Combination therapy for treating tumors and application thereof

Abstract

The invention relates to the technical field of medicines, and particularly provides a combined therapy for treating tumors and application thereof. The combination therapy comprises combination of polyethylene glycol modified IL-2 and rays, by using the combination therapy, the tumor volume can be effectively reduced, the tumor growth inhibition rate can be increased, and the effect is remarkably improved compared with that of a polyethylene glycol modified IL-2 single-use group and a ray single-use group.

Description

Technical Field

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

[0002] In recent years, the incidence and mortality of cancer have maintained an upward trend. The age of cancer onset is also tending to be younger, and most patients are already in the middle and late stages when diagnosed. Therefore, reducing the incidence and mortality of cancer is an urgent public health issue to be solved, and researching new and effective tumor treatment methods is still the main direction of tumor treatment.

[0003] Interleukin 2 (IL-2) is a cytokine that originates from multiple cells and has multidirectional effects; it plays an important role in the body's immune response and antiviral infection. IL-2 can activate T cells and stimulate the proliferation of T cells that have been activated by specific antigens, promote cytokine production; stimulate NK cell proliferation, enhance NK killing activity and cytokine production, induce LAK cell production; promote B cell proliferation and antibody secretion, and activate macrophages.

[0004] Since IL-2 can induce and enhance cytotoxic activity, the application of IL-2 to treat certain diseases, especially tumor treatment, has been widely carried out. The use of IL-2 alone or in combination with LAK cells to treat tumors has achieved certain therapeutic effects; 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 it was approved for the treatment of metastatic melanoma. However, due to its short half-life, narrow therapeutic window, large toxic side effects, and simultaneous stimulation of Treg activation, its clinical use is limited. In order to improve the pharmacokinetic properties and toxic side effects of IL-2, researchers have changed the affinity of IL-2 and different receptor subunits by amino acid mutation or chemical modification 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 treatment effect. For example:

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

[0007] Tumor radiotherapy is a local treatment method that uses radiation to treat tumors. Radiation includes α, β, γ rays produced by radioisotopes and X-rays, electron beams, fast neutrons, proton beams, negative pion beams or heavy particle beams produced by various X-ray therapy machines or accelerators. About 70% of cancer patients need radiotherapy during the treatment of cancer, and about 40% of cancers can be cured by radiotherapy. The role and status of radiotherapy in tumor treatment are becoming increasingly prominent, and it has become one of the main means 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. Summary of the invention

[0009] In order to solve the problems of the prior art, the present invention provides a method of combining PEG-modified IL-2 with radiotherapy to treat 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 that binds to the α subunit of the IL-2 receptor, 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 and weaken the immunosuppression caused by Treg, thereby using the patient's own immune system to fight cancer; radiotherapy mainly destroys the DNA structure of tumor cells through radiation, thereby inhibiting the reproduction 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 prolonging 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 is greatly reduced, effectively reducing side effects and the degree of pain of patients. The specific technical plan is as follows:

[0010] The first aspect of the present invention provides an application of polyethylene glycol-modified IL-2 in combination with radiation in the preparation of a product for treating tumors.

[0011] Preferably, the polyethylene glycol-modified IL-2 has the following structure (I):

[0012]

[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 having the same function 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 amino group at the N-terminal end 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, especially the side chain amino group of the lysine residue in the peptide chain.

[0024] Preferably, there are one or more than two 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 in 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 the IL-2 in which the binding site for binding to the IL-2 receptor α subunit is modified by polyethylene glycol.

[0028] Preferably, the molecular weight of PEG is 300-100000 Daltons, more preferably 10000-60000 Daltons, for example 300, 500, 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000 or 100000 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 a structure as shown in the following formula (II):

[0031]

[0032] 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).

[0033] In another embodiment of the present invention, PEG has a structure as shown in the following formula (III) or formula (IV):

[0034]

[0035]

[0036] 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).

[0037] In another embodiment of the present invention, the above-mentioned PEG has a structure shown in the following formula (V):

[0038]

[0039] in:

[0040] 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);

[0041] j is an integer from 3 to 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12);

[0042] 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.

[0043] In some embodiments of the present invention, PEG has a structure as shown in the following formula (VI):

[0044]

[0045] wherein q, s, t and z are independently selected from integers of 2-340 (e.g., 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 310, 320, 330, 340),

[0046] y is an integer of 1-5 (eg, 1, 2, 3, 4, 5).

[0047] In other embodiments of the present invention, PEG has a structure as shown in the following formula (VII):

[0048]

[0049] 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),

[0050] x is an integer of 1-5 (eg, 1, 2, 3, 4, 5).

[0051] In some embodiments of the present invention, the polyethylene glycol-modified IL-2 has the following structure:

[0052]

[0053] Among them, n can be 1, 2, 3, or 4.

[0054] In one embodiment of the present invention, n is 2.

[0055] Specifically, the molecular weight of the polyethylene glycol residue in the polyethylene glycol-modified IL-2 represented by Formula VIII may be 10,000-60,000 Daltons, for example, 20,000 Daltons.

[0056] Specifically, the above-mentioned polyethylene glycol-modified IL-2 can be prepared (and separated) 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 with shaking at room temperature.

[0057] 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.

[0058] In some embodiments of the present invention, the polyethylene glycol modifier is For example The molecular weight may be 300-100000 Daltons, preferably 10000-60000 Daltons, for example 300, 500, 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000 or 100000 Daltons.

[0059] Preferably, the radiation includes one or more of the following groups:

[0060] 1) Radiation produced by natural radioactive isotopes;

[0061] 2) X-rays; and / or,

[0062] 3) Electron beam, fast neutron beam, proton beam, negative pion beam or heavy particle beam.

[0063] Preferably, the rays produced by the 1) natural radioactive isotopes include one or more of α rays, β rays or γ rays.

[0064] Preferably, the radiation produced by the natural radioactive isotopes described in 1) is irradiated internally or externally.

[0065] Preferably, the 2) X-rays or 3) electron beams, fast neutrons, proton beams, negative pion beams or heavy particle beams are irradiated in vitro.

[0066] Preferably, the heavy particle beam includes but is not limited to heavy particle beams such as helium, carbon, nitrogen, oxygen, and neon.

[0067] In a specific embodiment of the present invention, the rays are X-rays.

[0068] 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, 1000 mg / kg.

[0069] 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.

[0070] Preferably, the radiation is used 1-50 times, preferably 1-30 times, and further 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.

[0071] As is known to all, the dosage of a drug can be adjusted according to the physician's experience and the condition of the patient. The dosage for humans and non-human animals can also be calculated according to conventional conversion methods in the prior art.

[0072] Preferably, the tumor includes solid tumors and / or non-solid tumors.

[0073] Preferably, the tumor includes a benign tumor or a malignant tumor.

[0074] Preferably, the tumor includes one or more of lymphoma, cervical cancer, leukemia, 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.

[0075] 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.

[0076] More preferably, the skin cancer includes one or more of squamous cell carcinoma, basal cell carcinoma, melanoma or eczematoid carcinoma.

[0077] Further preferably, the lung cancer includes but is not limited to non-small cell lung cancer (eg Lewis lung cancer) or small cell lung cancer.

[0078] Further preferably, the glioma includes but is not limited to glioma or glioblastoma.

[0079] 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.

[0080] Further preferably, the lymphoma includes but is not limited to Hodgkin's lymphoma and non-Hodgkin's lymphoma.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] The second aspect of the present invention provides an application of polyethylene glycol-modified IL-2, lymphokine-activated killer cells (LAK cells) and radiation in the preparation of a product for treating tumors.

[0085] 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.

[0086] The third aspect of the present invention provides a method for treating tumors.

[0087] 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).

[0088] 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.

[0089] In a specific embodiment of the present invention, the dosage form of the polyethylene glycol-modified IL-2 is an injection.

[0090] Preferably, the radiation exposure is radiotherapy.

[0091] 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.

[0092] Preferably, the radiation includes one or more of the following groups:

[0093] 1) Radiation produced by natural radioactive isotopes;

[0094] 2) X-rays; and / or,

[0095] 3) Electron beam, fast neutron beam, proton beam, negative pion beam or heavy particle beam.

[0096] Preferably, the rays produced by the 1) natural radioactive isotopes include one or more of α rays, β rays or γ rays.

[0097] Preferably, the radiation produced by the natural radioactive isotopes described in 1) is irradiated internally or externally.

[0098] Preferably, the 2) X-rays or 3) electron beams, fast neutrons, proton beams, negative pion beams or heavy particle beams are irradiated in vitro.

[0099] Preferably, the heavy particle beam includes but is not limited to heavy particle beams such as helium, carbon, nitrogen, oxygen, and neon.

[0100] In a specific embodiment of the present invention, the rays are X-rays.

[0101] 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, 1000 mg / kg.

[0102] 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.

[0103] Preferably, the radiation is used 1-50 times, preferably 1-30 times, and further 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.

[0104] Preferably, the treatment cycle of the treatment method includes:

[0105] 1) PEGylated IL-2, once a week or once every two weeks for 2-4 weeks;

[0106] 2) Radiation exposure five times a week or three times a week (with one day between each exposure) or once a week for 2-3 weeks.

[0107] Preferably, the treatment method also includes combination with other tumor treatment methods, such as chemotherapy, LAK cells, etc.

[0108] The definitions of PEG-modified IL-2, radiation and tumor are the same as those in the first aspect of the present invention.

[0109] 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.

[0110] 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.

[0111] The term "alkyl" of the present invention refers to a straight or branched hydrocarbon chain radical that does not contain an unsaturated bond, and the hydrocarbon chain radical 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, etc.

[0112] The term "alkylene" refers to a hydrocarbon group (divalent alkyl group) formed by losing two hydrogen atoms from an alkane molecule, which may be a straight chain or a branched chain and which is connected to the rest of the molecule by a single bond. A typical alkylene group contains 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.

[0113] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, and other specified components or steps that will not be substantially affected.

[0114] The "and / or" described in the present invention includes all combinations of 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".

[0115] The "effective amount" described in 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 an organ, such as the amount or dose of polyethylene glycol-modified IL-2, and / or the dose of radiation irradiation.

[0116] As used herein, "treating" means to slow down, terminate, prevent, control, stop, alleviate or reverse 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-related signs, symptoms, conditions, or disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0118] Figure 1 : HPLC detection spectrum of PEGylation reaction product;

[0119] Figure 2 : CM chromatography separation spectrum of PEGylation reaction products;

[0120] Figure 3 :2PEG-IL2 sample purity analysis chart;

[0121] Figure 4 :The changes of colon cancer tumor volume in each group of mice;

[0122] Figure 5 :Changes in lung cancer tumor volume of mice in each group;

[0123] Figure 6 : Changes in lymphoma tumor volume in each group of mice. DETAILED DESCRIPTION

[0124] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0125] Example 1: Preparation of PEGylation Reaction Mixture

[0126] Take an appropriate amount of IL-2 protein (provided by Beijing Jiankai Technology Co., Ltd.), exchange it into 5mM PB pH 7.0 buffer, add a certain amount of PEG modifier (provided by Beijing Jiankai Technology Co., Ltd.) according to a certain reaction molar ratio (IL2 protein: PEG modifier is 1:5.3), mix well, and react at room temperature for 2 hours. After the reaction, the preparation of the reaction mixture is detected by HPLC.

[0127] After the PEGylation reaction was completed, the content of each component was detected by HPLC analysis method. The results are shown in Table 1 and Figure 1 As shown, 2PEG-IL2 accounts for about 37%.

[0128] The structure of the PEG modifier used is shown below, with a molecular weight of 20,000 Daltons:

[0129]

[0130] Table 1: Analysis of reaction mixture components (UV-0217-0002)

[0131] Peak Peak name Retention time Peak height Peak area content% 1 4PEG-IL2 28.457 7663.378 259121.828 1.8749 2 3PEG-IL2 29.333 43000.484 1744956.750 12.6260 3 2PEG-IL2 30.470 121325.367 5250551.500 37.9913 4 1PEG-IL2 32.112 118941.758 4828735.000 34.9392 5 IL-2 34.543 86735.430 1737025.500 12.5686

[0132] Example 2: Separation of 2PEG-IL2 by CM chromatography

[0133] (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.

[0134] (2) The reaction mixture obtained in Example 1 was diluted with water and the pH was adjusted to 3.0 and the conductivity was 1.5 mS / cm. The mixture was loaded onto a equilibrated CM column using an AKTA chromatography system. Phase B gradient elution was performed and the eluted fractions at a phase B concentration of 8% were collected. The fractions were analyzed for purity by HPLC to obtain a sample of 2PEG-IL2.

[0135] (3) The separated 2PEG-IL2 sample was exchanged into 5 mM PB pH 7.0 buffer using a G25 desalting column for later use.

[0136] CM chromatography separation spectrum Figure 2 shown.

[0137] Using CM ion exchange column chromatography resin, samples coupled with different PEGs were separated by increasing salt concentration step elution. The peaks from left to right were >3PEG-IL2 (e.g. 3PEG-IL2, 4PEG-IL2, etc.), 2PEG-IL2, 1PEG-IL2, IL-2; combined with HPLC detection method, the purity of samples from different collection tubes was determined. The 2PEG-IL2 sample was mainly concentrated in the second peak on the left.

[0138] Example 3: Alpha affinity chromatography

[0139] (1) Equilibrate the packing with 5 mM PB pH 7.0 buffer;

[0140] (2) loading the sample obtained in Example 2 onto the affinity filler;

[0141] (3) eluting with the configured elution and collecting the elution sample;

[0142] (4) The collected eluted sample was exchanged into 5 mM PB buffer (pH 7.0) using a G25 desalting column;

[0143] (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 spectrum of the 2PEG-IL2 sample is shown in FIG. Figure 3 shown.

[0144] Example 4: Effect of 2PEG-IL2 combined with radiotherapy on CT26 cell transplanted tumor model in mice

[0145] 1. CT26 cells (colon cancer cells) were cultured in RPMI-1640 medium until the logarithmic growth phase, digested with trypsin and centrifuged, and the cell concentration was adjusted to 5×10 with PBS buffer solution. 6 / mL.

[0146] 2. Take 100 Balb / c mice, inoculate 0.1 mL of tumor cell solution subcutaneously on the right back of each mouse, and keep them in an SPF clean room. When the tumor grows to 100-150 mm 3 The animals were randomly divided into 6 groups, each with 10 mice. The specific grouping and administration methods are shown in Table 2 below, where the solvent control group was given normal saline, and the radiotherapy was performed by irradiating the mouse tumor with X-rays in vitro.

[0147] Table 2: Experimental groups and drug administration methods

[0148]

[0149] Among them, RT: radiation therapy; H: high dose; L: low dose; IV: intravenous injection; QW: once a week; Q2W: once every two weeks.

[0150] 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 killed, the tumor was peeled off, and the blood, fat and other non-tumor tissues were removed from the peeled 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%.

[0151] Experimental results:

[0152] The changes of tumor volume in each group of mice were as follows Figure 4 The tumor growth inhibition rate of each group of mice on the thirteenth day after treatment is shown in Table 3 below.

[0153] Table 3: Tumor growth inhibition rate of mice in each group on the thirteenth day

[0154]

[0155]

[0156] 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 drug treatment, the tumor volume of mice in the 2PEG-IL2 combined with radiotherapy group was significantly smaller than that of the 2PEG-IL2 alone group and the radiotherapy alone group. The tumor growth inhibition rate of the 2PEG-IL2 combined with radiotherapy group was significantly higher than that of 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.

[0157] From the perspective of safety: radiotherapy only acts on the local tumor and causes little damage to other tissues and organs. 2PEG-IL2 can regulate the body's immune system, killing tumor tissue while also effectively preventing tumor cell metastasis.

[0158] Example 5: Effect of 2PEG-IL2 combined with radiotherapy on LLC mouse transplant tumor model

[0159] 1. LLC cells (mouse lung cancer cells) were inoculated in the axilla of Balb / c mice to construct a subcutaneous transplant tumor model. The cell inoculation amount was 5×10 5 / mouse.

[0160] 2. When the tumor grows to 100-200 mm 3 Start group dosing.

[0161] 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.

[0162] 4. Regularly observe the condition of the mice, weigh the mice, and measure the tumor volume.

[0163] The specific grouping and administration methods are shown in Table 4 below, where radiotherapy uses X-rays:

[0164] Table 4: Experimental groups and drug administration methods

[0165]

[0166]

[0167] Among them, RT: radiation therapy; H: high dose; L: low dose; IV: intravenous injection; QW: once a week; Q2W: once every two weeks.

[0168] Experimental results:

[0169] The changes of tumor volume in each group of mice were as follows Figure 5 The tumor growth inhibition rate of each group of mice on the thirteenth day after treatment is shown in Table 5 below.

[0170] Table 5 TGI values ​​of LLC mouse model 13 days after grouping and administration

[0171] Group TGI(D13) Solvent Control - 2PEG-IL2 62.72％ RT(L) 25.77％ RT(H) 17.60％ 2PEG-IL2+RT(L) 80.92％ 2PEG-IL2+RT(H) 70.90％

[0172] 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 of the 2PEG-IL2 alone group and the radiotherapy alone group, and the tumor growth inhibition rate of the 2PEG-IL2 combined with radiotherapy group was significantly higher than that of 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.

[0173] From the perspective of safety: radiotherapy only acts on the local tumor and causes little damage to other tissues and organs. 2PEG-IL2 can regulate the body's immune system, killing tumor tissue while also effectively preventing tumor cell metastasis.

[0174] Example 6: Effect of 2PEG-IL2 combined with radiotherapy on A20 mouse transplant tumor model

[0175] 1. A20 cells (mouse lymphoma cells) were inoculated in the axilla of Balb / c mice to construct a subcutaneous transplant tumor model. The cell inoculation amount was 5×10 5 / mouse.

[0176] 2. When the tumor grows to 100-200 mm 3 Start group dosing.

[0177] 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.

[0178] 4. Regularly observe the condition of the mice, weigh the mice, and measure the tumor volume.

[0179] The specific grouping and administration methods are shown in Table 6 below, where radiotherapy uses X-rays:

[0180] Table 6: Experimental groups and drug administration methods

[0181]

[0182] Among them, RT: radiation therapy; H: high dose; L: low dose; IV: intravenous injection; QW: once a week; Q2W: once every two weeks.

[0183] Experimental results:

[0184] The changes of tumor volume in each group of mice were as follows Figure 6 The tumor growth inhibition rate of each group of mice on the twelfth day after treatment is shown in Table 7 below.

[0185] Table 7 TGI values ​​of A20 mouse model grouping and administration 12 days later

[0186] Group TGI(D12) Solvent Control - 2PEG-IL2 83.42％ RT(L) 81.71％ RT(H) 61.17％ 2PEG-IL2+RT(L) 96.73％ 2PEG-IL2+RT(H) 88.79％

[0187] 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 of the 2PEG-IL2 alone group and the radiotherapy alone group, and the tumor growth inhibition rate of the 2PEG-IL2 combined with radiotherapy group was significantly higher than that of 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.

[0188] From the perspective of safety: radiotherapy only acts on the local tumor and causes little damage to other tissues and organs. 2PEG-IL2 can regulate the body's immune system, killing tumor tissue while also effectively preventing tumor cell metastasis.

[0189] 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0190] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable 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, preferably including one or more of α-rays, β-rays or γ-rays; 2) X-rays; and / or, 3) Electron beam, fast neutron beam, proton beam, negative pion beam or heavy particle beam; Preferably, the radiation generated by the natural radioactive isotopes is irradiated internally or externally; Preferably, the 2) X-rays or 3) electron beams, fast neutrons, proton beams, negative pion beams or heavy particle beams are irradiated in vitro.

9. The use according to any one of claims 1 to 8, 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.

10. The use according to any one of claims 1 to 9, characterized in that: The radiation dose is 1-100 Gy / time, preferably 1-60 Gy / time, and more preferably 1-20 Gy / time; Preferably, the radiation is used 1-50 times, more preferably 1-30 times, and even more preferably 1-10 times.

11. The use according to any one of claims 1 to 10, characterized in that: The tumor includes solid tumor and / or non-solid tumor.

12. The use according to any one of claims 1 to 10, characterized in that: The tumor includes one or more of lymphoma, cervical cancer, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, digestive tract tumor, 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; 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; Preferably, the lymphoma includes one or more of B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, T-cell lymphoma or Waldenstrom's macroglobulinemia; Preferably, the lung cancer includes non-small cell lung cancer or small cell lung cancer.

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