Interleukin complex and use thereof

Abstract

The invention provides application of an interleukin complex in preparation of drugs for treating lung cancer, liver cancer, sarcoma, colorectal cancer, pancreatic cancer, prostate cancer and melanoma of subjects in need. Compared with wild type IL-2, the interleukin complex provided by the invention has the advantages that immune cells such as CD8 + T cells and NK cells for killing tumors can be more strongly activated, meanwhile, the growth of Treg cells for inhibiting immune reaction can be less activated, the administration convenience is more excellent, and the application prospect is wider. The effect of better inhibiting the growth and reproduction of tumor cells within the acceptable toxic dose of an organism can be realized, and the growth of tumor cells of more types of tumors can be inhibited.

Description

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] Tumors are a major threat to human health and a cause of death. Traditional treatments include surgery, radiotherapy, chemotherapy, targeted drug therapy, etc. However, local treatments such as surgery and radiotherapy are only suitable for larger lesions and cannot achieve the same effect as systemic treatment. Tumors are prone to metastasis and recurrence. Systemic treatments such as chemotherapy and targeted drugs can treat smaller, less-detectable tumor lesions, but due to the genetic heterogeneity of tumor cells, after using specific chemotherapy and targeted drugs to kill tumor cells, tumor cells that are insensitive to the drugs 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 method to inhibit the growth and reproduction of tumor cells. Different tumor cells can be identified by the immune system, and comprehensive killing can be carried out to reduce the metastasis and recurrence rate of tumors.

[0003] Interleukin-2 (IL-2), discovered in 1976, was then called T cell growth factor (TCGF). It 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 15kD. IL-2 is mainly produced by activated T cells. It can promote the proliferation and differentiation of T cells and maintain T cell activity; stimulate the generation, proliferation and activation of natural killer (NK) cells, induce the generation of cytotoxic T lymphocytes (CTL), and induce and activate lymphokine-activated killer cells (LAK) and tumor-infiltrating lymphocytes; promote T cells to express cytokines and cytolytic molecules, and promote the proliferation of B cells (Waldmann et al., Nat Rev Immunol 6, 595-601 (2009)); these cells have the function of directly or indirectly killing cells infected with exogenous microorganisms and cancerous cells, so IL-2 has good antiviral and anticancer effects and a wide range of clinical application potential.

[0004] The method of using interleukin 2 (IL-2) to activate the human immune system to inhibit tumor growth has been disclosed in the 1990s. The wild-type IL-2 drug is called aldesleukin, which was approved for marketing by the U.S. Food and Drug Administration in 1992 and 1998, respectively, for the treatment of metastatic renal cell carcinoma and metastatic melanoma, showing definite efficacy. However, this technical solution has serious limitations, which is reflected in the fact that due to the high toxicity of the drug, the dosage is very low, below 37μg (drug weight) / kg (body weight). Higher doses of IL-2 can cause severe toxicity, including vascular leak syndrome, pulmonary edema, hypotension and cardiac toxicity. When used at clinical doses, aldesleukin has limited ability to inhibit tumor growth, and the overall tumor inhibition rate is relatively low. Moreover, the approved indications for aldesleukin are relatively narrow, only 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 of suppressing tumors, such as using toxic chemicals and radiation, are prone to drug resistance and recurrence of tumors if they cannot completely kill all tumor cells. There is a lack of a safe and effective immune stimulation method to stimulate the body's own immunity to inhibit tumor growth, thereby achieving long-term survival benefits for the body. Currently, there is a need for an immune stimulation method that can activate immune cells such as CD8+T cells that kill tumors and minimize the activation of regulatory T cells that inhibit immune responses. At the same time, the immune stimulation method has low toxicity enough for animals (including humans) to tolerate, thereby utilizing the stimulation of the animal's own immune system to inhibit the growth of a variety of different tumors. Summary of the invention

[0006] In order to improve the above technical problems, the present invention provides the use of an interleukin complex, which uses an appropriate dose to maximize the activation of tumor-killing immune cells such as CD8+T cells and minimize the growth of activated regulatory T cells that inhibit immune responses. Combined with the low toxicity of the complex, it is possible to activate 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.

[0007] The invention of the present invention is to use a new interleukin complex specific dosage range to achieve the inhibition of tumor growth in animal models of various tumors. The dosage range in animals can be converted to the dosage used in other species by the affinity of the interleukin complex with receptors of different species.

[0008] On the one hand, the present invention discloses 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 a sushi domain of an IL15Rα receptor as shown in SEQ ID NO.2.

[0009] 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.

[0010] In one embodiment of the present invention, the aforementioned non-human primate is a monkey or a chimpanzee.

[0011] 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;

[0012] Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

[0013] 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;

[0014] Preferably, the content of the interleukin complex is 75-250 mg.

[0015] 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;

[0016] Preferably, the content of the interleukin complex is 150-250 mg.

[0017] 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;

[0018] Preferably, the content of the interleukin complex is 150-250 mg.

[0019] 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;

[0020] Preferably, the content of the interleukin complex is 50-105 mg.

[0021] 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;

[0022] Preferably, the content of the interleukin complex is 50-105 mg.

[0023] 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;

[0024] Preferably, the content of the interleukin complex is 50-105 mg.

[0025] 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;

[0026] Preferably, the content of the interleukin complex is 15-95 mg.

[0027] 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;

[0028] Preferably, the content of the interleukin complex is 50-105 mg.

[0029] In one embodiment of the present invention, the aforementioned drug for treating colorectal cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage administered to the subject each time is 0.3-10 mg / kg;

[0030] Preferably, the dose administered to the subject each time is 1.5-5 mg / kg;

[0031] Preferably, the drug is administered to the subject once or twice a week;

[0032] Preferably, the drug is administered to the subject a total of three, five or six times.

[0033] In one embodiment of the present invention, the aforementioned drug for treating liver cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage administered to the subject each time is 1.5-5 mg / kg;

[0034] Preferably, the dose administered to the subject each time is 3-5 mg / kg / w;

[0035] Preferably, the drug is administered to the subject once a week for a total of three times.

[0036] In one embodiment of the present invention, the aforementioned drug for treating pancreatic cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage administered to the subject each time is 1.5-5 mg / kg;

[0037] Preferably, the dose administered to the subject each time is 3-5 mg / kg / w;

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

[0039] In one embodiment of the present invention, the aforementioned drug for treating sarcoma is calculated based on the mass of the aforementioned interleukin complex, and the dosage administered to the subject each time is 0.03-10 mg / kg / w;

[0040] Preferably, the dose administered to the subject each time is 0.3-2.1 mg / kg / w;

[0041] Preferably, the drug is administered to the subject once or twice a week;

[0042] Preferably, the drug is administered to the subject a total of three, four or six times.

[0043] In one embodiment of the present invention, the aforementioned drug for treating prostate cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage administered to the subject each time is 0.03-10 mg / kg / w;

[0044] Preferably, the dose administered to the subject each time is 0.3-2.1 mg / kg / w;

[0045] Preferably, the drug is administered to the subject once or twice a week;

[0046] Preferably, the drug is administered to the subject a total of three, four or six times.

[0047] In one embodiment of the present invention, the aforementioned drug for treating lung cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage administered to the subject each time is 0.03-10 mg / kg;

[0048] Preferably, the dose administered to the subject each time is 0.3-2.1 mg / kg;

[0049] Preferably, the drug is administered to the subject once or twice a week;

[0050] Preferably, the drug is administered to the subject a total of three, four or six times.

[0051] In one embodiment of the present invention, the drug for treating melanoma prepared by using the aforementioned interleukin complex is calculated according to the mass of the aforementioned interleukin complex, and the dosage administered to the subject each time is 0.3-1.9 mg / kg or 2.1-10 mg / kg;

[0052] Preferably, the dose administered to the subject each time is 0.3-1.9 mg / kg;

[0053] Preferably, the drug is administered to the subject once or twice a week for a total of five times.

[0054] In one embodiment of the present invention, the drug for treating breast cancer prepared by using the aforementioned interleukin complex is calculated according to the mass of the aforementioned interleukin complex, and the dosage administered to the subject each time is 0.03-2.1 mg / kg;

[0055] Preferably, the dose administered to the subject each time is 1-2.1 mg / kg;

[0056] Preferably, the drug is administered to the subject once a week for a total of three times.

[0057] 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 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.

[0058] 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;

[0059] Preferably, the digestive tract solid tumor is selected from liver cancer, colorectal cancer and pancreatic cancer.

[0060] 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;

[0061] Preferably, the content of the interleukin complex is 2.625-8.75 mg.

[0062] 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;

[0063] Preferably, the content of the interleukin complex is 5.25-8.75 mg.

[0064] 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;

[0065] Preferably, the content of the interleukin complex is 5.25-8.75 mg.

[0066] 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;

[0067] Preferably, the content of the interleukin complex is 1.75-3.675 mg.

[0068] 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;

[0069] Preferably, the content of the interleukin complex is 1.75-3.675 mg.

[0070] 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;

[0071] Preferably, the content of the interleukin complex is 1.75-3.675 mg.

[0072] 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;

[0073] Preferably, the content of the interleukin complex is 0.525-3.325 mg.

[0074] 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;

[0075] Preferably, the content of the interleukin complex is 1.75-3.675 mg.

[0076] In one embodiment of the present invention, the aforementioned drug for treating colorectal cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage for each administration to a human is 0.01-0.35 mg / kg;

[0077] Preferably, the dosage for each administration to humans is 0.0525-0.175 mg / kg;

[0078] Preferably, the drug is administered to humans once or twice a week;

[0079] Preferably, the drug is administered to a human a total of three, five or six times.

[0080] In one embodiment of the present invention, the aforementioned drug for treating liver cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage for each administration to humans is 0.0525-0.175 mg / kg;

[0081] Preferably, the dosage for each administration to humans is 0.105-0.175 mg / kg;

[0082] Preferably, the drug is administered to humans once a week for a total of three times.

[0083] In one embodiment of the present invention, the aforementioned drug for treating pancreatic cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage for each administration to a human is 0.0525-0.175 mg / kg;

[0084] Preferably, the dosage for each administration to humans is 0.105-0.175 mg / kg;

[0085] Preferably, the drug is administered to humans once a week for a total of three times.

[0086] In one embodiment of the present invention, the aforementioned drug for treating sarcoma is calculated based on the mass of the aforementioned interleukin complex, and the dosage for each administration to humans is 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 a total of three, four or six times.

[0090] In one embodiment of the present invention, the aforementioned drug for treating prostate cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage for each administration to humans is 0.001-0.35 mg / kg;

[0091] Preferably, the dosage for each administration to humans is 0.01-0.0735 mg / kg;

[0092] Preferably, the drug is administered to humans once or twice a week;

[0093] Preferably, the drug is administered to a human a total of three, four or six times.

[0094] In one embodiment of the present invention, the aforementioned drug for treating lung cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage for each administration to a human is 0.001-0.35 mg / kg;

[0095] Preferably, the dosage for each administration to humans is 0.01-0.0735 mg / kg;

[0096] Preferably, the drug is administered to humans once or twice a week;

[0097] Preferably, the drug is administered to a human a total of three, four or six times.

[0098] In one embodiment of the present invention, the aforementioned drug for treating melanoma is calculated based on the mass of the aforementioned interleukin complex, and the dosage per administration to a human is 0.01-0.0665 mg / kg or 0.0735-0.35 mg / kg;

[0099] Preferably, the dosage for each administration to humans is 0.01-0.0665 mg / kg;

[0100] Preferably, the drug is administered to humans once or twice a week for a total of five times.

[0101] In one embodiment of the present invention, the aforementioned drug for treating breast cancer is calculated based on the mass of the aforementioned interleukin complex, and the dosage for each administration to a human is 0.001-0.0735 mg / kg;

[0102] Preferably, the dosage for each administration to humans is 0.035-0.0735 mg / kg;

[0103] Preferably, the drug is administered to humans once a week for a total of three times.

[0104] In another aspect, the present invention provides a method of administering the aforementioned drug, which includes intravenous, intradermal, subcutaneous, oral inhalation, transdermal, transmucosal administration, and enteral administration such as rectal administration.

[0105] Beneficial Effects

[0106] The interleukin complex provided by the present invention can more strongly activate tumor-killing immune cells such as CD8+T cells and NK cells compared to wild-type IL-2, while less effectively activating the growth of Treg cells that inhibit 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

[0107] Figure 1The antitumor 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 CT26 colon cancer subcutaneous transplantation model in mice is shown. The figure shows the tumor size at different days after administration.

[0108] Figure 2 The figure shows the anti-tumor effect of the modified interleukin complex injected intraperitoneally at a dose of 1.5, 3.0, and 5.0 mg (drug weight) / kg (body weight) in a subcutaneous transplantation model of CT26 colon cancer mice. The figure shows the tumor size at different days after administration.

[0109] Figure 3 : The antitumor 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 MC38 colon cancer subcutaneous transplantation model in mice. The figure shows the tumor size at different days after administration.

[0110] Figure 4 The figure 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 the Hepa 1-6 liver cancer subcutaneous transplantation model in mice. The figure shows the tumor size at different days after administration.

[0111] Figure 5 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 the Pan02 pancreatic cancer mouse subcutaneous transplantation model is shown. The figure shows the tumor size at different days after administration.

[0112] Figure 6 The antitumor 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 S180 sarcoma subcutaneous transplantation model in mice is shown. The tumor size at different days after administration is shown in the figure.

[0113] Figure 7 The antitumor 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 B16F10 melanoma subcutaneous transplantation model in mice is shown. The tumor size at different days after administration is shown in the figure.

[0114] Figure 8The antitumor 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 is shown. The figure shows the tumor size at different days after administration.

[0115] Fig. 9 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 is shown. The figure shows the tumor size at different days after administration.

[0116] Fig.10 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 is shown. The figure shows the tumor size at different days after administration. DETAILED DESCRIPTION

[0117] definition

[0118] It should be understood that the aforementioned interleukin complexes will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into formulations 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)), especially Chapter 87 of Blaug, Seymour therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (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, emulsions of polyethylene glycol (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 treatments or therapies according to the present application, provided that the active ingredients in the formulation are not inactivated by the formulation and that the formulation is physiologically compatible and tolerated by the route of administration.

[0119] 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., cattle, 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.

[0120] As used herein, a "unit dose formulation" may be a packaged formulation, such as a tablet, capsule or powder packaged in a bottle, bag, box or ampoule. A unit dose formulation may be a capsule, tablet or any dosage form in an appropriate number in a packaged form. A unit dose refers to a unit suitable for a single dose for a human or other mammal.

[0121] 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 drug administration. 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.

[0122] Preparations to be used for clinical in vivo administration must be sterile. This can be easily achieved by filtration through sterile filtration membranes.

[0123] The aforementioned drugs are formulated to be compatible with their intended routes 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: sterile diluents for injection such as water, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol, methyl parabens, phenol, or metacresol; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents for regulating osmotic pressure such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations 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 replacement of the drug cartridge, so 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, you can replace the cartridge and continue to use it.

[0124] The medicine suitable for injection purposes includes sterile aqueous solution (water-soluble here) or dispersion and sterile powder for instant preparation of sterile injection or dispersion. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and the fluidity should reach the degree of easy injection. 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 required particle size in the case of dispersion, 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.

[0125] As required, sterile injection solutions can be prepared by incorporating the drug of the present invention into a suitable solvent with one or a combination of the above-listed components (as required) in the required amount, and then filtering and sterilizing. Generally speaking, dispersions are prepared by incorporating the drug of the present invention into a sterile carrier containing a dispersion medium and those other required components listed above. With regard to the sterile powders for the preparation of sterile injection solutions, the preparation method is to obtain vacuum drying and freeze drying of the powder, which comprises the active ingredient and any other desired components, which are derived from the aseptically filtered solutions of the aforementioned components.

[0126] For administration by inhalation, the drug is delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

[0127] It is also possible to administer the drug systemically via mucosal or transdermal administration. For mucosal or transdermal administration, a penetrant suitable for permeation of the barrier is used in the formulation. Such penetrants are generally known in the art and include detergents, bile salts and fusidic acid derivatives such as those used for mucosal administration. Mucosal 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 ointment, a gel or a cream as is generally known in the art.

[0128] 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.

[0129] 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 release / 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.

[0130] 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.

[0131] Sustained release preparations can be prepared. Examples of suitable sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the drug of the present invention, which are in the form of molded 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 DEPOTTM (microspheres for injection consisting of lactic acid-glycolic acid copolymers and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for a shorter time. Polylactic acid (PLA) and polylactic-glycolic acid copolymers (PLGA) are hot topics in recent years. In addition, there are albumin microspheres, chitosan microspheres, gelatin microspheres, etc.

[0132] Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example as described in US Pat. No. 4,522,811.

[0133] 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 unit dosages for the subject to be treated; each unit contains a predetermined amount of the drug of the present invention calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the embodiments are dictated by and directly dependent on the unique characteristics of the drug of the present invention and the specific therapeutic effect to be achieved, and the limitations inherent in the art of formulating such drugs of the present invention for treating individuals.

[0134] The medicament can be included in a container, pack, or dispenser together with instructions for administration.

[0135] As used herein, "effective amount" refers to an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic amount is an amount that achieves a 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 symptom. An effective amount may be administered in one or more administrations, applications, or dosages. The "therapeutically effective amount" (i.e., effective dose) of a therapeutic drug depends on the selected therapeutic drug. The administration of the composition may be from one or more times per day to one or more times per week; including once every other day. Those skilled in the art will appreciate that certain factors may affect the dose 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.

[0136] As used herein, the term "treatment" generally refers to the administration or administration of a therapeutic agent to a patient, or to a tissue or cell line isolated from a patient, who suffers from a disease, has disease symptoms or disease tendencies, with the purpose of treating, curing, alleviating, relieving, altering, remedying, improving, enhancing or influencing the disease, disease symptoms or disease tendencies. This may include improving the disease state, eliminating lesions or improving prognosis.

[0137] 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 the growth of 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.

[0138] As used herein, "solid tumor" refers to all neoplastic cell growth and proliferation, and all precancerous and cancerous cells and tissues, except hematological cancers (e.g., lymphoma, leukemia, and multiple myeloma). 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 carcinoma, 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.

[0139] 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.

[0140] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions 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 included in the scope that the present invention is intended to protect.

[0141] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0142] Example 1: Preparation of interleukin complexes engineered from mammalian cells

[0143] 1.1 Synthesis and construction of expression plasmid

[0144] Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the target gene fragments of the two proteins, interleukin (SEQ ID NO.1) and sushi-hIgG4Fc (SEQ ID NO.2), and clone them into 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.

[0145] 1.2 Plasmid extraction and HEK293 cell preparation

[0146] 1.2.1 Plasmid extraction

[0147] The above plasmid preparation was performed according to the operating procedures mentioned in "Qiagen Mini-prep Kit" and "Qiagen Endofree Maxi-prep Kit".

[0148] 1.2.2 HEK293 cell preparation

[0149] Use a density of 1-1.2 × 10 6 HEK293 cells (National Research Council, Canada) at 4% paraformaldehyde / mL were used for transient expression.

[0150] 1.3 HEK293 transient expression

[0151] 1.3.1 Reagent preparation

[0152] A) G418 solution: weigh 250 mg GeneticinTM and dissolve it in 4.5 mL ultrapure water, dilute to 5 mL with ultrapure water, filter with 0.22 μm filter membrane, and store at -20°C;

[0153] B) PEI solution: weigh 50 mg PEI and add 45 mL ultrapure water to dissolve, adjust the pH to 7.0 with 1 M NaOH, make up to 50 mL with ultrapure water, filter with 0.22 μm filter membrane, and store at -20°C;

[0154] C) Culture medium: Add 10 mL of 10% PluronicdTM F-68 and 500 μL of G418 to 1 liter of FreeStyleTM 293 Expression Medium;

[0155] D) The plasmid is prepared in advance in a 2 mL endotoxin-free centrifuge tube;

[0156] E) Prepare fresh passages to 1-1.2×10 cells / mL according to the volume required for transfection. 6 / mL of cell suspension.

[0157] 1.3.2 Preparation of transfection reagent-plasmid complex

[0158] Solution A: Plasmid 1μg / mL + Opti-MEMTM 33.3μL / mL

[0159] Solution B: PEI 2μg / mL+Opti-MEMTM 33.3μL / mL

[0160] Pour 1 mL of solution B into 1 mL of solution A, mix well, incubate for 10 minutes, and then add the cell suspension.

[0161] The modified interleukin and sushi-hIgG4Fc were mixed at a mass ratio of 1:1 (total amount of 1 μg / mL) and then transfected.

[0162] 1.3.3 Fluid exchange

[0163] After culturing for 4 small trials at 115 rpm, 36.8°C, 5% CO2, the culture medium was centrifuged at 800 g for 5 minutes and replaced with FreeStyleTM 293 Expression medium without PluronicdTM F-68 and G418.

[0164] 1.3.4 Expression culture and harvest

[0165] 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.

[0166] 1.4 Purification and preparation

[0167] The modified interleukin and sushi-hIgG4Fc were co-expressed, and a heterodimer complex was formed during the expression process, which was named the modified interleukin complex, which was labeled with an Fc tag. Mabselect sure (ProteinA, GE Healthcare) was used for affinity purification, and the purification method was referred to the purification filler instructions; then it was further purified by gel filtration chromatography (Superdex200pg, GE Healthcare) to obtain a protein with higher purity and remove aggregates. Purity analysis was performed using a high-performance liquid chromatography molecular sieve column (SEC-HPLC). If the main peak of the purified protein SEC was a single peak, it indicated that the complex was successfully formed.

[0168] Example 2: Modified interleukin complex activates immune cell signaling pathway

[0169] Based on the research results of modern immunology, tumor-killing T cells, such as CD8+T cells, play a role in killing tumor cells in the process of inhibiting tumor growth, which is beneficial to inhibiting tumor growth. Treg cells negatively regulate the immune system, inhibit the growth of immune cells, and are beneficial to the growth of tumor cells. To achieve the purpose of inhibiting tumors by activating the immune system of animals through interleukins, it is necessary to use a cytokine that can activate killer T cells, such as CD8+T cells, as much as possible and activate Treg cells as little as possible.

[0170] 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 and then form homodimers or heterodimers that translocate to the nucleus where they act as transcriptional activators. STAT5 is activated and mediates responses by many cellular ligands, such as IL-2, IL-3, IL-7, GM-CSF, erythropoietin, thrombopoietin, and different growth hormones. Testing the phosphorylation of STAT5 at tryptophan 694 is a routine quantitative method for testing cytokine receptor activation.

[0171] CD8+T cells and Treg cells were resuspended in 20 mL of complete 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 the culture flask was placed in a 37°C, 5% CO2 incubator for overnight culture.

[0172] On the second day, transfer the overnight cultured CD8+T cells and Treg cells to 50 mL centrifuge tubes (NEST, catalog number: 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, 50μL / well were added to 96-well plates (NEST, catalog number: 701101). Wild-type IL-2 was diluted to a starting concentration of 10nM, wild-type IL-2, IL-15 and modified interleukin complex were diluted to a starting concentration of 100nM, and then 5-fold serial dilutions were performed for a total of 10 concentrations, and 50μL / well were added to the paved CD8+T cells and Treg cells. The 96-well plate was placed in a 37°C, 5% CO2 incubator and incubated for 15 minutes. After incubation, 100μL Fixbuffer I (BD, catalog number: 557870) was immediately added to each well and fixed in a 37°C, 5% CO2 incubator for 10 minutes. 100μL stainbuffer (BD, catalog number: 554656) was added to each well, centrifuged at 300g for 5 minutes, and the supernatant was discarded.

[0173] Surface antibodies against CD8+T cells (tube 1) and Treg cells (tube 2) were prepared respectively. The types of antibodies are as follows: tube 1: APC-H7mouse Anti-human CD3 (BD, catalog number: 560176) and BV605mouse Anti-humanCD8 (BD, catalog number: 564116); tube 2: APC-H7mouse Anti-human CD3 (BD, catalog number: 560176), BV650mouse Anti-humanCD4 (BD, catalog number: 563875), PE mouse anti-human CD25 (BD, catalog number: 555432); after mixing, 100 μL / well was added to the corresponding cells, incubated at 4°C in the dark for 30 minutes, 100 μL stain buffer was added to each well, centrifuged at 300g for 5 minutes, and the supernatant was discarded. Add 100 μL Perm Buffer III (BD, Catalog No.: 558050) to each well and incubate on ice for 30 minutes. After incubation, add 100 μL stain buffer to each well, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0174] Intracellular antibodies targeting CD8+T cells (tube 3) and Treg cells (tube 4) were prepared respectively. The types of antibodies are as follows: tube 3: AF647Mouse Anti-Stat5 (pY694) (BD, catalog number: 562076); tube 4: AF647Mouse Anti-Stat5 (pY694) (BD, catalog number: 562076) and V450mouse Anti-human Foxp3 (BD, catalog number: 561182); mix well, add 100μL to each well of the corresponding cells, incubate at 4℃ in the dark overnight, add 100μL stain buffer to each well the next day, centrifuge at 300g for 5min, and discard the supernatant. Add 80μL stain buffer to each well to resuspend the cells, set compensation, and use flow cytometer for detection, use Flowjo [v10.8] for data analysis, and Graphpad Prism [v9.0] for plotting.

[0175] Table 1 EC50 of interleukin activation of START5 signaling pathway in immune cells

[0176]

[0177]

[0178] 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 achieve the activation of CD8+T cells as much as possible and the activation of Treg cells as little as possible, the ratio of CD8+T EC50 / Treg EC50 needs to be as low as possible. At this time, the cytokine can activate the proliferation of CD8+T cells at a lower concentration, and the activity of stimulating Treg cell proliferation at this concentration is relatively smaller. From the data in Table 1, it can be seen that the selected modified interleukin complex can preferentially stimulate the cell proliferation signaling pathway of CD8+T cells relative to IL-2 and IL-15. In summary, by selectively stimulating the proliferation of immune cells, the effect of inhibiting tumor cell growth is achieved.

[0179] Example 3: Modified interleukin complex activates immune cell proliferation

[0180] As described in Example 2, to achieve the purpose of inhibiting tumors by activating the immune system of animals through interleukin, it is necessary to use a cytokine that can activate killer T cells, such as CD8+T cells, as much as possible and activate Treg cells as little as possible. In addition to quantitatively detecting immune cell signaling pathways, the activation and proliferation of different immune cells by interleukin can also be determined by quantitatively detecting the proliferation of activated immune cells.

[0181] CD8+ T cells and Treg cells were revived by adding 10 mL of complete 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, 50μL / well were added to 96-well white bottom plates (NEST, catalog number: 701301), and 2 replicates were made for each sample. Wild-type IL-2 and IL-15 and modified interleukin complexes were diluted to a starting concentration of 1000nM, and then 5-fold serial dilutions were performed for a total of 10 concentrations, and 50μL / well were added to the paved CD8+T cells and Treg cells. After mixing, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 4 days. After incubation, 50μL CELLTiter-Glo-2.0Assay (Promega, catalog number: G9243) was added to each well, mixed and placed on a horizontal shaker at 550rpm for 5 minutes. After standing at room temperature for 10 minutes, the chemiluminescence value (RLU) was detected using an enzyme reader. The detection principle is shown in the CELLTiter-Glo-2.0Assay instruction manual. The chemiluminescence value can be used to quantitatively characterize the viability of cells (Cell viability).

[0182] Table 2 EC50 of interleukin activating immune cell proliferation

[0183]

[0184] The lower the EC50, the lower the dose at which immune cell proliferation can be activated. To achieve the goal of activating CD8+T cells as much as possible and activating Treg cells as little as possible, the ratio of CD8+T EC50 / Treg EC50 needs to be as low as possible. At this time, the cytokine can activate the proliferation of CD8+T cells at a lower concentration, and the activity of stimulating Treg cell proliferation at this concentration is relatively smaller. From the data in Table 2, it can be seen that the selected modified interleukin complex can preferentially activate CD8+T cells relative to IL-2 and IL-15. In combination, it can achieve the effect of inhibiting tumor cell growth by selectively stimulating the proliferation of immune cells.

[0185] Example 4: Antitumor effect in subcutaneous colorectal cancer transplantation model

[0186] Subcutaneous tumor transplantation animal models play a vital role in basic research on tumor occurrence, development, metastasis, and drug development and treatment. Tumor research is inseparable from the establishment of animal models and drug efficacy evaluation. Tumor cell lines are transplanted into the subcutaneous tissue of the same or xenogeneic animals to form tumor masses, which are generally used for screening and testing of drugs that inhibit tumor cell proliferation and have been widely recognized and used in the pharmaceutical industry.

[0187] 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 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 administration was done on D0, D3, D7, D10, and D14. The subcutaneous transplanted tumor model of colorectal cancer was studied, the tumor volume was observed, measured, and recorded, and the efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0188] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0189] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0190] On the 17th day after the start of drug administration, the average tumor volume of the 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.

[0191] Table 3 Average tumor volume and TGI% of different doses of modified interleukin complexes in the CT26 colon cancer mouse subcutaneous transplantation model on day 17 after administration

[0192]

[0193] Depend on Figure 1 From the information in Table 3, it can be seen that the difference in average tumor volume between each dosage group and the solvent control group has been shown around the 6th to 10th day after administration. Except for the lowest dose, i.e., the 0.03 mg (drug weight) / kg (body weight) group, the tumor growth was faster than that of the control group, and the other dosage groups all showed tumor inhibition effects. By the 17th day, except for the 0.03 mg (drug weight) / kg (body weight) group, the tumor inhibition effect of each dosage group was more obvious. The 0.3 mg (drug weight) / kg (body weight) group can be observed to have a TGI% of 14%, which has shown a tumor inhibition effect. And the 10 mg (drug weight) / kg (body weight) group, the TGI% has reached 76%, showing a very prominent tumor inhibition effect. It can be clearly seen that with the increase in dose, the effect of inhibiting tumor growth has gradually increased, forming a clear dose-dependent trend of inhibiting tumor growth. It can be reasonably inferred that at a higher dose greater than 10 mg (drug weight) / kg (body weight) and tolerable to mice, the interleukin complex can also exert an effect of inhibiting tumor growth.

[0194] Example 5: Antitumor effect in subcutaneous colorectal cancer transplantation model

[0195] 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 CT26 cells were inoculated with cancer cells. 7 days after cancer cells were inoculated, 40 mice were selected and divided into 4 groups, each with 10 mice, which were the modified interleukin complex at a dose of 1.5 mg (drug weight) / kg (body weight), 3 mg (drug weight) / kg (body weight), 5 mg (drug weight) / kg (body weight) single drug administration group and solvent control group, intraperitoneal injection of 4 μL / g (body weight) administration, counted as day 0 (D0) after administration, once a week, a total of 3 weeks (3 times), the mouse CT26 colorectal cancer model was studied. The efficacy was evaluated according to the relative tumor inhibition rate (TGI).

[0196] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0197] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0198] 24 days after the start of drug administration, the average tumor volume of the solvent control group (Group 1) was 2499mm 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 after administration in the CT26 colon cancer mouse subcutaneous transplantation model

[0199]

[0200] Depend on Figure 2 From the information in Table 4, it can be seen that around the 5th to 7th day after administration, the difference in average tumor volume between each dosage group and the solvent control group was very obvious. By the 24th day, the tumor inhibition effect of each dosage group was more obvious. A TGI% of 36% can be 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, and the TGI% exceeded 50%, showing a very prominent tumor inhibition effect. Statistical analysis showed that the relative tumor volumes of the three dosage 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 were well tolerated throughout the experiment, and there was no mouse death or obvious weight loss.

[0201] Example 6: Antitumor effect in subcutaneous colorectal cancer transplantation model

[0202] In this example, colon cancer MC38 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 anterior 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 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 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).

[0203] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0204] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0205] On the 18th day 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.

[0206] Table 5 Average tumor volume and TGI% of different doses of modified interleukin complexes on day 18 after administration in the subcutaneous transplantation model of MC38 colon cancer mice

[0207]

[0208] Depend on Figure 3From the information in Table 5, it can be seen that around the 8th to 11th day after administration, the difference in average tumor volume between each dosage group and the solvent control group has already appeared. Except for the lowest dose, i.e., the 0.03 mg (drug weight) / kg (body weight) group, the tumor growth was faster than that of the control group, and the remaining dosage groups all showed tumor inhibition effects. By the 18th day, except for the 0.03 mg (drug weight) / kg (body weight) group, the tumor inhibition effect of each dosage group was more obvious. The 0.3 mg (drug weight) / kg (body weight) group can be observed to have a TGI% of 15%, which has shown a tumor inhibition effect. In the 10 mg (drug weight) / kg (body weight) group, a trend of tumor shrinkage can be observed, and the TGI% has reached 96%, showing a very prominent tumor inhibition effect. It can be clearly seen that with the increase in dose, the effect of inhibiting tumor growth has gradually increased, forming an obvious dose-dependent trend of inhibiting tumor growth. It can be reasonably speculated that the interleukin complex can also exert the effect of inhibiting tumor growth at a higher dose greater than 10 mg (drug weight) / kg (body weight) that mice can tolerate.

[0209] Example 7: Antitumor effect in a subcutaneous liver cancer transplantation model

[0210] In this example, Hepa 1-6 hepatocellular carcinoma cells in the exponential growth phase were resuspended in PBS solution to 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 The mice were randomly divided into groups (40 mice, 10 mice / group) according to the tumor size and mouse weight, and the modified interleukin complex was administered at a dose of 1.5 mg (drug weight) / kg (body weight), 3 mg (drug weight) / kg (body weight), and 5 mg (drug weight) / kg (body weight) at a single drug group and a solvent control group. The animals were administered once a week for 3 consecutive weeks, for a total of 3 times. The mouse Hepa 1-6 liver cancer model was studied by intraperitoneal injection of 4 μL / g (body weight), which was counted as day 0 (D0) after administration, and the drug was administered once a week for a total of 3 weeks (3 times). The efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0211] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0212] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0213] 58 days after the start of drug administration, the average tumor volume of the solvent control group (Group 1) was 3515mm 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 different doses in the Hepa 1-6 liver cancer mouse subcutaneous transplantation model on day 58 after administration

[0214]

[0215] Depend on Figure 4 From the information in Table 6, it can be seen that within the 7th day after administration, the difference in the average tumor volume between each dosage group and the solvent control group was very obvious. By the 58th day, the tumor inhibition effect of each dosage group was very obvious. A TGI% of 90% can be observed in the 1.5 mg (drug weight) / kg (body weight) group, showing an excellent tumor inhibition effect. The tumor inhibition effect of the 3.0 and 5.0 mg (drug weight) / kg (body weight) groups was similar, and the TGI% reached 100%, achieving complete remission, showing a very outstanding tumor inhibition effect. Statistical analysis showed that the relative tumor volume of the three dosage groups showed significant differences compared with the solvent control group (p<0.05). It can be clearly seen that with the increase in dose, the effect of inhibiting tumor growth also gradually increased, forming an obvious dose-dependent trend of inhibiting tumor growth, and complete remission of the tumor was achieved at a dose greater than 3.0 mg (drug weight) / kg (body weight). It can be reasonably inferred that the interleukin complex can also inhibit tumor growth at a higher dose greater than 5.0 mg (drug weight) / kg (body weight) that mice can tolerate. The experimental mice tolerated the treatment well during the experiment described in this example, and no mice died. During the experiment, the mice in the administration group of the modified interleukin complex at the set dose were well tolerated throughout the experiment, and no mice died or lost significant weight.

[0216] Example 8: Antitumor Effect in a Subcutaneous Pancreatic Cancer Transplant Model

[0217] 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 anterior 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 ) were divided into 4 groups, 10 in each group, which were the modified interleukin complex single drug administration group at a dose of 1.5, 3 and 5 mg (drug weight) / kg (body weight) and the solvent control group, and intraperitoneal injection of 4 μL / g (body weight) was used for administration once a week for a total of 3 times to study the mouse Pan02 pancreatic cancer model. The efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0218] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0219] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0220] 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.

[0221] 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

[0222]

[0223] Depend on Figure 5 From the information in Table 7, it can be seen that on days 7-14 after administration, the difference in average tumor volume between each dosage group and the solvent control group was manifested. By day 28, the tumor inhibition effect of each dosage group was very obvious. A TGI% of 16% can be 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%, showing an outstanding tumor inhibition effect. Statistical analysis showed that the relative tumor volumes of the three dosage groups were significantly different from those of 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, and there was no mouse death or significant weight loss.

[0224] Example 9: Antitumor Effect in a Subcutaneous Sarcoma Transplant Model

[0225] 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 anterior back of BALB / c mice. 6 S180 cells were used to establish a subcutaneous transplant tumor model of sarcoma. 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 single-drug administration 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 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 comprehensively based on the relative tumor inhibition rate (TGI).

[0226] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0227] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0228] On the 19th day after the start of drug administration, the average tumor volume of mice in the solvent control group (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 on day 19 after administration in the S180 sarcoma subcutaneous transplantation model in mice

[0229]

[0230] Depend on Figure 6 From the information in Table 8, it can be seen that around the 5th to 9th day after administration, the difference in average tumor volume between each dosage group and the solvent control group has been shown. By the 19th day, the tumor inhibition effect of each dosage group has been shown. Even at the lowest dose, i.e., the 0.03 mg (drug weight) / kg (body weight) group, a TGI% of 2% can be observed, which has shown a tumor inhibition effect. In the 10 mg (drug weight) / kg (body weight) group, a trend of tumor shrinkage can be observed, and the TGI% reached 93%, showing a very prominent tumor inhibition effect. It can be clearly seen that with the increase in dose, the effect of inhibiting tumor growth also gradually increases, forming a clear dose-dependent trend of inhibiting tumor growth. It can be reasonably inferred that at a higher dose 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.

[0231] Example 10: Anti-tumor effect in a subcutaneous melanoma transplantation model

[0232] 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 anterior back of C57BL / 6J mice. 5B16F10 cells were used to establish a subcutaneous melanoma transplant tumor model. 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 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 administration on the day of grouping was counted as day 0 (D0) after administration, and the administration was done on D0, D3, D7, D10, and D14. The small melanoma 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).

[0233] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0234] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0235] On the 14th day after the start of drug administration, the average tumor volume of the solvent control group mice (Group 1) was 3761mm 3 The average tumor volume and TGI% of the modified interleukin complex at various doses are shown in the following table.

[0236] Table 9 Average tumor volume and TGI% of different doses of modified interleukin complexes in the B16F10 melanoma mouse subcutaneous transplantation model on day 14 after administration

[0237]

[0238] Depend on Figure 7From the information in Table 9, it can be seen that around the 7th to 10th day after administration, the difference in average tumor volume between each dosage group and the solvent control group has already been shown. Except for the lowest dose, i.e., the 0.03 mg (drug weight) / kg (body weight) group, the tumor growth was faster than that of the control group, and the other dosage groups all showed tumor inhibition effects. By the 14th day, except for the 0.03 mg (drug weight) / kg (body weight) group, the tumor inhibition effect of each dosage group was more obvious. The 0.3 mg (drug weight) / kg (body weight) group can be observed to have a TGI% of 35%, which has shown a tumor inhibition effect. And the 10 mg (drug weight) / kg (body weight) group, the TGI% has reached 69%, showing a very prominent tumor inhibition effect. It can be clearly seen that with the increase in dose, the effect of inhibiting tumor growth has gradually increased, forming a clear dose-dependent trend of inhibiting tumor growth. It can be reasonably inferred that at a higher dose greater than 10 mg (drug weight) / kg (body weight) and tolerable to mice, the interleukin complex can also exert an effect of inhibiting tumor growth.

[0239] Example 11: Antitumor effect in a subcutaneous prostate cancer transplant 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 anterior 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 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 administration on the day of grouping 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, the tumor volume was observed, measured, and recorded, and the efficacy was evaluated comprehensively based on the relative tumor inhibition rate (TGI).

[0241] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0242] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0243] On the 13th day 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 mouse subcutaneous transplantation model on day 13 after administration

[0245]

[0246] By the attached Figure 8 From the information in Table 10, it can be seen that around the 6th to 10th day after administration, the difference in average tumor volume between each dosage group and the solvent control group has already appeared. On the 13th day after administration, the TGI% of 7% can be observed in the 0.03 mg (drug weight) / kg (body weight) group, which has shown a tumor inhibitory effect. In the 10 mg (drug weight) / kg (body weight) group, the TGI% has reached 81%, showing a very prominent tumor inhibitory effect. It can be clearly seen that with the increase in dosage, the effect of inhibiting tumor growth also gradually increases, forming an obvious dose-dependent trend of inhibiting tumor growth. It can be reasonably inferred that at a higher dose 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.

[0247] Example 12: Antitumor Effect in a Subcutaneous Lung Cancer Transplantation Model

[0248] In this example, lung cancer NCI-H460 cells in the exponential growth phase were resuspended in PBS solution to 1.0×10 8 1.0×10 cells / mL were inoculated subcutaneously on the right anterior back of NCG mice. 7NCI-H460 cells were used to establish a subcutaneous transplant 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 single-drug administration 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 administration on the day of grouping was counted as day 0 (D0) after administration, and the drugs were administered on D0, D7, and D14. 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).

[0249] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0250] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0251] On the 15th day after the start of drug administration, the average tumor volume of mice in the solvent control group (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.

[0252] Table 11 Average tumor volume and TGI% of different doses of modified interleukin complexes in NCI-H460 lung cancer mouse subcutaneous transplantation model on day 19 after administration

[0253]

[0254] Depend on Fig. 9From the information in Table 11, it can be seen that around the 4th to 8th day after administration, the difference in average tumor volume between each dosage group and the solvent control group has already appeared. By the 15th day, the tumor inhibition effect of each dosage group has been clearly shown. Even at the lowest dose, i.e., the 0.03 mg (drug weight) / kg (body weight) group, a TGI% of 6% can be observed, which has shown a tumor inhibition effect. In the 10 mg (drug weight) / kg (body weight) group, a TGI% of 32% can be observed, showing a very prominent tumor inhibition effect. It can be clearly seen that with the increase in dose, the effect of inhibiting tumor growth also gradually increases, forming an obvious dose-dependent trend of inhibiting tumor growth. It can be reasonably inferred that at a higher dose 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.

[0255] Example 13: Anti-tumor effect in a subcutaneous breast cancer transplant model

[0256] 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 anterior back of BALB / c mice. 5 EMT6 cells were used to establish a subcutaneous transplant 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 administration on the day of grouping was counted as day 0 (D0) after administration, and the administration was done on D0, D7, and D14. The subcutaneous transplant tumor model of breast cancer in mice was studied, the tumor volume was observed, measured, and recorded, and the efficacy was evaluated based on the relative tumor inhibition rate (TGI).

[0257] After tumor inoculation, the animals were checked for tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors on a daily basis, and humane endpoints were given accordingly. After group administration, the tumor volume of the animals was measured twice a week. The 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 The tumor volume was calculated. a and b represent the long diameter and short diameter of the tumor, respectively.

[0258] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T / C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the solvent control group; RTV = Vt-V0, V0 is the tumor volume of the animal when grouping, and Vt is the tumor volume of the animal after treatment).

[0259] On the 20th day after the start of drug administration, the average tumor volume of 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.

[0260] Table 12 Average tumor volume and TGI% of different doses of modified interleukin complexes on day 20 after administration in the subcutaneous transplantation model of EMT6 breast cancer mice

[0261]

[0262]

[0263] Depend on Fig.10 From the information in Table 12, it can be seen that around the 9th to 13th day after administration, the difference in average tumor volume between each dosage group and the solvent control group has already appeared. Except for the 10 mg (drug weight) / kg (body weight) group, where the tumor grew faster than the control group, the other groups all showed tumor inhibition effects. On the 20th day after administration, a TGI% of 5% was observed in the 0.03 mg (drug weight) / kg (body weight) group, which has shown a tumor inhibition effect. All groups with 0.3-2.1 mg (drug weight) / kg (body weight) showed a good tumor inhibition effect. Only the 10 mg (drug weight) / kg (body weight) group had a TGI% of -8%, which did not show a tumor inhibition effect.

[0264] Example 14: Affinity of modified interleukin complexes for receptors of different species

[0265] There is a connection 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 reaches the same binding ratio with homologous receptors of different species, the activation of immune cells caused by cytokines is also similar because the signaling pathways downstream of immune cells are conserved and similar. In order to study the dosage relationship of modified interleukin complexes between different species, this example studies its affinity with IL-2 receptors of different species. Through the ratio of affinity, the dosage used in mice can be estimated to correspond to the dosage used in other species.

[0266] The receptors of IL-2 include CD25, CD122 and CD132, wherein 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 the ratio between the KD values ​​between different species, the ratio of the affinity of the modified interleukin complex to the CD122 receptor 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, and accordingly, the dosage of the complex in species B is 10 times lower than that in species A.

[0267] This experiment uses biolayer interferometry (BLI) technology to determine the equilibrium dissociation constant (KD) between IL-2 and its receptor CD122. The measuring instrument is Octet RED96e (Sartorius), and the measuring method is carried out according to the instrument manufacturer's instructions. The specific experimental operation is as follows:

[0268] Half an hour before the start of the experiment, according to the number of samples, take an appropriate number of Protein A sensors (Sartorius, Catalog No. 18-5010, for receptor proteins containing Fc) or HIS1K sensors (Sartorius, Catalog No. 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) for pre-wetting.

[0269] The sensor position was selected according to 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), 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-volume microplate (Greiner, Cat. No. 675076). The instrument experimental procedure is as follows: Baseline 60s, Loading 1nm, Baseline 60s, Association 200s, Dissociation 400s, rotation speed 400rpm, temperature 30°C. The KD value was analyzed using ForteBio analysis software, where KD = Koff / Kon. The results are shown in the following table:

[0270] Table 13 Affinity of modified interleukin complexes to IL-2 receptor CD122 of different species

[0271]

[0272] Based on the ratio of the KD value (affinity) information listed in the table, the dose conversion relationship between different species can be estimated.

[0273] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. 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.

2. The use according to claim 1, wherein the subject is a mammal, including humans, non-human primates, cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice; 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 use according to claim 3, 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.

5. The use according to claim 3, 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.

6. The use according to claim 3, 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.

7. The use according to claim 3, 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.

8. The use according to claim 3, 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.

9. Use of an interleukin complex in the preparation of a medicament for treating solid tumors 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.

10. The use according to claim 9, 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.

11. The use according to claim 10, 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.

12. The use according to claim 10, 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.

13. The use according to claim 10, 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.

14. The use according to claim 10, 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.

15. The use according to claim 10, 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.

16. The use according to claim 3, wherein the drug for treating colorectal cancer is calculated based on the mass of the interleukin complex, and the dosage of each administration to the subject is 0.3-10 mg / kg; Preferably, the dose administered to the subject each time is 1.5-5 mg / kg; Preferably, the drug is administered to the subject once or twice a week; Preferably, the drug is administered to the subject a total of three, five or six times.

17. The use according to claim 3, wherein the drug for treating liver cancer or pancreatic cancer is calculated based on the mass of the interleukin complex, and the dosage administered to the subject each time is 1.5-5 mg / kg; Preferably, the dose administered to the subject each time is 3-5 mg / kg; Preferably, the drug is administered to the subject once a week for a total of three times.

18. The use according to claim 3, wherein the drug for treating sarcoma, prostate cancer or lung cancer is calculated based on the mass of the interleukin complex, and the dosage administered to the subject each time is 0.03-10 mg / kg; Preferably, the dose administered to the subject each time is 0.3-2.1 mg / kg; Preferably, the drug is administered to the subject once or twice a week; Preferably, the drug is administered to the subject a total of three, four or six times.

19. The use according to claim 3, wherein the drug for treating melanoma is calculated based on the mass of the interleukin complex, and the dosage of each administration to the subject is 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.

20. The use according to claim 3, wherein the drug for treating breast cancer is calculated based on the mass of the interleukin complex, and the dosage of each administration to the subject is 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.

21. The use according to claim 10, wherein the drug for treating colorectal cancer is calculated based on the mass of the interleukin complex, and the dosage for each administration to humans is 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.

22. The use according to claim 10, wherein the drug for treating liver cancer or pancreatic cancer is calculated based on the mass of the interleukin complex, and the dosage per administration to a human 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.

23. The use according to claim 10, wherein the drug for treating sarcoma, prostate cancer or lung cancer is calculated based on the mass of the interleukin complex, and the dosage for each administration to humans is 0.001-0.35 mg / kg; 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.

24. The use according to claim 10, wherein the drug for treating melanoma is calculated based on the mass of the interleukin complex, and the dosage per administration to a human 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.

25. The use according to claim 10, wherein the drug for treating breast cancer is calculated based on the mass of the interleukin complex, and the dosage per administration to a human 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.

26. The use according to any one of claims 1 to 25, 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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