosage group and the solvent control group were already evident. Except for the 10 mg (drug weight) / kg (body weight) group, where tumor growth was faster than that of the control group, all other groups showed a tumor inhibitory effect. On day 20 after administration, a TGI% of 5% was observed in the 0.03 mg (drug weight) / kg (body weight) group, indicating a tumor inhibitory effect. All groups with 0.3-2.1 mg (drug weight) / kg (body weight) showed a good tumor inhibitory effect. Only the 10 mg (drug weight) / kg (body weight) group had a TGI% of -8%, indicating no tumor inhibitory effect.

[0260] Example 14: Affinity of the engineered interleukin complex for receptors of different species

[0261] There is a relationship between the dosage used in mice and the dosage used in other species, and the affinity of the same cytokine for homologous receptors of different species can be used to estimate it. Because when the same cytokine achieves the same binding ratio with homologous receptors of different species, the activation of immune cells caused by the cytokine is also similar because the signaling pathways downstream of immune cells are conserved and similar. In order to study the dosage relationship of the modified interleukin complex between different species, this example studied its affinity for IL-2 receptors of different species. By using the ratio of the affinities, it is possible to estimate the dosage used in mice and the corresponding dosage used in other species.

[0262] The receptors for IL-2 include CD25, CD122, and CD132, of which CD132 cannot bind to IL-2 alone. The modified interleukin complex involved in the present invention does not bind to CD25, and the data has been disclosed in CN 114380919A. Therefore, this study focused on the KD value of the modified interleukin complex binding to CD122 of different species. By comparing the KD values ​​between different species, the ratio of the affinity of the modified interleukin complex to the CD122 receptors of different species can be estimated, thereby estimating the dose conversion ratio in different species. For example, the KD value in species A is 10 times that in species B, indicating that the affinity of the interleukin complex to species B is 10 times stronger than that of species A. Accordingly, the dosage of the complex in species B is 10 times lower than that in species A.

[0263] This experiment used biolayer interferometry (BLI) to determine the equilibrium dissociation constant (KD) between IL-2 and its receptor CD122. The instrument used was an Octet RED96e (Sartorius), and the assay was performed according to the instrument manufacturer's instructions. The specific experimental procedures are as follows:

[0264] Half an hour before the start of the experiment, according to the number of samples, take an appropriate number of Protein A sensors (Sartorius, product number 18-5010, for receptor proteins containing Fc) or HIS1K sensors (Sartorius, product number 18-5120, for receptor proteins without Fc) and soak them in SD buffer (phosphate-buffered saline, 0.1% bovine serum albumin, 0.05% Tween-20) to pre-wet.

[0265] The sensor position was selected based on the sample position layout. A total volume of 200 μL of SD buffer, 20 nM modified interleukin dissolved in SD buffer, 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 nM human CD122 (SinoBiological, Cat. No. 10696-H02H), monkey CD122 (SinoBiological, Cat. No. 90328-C02H), rat CD122 (SinoBiological, Cat. No. 80340-R08H), and mouse CD122 (SinoBiological, Cat. No. 50792-M02H) dissolved in SD buffer, and dissociation buffer (10 mM glycine, pH 2.5) were added to a 96-well black polystyrene half-well microplate (Greiner, Cat. No. 675076). The instrument experimental procedure was as follows: Baseline 60 s, Loading 1 nm, Baseline 60 s, Association 200 s, Dissociation 400 s, rotation speed 400 rpm, temperature 30°C. KD values ​​were analyzed using ForteBio analysis software, where KD = Koff / Kon. The results are shown in the following table:

[0266] Table 13 Affinity of the modified interleukin complexes to IL-2 receptor CD122 of different species

[0267] Based on the ratio of the KD value (affinity) information listed in the table, the dose conversion relationship between different species can be estimated.

[0268] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for treating a solid tumor in a subject in need thereof, comprising administering an interleukin complex to the subject, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1, and a sushi domain of an IL15Rα receptor as shown in SEQ ID NO.

2.

2. The method of claim 1, wherein the subject is a mammal, including a human, non-human primate, cow, pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat or mouse; Preferably, the non-human primate is a monkey or a chimpanzee.

3. The use according to claim 1 or 2, wherein the solid tumor is selected from lung cancer, sarcoma, prostate cancer, digestive tract solid tumor and melanoma; Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

4. The method according to claim 3, wherein the method for treating colorectal cancer comprises administering the interleukin complex to the subject, with each administration being at a dose of 0.3-10 mg / kg; Preferably, the dose administered to the subject each time is 1.5-5 mg / kg; Preferably, the subject is administered once or twice a week; Preferably, the drug is administered to the subject three, five or six times in total.

5. The method according to claim 3, wherein the method for treating liver cancer or pancreatic cancer comprises administering the interleukin complex to the subject at a dose of 1.5-5 mg / kg per administration; Preferably, the dose administered to the subject each time is 3-5 mg / kg / w; Preferably, the drug is administered to the subject once a week, for a total of three times.

6. The method according to claim 3, wherein the method for treating sarcoma, prostate cancer or lung cancer comprises administering the interleukin complex to the subject at a dose of 0.03-10 mg / kg / w per administration; Preferably, the dosage administered to the subject each time is 0.3-2.1 mg / kg / w; Preferably, the subject is administered once or twice a week; Preferably, the drug is administered to the subject three, four or six times in total.

7. The method according to claim 3, wherein the method for treating melanoma comprises administering the interleukin complex to the subject, with each administration dose being 0.3-1.9 mg / kg or 2.1-10 mg / kg; Preferably, the dose administered to the subject each time is 0.3-1.9 mg / kg; Preferably, the drug is administered to the subject once or twice a week, for a total of five times.

8. The method according to claim 3, wherein the method for treating breast cancer comprises administering the interleukin complex to the subject, with each administration dose being 0.03-2.1 mg / kg; Preferably, the dose administered to the subject each time is 1-2.1 mg / kg; Preferably, the drug is administered to the subject once a week, for a total of three times.

9. Use of an interleukin complex in the preparation of a medicament for treating a solid tumor in a subject in need thereof, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1, and a sushi domain of an IL15Rα receptor as shown in SEQ ID NO.

2.

10. The use according to claim 9, wherein the subject is a mammal, including a human, a non-human primate, a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat or a mouse; Preferably, the non-human primate is a monkey or a chimpanzee.

11. The use according to claim 9 or 10, wherein the solid tumor is selected from lung cancer, sarcoma, prostate cancer, digestive tract solid tumor and melanoma; Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

12. The use according to claim 11, wherein the drug for treating colorectal cancer is a unit dose preparation, wherein the content of the interleukin complex is 15-500 mg; Preferably, the content of the interleukin complex is 75-250 mg.

13. The use according to claim 11, wherein the drug for treating liver cancer or pancreatic cancer is a unit dose preparation, wherein the content of the interleukin complex is 75-250 mg; Preferably, the content of the interleukin complex is 150-250 mg.

14. The use according to claim 11, wherein the drug for treating sarcoma, prostate cancer or lung cancer is a unit dose preparation, wherein the content of the interleukin complex is 1.5-500 mg; Preferably, the content of the interleukin complex is 50-105 mg.

15. The use according to claim 11, wherein the drug for treating melanoma is a unit dose preparation, wherein the content of the interleukin complex is 15-95 mg or 105-500 mg; Preferably, the content of the interleukin complex is 15-95 mg.

16. The use according to claim 11, wherein the drug for treating breast cancer is a unit dose preparation, wherein the content of the interleukin complex is 1.5-105 mg; Preferably, the content of the interleukin complex is 50-105 mg.

17. A method for treating lung cancer, liver cancer, sarcoma, colorectal cancer, pancreatic cancer, prostate cancer, and melanoma in a subject in need thereof using an interleukin complex, comprising determining the dosage to be administered to the subject using the difference in equilibrium dissociation constant (KD) of the interleukin complex for the CD122 receptor of the subject belonging to different species, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1, and a sushi domain of the IL15Rα receptor as shown in SEQ ID NO.

2.

18. A method for treating a solid tumor in a human in need thereof, comprising administering to the human an interleukin complex, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1, and a sushi domain of an IL15Rα receptor as shown in SEQ ID NO.

2.

19. The use according to claim 18, wherein the solid tumor is selected from lung cancer, sarcoma, prostate cancer, digestive tract solid tumors and melanoma; Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

20. The method according to claim 19, wherein the method for treating colorectal cancer comprises administering the interleukin complex to a human being, with each administration being at a dose of 0.01-0.35 mg / kg; Preferably, the dosage for each administration to humans is 0.0525-0.175 mg / kg; Preferably, the drug is administered to humans once or twice a week; Preferably, the drug is administered to a human a total of three, five or six times.

21. The method according to claim 19, wherein the method for treating liver cancer or pancreatic cancer comprises administering the interleukin complex to a human being, wherein the dosage of each administration is 0.0525-0.175 mg / kg; Preferably, the dosage for each administration to humans is 0.105-0.175 mg / kg; Preferably, the drug is administered to humans once a week for a total of three times.

22. The method according to claim 19, wherein the method for treating sarcoma, prostate cancer, and lung cancer comprises administering the interleukin complex to a human being at a dose of 0.001-0.35 mg / kg per administration; Preferably, the dosage for each administration to humans is 0.01-0.0735 mg / kg; Preferably, the drug is administered to humans once or twice a week; Preferably, the drug is administered to a human a total of three, four or six times.

23. The method according to claim 19, wherein the method for treating melanoma comprises administering the interleukin complex to a human, wherein the dosage of each administration is 0.01-0.0665 mg / kg or 0.0735-0.35 mg / kg; Preferably, the dosage for each administration to humans is 0.01-0.0665 mg / kg; Preferably, the drug is administered to humans once or twice a week for a total of five times.

24. The method according to claim 19, wherein the method for treating breast cancer comprises administering the interleukin complex to a human, wherein the dosage of each administration is 0.001-0.0735 mg / kg; Preferably, the dosage for each administration to humans is 0.035-0.0735 mg / kg; Preferably, the drug is administered to humans once a week for a total of three times.

25. Use of an interleukin complex in the preparation of a medicament for treating a solid tumor in a person in need thereof, wherein the interleukin complex comprises a modified IL-2 molecule as shown in SEQ ID NO.1, and a sushi domain of an IL15Rα receptor as shown in SEQ ID NO.

2.

26. The use according to claim 25, wherein the solid tumor is selected from lung cancer, sarcoma, prostate cancer, solid tumors of the digestive tract and melanoma; Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

27. The use according to claim 25, wherein the drug for treating colorectal cancer is a unit dose preparation, wherein the content of the interleukin complex is 0.525-17.5 mg; Preferably, the content of the interleukin complex is 2.625-8.75 mg.

28. The use according to claim 25, wherein the drug for treating liver cancer or pancreatic cancer is a unit dose preparation, wherein the content of the interleukin complex is 2.625-8.75 mg; Preferably, the content of the interleukin complex is 5.25-8.75 mg.

29. The use according to claim 25, wherein the drug for treating sarcoma, prostate cancer or lung cancer is a unit dose preparation, wherein the content of the interleukin complex is 0.0525-17.5 mg; Preferably, the content of the interleukin complex is 1.75-3.675 mg.

30. The use according to claim 25, wherein the drug for treating melanoma is a unit dose preparation, wherein the content of the interleukin complex is 0.525-3.325 mg or 3.675-17.5 mg; Preferably, the content of the interleukin complex is 0.525-3.325 mg.

31. The use according to claim 25, wherein the drug for treating breast cancer is a unit dose preparation, wherein the content of the interleukin complex is 0.0525-3.675 mg; Preferably, the content of the interleukin complex is 1.75-3.675 mg.

32. The method according to claims 1-8, 17-24 or the use according to claims 9-16, 25-31, wherein the administration of the drug includes intravenous, intradermal, subcutaneous, oral inhalation, transdermal, transmucosal administration and enteral administration such as rectal administration.

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