Combination therapy for treating tumors and use thereof

Abstract

Provided are a combination therapy for treating tumors and a use thereof. The combination therapy comprises combined use of polyethylene glycol-modified IL-2 and rays. The use of the combination therapy can effectively reduce the tumor volume and increase the tumor growth inhibition rate, and has a significantly improved effect compared with single use of polyethylene glycol-modified IL-2 and single use of rays.

Description

A combined therapy for treating tumors and its application Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a combined therapy for treating tumors and applications thereof. Background Art

[0002] In recent years, both the incidence and mortality rates of cancer have continued to rise. The age of cancer onset is also trending younger, with most patients diagnosed in the advanced stages of the disease. Therefore, reducing the incidence and mortality rates of cancer is an urgent public health issue, and research into new and effective cancer treatments remains a major focus of cancer treatment.

[0003] Interleukin-2 (IL-2) is a cytokine derived from multiple cells and possessing pleiotropic effects. It plays a crucial role in the body's immune response and resistance to viral infection. IL-2 can activate T cells and stimulate the proliferation of T cells primed by specific antigens, promoting cytokine production. It can also stimulate the proliferation of NK cells, enhancing their cytotoxic activity and cytokine production, and induce the production of LAK cells. It can also promote B cell proliferation and antibody secretion, and activate macrophages.

[0004] Because IL-2 can induce and enhance cytotoxic activity, research on the use of IL-2 to treat certain diseases, especially tumors, has been widely conducted. The use of IL-2 alone or in combination with LAK cells has achieved certain therapeutic effects in treating tumors. IL-2 is also expected to be used in the treatment of viral infections, immunodeficiency diseases, and autoimmune diseases. In 1991, the recombinant IL-2 drug Proleukin (Aldesleukin), jointly developed by Novartis and Linigen, was approved by the FDA for the treatment of metastatic renal cancer and in 1998 for the treatment of metastatic melanoma. However, its short half-life, narrow therapeutic window, significant toxic side effects, and simultaneous stimulation of Treg activation have limited its clinical use. To improve the pharmacokinetic properties and toxic side effects of IL-2, researchers have altered the affinity between IL-2 and different receptor subunits through amino acid mutations or chemical modifications to increase activity and reduce toxicity.

[0005] In addition, the prior art also discloses that IL-2 can be used in combination with other tumor treatment methods to improve the therapeutic effect. For example:

[0006] Patent document (CN114767835A) discloses a pharmaceutical composition for treating colorectal cancer and its use. The active ingredients of the pharmaceutical composition include or consist of: polyethylene glycol-modified IL-2 and capecitabine. The combination of polyethylene glycol-modified IL-2 and capecitabine can be used to treat colon cancer. This technology combines IL-2 with chemotherapy for cancer treatment.

[0007] Radiation therapy is a localized treatment method for tumors that uses radiation. Radiation includes alpha, beta, and gamma rays produced by radioisotopes, X-rays from various X-ray therapy machines or accelerators, electron beams, fast neutrons, proton beams, negative pion beams, and heavy particle beams. Approximately 70% of cancer patients require radiation therapy during treatment, and approximately 40% of cancers are curable with radiation therapy. The role and status of radiation therapy in tumor treatment are becoming increasingly prominent, and it has become one of the mainstays of treating malignant tumors.

[0008] However, although radiotherapy can also cure tumors, large doses or long-term radiotherapy can also cause many side effects, such as increased intracranial pressure, nausea, vomiting, headache, itchy skin, dry skin, hair loss, decreased white blood cell count, decreased platelet activity, difficulty breathing, bleeding, redness and swelling of the oral or intestinal mucosal tissue, etc.

[0009] Summary of the Invention

[0010] To solve the problems of the prior art, the present invention provides a method for combining PEG-modified IL-2 with radiotherapy for the treatment of specific solid tumors (especially digestive tract tumors, lung cancer, lymphoma, skin cancer or sarcoma). Polyethylene glycol is modified at the binding site of IL-2 receptor α subunit, thereby changing the affinity between IL-2 and different receptor subunits. The PEG-modified IL-2 acts on CD8 + Effector T cells stimulate the proliferation of tumor-killing T cells while weakening the immunosuppression caused by Tregs, thereby utilizing the patient's own immune system to fight cancer; radiotherapy mainly destroys the DNA structure of tumor cells through radiation, thereby inhibiting the proliferation and growth of tumor cells and achieving the effect of alleviating the disease. The two treatment methods can complement each other and achieve synergistic therapeutic effects, such as greatly extending the half-life in the body, effectively prolonging the duration of action, and reducing toxic side effects. Moreover, the total dose, single dose, or number of radiotherapy in the combination therapy are greatly reduced, effectively reducing side effects and the degree of pain of the patient. The specific technical solution is as follows:

[0011] The first aspect of the present invention provides a use of polyethylene glycol-modified IL-2 in combination with radiation in the preparation of a product for treating tumors.

[0012] Preferably, the polyethylene glycol-modified IL-2 has the following structure:

[0013] Wherein, PEG represents polyethylene glycol residue;

[0014] L represents the linking group between PEG and D;

[0015] D represents the residue of IL-2;

[0016] n is an integer of 1-12, preferably n is an integer of 1-7, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0017] Preferably, the L has the following structure: Wherein, L1 is selected from: single bond, C1-6 alkylene, -(C1-6 alkylene)-NR L1 -, -(C1-6 alkylene)-O-, -(C1-6 alkylene)-S-, -(C1-6 alkylene)-NR L1 CO-, -(C1-6 alkylene)-CONR L1 -, -(C1-6 alkylene)-CO-, -(C1-6 alkylene)-OCO-, -(C1-6 alkylene)-NHCONR L1 -, -(C1-6 alkylene)-SO2-, -(C1-6 alkylene)-SO-; L2 is selected from: single bond, C1-6 alkylene, cycloalkylene, arylene, heterocyclylene; L3 is selected from: C1-6 alkylene, -(C1-6 alkylene)-NR L2 -, -(C1-6 alkylene)-NR L2 CO-, -(C1-6 alkylene)-CO-, -(C1-6 alkylene)-OCO-; R L1 and R L2 Independently selected from: H, C1-6 alkyl.

[0018] In some embodiments of the present invention, R L1 and R L2 Both are H.

[0019] In some embodiments of the present invention, L3 is -(C1-6 alkylene)-CO-.

[0020] In some embodiments of the present invention, L2 is a single bond.

[0021] In some embodiments of the present invention, L1 is selected from: a single bond, -(C1-6 alkylene)-NH-, -(C1-6 alkylene)-NHCO-, -(C1-6 alkylene)-CONH-, -(C1-6 alkylene)-CO-, and -(C1-6 alkylene)-OCO-.

[0022] In some embodiments of the present invention, L is -(C1-6 alkylene)-CO-, in particular -CH2CH2-CO-.

[0023] Preferably, the IL-2 is natural IL-2, recombinant IL-2, or a mutant that also possesses the functions of natural IL-2. Specifically, the attachment site of polyethylene glycol modification on IL-2 is an amino group (or amine group), such as one or more of the following: the N-terminal amino group of the peptide chain, the side chain amino group of a lysine residue within the peptide chain, the side chain amino group of an arginine residue, the secondary amino group on the imidazole ring of a histidine residue, and the secondary amino group on the indole ring of a tryptophan residue, particularly the side chain amino group of a lysine residue within the peptide chain.

[0024] Preferably, there are one or more connection sites.

[0025] In a specific embodiment of the present invention, there are two connection sites.

[0026] Preferably, the polyethylene glycol modifies the binding site of IL-2 that binds to the IL-2 receptor α subunit.

[0027] It should be understood that the polyethylene glycol-modified IL-2 refers to IL-2 in which the binding site for the IL-2 receptor α subunit is modified with polyethylene glycol.

[0028] Preferably, the molecular weight of PEG is 300-100,000 Daltons, more preferably 10,000-60,000 Daltons, for example 300, 500, 1,000, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000 or 100,000 Daltons.

[0029] Specifically, PEG can be a linear, Y-shaped, or multi-branched (eg, four-arm, six-arm, eight-arm) polyethylene glycol residue.

[0030] In one embodiment of the present invention, PEG has the structure shown in the following formula (II):

[0031] wherein i is an integer from 5 to 2500 (e.g., 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2500).

[0032] In another embodiment of the present invention, PEG has a structure as shown in the following formula (III) or formula (IV):

[0033] wherein h is an integer from 3 to 1200 (e.g., 3, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200).

[0034] In another embodiment of the present invention, the PEG has a structure as shown in the following formula (V):

[0035] in:

[0036] k is an integer from 1 to 800 (e.g., 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800);

[0037] j is an integer from 3 to 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12);

[0038] R is a core molecule of multi-branched polyethylene glycol, which can be selected from the residues of pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerol or polyglycerol.

[0039] In some embodiments of the present invention, PEG has the structure shown in the following formula (VI):

[0040] wherein q, s, t, and z are independently selected from integers ranging from 2 to 340 (e.g., 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 310, 320, 330, 340),

[0041] y is an integer of 1-5 (eg, 1, 2, 3, 4, 5).

[0042] In other embodiments of the present invention, PEG has the structure shown in the following formula (VII):

[0043] wherein u, v, and w are independently selected from integers of 2-460 (e.g., 2, 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 460),

[0044] x is an integer of 1-5 (eg, 1, 2, 3, 4, 5).

[0045] In some embodiments of the present invention, the polyethylene glycol-modified IL-2 has the following structure:

[0046] Here, n can be 1, 2, 3, or 4.

[0047] In one embodiment of the present invention, n is 2.

[0048] Specifically, the molecular weight of the polyethylene glycol residue in the polyethylene glycol-modified IL-2 represented by Formula VIII can be 10,000-60,000 Daltons, for example, 20,000 Daltons.

[0049] Specifically, the above-mentioned polyethylene glycol-modified IL-2 can be prepared (and isolated) by reacting a polyethylene glycol modifier with an IL-2 protein (especially a polyethylene glycol modifier and an IL-2 protein at a certain molar ratio), for example, in a pH neutral buffer solution with shaking at room temperature.

[0050] Preferably, the molar ratio of IL-2 protein to polyethylene glycol modifier includes 1:(3-10), preferably 1:(5-6), for example 1:3, 1:4, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0051] In some embodiments of the present invention, the polyethylene glycol modifier is For example Its molecular weight may be 300-100,000 Daltons, preferably 10,000-60,000 Daltons, for example 300, 500, 1,000, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000 or 100,000 Daltons.

[0052] Preferably, the radiation includes one or more of the following groups:

[0053] 1) Radiation produced by natural radioactive isotopes;

[0054] 2) X-rays; and / or,

[0055] 3) Electron beam, fast neutron beam, proton beam, negative pion beam or heavy particle beam.

[0056] Preferably, the rays produced by the natural radioactive isotopes 1) include one or more of α rays, β rays or γ rays.

[0057] Preferably, the radiation generated by the natural radioactive isotopes in 1) is irradiated internally or externally.

[0058] Preferably, the 2) X-rays or 3) electron beams, fast neutrons, proton beams, negative pion beams or heavy particle beams are irradiated in vitro.

[0059] Preferably, the heavy particle beam includes but is not limited to heavy particle beams such as helium, carbon, nitrogen, oxygen, and neon.

[0060] In a specific embodiment of the present invention, the rays are X-rays.

[0061] Preferably, the single dose of the polyethylene glycol-modified IL-2 is any value between 0.1 μg / kg and 1000 mg / kg, preferably any value between 1 μg / kg and 100 mg / kg, for example, 0.1 μg / kg, 1 μg / kg, 10 μg / kg, 50 μg / kg, 100 μg / kg, 200 μg / kg, 300 μg / kg, 400 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 800 μg / kg, 900 μg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, and 1000 mg / kg.

[0062] Preferably, the dose of the radiation is any value in the range of 1-100 Gy / time, preferably any value in the range of 1-60 Gy / time, and further preferably any value in the range of 1-20 Gy / time, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 Gy / time.

[0063] Preferably, the radiation is used 1-50 times, preferably 1-30 times, and more preferably 1-10 times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 times.

[0064] As is well known, the dosage of a drug can be adjusted based on the physician's experience and the patient's condition. The dosage for humans and non-human animals can also be calculated based on conventional conversion methods in the prior art.

[0065] Preferably, the tumor includes solid tumors and / or non-solid tumors.

[0066] Preferably, the tumor includes a benign tumor or a malignant tumor.

[0067] Preferably, the tumor includes one or more of lymphoma, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, digestive tract cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioma, astrocytoma, skin cancer, myelodysplastic syndrome or sarcoma.

[0068] Further preferably, the digestive tract tumor includes one or more of esophageal cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, gallbladder cancer or pancreatic cancer.

[0069] More preferably, the skin cancer includes one or more of squamous cell carcinoma, basal cell carcinoma, melanoma or eczematoid carcinoma.

[0070] Further preferably, the lung cancer includes but is not limited to non-small cell lung cancer (such as Lewis lung cancer) or small cell lung cancer.

[0071] Further preferably, the glioma includes but is not limited to glioma or glioblastoma.

[0072] Further preferably, the leukemia includes but is not limited to one or more of acute lymphocytic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia or chronic myeloid leukemia.

[0073] Further preferably, the lymphoma includes but is not limited to Hodgkin's lymphoma and non-Hodgkin's lymphoma.

[0074] Further preferably, the lymphoma includes but is not limited to one or more of B cell lymphoma (such as diffuse large B cell lymphoma or marginal zone B cell lymphoma), follicular lymphoma, mantle cell lymphoma, T cell lymphoma or Waldenstrom's macroglobulinemia.

[0075] Further preferably, the sarcoma includes but is not limited to one or more of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma or chondrosarcoma.

[0076] In a specific embodiment of the present invention, the tumor includes one or more of breast cancer (especially primary or metastatic breast cancer), lymphoma (especially B-cell lymphoma), lung cancer (especially non-small cell lung cancer, such as Lewis lung cancer), digestive tract tumors (especially rectal cancer, colon cancer, gastric cancer), skin cancer (especially melanoma) or sarcoma.

[0077] The second aspect of the present invention provides a use of polyethylene glycol-modified IL-2, lymphokine-activated killer cells (LAK cells) and radiation in combination in the preparation of a product for treating tumors.

[0078] Preferably, the definitions of polyethylene glycol-modified IL-2, radiation, and tumor are the same as those in the first aspect of the present invention.

[0079] The third aspect of the present invention provides a method for treating tumors.

[0080] The treatment method comprises administering an effective amount of polyethylene glycol-modified IL-2 to a subject in need thereof, and combining the method with radiation irradiation (preferably local irradiation).

[0081] Preferably, the dosage form of the polyethylene glycol-modified IL-2 can be an oral dosage form, such as tablets (for example, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, etc.), injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), etc.

[0082] In a specific embodiment of the present invention, the dosage form of the polyethylene glycol-modified IL-2 is an injection.

[0083] Preferably, the radiation exposure is radiotherapy.

[0084] In a specific embodiment of the present invention, the treatment method comprises administering an effective amount of polyethylene glycol-modified IL-2 to a subject in need thereof, and performing radiotherapy on the subject in need thereof.

[0085] Preferably, the radiation includes one or more of the following groups:

[0086] 1) Radiation produced by natural radioactive isotopes;

[0087] 2) X-rays; and / or,

[0088] 3) Electron beam, fast neutron beam, proton beam, negative pion beam or heavy particle beam.

[0089] Preferably, the rays produced by the natural radioactive isotopes 1) include one or more of α rays, β rays or γ rays.

[0090] Preferably, the radiation generated by the natural radioactive isotopes in 1) is irradiated internally or externally.

[0091] Preferably, the 2) X-rays or 3) electron beams, fast neutrons, proton beams, negative pion beams or heavy particle beams are irradiated in vitro.

[0092] Preferably, the heavy particle beam includes but is not limited to heavy particle beams such as helium, carbon, nitrogen, oxygen, and neon.

[0093] In a specific embodiment of the present invention, the rays are X-rays.

[0094] Preferably, the single dose of the polyethylene glycol-modified IL-2 is any value between 0.1 μg / kg and 1000 mg / kg, preferably any value between 1 μg / kg and 100 mg / kg, for example, 0.1 μg / kg, 1 μg / kg, 10 μg / kg, 50 μg / kg, 100 μg / kg, 200 μg / kg, 300 μg / kg, 400 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 800 μg / kg, 900 μg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, and 1000 mg / kg.

[0095] Preferably, the dose of the radiation is any value in the range of 1-100 Gy / time, preferably any value in the range of 1-60 Gy / time, and further preferably any value in the range of 1-20 Gy / time, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 Gy / time.

[0096] Preferably, the radiation is used 1-50 times, preferably 1-30 times, and more preferably 1-10 times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 times.

[0097] Preferably, the treatment cycle of the treatment method includes:

[0098] 1) PEG-modified IL-2, once a week or once every two weeks for 2-4 weeks;

[0099] 2) Radiation exposure five times a week or three times a week (with one day between each treatment) or once a week for 2-3 weeks.

[0100] Preferably, the treatment method also includes combination with other tumor treatment methods, such as chemotherapy, LAK cells, etc.

[0101] The definitions of polyethylene glycol-modified IL-2, radiation and tumor are the same as those in the first aspect of the present invention.

[0102] The subject of the present invention can be a human or a non-human animal, and the non-human animal includes monkeys, pigs, cows, horses, sheep, dogs, cats, mice, etc.

[0103] The polyethylene glycol-modified IL-2 described in the present invention is the same as PEG-IL2. The abbreviations of IL-2 modified with different numbers of PEG are as follows: 1 PEG-modified IL-2 is abbreviated as 1PEG-IL2, 2 PEG-modified IL-2 are abbreviated as 2PEG-IL2, and so on.

[0104] The term "alkyl" herein refers to a straight or branched hydrocarbon chain radical that does not contain an unsaturated bond and is connected to the rest of the molecule by a single bond. Typical alkyl groups contain 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, and the like.

[0105] The term "alkylene" refers to a hydrocarbon group (divalent alkyl group) formed by losing two hydrogen atoms from an alkane molecule, which can be a straight chain or branched chain and is connected to the rest of the molecule by a single bond. Typical alkylene groups contain 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably 1 to 6 carbon atoms, such as methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-), butylene, etc.

[0106] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, as well as other specified components or steps that do not substantially affect them.

[0107] The term "and / or" as used herein includes all combinations of the items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B, and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0108] The "effective amount" of the present invention refers to the amount or dose that provides the desired treatment after single or multiple doses are administered to a patient or organ, such as the amount or dose of polyethylene glycol-modified IL-2 and / or the dose of radiation.

[0109] As used herein, "treating" means slowing, stopping, arresting, controlling, halting, lessening, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0111] Figure 1: HPLC detection spectrum of PEGylation reaction products;

[0112] Figure 2: CM chromatography separation profile of PEGylation reaction products;

[0113] Figure 3: 2PEG-IL2 sample purity analysis chart;

[0114] Figure 4: Changes in colon cancer tumor volume in mice of each group;

[0115] Figure 5: Changes in lung cancer tumor volume in each group of mice;

[0116] Figure 6: Changes in lymphoma tumor volume in mice in each group. DETAILED DESCRIPTION

[0117] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0118] Example 1: Preparation of PEGylation Reaction Mixture

[0119] An appropriate amount of IL-2 protein (provided by Beijing Jiankai Technology Co., Ltd.) was exchanged into 5 mM PB pH 7.0 buffer, and a certain amount of PEG modifier (provided by Beijing Jiankai Technology Co., Ltd.) was added according to a certain reaction molar ratio (IL2 protein: PEG modifier was 1:5.3). The mixture was mixed and shaken at room temperature for 2 hours. After the reaction, the preparation of the reaction mixture was detected by HPLC.

[0120] After the PEGylation reaction, the content of each component was detected by HPLC analysis method. The results are shown in Table 1 and Figure 1 below. Among them, 2PEG-IL2 accounts for about 37%.

[0121] The structure of the PEG modifier used is shown below, with a molecular weight of 20,000 Daltons:

[0122] Table 1: Analysis of Reaction Mixture Components (UV-0217-0002)

[0123] Example 2: Separation of 2PEG-IL2 by CM chromatography

[0124] (1) 10 mM sodium acetate buffer at pH 3.0 was used as phase A, and 10 mM sodium acetate buffer at pH 3.0 containing 1 M NaCl was used as phase B.

[0125] (2) The reaction mixture obtained in Example 1 was diluted with water and the pH was adjusted to 3.0 and the conductivity to 1.5 mS / cm. The sample was loaded onto a equilibrated CM column using an AKTA chromatography system. Phase B gradient elution was performed, and the elution fraction at a phase B concentration of 8% was collected. The purity was analyzed by HPLC to obtain a sample of 2PEG-IL2.

[0126] (3) The separated 2PEG-IL2 sample was exchanged into 5 mM PB pH 7.0 buffer using a G25 desalting column for later use.

[0127] The CM chromatography separation spectrum is shown in Figure 2.

[0128] Using CM ion exchange column chromatography resin, samples conjugated with different PEGs were separated by stepwise elution using increasing salt concentrations. The peaks from left to right are >3PEG-IL2 (e.g., 3PEG-IL2, 4PEG-IL2, etc.), 2PEG-IL2, 1PEG-IL2, and IL-2. The purity of samples collected from different tubes was determined using HPLC. The 2PEG-IL2 sample was primarily concentrated in the second peak on the left.

[0129] Example 3: Alpha affinity chromatography

[0130] (1) Equilibrate the packing with 5 mM PB pH 7.0 buffer;

[0131] (2) Loading the sample obtained in Example 2 onto the affinity filler;

[0132] (3) eluting with the configured elution and collecting the elution sample;

[0133] (4) The collected eluted sample was exchanged into 5 mM PB buffer (pH 7.0) using a G25 desalting column;

[0134] (5) The sample obtained in the previous step was concentrated, sterilized by filtration with a 0.22 μm filter membrane, and the purity was detected by HPLC to obtain the final desired 2PEG-IL2. The purity analysis chart of the 2PEG-IL2 sample is shown in FIG3 .

[0135] Example 4: Effects of PEG-IL2 combined with radiotherapy on a CT26 cell transplanted tumor model in mice

[0136] 1. CT26 cells (colon cancer cells) were cultured in RPMI-1640 medium until the logarithmic growth phase, trypsinized and centrifuged, and the cell concentration was adjusted to 5×10 6 / mL.

[0137] 2. Take 100 Balb / c mice, inoculate 0.1 mL of tumor cell solution subcutaneously on the right side of each mouse, and keep them in an SPF clean room. 3 The animals were randomly divided into 6 groups, with 10 mice in each group. The specific grouping and administration methods are shown in Table 2 below. The solvent control group was given normal saline, and the radiotherapy was performed by irradiating the mouse tumor with X-rays in vitro.

[0138] Table 2: Experimental groups and drug administration methods

[0139] RT: radiation therapy; H: high dose; L: low dose; IV: intravenous injection; QW: once a week; Q2W: once every two weeks.

[0140] 3. During the experiment, the tumor growth was dynamically observed and weighed. The long diameter (a) and short diameter (b) of the tumor were measured with a vernier caliper. At the end of the experiment, the tumor-bearing mice were sacrificed, the tumor was removed, and the blood, fat and other non-tumor tissues were removed from the removed tumor. The tumor tissue was preserved in liquid nitrogen and 4% paraformaldehyde respectively. The tumor volume and tumor volume inhibition rate were calculated according to the formula: Average tumor volume (mm 3 )=ab 2 / 2; tumor volume inhibition rate (%) = (1-average tumor volume of experimental group / average tumor volume of negative control group) × 100%.

[0141] Experimental results:

[0142] The changes in tumor volume of mice in each group are shown in FIG4 , and the tumor growth inhibition rates of mice in each group on the thirteenth day after treatment are shown in Table 3 below.

[0143] Table 3: Tumor growth inhibition rate of mice in each group on the thirteenth day

[0144] From the perspective of drug efficacy: (1) During the experiment, the tumor volume of mice in the solvent control group showed a gradual upward trend, indicating that the mouse colon cancer model was successfully constructed and can be used for drug efficacy evaluation. The tumor growth rate of all treatment groups was slower than that of the solvent control group, indicating that these treatment methods can inhibit tumor growth. (2) On the 13th day after treatment, the tumor volume of mice in the 2PEG-IL2 combined with radiotherapy group was significantly smaller than that in the 2PEG-IL2 alone group and the radiotherapy alone group. The tumor growth inhibition rate in the 2PEG-IL2 combined with radiotherapy group was significantly higher than that in the 2PEG-IL2 alone group and the radiotherapy alone group, and the tumor growth inhibition effect showed a significant dose-response relationship with the dose of radiotherapy. The above results show that the anti-tumor efficacy of 2PEG-IL2 combined with radiotherapy is significantly better than that of the two treatment methods alone.

[0145] From a safety perspective: radiotherapy only affects the local tumor site, causing minimal damage to other tissues and organs. 2PEG-IL2, on the other hand, can regulate the body's immune system, effectively preventing tumor cell metastasis while killing tumor tissue.

[0146] Example 5: Effects of PEG-IL2 combined with radiotherapy on LLC mouse transplant tumor model

[0147] 1. LLC cells (mouse lung cancer cells) were inoculated into the axilla of Balb / c mice to establish a subcutaneous transplant tumor model. The cell inoculation volume was 5×10 5 / mouse.

[0148] 2. When the tumor grows to 100-200 mm 3 The group dosing started at 14:00.

[0149] 3. 2PEG-IL2 was injected into the tail vein, and the solvent control group was given normal saline. Radiotherapy was performed using an irradiator to irradiate the local tumor of the mice.

[0150] 4. Regularly observe the condition of the mice, weigh them, and measure the tumor volume.

[0151] The specific groups and administration methods are shown in Table 4 below, where radiotherapy uses X-rays:

[0152] Table 4: Experimental groups and drug administration methods

[0153] RT: radiation therapy; H: high dose; L: low dose; IV: intravenous injection; QW: once a week; Q2W: once every two weeks.

[0154] Experimental results:

[0155] The changes in tumor volume of mice in each group are shown in FIG5 , and the tumor growth inhibition rates of mice in each group on the thirteenth day after treatment are shown in Table 5 below.

[0156] Table 5 TGI values ​​of LLC mouse model 13 days after administration

[0157] From the perspective of drug efficacy: (1) During the experiment, the tumor volume of mice in the solvent control group showed a gradual upward trend, indicating that the mouse lung cancer model was successfully constructed and can be used for drug efficacy evaluation. The tumor growth rate of all treatment groups was slower than that of the solvent control group, indicating that these treatment methods can inhibit tumor growth. (2) On the 13th day after treatment, the tumor volume of mice in the 2PEG-IL2 combined with radiotherapy group was significantly smaller than that in the 2PEG-IL2 alone group and the radiotherapy alone group, and the tumor growth inhibition rate in the 2PEG-IL2 combined with radiotherapy group was significantly higher than that in the 2PEG-IL2 alone group and the radiotherapy alone group. The above results show that the anti-tumor efficacy of 2PEG-IL2 combined with radiotherapy is significantly better than that of the two treatment methods alone.

[0158] From a safety perspective, radiotherapy only targets the tumor locally, causing minimal damage to other tissues and organs. 2PEG-IL2, on the other hand, can modulate the systemic immune system, effectively preventing tumor cell metastasis while simultaneously killing tumor tissue.

[0159] Example 6: Effects of PEG-IL2 combined with radiotherapy on the A20 mouse transplant tumor model

[0160] 1. A20 cells (mouse lymphoma cells) were inoculated into the axilla of Balb / c mice to establish a subcutaneous transplant tumor model. The cell inoculation volume was 5×10 5 / mouse.

[0161] 2. When the tumor grows to 100-200 mm 3 The group dosing started at 14:00.

[0162] 3. 2PEG-IL2 was injected into the tail vein, and the solvent control group was given normal saline. Radiotherapy was performed using an irradiator to irradiate the local tumor of the mice.

[0163] 4. Regularly observe the condition of the mice, weigh them, and measure the tumor volume.

[0164] The specific groups and administration methods are shown in Table 6 below, where radiotherapy uses X-rays:

[0165] Table 6: Experimental groups and drug administration methods

[0166] RT: radiation therapy; H: high dose; L: low dose; IV: intravenous injection; QW: once a week; Q2W: once every two weeks.

[0167] Experimental results:

[0168] The changes in tumor volume of mice in each group are shown in FIG6 , and the tumor growth inhibition rates of mice in each group on the twelfth day after treatment are shown in Table 7 below.

[0169] Table 7 TGI values ​​of A20 mouse model 12 days after group administration

[0170] From the perspective of drug efficacy: (1) During the experiment, the tumor volume of mice in the solvent control group showed a gradual upward trend, indicating that the mouse lymphoma model was successfully constructed and can be used for drug efficacy evaluation. The tumor growth rate of all treatment groups was slower than that of the solvent control group, indicating that these treatment methods can inhibit tumor growth. (2) On the 12th day after treatment, the tumor volume of mice in the 2PEG-IL2 combined with radiotherapy group was significantly smaller than that in the 2PEG-IL2 alone group and the radiotherapy alone group, and the tumor growth inhibition rate in the 2PEG-IL2 combined with radiotherapy group was significantly higher than that in the 2PEG-IL2 alone group and the radiotherapy alone group. The above results show that the anti-tumor efficacy of 2PEG-IL2 combined with radiotherapy is significantly better than that of the two treatment methods alone.

[0171] From a safety perspective: radiotherapy only affects the local tumor site, causing minimal damage to other tissues and organs. 2PEG-IL2, on the other hand, can regulate the body's immune system, effectively preventing tumor cell metastasis while killing tumor tissue.

[0172] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0173] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An application of polyethylene glycol-modified IL-2 in combination with radiation in the preparation of products for treating tumors.

2. The use according to claim 1, characterized in that: The polyethylene glycol-modified IL-2 has the following structure (I): Wherein, PEG represents polyethylene glycol residue; L represents the linking group between PEG and D; D represents the residue of IL-2; n is an integer of 1-12; preferably, n is an integer of 1-7.

3. The use according to any one of claims 1-2, characterized in that: The L has the following structure: Wherein, L1 is selected from: single bond, C1-6 alkylene, -(C1-6 alkylene)-NR L1 -, -(C1-6 alkylene)-O-, -(C1-6 alkylene)-S-, -(C1-6 alkylene)-NR L1 CO-, -(C1-6 alkylene)-CONR L1 -, -(C1-6 alkylene)-CO-, -(C1-6 alkylene)-OCO-, -(C1-6 alkylene)-NHCONR L1 -, -(C1-6 alkylene)-SO2-, -(C1-6 alkylene)-SO-; L2 is selected from: single bond, C1-6 alkylene, cycloalkylene, arylene, heterocyclylene; L3 is selected from: C1-6 alkylene, -(C1-6 alkylene)-NR L2 -、-(C1-6 alkylene)-NR L2 CO-, -(C1-6 alkylene)-CO-, -(C1-6 alkylene)-OCO-; R L1 and R L2 Independently selected from: H, C1-6 alkyl; Preferably, L3 is -(C1-6 alkylene)-CO-; More preferably, L is -(C1-6 alkylene)-CO-.

4. The use according to any one of claims 1 to 3, characterized in that: The molecular weight of PEG is 300-100000 Daltons, preferably 10000-60000 Daltons; Preferably, PEG has a structure as shown in the following formula (II): wherein i is an integer from 5 to 2500; or PEG has a structure as shown in the following formula (III) or formula (IV): wherein h is an integer from 3 to 1200; or PEG has the structure shown in the following formula (V): in: k is an integer from 1 to 800; j is an integer from 3 to 12; R is a core molecule of a multi-branched polyethylene glycol, which may be selected from the residues of pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerol or polyglycerol; More preferably, PEG has the structure shown in the following formula (VI): wherein q, s, t and z are independently selected from integers of 2-340, y is an integer from 1 to 5; or, PEG has the structure shown in the following formula (VII): wherein u, v and w are independently selected from integers of 2-460, x is an integer from 1 to 5.

5. The use according to any one of claims 1 to 4, characterized in that: The polyethylene glycol-modified IL-2 has the following structure: Preferably, n is 1, 2, 3, or 4.

6. The use according to any one of claims 1 to 5, characterized in that: The IL-2 is natural IL-2, recombinant IL-2 or a mutant having the same function as natural IL-2.

7. The use according to any one of claims 1 to 6, characterized in that: The attachment site of polyethylene glycol modification on IL-2 is an amino group. Preferably, the attachment site on IL-2 includes one or more of the following: the N-terminal amino group of the peptide chain, the side chain amino group of the lysine residue in the peptide chain, the side chain amino group of the arginine residue, the secondary amine group on the imidazole ring of the histidine residue, and the secondary amine group on the indole ring of the tryptophan residue. More preferably, it is the side chain amino group of the lysine residue in the peptide chain. Preferably, the polyethylene glycol modifies the binding site in IL-2 that binds to the IL-2 receptor α subunit.

8. The use according to any one of claims 1 to 7, characterized in that: The rays include one or more of the following groups: 1) Radiation produced by natural radioactive isotopes; 2) X-rays; and / or, 3) Electron beam, fast neutron beam, proton beam, negative pion beam or heavy particle beam.

9. The use according to claim 8, characterized in that: The rays produced by the 1) natural radioactive isotopes include one or more of α rays, β rays or γ rays.

10. The use according to claim 8 or 9, characterized in that: The radiation produced by the natural radioactive isotopes described in 1) is irradiated internally or externally.

11. The use according to claim 8, characterized in that: The 2) X-rays or 3) electron beams, fast neutrons, proton beams, negative pion beams or heavy particle beams are irradiated outside the body.

12. The use according to any one of claims 1 to 11, characterized in that: The single dose of the polyethylene glycol-modified IL-2 is 0.1 μg / kg-1000 mg / kg, preferably 1 μg / kg-100 mg / kg.

13. The use according to any one of claims 1 to 12, characterized in that: The radiation dosage is 1-100 Gy / time, preferably 1-60 Gy / time, and more preferably 1-20 Gy / time.

14. The use according to claim 13, characterized in that The radiation is used 1-50 times, preferably 1-30 times, and more preferably 1-10 times.

15. The use according to any one of claims 1 to 14, characterized in that: The tumor includes solid tumor and / or non-solid tumor.

16. The use according to any one of claims 1 to 14, characterized in that: The tumor includes one or more of lymphoma, cervical cancer, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, digestive tract tumors, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioma, astrocytoma, skin cancer, myelodysplastic syndrome or sarcoma.

17. The use according to claim 16, characterized in that The digestive tract tumors include one or more of esophageal cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, gallbladder cancer or pancreatic cancer.

18. The use according to claim 16, characterized in that The lymphoma includes one or more of B cell lymphoma, follicular lymphoma, mantle cell lymphoma, T cell lymphoma or Waldenstrom's macroglobulinemia.

19. The use according to claim 16, characterized in that: The lung cancer includes non-small cell lung cancer or small cell lung cancer.

20. A method for treating a tumor, characterized in that: The treatment method comprises administering an effective amount of polyethylene glycol-modified IL-2 to a subject in need thereof, and combining the method with radiation irradiation.

21. The method of claim 20, wherein: The rays include one or more of the following groups: 1) Radiation produced by natural radioactive isotopes; 2) X-rays; and / or, 3) Electron beam, fast neutron beam, proton beam, negative pion beam or heavy particle beam.

22. The method of claim 21, characterized in that The rays produced by the 1) natural radioactive isotopes include one or more of α rays, β rays or γ rays.

23. The method of treatment according to claim 21 or 22, characterized in that The radiation produced by the natural radioactive isotopes described in 1) is irradiated internally or externally.

24. The method of claim 21, wherein: The 2) X-rays or 3) electron beams, fast neutrons, proton beams, negative pion beams or heavy particle beams are irradiated outside the body.

25. The method of any one of claims 20-24, characterized in that: The single dose of the polyethylene glycol-modified IL-2 is 0.1 μg / kg-1000 mg / kg, preferably 1 μg / kg-100 mg / kg.

26. The method of any one of claims 20-25, characterized in that: The radiation dosage is 1-100 Gy / time, preferably 1-60 Gy / time, and more preferably 1-20 Gy / time.

27. The method of claim 26, wherein: The radiation is used 1-50 times, preferably 1-30 times, and more preferably 1-10 times.

28. The method of any one of claims 20-27, characterized in that: The tumor includes solid tumor and / or non-solid tumor.

29. The method of any one of claims 20-27, characterized in that: The tumor includes one or more of lymphoma, cervical cancer, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, digestive tract tumors, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioma, astrocytoma, skin cancer, myelodysplastic syndrome or sarcoma.

30. The method of claim 29, wherein: The digestive tract tumors include one or more of esophageal cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, gallbladder cancer or pancreatic cancer.

31. The method of claim 29, wherein: The lymphoma includes one or more of B cell lymphoma, follicular lymphoma, mantle cell lymphoma, T cell lymphoma or Waldenstrom's macroglobulinemia.

32. The method of claim 29, wherein: The lung cancer includes non-small cell lung cancer or small cell lung cancer.

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