WO2020135683A1 - 一种制备结合位点可控的peg化生物分子的方法 - Google Patents

一种制备结合位点可控的peg化生物分子的方法 Download PDF

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WO2020135683A1
WO2020135683A1 PCT/CN2019/129052 CN2019129052W WO2020135683A1 WO 2020135683 A1 WO2020135683 A1 WO 2020135683A1 CN 2019129052 W CN2019129052 W CN 2019129052W WO 2020135683 A1 WO2020135683 A1 WO 2020135683A1
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peg
receptor
preparation
receptors
integer
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French (fr)
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王庆彬
齐杰
李玉
赵宣
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天津键凯科技有限公司
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Priority to JP2021556649A priority Critical patent/JP2022515298A/ja
Priority to EP19904564.2A priority patent/EP3896079A4/en
Priority to US17/418,671 priority patent/US20220125939A1/en
Publication of WO2020135683A1 publication Critical patent/WO2020135683A1/zh

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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
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Definitions

  • the invention belongs to the technical field of medicine, and in particular relates to a method for preparing PEGylated biomolecules with controllable binding sites.
  • Interleukin-2 is produced when mitogens such as phytohemagglutinin (PHA) stimulate lymphocyte proliferation.
  • PHA phytohemagglutinin
  • IL-2 is mostly produced by CD4+ T helper cells (Th), and a few are produced by CD8+ and other immune cells. These cells can secrete IL-2 under the action of IL-1.
  • IL-2 produced by lymphocytes acts on cells with IL-2 receptors on the surface, so that the cells proliferate to exert their biological functions, and can act on the precursor cells of normal T, B lymphocytes, NK cells, and cytotoxic T cells (CTL) and precursor cells of lymphokine-activated killer cells.
  • CTL cytotoxic T cells
  • the biological functions of IL-2 include: 1. Maintain long-term survival of activated T cells in vitro and maintain their biological functional activity. Some T cells activated by tumor antigens can enhance their ability to kill tumor cells under the action of IL-2. Function, strengthen anti-tumor effect; 2.
  • IL-2 is the earliest immunotherapy drug. As early as 1992 and 1998, the US FDA approved the IL-2 drug Aldesleukin for advanced kidney cancer and malignant melanoma, with an effective rate of 15%-20%, which can make nearly 10%-15% The patient's survival period exceeds 5 years. However, due to the high doses used clinically, IL-2 has significant side effects, such as severe hypotension and vascular leakage syndrome. At that time, patients treated with IL-2 had to be hospitalized and watched closely for 24 hours. If they were not careful, they would be in danger of life. Therefore, huge side effects limit the use of IL-2, and most clinicians do not dare to touch it easily.
  • IL-2 is mainly produced by CD4+ T cells stimulated by antigen, CD8+ T cells, NK cells and DC cells can also be secreted. High doses of IL-2 can promote the differentiation of CD4+ T cells, the proliferation of CD8+ T cells and NK cells, increase their lethality and thereby tune the body’s immune response. In turn, low-dose IL-2 will promote the proliferation of regulatory T cells and suppress the immune system, so low-dose IL-2 is also often used to treat autoimmune diseases.
  • IL-2 receptors have three subunits that constitute ⁇ , ⁇ , and ⁇ subunits.
  • CD8 positive T cells and NK cells and other killer cell surface receptors are mainly composed of ⁇ and ⁇ subunits (IL-2R ⁇ ).
  • This receptor has a low affinity for IL-2 and requires a large dose of IL-2 to activate.
  • the surface of regulatory T cells is mostly high-affinity antibody (IL-2R ⁇ ) composed of three subunits of ⁇ , ⁇ , and ⁇ , and IL-2 is easily activated by low dose to play an immunosuppressive effect.
  • IL-2 was approved for the treatment of renal cell tumors and metastatic melanoma as early as more than 20 years ago, but it has been in the cold palace of immunotherapy and is rarely used. The main reasons are as follows: 1. The efficiency is low, the effective rate of IL-2 alone can only reach about 15-20%; 2. The functional duality of IL-2, low dose IL-2 promotes regulation The proliferation of T cells causes immunosuppression, and how to reconcile these two functions remains to be resolved; 3. The half-life is short, only a few minutes, and large doses must be given to continue to work; 4. Serious adverse reactions, over-activated immune system It may attack your own organs and cause organ failure. The dose window is relatively narrow.
  • NKTR-214 was born.
  • NKTR-214 is not mysterious. It is essentially an upgraded version of the old drug IL-2 that was launched more than 20 years ago. It has lower side effects and better specificity (it is easier to activate CD8 instead of Treg cells) and can be activated more efficiently. immune system. Specifically, 6 polyethylene glycol (PEG) modifications were added to the IL-2 molecule to form an inactive drug; magically, after injection to tumor patients, these 6 PEG modifications will gradually fall off, forming a Active 2-PEG and 1-PEG forms.
  • PEG polyethylene glycol
  • NKTR-214 is PEGylated IL-2, the activity of the original drug is very low. Six PEG chains are cleverly connected to the IL-2R ⁇ high affinity antibody ⁇ subunit in IL-2, making the PEGylated IL-2 more likely to bind IL-2R ⁇ in killer immune cells Receptor-like. As PEG is degraded one by one, the activity of the drug is slowly released. NKTR-214 solves the two major pain points of IL-2 in tumor treatment: one is the slow release of PEG to achieve the effect of drug release, which greatly improves tolerance; the second is to improve the selectivity of killer immune cells , Increase the expression of immune cell PD-1 in the microenvironment.
  • NKTR-214 Nektar announced on Nasdaq on October 7, 2015 that it has submitted a new drug application for NKTR-214 to the US Food and Drug Administration (FDA). The company has completed pre-clinical studies of NKTR-214 and plans to initiate phase I and phase II clinical studies of the drug by the end of 2015.
  • FDA US Food and Drug Administration
  • NKTR-214 compared with the existing IL-2 treatment drug adileukin, after a single dose administration, the AUC value in NKTR-214 tumors is nearly 400 times that of adileukin.
  • the data provided by Nektar Company shows that NKTR-214 shows long-term anti-tumor immunotherapy effect in multiple preclinical animal models; in tumor infiltrating lymphocyte model, after treatment with NKTR-214, CD8 is positive
  • the ratio of T cells to CD4 positive T cells is about 450:1, showing a potent tumor killing effect.
  • NKTR-214 is an anti-cancer drug of the immune stimulating factor class, which has a therapeutic effect similar to that of antibodies, and can stimulate the growth of T cells, thereby achieving the killing effect on tumors.
  • the results of pre-clinical experiments show that the drug has a long-acting anti-tumor therapeutic effect and is a highly anticipated anti-cancer drug.
  • the normal IL-2 molecule is chemically modified by adding 6 PEGs to form NKTR-214, thereby achieving IL-2 selective binding to IL-2R ⁇ receptors.
  • the applicant provides a method for preparing a PEGylated biomolecule with a controllable binding site, and human intervention is performed on the binding site of the biomolecule in vitro to keep the binding site exposed and not PEGylated In order to make biomolecules more targeted to enter the body, the therapeutic effect is more rapid. Specifically, when modifying IL-2, by regulating the binding site of IL-2, only one or two PEGs are added for PEGylation, so that IL-2 selectively binds to IL-2R ⁇ receptors.
  • the object of the present invention is to provide a method for preparing PEGylated biomolecules with controllable binding sites. Specifically, the present invention provides a method for preparing PEGylated IL-2 with controllable binding sites.
  • a method for preparing PEGylated biomolecules with controllable binding sites includes the following steps:
  • the barrier binds to at least one binding site in the biomolecule
  • the biomolecule in the step (1) is selected from biomacromolecules or small molecules having a specific binding effect with the barrier, and the biomolecule has at least one binding site of the barrier.
  • the barrier and the biomolecule are selected from the following combinations: ligand and receptor, DNA and its complementary DNA or RNA, enzyme and its substrate, enzyme and its competitive inhibitor, enzyme and its coenzyme factor, vitamin and its specific binding protein, glycoprotein Corresponding to the lectin and so on.
  • the ligand and receptor are selected from hormones and receptors, drugs and receptors, and the like.
  • the DNA and the complementary DNA or RNA are selected from the group consisting of a gene sequence whose gene probe is complementary to a base.
  • the enzyme and its substrate are selected from: protease and protein, amylase and starch, nuclease and nucleic acid, lactate dehydrogenase and lactic acid, oxaloacetate decarboxylase and oxaloacetate.
  • the enzyme and its competitive inhibitors are selected from the group consisting of succinate dehydrogenase and malonate, dihydrofolate synthase and sulfonamides, cholinesterase and organophosphorus pesticides, thiolase and Lewis gas.
  • the enzyme and its coenzyme factors are selected from pyruvate dehydrogenase and Mn2 + , catalase and oxidoreductase and Fe2 + /Fe3 + .
  • the hormone and receptor are selected from the group consisting of estrogen and estrogen receptor, androgen and androgen receptor, mineralocorticoid and mineralocorticoid receptor, thyroid hormone and thyroid hormone receptor, progesterone and progesterone receptor .
  • the drugs and receptors are selected from the group consisting of insulin, insulin-like growth factor, epithelial growth factor, platelet growth factor and lymphokine and receptors with tyrosine kinase activity, epinephrine, dopamine, serotonin, M-acetylcholine, Opioids, purines, prostaglandins and peptide hormone drugs and G-protein coupled receptors.
  • the drugs and receptors are selected from folate and folate receptors, interleukins and interleukin receptors.
  • the drugs and receptors are selected from: interleukin-2 and interleukin-2 receptors.
  • Specific interleukin-2 receptors include interleukin ⁇ , ⁇ , and ⁇ receptors.
  • the vitamin and its specific binding protein are selected from: vitamin A and retinol binding protein, vitamin D and vitamin D binding protein, ⁇ -tocopherol and ⁇ -tocopherol transport protein, vitamin K and lipoprotein.
  • glycoprotein and its corresponding lectin are selected from: horseradish peroxidase and concanavalin lectin A, lentil lectin and pea lectin, lignin and galactose, amaranthaceae lectin and N-acetylgalactose Amine, dimer chitin binding lectin and N-acetylglucosamine oligosaccharide, lentin and blood group A substance, Jingdou lectin and blood group O substance 2-L-fucose.
  • the PEGylated biomolecule in the step (2) has the following structure:
  • X is a linking group between PEG and biomolecule, selected from: -(CH 2 )a-, -(CR 1 R 2 )a-, -(CH 2 )aNH-, -NHCO(CH 2 )a- , - (CH 2) aCONH - , - (CH 2) aCO -, - CO (CH 2) a -, - (CH 2) aCONH (CH 2) a -, - (CH 2) aSS- (CH 2) Combination of one or more of a-, -(CH 2 )aCOO(CH 2 )a-, -(CH 2 )aS-(CH 2 )a-.
  • a is an integer of 0-10.
  • a is an integer of 0-5, such as 0, 1, 2, 3, 4 or 5. More preferably, it is an integer of 0-3.
  • n is an integer of 1-12, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
  • R 1 and R 2 are independently selected from: -H, C 1-6 alkyl, -OR', -NHR', -N(R') 2 , -CN, -F, -Cl, -Br,- A combination of one or more of I, -COR', -COOR', -OCOR', -CONHR', or -CON(R') 2 .
  • R 1 and R 2 are independently selected from: H, C 1-3 alkyl, specifically may be methyl, ethyl, n-propyl or isopropyl, -OH, C 1-3 alkoxy A combination of one or more of the group, -NH 2 , -F, -Cl, -Br, and -I. More preferably from: H, -CH 3 , -OH, -OCH 3 and -OCH 2 CH 3 . In one embodiment of the present invention, R 1 is -H, and R 2 is selected from: -CH 3 , -OH, -OCH 3 and -OCH 2 CH 3 .
  • R' is selected from: -H, C 1-6 alkyl, -F, -Cl, -Br or -I.
  • R' is selected from the group consisting of: -H and C 1-3 alkyl, which may specifically be methyl, ethyl, n-propyl or isopropyl.
  • the X is selected from: -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CONHCH 2 -, -CH 2 CONHCH 2 CH 2 -, -CH 2 CONHCH 2 CH 2 NH-, -CH 2 CH 2 CONHCH 2 -, -CH 2 CO-, -CH 2 CH 2 CO-, -CH 2 CH 2 CONHCH 2 CH 2 -, -CH 2 NH-, -CH 2 CONH -, -COCH 2 -, -COCH 2 CH 2 -, -COCH 2 CH 2 CH 2 -, -CH 2 -SS-CH 2 -, -CH 2 COOCH 2 -, -CH 2 -S-CH 2- One or more combinations.
  • X is selected from: -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CONHCH 2 -, -CH 2 CH 2 CO-, -CH 2 COOCH 2 -.
  • the PEG is a linear, Y-shaped, multi-branched polyethylene glycol residue, including, for example, monomethoxy polyethylene glycol (mPEG), linear double-ended PEG, Y-shaped PEG, 4-arm branched PEG, 6-arm branched PEG or 8-arm branched PEG, etc.
  • mPEG monomethoxy polyethylene glycol
  • Y-shaped PEG 4-arm branched PEG
  • 6-arm branched PEG 6-arm branched PEG or 8-arm branched PEG, etc.
  • the molecular weight of the PEG may be 1-100KDa, such as 1-10KDa (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10KDa), 10-50KDa (specifically 10, 15, 20, 25, 30, 35, 40, 45 or 50KDa) or 50-100KDa (specifically 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100KDa), etc. ; Further preferably 10-50KDa.
  • the PEG is a linear polyethylene glycol residue and has a structure represented by Formula II or III:
  • p and q are independently selected integers of 1-2280, preferably integers of 220-1140,
  • Y is an end-capping group, selected from: H, C 1-6 alkyl, specifically methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl , N-hexyl, etc., C 3-6 cycloalkyl, specifically cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., C 6-10 aryl, specifically phenyl, naphthyl, etc.,- LT,
  • L is a linking group between oxygen (O) and terminal group T, selected from: -(CH 2 ) b -, -(CR 3 R 4 ) b -, -(CH 2 ) b NH-, -NHCO( CH 2 ) b -, -(CH 2 ) b CONH- and -CO(CH 2 ) b -one or more combinations, b is an integer of 0-10.
  • R 3 and R 4 are independently selected from: -H, C 1-6 alkyl, -OR", -NHR", -N(R") 2 , -CN, -F, -Cl, -Br,- A combination of one or more of I, -COR', -COOR", -OCOR", -CONHR", and -CON(R") 2 .
  • R" is selected from: -H, C 1-6 alkyl, -F, -Cl, -Br and -I.
  • L is selected from: -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CONH-, -NH-, -CO-, -CONHCH 2 -, -CH 2 NH- , -CH 2 CONH- and -COCH 2 -one or more combinations.
  • T is an end group, selected from: H, C 1-6 alkyl, C 3-6 cycloalkyl, C 6-10 aryl, monosaccharide, specifically glucose, fructose, galactose, ribose, deoxyribose, etc. , Oligosaccharides, specifically can be disaccharide (sucrose, lactose, etc.), trisaccharide (gentiotriose, raffinose, etc.) residues.
  • T is selected from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl,
  • the Y is selected from the group consisting of methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl,
  • Y is methyl
  • the Y is
  • the PEG is a Y-shaped polyethylene glycol residue and has a structure represented by the general formula IV or V:
  • i and h are independently integers selected from 1-1140, preferably integers from 110-570.
  • Y has the same definition as in the above general formula II, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • the PEG is a multi-branched polyethylene glycol residue and has a structure represented by Formula VI:
  • k is an integer of 1-760, preferably an integer of 70-380.
  • j is an integer of 3-8.
  • Q is the core molecule of multi-branched polyethylene glycol.
  • Q is selected from the group consisting of pentaerythritol, oligomeric pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerol and polyglycerol.
  • Q is selected from: Pentaerythritol, dipentaerythritol, and tripentaerythritol.
  • the multi-branched polyethylene glycol residue has the following structure:
  • Q, k and j have the same definitions as the above general formula VI
  • Y has the same definition as the above general formula II, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, Cyclohexyl, benzyl, More preferably from: methyl,
  • the multi-branched polyethylene glycol residue has the following structure:
  • w is an integer of 1-570, preferably an integer of 55-285;
  • t is an integer of 1-10 (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably an integer of 1-6;
  • Y has the same definition as in the above general formula II, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • the multi-branched polyethylene glycol residue has the following structure:
  • s is an integer of 1-280, preferably an integer of 28-140.
  • y is an integer of 1-10, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably an integer of 1-5, and more preferably an integer of 1-3.
  • Y has the same definition as in the above general formula II, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • the method for separating the barrier from the biomolecule in the step (3) includes one of centrifugal separation, precipitation separation, filtration separation, foam separation, extraction separation, membrane separation, chromatography separation, electrophoretic separation, and gradient elution Or a combination of two or more.
  • a PEGylated biomolecule with a controllable binding site prepared as described above.
  • a pharmaceutical composition prepared by a PEGylated biomolecule with a controllable binding site and a pharmaceutically acceptable carrier prepared as described above.
  • a pharmaceutical composition prepared by a PEGylated biomolecule with a controllable binding site and a pharmaceutically acceptable carrier prepared as described above is used for preparing a medicine for treating and/or preventing diseases.
  • the biomolecule is IL-2
  • the barrier is IL-2 ⁇ receptor (IL-2R ⁇ ).
  • the present invention provides a method for preparing PEGylated IL-2 with a controllable binding site, including the following steps:
  • the present invention provides a method for preparing PEGylated IL-2 with a controllable binding site, including the following steps:
  • a PEGylated IL-2 with a controllable binding site prepared as described above has the following structure:
  • n 1 or 2; preferably, n is 1.
  • X and PEG are the same as defined above.
  • X is a linking group between PEG and IL-2, selected from: -(CH 2 )a-, -(CR 1 R 2 )a-, -(CH 2 )aNH-, -NHCO(CH 2 )a -, - (CH 2) aCONH -, - (CH 2) aCO -, - CO (CH 2) a -, - (CH 2) aCONH (CH 2) a -, - (CH 2) aSS- (CH 2 ) A-, -(CH 2 )aCOO(CH 2 )a-, -(CH 2 )aS-(CH 2 )a- One or more combinations.
  • a is an integer of 0-10.
  • a is an integer of 0-5, such as 0, 1, 2, 3, 4 or 5. More preferably, it is an integer of 0-3.
  • R 1 and R 2 are independently selected from: -H, C 1-6 alkyl, -OR', -NHR', -N(R') 2 , -CN, -F, -Cl, -Br,- A combination of one or more of I, -COR', -COOR', -OCOR', -CONHR', or -CON(R') 2 .
  • R 1 and R 2 are independently selected from: H, C 1-3 alkyl, specifically may be methyl, ethyl, n-propyl or isopropyl, -OH, C 1-3 alkoxy A combination of one or more of the group, -NH 2 , -F, -Cl, -Br, and -I. More preferably from: H, -CH 3 , -OH, -OCH 3 and -OCH 2 CH 3 . In one embodiment of the present invention, R 1 is -H, and R 2 is selected from: -CH 3 , -OH, -OCH 3 and -OCH 2 CH 3 .
  • R' is selected from: -H, C 1-6 alkyl, -F, -Cl, -Br or -I.
  • R' is selected from the group consisting of: -H and C 1-3 alkyl, which may specifically be methyl, ethyl, n-propyl or isopropyl.
  • the X is selected from: -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CONHCH 2 -, -CH 2 CONHCH 2 CH 2 -, -CH 2 CONHCH 2 CH 2 NH-, -CH 2 CH 2 CONHCH 2 -, -CH 2 CO-, -CH 2 CH 2 CO-, -CH 2 CH 2 CONHCH 2 CH 2 -, -CH 2 NH-, -CH 2 CONH -, -COCH 2 -, -COCH 2 CH 2 -, -COCH 2 CH 2 CH 2 -, -CH 2 -SS-CH 2 -, -CH 2 COOCH 2 -, -CH 2 -S-CH 2- One or more combinations.
  • the X is selected from: -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CONHCH 2 -, -CH 2 CH 2 CO-, -CH 2 COOCH 2 -.
  • the PEG is a linear, Y-shaped, multi-branched polyethylene glycol residue, including, for example, monomethoxy polyethylene glycol (mPEG), linear double-ended PEG, Y-shaped PEG, 4-arm branched PEG, 6-arm branched PEG or 8-arm branched PEG, etc.
  • mPEG monomethoxy polyethylene glycol
  • Y-shaped PEG 4-arm branched PEG
  • 6-arm branched PEG 6-arm branched PEG or 8-arm branched PEG, etc.
  • the molecular weight of the PEG may be 1-100KDa, such as 1-10KDa (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10KDa), 10-50KDa (specifically 10, 15, 20, 25, 30, 35, 40, 45 or 50KDa) or 50-100KDa (specifically 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100KDa), etc. ; Further preferably 10-50KDa.
  • the PEG is a linear polyethylene glycol residue and has a structure represented by Formula II or III:
  • p and q are independently selected integers of 1-2280, preferably integers of 220-1140,
  • Y is an end-capping group, selected from: H, C 1-6 alkyl, specifically methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl , N-hexyl, etc., C 3-6 cycloalkyl, specifically cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., C 6-10 aryl, specifically phenyl, naphthyl, etc.,- LT,
  • L is a linking group between oxygen (O) and terminal group T, selected from: -(CH 2 ) b -, -(CR 3 R 4 ) b -, -(CH 2 ) b NH-, -NHCO( CH 2 ) b -, -(CH 2 ) b CONH- and -CO(CH 2 ) b -one or more combinations, b is an integer of 0-10.
  • R 3 and R 4 are independently selected from: -H, C 1-6 alkyl, -OR", -NHR", -N(R") 2 , -CN, -F, -Cl, -Br,- A combination of one or more of I, -COR', -COOR", -OCOR", -CONHR", and -CON(R") 2 .
  • R" is selected from: -H, C 1-6 alkyl, -F, -Cl, -Br and -I.
  • L is selected from: -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CONH-, -NH-, -CO-, -CONHCH 2 -, -CH 2 NH- , -CH 2 CONH- and -COCH 2 -one or more combinations.
  • T is an end group, selected from: H, C 1-6 alkyl, C 3-6 cycloalkyl, C 6-10 aryl, monosaccharide, specifically glucose, fructose, galactose, ribose, deoxyribose, etc. , Oligosaccharides, specifically can be disaccharide (sucrose, lactose, etc.), trisaccharide (gentiotriose, raffinose, etc.) residues.
  • T is selected from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl,
  • the Y is selected from the group consisting of methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl,
  • Y is methyl
  • the Y is
  • the PEG is a Y-shaped polyethylene glycol residue and has a structure represented by the general formula IV or V:
  • i and h are independently integers selected from 1-1140, preferably integers from 110-570.
  • Y has the same definition as in the above general formula II, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • the PEG is a multi-branched polyethylene glycol residue and has a structure represented by Formula VI:
  • k is an integer of 1-760, preferably an integer of 70-380.
  • j is an integer of 3-8.
  • Q is the core molecule of multi-branched polyethylene glycol.
  • Q is selected from the group consisting of pentaerythritol, oligomeric pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerol and polyglycerol.
  • Q is selected from: Pentaerythritol, dipentaerythritol, and tripentaerythritol.
  • the multi-branched polyethylene glycol residue has the following structure:
  • Q, k and j have the same definitions as the above general formula VI
  • Y has the same definition as the above general formula II, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, Cyclohexyl, benzyl, More preferably from: methyl,
  • the multi-branched polyethylene glycol residue has the following structure:
  • w is an integer of 1-570, preferably an integer of 55-285,
  • t is an integer of 1-10 (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably an integer of 1-6,
  • Y has the same definition as in the above general formula II, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • the multi-branched polyethylene glycol residue has the following structure:
  • s is an integer of 1-280, preferably an integer of 28-140.
  • y is an integer of 1-10, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably an integer of 1-5, and more preferably an integer of 1-3.
  • Y has the same definition as in the above general formula II, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • a pharmaceutical composition prepared by a PEGylated IL-2 with a controllable binding site and a pharmaceutically acceptable carrier prepared as described above is used for preparing a medicine for treating and/or preventing diseases.
  • the disease is a tumor, autoimmune disease, viral disease or bacterial disease.
  • the tumor diseases are renal cell carcinoma, melanoma, malignant hemangioendothelioma, cutaneous T cell tumor, ovarian cancer, breast cancer, bladder cancer, lung cancer, glioma, neuroblastoma, liver cancer, hairy cell leukemia, Myeloid leukemia, colon cancer, cancerous pleural and peritoneal effusion or non-Hodgkin's lymphoma.
  • the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, or Sjogren's syndrome.
  • the viruses are hepatitis virus, papilloma virus, simple herpes simplex virus (HSV), human immunodeficiency virus (HIV), human herpes virus 4 (EBv), coronavirus and influenza virus, more preferably hepatitis virus, such as Hepatitis B virus (HBV) or Hepatitis C virus (HCV).
  • HSV simple herpes simplex virus
  • HAV human immunodeficiency virus
  • EBv human herpes virus 4
  • coronavirus and influenza virus more preferably hepatitis virus, such as Hepatitis B virus (HBV) or Hepatitis C virus (HCV).
  • the bacterial disease is leprosy or tuberculosis.
  • interleukin 2 can be natural interleukin 2, recombinant protein (such as recombinant human interleukin 2) or the same Mutants with natural IL-2 function (such as "cloning of recombinant human interleukin-2 (IL-2) mutant and expression and purification in Pichia pastoris system", Liu Yan, PhD thesis, Zhongsuo
  • IL-2-C125A/L18M/L19S also includes products obtained by tissue culture, protein synthesis, cell culture (natural, recombinant cells or mutants) methods. Methods for extraction and isolation of natural, recombinant IL-2 or mutants are well known to those skilled in the art.
  • the term "pharmaceutically acceptable” refers to physiological compatibility after administration to humans and does not cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions.
  • the additive may be any of excipients, disintegrants, binders, lubricants, suspending agents, stabilizers, and the like.
  • excipients include lactose, mannitol, isose, microcrystalline cellulose, siliconized microcrystalline cellulose, powdered cellulose, and the like.
  • disintegrants include low-substituted hydroxypropyl cellulose, crospovidone, sodium starch glycolate, croscarmellose sodium, starch, and the like.
  • binders include hydroxypropyl cellulose, hypromellose, povidone, copovidone, pregelatinized starch, and the like.
  • lubricants include stearic acid, magnesium stearate, sodium fumarate and the like;
  • wetting agents include polyoxyethylene sorbitan fatty acid esters, poloxamers, polyoxyethylene castors Sesame oil derivatives and so on.
  • suspending agents include hypromellose, hydroxypropyl cellulose, povidone, copovidone, sodium carboxymethyl cellulose, methyl cellulose, and the like.
  • stabilizers include citric acid, fumaric acid, succinic acid, and the like.
  • the pharmaceutical composition of the present invention may further include any of anticoagulants, flavor enhancers, emulsifiers, preservatives, and the like.
  • the pharmaceutical composition of the present invention may be tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, oral disintegrating tablets, oral tablets, etc.), pills, powders, granules, capsules (Including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled release formulations (e.g., instant release formulations, sustained release formulations, sustained release microcapsules), aerosols, film formulations ( For example, oral disintegrating film, oral mucosa-adhesive film), injection (for example, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous infusion, transdermal absorption preparation, ointment, washing Agents, adhesive preparations, suppositories (eg, rectal suppositories, vaginal suppositories), small pills, nasal preparations, lung preparations (inhalation), eye drops, etc., oral or parenteral preparations (eg, intravenous
  • the pharmaceutically acceptable auxiliary materials of the present invention are preferably pharmaceutically acceptable injection auxiliary materials, such as isotonic sterile saline solution (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, chloride Calcium, magnesium chloride, etc., or a mixture of the above salts), or dried, for example, a freeze-dried composition, which suitably forms an injectable solute by adding sterile water or physiological saline.
  • isotonic sterile saline solution sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, chloride Calcium, magnesium chloride, etc., or a mixture of the above salts
  • a freeze-dried composition which suitably forms an injectable solute by adding sterile water or physiological saline.
  • the PEGylated IL-2 of the present invention can be abbreviated as PEG-IL-2.
  • Figure 1 The chromatogram of PEGylation of IL-2 on the receptor affinity column.
  • FIG. 1 Electrophoresis results of PEG-IL-2, where 1 is PEG-IL-2 and 2 is protein standard.
  • FIG. 4 SDS-PAGE results of PEG-IL-2, where 1 is protein standard, 2 is IL-2, 3 is peak 2, and 4 is peak 1.
  • Tris-HCl tris(hydroxymethyl)aminomethane
  • S10 Fill sepharose resin into a 5ml empty column and store in a refrigerator at 4°C.
  • Phase A 5mM PBS, pH 7.0
  • Phase B 0.2M acetic acid solution containing 0.2M NaCl
  • Phase A is balanced to a stable UV curve
  • the gel filtration chromatography settings are as follows: mobile phase: 5MmPBS, flow rate: 0.75ml/min, column: superdex200increase10/300GL.
  • the peak 1 (PEG-IL-2) and peak 2 (IL-2) samples in Figure 4 were collected separately, and after concentration, the affinity test was performed with an SPR instrument.
  • the receptor proteins are IL-2 ⁇ receptor and IL-2 ⁇ receptor. The test results are as follows:

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Abstract

本发明公开了一种制备结合位点可控的PEG化生物分子的方法，包括如下步骤：(1)阻隔物与生物分子结合；(2)PEG化生物分子；(3)将阻隔物与生物分子分离。另一方面，本发明公开了一种制备结合位点可控的PEG化IL-2的方法，包括如下步骤：(1)将IL-2与IL-2α受体结合，封闭IL-2的α结合位点；(2)PEG化，PEG与IL-2偶联；(3)将IL-2与IL-2α受体分离。通过对IL-2的结合位点进行调控，PEG化过程中只添加1或2个PEG，使IL-2选择性的结合IL-2Rβγ类受体。

Description

一种制备结合位点可控的PEG化生物分子的方法 技术领域
本发明属于医药技术领域，具体的涉及一种制备结合位点可控的PEG化生物分子的方法。
背景技术
白细胞介素-2(IL-2)是由植物血凝素(PHA)等分裂原刺激淋巴细胞增殖时产生的。近来对该因子及其受体的研究日益深入，发现它在细胞免疫、体液免疫及免疫调节中起重要作用。特别是在肿瘤免疫治疗中的作用及临床意义受到许多作者的重视。
IL-2大多数由CD4+的T辅助细胞(Th)产生的，少数由CD8+及其它免疫细胞产生。这些细胞在IL-1作用下能分泌IL-2。淋巴细胞产生的IL-2作用于表面带IL-2受体的细胞，使细胞增殖而发挥其生物学功能，可作用于正常T、B淋巴细胞、NK细胞的前体细胞、细胞毒T细胞(CTL)以及淋巴因子活化的杀伤细胞的前体细胞等。IL-2的生物学功能包括：1，维持活化后T细胞体外长期生存，并保持其生物学功能活性，一些受肿瘤抗原激活的T细胞在IL-2作用下，可增强其杀伤肿瘤细胞的功能，加强抗肿瘤作用；2，促进B细胞增殖，分化并产生抗体；3，刺激粒细胞-巨噬细胞集落刺激因子及γ-干扰素等的分泌，促进淋巴细胞表达这些受体；维特亚剂量促分裂原存在时胸腺的增殖；促进IL-2受体表达，参与机体免疫调节系统，维持免疫平衡和稳定。
IL-2是最早的免疫治疗药物。早在1992和1998年，美国FDA就批准了IL-2药物阿地白介素(Aldesleukin)用于晚期肾癌和恶性黑色素瘤，有效率在15％-20％，能使近10％-15％的患者存活期超过5年。不过，由于临床使用的剂量很高，IL-2的副作用很大，比如严重的低血压和血管渗漏综合征。在当时，使用IL-2治疗的患者必须住院，24小时密切观察，稍有不慎就有生命危险。所以，巨大的副作用限制了IL-2的使用，多数临床医生并不敢轻易碰它。
IL-2的作用机理如下：IL-2主要由经抗原刺激后的CD4+T细胞产生，CD8+T细胞，NK细胞和DC细胞也可以分泌。高剂量的IL-2可以促进CD4+T细胞的分化，CD8+T 细胞和NK细胞的增殖，增加他们的杀伤力从而调动机体的免疫反应。反过来，低剂量的IL-2会促进调节性T细胞的增殖，对免疫系统起到抑制的作用，因此低剂量的IL-2也常常用来治疗自身免疫性疾病。
之所以有此不同主要是因为免疫细胞表面IL-2受体分布不同，IL-2受体共有三个亚基构成α，β，γ亚基。CD8阳性T细胞和NK细胞等起杀伤性细胞表面受体主要是β和γ亚基构成(IL-2Rβγ)，该受体对IL-2亲和力较低，需要大剂量IL-2才能激活。而调节性T细胞表面多为α，β，γ三个亚基组成的高亲和抗体(IL-2Rαβγ)，低剂量的IL-2更容易激活他们从而起到免疫抑制作用。
IL-2早在20多年前就被批准治疗肾细胞瘤和转移性黑色素瘤，却一直身处免疫治疗的冷宫，鲜有使用。究其原因主要是以下几点：1，有效率低，单用IL-2的有效率只能达到15-20％左右；2，IL-2功能双面性，低剂量IL-2促进调节性T细胞的增殖引起免疫抑制，如何调和这两个功能尚待解决；3，半衰期短，只有几分钟，想要持续起效必须要大剂量给药；4，不良反应严重，过度激活的免疫系统有可能会攻击自身脏器造成器官衰竭，剂量窗口比较窄。为了提高IL-2的疗效，降低不良反应，科学家们还尝试了各种联用方案，比如IL-2+干扰素，IL-2+淋巴因子激活的杀伤(LAK)细胞疗法，IL-2+化疗等，效果均不理想。
最近几年，科学家们发现：在正常的IL-2分子上面添加化学修饰，可以维持IL-2的效果，同时降低使用浓度，也降低副作用。于是，NKTR-214就诞生了。NKTR-214并不神秘，本质上是一个20多年前就上市的老药IL-2的升级版，副作用更低，特异性更好(更容易激活CD8而不是Treg细胞)，可以更高效的激活免疫系统。具体的是在IL-2分子上添加6个聚乙二醇(PEG)修饰，形成了无活性的药物；神奇的是，给肿瘤患者注射之后，这6个PEG修饰会逐渐脱落，形成了有活性的2-PEG和1-PEG的形式。
NKTR-214是PEG化的IL-2，原药的活性很低。六个PEG链被巧妙的连接在IL-2中可以结合IL-2Rαβγ高亲和抗体α亚基的部位，使得PEG化后的IL-2更倾向于结合在杀伤性免疫细胞中的IL-2Rβγ类受体。随着PEG逐个被降解掉，药物的活性得到缓慢 的释放。NKTR-214解决了IL-2在肿瘤治疗中的两大痛点：一是通过PEG降级达到了药物缓释的效果，从而大大提高了耐受性；二是提高了对杀伤性免疫细胞的选择性，增加了微环境中免疫细胞PD-1的表达量。
Nektar公司于2015年10月7日在纳斯达克宣布，已向美国食品药品监督管理局(FDA)提交NKTR-214的新药申请。该公司已完成NKTR-214的临床前研究，并计划在2015年底启动该药物的一期和二期临床研究。
在临床前研究中，NKTR-214与现有的IL-2治疗药物阿地白介素相比，单剂量给药后，NKTR-214肿瘤内的AUC值是阿地白介素的近400倍。此外，Nektar公司提供的数据表明，NKTR-214在多个临床前动物模型中均表现出长效的抗肿瘤免疫治疗效果；在肿瘤浸润型淋巴细胞模型中，经NKTR-214处理后，CD8阳性T细胞与CD4阳性T细胞的比值约为450:1，表现出了强效的肿瘤杀伤作用。因此，NKTR-214是免疫刺激因子类抗癌药物，有着与抗体相近的治疗效应，可刺激T细胞的生长，从而实现对肿瘤的杀伤作用。临床前实验结果表明该药物具有长效抗肿瘤的治疗效果，是一种极具期待性的抗癌药物。
但是在实际生产过程中，正常的IL-2分子上面通过化学修饰添加6个PEG形成NKTR-214，从而实现IL-2选择性的结合IL-2Rβγ类受体。在本发明中，申请人提供一种制备结合位点可控的PEG化生物分子的方法，在体外对生物分子的结合位点进行人为干预，保持结合位点暴露且不被PEG化的状态下，使生物分子进入体内靶向性更强，发挥治疗效果更迅速。具体的在对IL-2进行修饰时，通过对IL-2的结合位点进行调控，PEG化只添加1或2个PEG，使IL-2选择性的结合IL-2Rβγ类受体。
发明内容
本发明的目的是提供一种制备结合位点可控的PEG化生物分子的方法，具体的，本发明提供一种制备结合位点可控的PEG化IL-2的方法。
本发明的目的是通过以下技术方案实现的：
一种制备结合位点可控的PEG化生物分子的方法，包括如下步骤：
(1)阻隔物与生物分子中至少一个结合位点结合；
(2)PEG化生物分子；
(3)将阻隔物与生物分子分离。
所述步骤(1)中生物分子选自与阻隔物具有特异性结合作用的生物大分子或小分子，所述生物分子上具有至少一个阻隔物的结合位点。阻隔物与生物分子选自如下组合：配体与受体、DNA与其互补的DNA或RNA、酶与其底物、酶与其竞争性抑制剂、酶与其辅酶因子、维生素与其特异性结合蛋白、糖蛋白与其相应的凝集素等。优选的，所述配体与受体选自：激素与受体、药物与受体等。
所述DNA与其互补的DNA或RNA选自：基因探针与碱基互补的基因序列。
所述酶与其底物选自：蛋白酶与蛋白质、淀粉酶与淀粉、核酸酶与核酸、乳酸脱氢酶与乳酸、草酰乙酸脱羧酶与草酰乙酸。
所述酶与其竞争性抑制剂选自：琥珀酸脱氢酶与丙二酸、二氢叶酸合成酶与磺胺类药物、胆碱酯酶与有机磷农药、巯基酶与路易士气。
所述酶与其辅酶因子选自：丙酮酸脱氢酶与Mn2 +、过氧化氢酶和氧化还原酶与Fe2 +/Fe3 +。
所述激素与受体选自：雌激素与雌激素受体、雄激素与雄激素受体、盐皮质激素与盐皮质激素受体、甲状腺激素与甲状腺激素受体、孕激素与孕激素受体。
所述药物与受体选自：胰岛素、胰岛素样生长因子、上皮生长因子、血小板生长因子及淋巴因子与具有酪氨酸激酶活性的受体、肾上腺素、多巴胺、5-羟色胺、M-乙酰胆碱、阿片类、嘌呤类、前列腺素及多肽激素类药物与G-蛋白偶联受体。
优选的，所述药物与受体选自：叶酸与叶酸受体、白介素与白介素受体。
更优选的，所述药物与受体选自：白介素-2与白介素-2受体，具体的白介素-2受体有白介素α、β、γ受体。
所述维生素与其特异性结合蛋白选自：维生素A与视黄醇结合蛋白、维生素D与维生素D结合蛋白、α-生育酚与α-生育酚转运蛋白、维生素K与脂蛋白。
所述糖蛋白与其相应的凝集素选自：辣根过氧化物酶与刀豆凝集素A、扁豆凝集素和 豌豆凝集素、木菠萝素与半乳糖、苋科凝集素与N-乙酰半乳糖胺、二聚体几丁质结合凝集素与N-乙酰氨基葡萄糖寡聚糖、双花扁豆素与血型A物质、荆豆凝集素与血型O物质2-L-岩藻糖。
所述步骤(2)中PEG化的生物分子具有如下结构：
Figure PCTCN2019129052-appb-000001
X为PEG与生物分子之间的连接基团，选自：-(CH 2)a-、-(CR 1R 2)a-、-(CH 2)aNH-、-NHCO(CH 2)a-、-(CH 2)aCONH-、-(CH 2)aCO-、-CO(CH 2)a-、-(CH 2)aCONH(CH 2)a-、-(CH 2)a-S-S-(CH 2)a-、-(CH 2)aCOO(CH 2)a-、-(CH 2)a-S-(CH 2)a-中的一种或多种的组合。
a为0-10的整数。优选的，所述a为0-5的整数，如0、1、2、3、4或5。更优选为0-3的整数。
m为1-12的整数，具体可为1、2、3、4、5、6、7、8、9、10、11或12。
R 1和R 2独立地选自：-H、C 1-6的烷基、-OR'、-NHR'、-N(R') 2、-CN、-F、-Cl、-Br、-I、-COR'、-COOR'、-OCOR'、-CONHR'或-CON(R') 2中的一种或多种的组合。优选的，R 1和R 2独立地选自：H、C 1-3的烷基，具体可为甲基、乙基、正丙基或异丙基、-OH、C 1-3的烷氧基、-NH 2、-F、-Cl、-Br和-I中的一种或多种的组合。更优选自：H、-CH 3、-OH、-OCH 3和-OCH 2CH 3。在本发明的一个实施例中，R 1为-H，R 2选自：-CH 3、-OH、-OCH 3和-OCH 2CH 3。
R'选自：-H、C 1-6的烷基、-F、-Cl、-Br或-I。优选的，R'选自：-H和C 1-3的烷基，具体可为甲基、乙基、正丙基或异丙基。
优选的，所述X选自：-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CONHCH 2-、-CH 2CONHCH 2CH 2-、-CH 2CONHCH 2CH 2NH-、-CH 2CH 2CONHCH 2-、-CH 2CO-、-CH 2CH 2CO-、-CH 2CH 2CONHCH 2CH 2-、-CH 2NH-、-CH 2CONH-、-COCH 2-、-COCH 2CH 2-、-COCH 2CH 2CH 2-、-CH 2-S-S-CH 2-、-CH 2COOCH 2-、-CH 2-S-CH 2-中的一种或多种的组合。
更优选的，所述X选自：-CH 2-、-CH 2CH 2-、-CH 2CH 2CONHCH 2-、-CH 2CH 2CO-、 -CH 2COOCH 2-。
所述PEG为直链、Y型、多分支的聚乙二醇残基，例如包括单甲氧基聚乙二醇(mPEG)、直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG等。
优选地，所述的PEG的分子量可为1-100KDa，例如1-10KDa(具体可为1、2、3、4、5、6、7、8、9或10KDa)、10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)或50-100KDa(具体可为50、55、60、65、70、75、80、85、90、95或100KDa)等；进一步优选为10-50KDa。
在本发明的具体实施方式中，所述PEG为直链聚乙二醇残基，具有通式Ⅱ或Ⅲ所示的结构：
Figure PCTCN2019129052-appb-000002
其中，p和q独立的选自1-2280的整数，优选为220-1140的整数，
Y为封端基，选自：H、C 1-6烷基，具体可为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、正己基等、C 3-6环烷基，具体可为环丙基、环丁基、环戊基、环己基等、C 6-10芳基，具体可为苯基、萘基等、-L-T，
L为氧(O)和端基T之间的连接基团，选自：-(CH 2) b-、-(CR 3R 4) b-、-(CH 2) bNH-、-NHCO(CH 2) b-、-(CH 2) bCONH-和-CO(CH 2) b-中的一种或多种的组合，b为0-10的整数。
R 3和R 4独立地选自：-H、C 1-6的烷基、-OR”、-NHR”、-N(R”) 2、-CN、-F、-Cl、-Br、-I、-COR'、-COOR”、-OCOR”、-CONHR”和-CON(R”) 2中的一种或多种的组合。
R”选自：-H、C 1-6的烷基、-F、-Cl、-Br和-I。
优选的，L选自：-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CONH-、-NH-、-CO-、-CONHCH 2-、 -CH 2NH-、-CH 2CONH-和-COCH 2-中的一种或多种的组合。
T为端基，选自：H、C 1-6烷基、C 3-6环烷基、C 6-10芳基、单糖，具体可为葡萄糖、果糖、半乳糖、核糖、脱氧核糖等、低聚糖，具体可为二糖(蔗糖、乳糖等)、三糖(龙胆三糖、棉子糖等)残基。
优选的，T选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000003
Figure PCTCN2019129052-appb-000004
优选的，所述Y选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000005
在本发明的一个优选实施例中，所述Y为甲基。
在本发明的另一个优选实施例中，所述Y为
Figure PCTCN2019129052-appb-000006
Figure PCTCN2019129052-appb-000007
在本发明的具体实施方式中，所述PEG为Y型聚乙二醇残基，具有通式Ⅳ或Ⅴ所示的结构：
Figure PCTCN2019129052-appb-000008
其中，i和h独立的选自1-1140的整数，优选为110-570的整数。
Y具有与上述通式Ⅱ中相同的定义，优选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000009
更优选自：甲基、
Figure PCTCN2019129052-appb-000010
在本发明的具体实施方式中，所述PEG为多分支聚乙二醇残基，具有通式Ⅵ所示的结构：
Figure PCTCN2019129052-appb-000011
其中，k是1-760的整数，优选为70-380的整数。
j是3-8的整数。
Q是多分支聚乙二醇的核心分子，Q选自：季戊四醇、寡聚季戊四醇、甲基葡萄 糖苷、蔗糖、二甘醇、丙二醇、甘油和聚甘油的残基，优选的，Q选自：季戊四醇、二聚季戊四醇和三聚季戊四醇。
优选地，所述多分支聚乙二醇残基，其具有如下结构：
Figure PCTCN2019129052-appb-000012
其中，Q、k与j具有与上述通式Ⅵ相同的定义，Y具有与上述通式Ⅱ中相同的定义，优选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000013
Figure PCTCN2019129052-appb-000014
更优选自：甲基、
Figure PCTCN2019129052-appb-000015
在本发明一个优选实施例中，所述多分支聚乙二醇残基具有如下结构：
Figure PCTCN2019129052-appb-000016
其中，w是1-570的整数，优选为55-285的整数；
t为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-6的整数；
其中，Y具有与上述通式Ⅱ中相同的定义，优选自：甲基、乙基、异丙基、环丙基、 环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000017
更优选自：甲基、
Figure PCTCN2019129052-appb-000018
在本发明另一个优选实施例中，所述多分支聚乙二醇残基具有如下结构：
Figure PCTCN2019129052-appb-000019
其中，s是1-280的整数，优选为28-140的整数。
y为1-10的整数，具体可为1、2、3、4、5、6、7、8、9或10，优选为1-5的整数，更优选为1-3的整数。
Y具有与上述通式Ⅱ中相同的定义，优选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000020
更优选自：甲基、
Figure PCTCN2019129052-appb-000021
所述步骤(3)中将阻隔物与生物分子分离的方法包括：离心分离、沉淀分离、过滤分离、泡沫分离、萃取分离、膜分离、层析分离、电泳分离、梯度洗脱中的一种或两种以上的组合。
一种如上述方法制备的结合位点可控的PEG化生物分子。
一种如上述方法制备的结合位点可控的PEG化生物分子与药学上可接受的载体制备的药物组合物。
一种如上述方法制备的结合位点可控的PEG化生物分子与药学上可接受的载体制备的药物组合物在制备治疗和/或预防疾病的药物中的应用。
在本发明的一个优选实施方案中，所述生物分子为IL-2，所述阻隔物为IL-2α受体(IL-2Rα)。
具体的，本发明提供一种制备结合位点可控的PEG化IL-2的方法，包括如下步骤：
(1)将IL-2与IL-2α受体结合；
(2)PEG化，PEG与IL-2偶联；
(3)将IL-2与IL-2α受体分离。
优选的，本发明提供一种制备结合位点可控的PEG化IL-2的方法，包括如下步骤：
(1)制备IL-2α受体亲和柱；
(2)IL-2在亲和柱上与IL-2α受体结合；
(3)PEG化，PEG与IL-2偶联；
(4)通过梯度洗脱将IL-2与IL-2α受体分离。
一种如上述方法制备的结合位点可控的PEG化IL-2具有如下结构：
Figure PCTCN2019129052-appb-000022
n为1或2；优选的，n为1。
X和PEG与前述定义相同。X为PEG与IL-2之间的连接基团，选自：-(CH 2)a-、-(CR 1R 2)a-、-(CH 2)aNH-、-NHCO(CH 2)a-、-(CH 2)aCONH-、-(CH 2)aCO-、-CO(CH 2)a-、-(CH 2)aCONH(CH 2)a-、-(CH 2)a-S-S-(CH 2)a-、-(CH 2)aCOO(CH 2)a-、-(CH 2)a-S-(CH 2)a-中的一种或多种的组合。
a为0-10的整数。优选的，所述a为0-5的整数，如0、1、2、3、4或5。更优选为0-3的整数。
R 1和R 2独立地选自：-H、C 1-6的烷基、-OR'、-NHR'、-N(R') 2、-CN、-F、-Cl、-Br、-I、-COR'、-COOR'、-OCOR'、-CONHR'或-CON(R') 2中的一种或多种的组合。优选的，R 1和R 2独立地选自：H、C 1-3的烷基，具体可为甲基、乙基、正丙基或异丙基、-OH、C 1-3的烷氧基、-NH 2、-F、-Cl、-Br和-I中的一种或多种的组合。更优选自：H、-CH 3、-OH、-OCH 3和-OCH 2CH 3。在本发明的一个实施例中，R 1为-H，R 2选自：-CH 3、-OH、-OCH 3和-OCH 2CH 3。
R'选自：-H、C 1-6的烷基、-F、-Cl、-Br或-I。优选的，R'选自：-H和C 1-3的烷基，具体可为甲基、乙基、正丙基或异丙基。
优选的，所述X选自：-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CONHCH 2-、-CH 2CONHCH 2CH 2-、-CH 2CONHCH 2CH 2NH-、-CH 2CH 2CONHCH 2-、-CH 2CO-、-CH 2CH 2CO-、-CH 2CH 2CONHCH 2CH 2-、-CH 2NH-、-CH 2CONH-、-COCH 2-、-COCH 2CH 2-、-COCH 2CH 2CH 2-、-CH 2-S-S-CH 2-、-CH 2COOCH 2-、-CH 2-S-CH 2-中的一种或多种的组合。
更优选的，所述X选自：-CH 2-、-CH 2CH 2-、-CH 2CH 2CONHCH 2-、-CH 2CH 2CO-、-CH 2COOCH 2-。
所述PEG为直链、Y型、多分支的聚乙二醇残基，例如包括单甲氧基聚乙二醇(mPEG)、直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG 等。
优选地，所述的PEG的分子量可为1-100KDa，例如1-10KDa(具体可为1、2、3、4、5、6、7、8、9或10KDa)、10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)或50-100KDa(具体可为50、55、60、65、70、75、80、85、90、95或100KDa)等；进一步优选为10-50KDa。
在本发明的具体实施方式中，所述PEG为直链聚乙二醇残基，具有通式Ⅱ或Ⅲ所示的结构：
Figure PCTCN2019129052-appb-000023
其中，p和q独立的选自1-2280的整数，优选为220-1140的整数，
Y为封端基，选自：H、C 1-6烷基，具体可为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、正己基等、C 3-6环烷基，具体可为环丙基、环丁基、环戊基、环己基等、C 6-10芳基，具体可为苯基、萘基等、-L-T，
L为氧(O)和端基T之间的连接基团，选自：-(CH 2) b-、-(CR 3R 4) b-、-(CH 2) bNH-、-NHCO(CH 2) b-、-(CH 2) bCONH-和-CO(CH 2) b-中的一种或多种的组合，b为0-10的整数。
R 3和R 4独立地选自：-H、C 1-6的烷基、-OR”、-NHR”、-N(R”) 2、-CN、-F、-Cl、-Br、-I、-COR'、-COOR”、-OCOR”、-CONHR”和-CON(R”) 2中的一种或多种的组合。
R”选自：-H、C 1-6的烷基、-F、-Cl、-Br和-I。
优选的，L选自：-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CONH-、-NH-、-CO-、-CONHCH 2-、-CH 2NH-、-CH 2CONH-和-COCH 2-中的一种或多种的组合。
T为端基，选自：H、C 1-6烷基、C 3-6环烷基、C 6-10芳基、单糖，具体可为葡萄糖、果糖、半乳糖、核糖、脱氧核糖等、低聚糖，具体可为二糖(蔗糖、乳糖等)、三糖(龙胆 三糖、棉子糖等)残基。
优选的，T选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000024
Figure PCTCN2019129052-appb-000025
优选的，所述Y选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000026
在本发明的一个优选实施例中，所述Y为甲基。
在本发明的另一个优选实施例中，所述Y为
Figure PCTCN2019129052-appb-000027
Figure PCTCN2019129052-appb-000028
在本发明的具体实施方式中，所述PEG为Y型聚乙二醇残基，具有通式Ⅳ或Ⅴ所示的结构：
Figure PCTCN2019129052-appb-000029
Figure PCTCN2019129052-appb-000030
其中，i和h独立的选自1-1140的整数，优选为110-570的整数。
Y具有与上述通式Ⅱ中相同的定义，优选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000031
更优选自：甲基、
Figure PCTCN2019129052-appb-000032
在本发明的具体实施方式中，所述PEG为多分支聚乙二醇残基，具有通式Ⅵ所示的结构：
Figure PCTCN2019129052-appb-000033
其中，k是1-760的整数，优选为70-380的整数。
j是3-8的整数。
Q是多分支聚乙二醇的核心分子，Q选自：季戊四醇、寡聚季戊四醇、甲基葡萄糖苷、蔗糖、二甘醇、丙二醇、甘油和聚甘油的残基，优选的，Q选自：季戊四醇、二聚季戊四醇和三聚季戊四醇。
优选地，所述多分支聚乙二醇残基，其具有如下结构：
Figure PCTCN2019129052-appb-000034
其中，Q、k与j具有与上述通式Ⅵ相同的定义，Y具有与上述通式Ⅱ中相同的定义，优选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000035
Figure PCTCN2019129052-appb-000036
更优选自：甲基、
Figure PCTCN2019129052-appb-000037
在本发明一个优选实施例中，所述多分支聚乙二醇残基具有如下结构：
Figure PCTCN2019129052-appb-000038
其中，w是1-570的整数，优选为55-285的整数，
t为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-6的整数，
其中，Y具有与上述通式Ⅱ中相同的定义，优选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000039
更优选自：甲 基、
Figure PCTCN2019129052-appb-000040
在本发明另一个优选实施例中，所述多分支聚乙二醇残基具有如下结构：
Figure PCTCN2019129052-appb-000041
其中，s是1-280的整数，优选为28-140的整数。
y为1-10的整数，具体可为1、2、3、4、5、6、7、8、9或10，优选为1-5的整数，更优选为1-3的整数。
Y具有与上述通式Ⅱ中相同的定义，优选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
Figure PCTCN2019129052-appb-000042
更优选自：甲基、
Figure PCTCN2019129052-appb-000043
一种如上述方法制备的结合位点可控的PEG化IL-2与药学上可接受的载体制备的药物组合物。
一种如上述方法制备的结合位点可控的PEG化IL-2与药学上可接受的载体制备的药物组合物在制备治疗和/或预防疾病的药物中的应用。
优选的，所述的疾病为肿瘤、自身免疫疾病、病毒性疾病或细菌性疾病。
所述肿瘤疾病为肾细胞癌、黑色素瘤、恶性血管内皮细胞瘤、皮肤T细胞瘤、卵巢癌、乳腺癌、膀胱癌、肺癌、神经胶质瘤、神经母细胞瘤、肝癌、毛细胞白血病、髓样母细胞白血病、结肠癌、癌性胸腹腔积液或非霍奇金淋巴瘤。
所述自身免疫疾病为类风湿关节炎、系统性红斑狼疮或干燥综合征。
所述病毒为肝炎病毒、乳头状病毒、单纯孢疹病毒(HSV)、人类免疫缺陷病毒(HIV)、人类孢疹病毒4型(EBv)、冠状病毒和流感病毒，更优选为肝炎病毒，如乙型肝炎病毒(HBV)或丙型肝炎病毒(HCV)。
所述细菌性疾病为麻风病或肺结核。
除非另有定义，本发明中所使用的所有的技术和科学术语具有与本发明涉及领域的技术人员通常理解的相同的含义，如：
本发明中术语“白细胞介素2”、“白介素2”、“IL-2”具有相同的含义，可互换使用，其可以是天然的白介素2、重组蛋白(如重组人白介素2)或同样具有天然IL-2功能的突变体(如“重组人白介素-2(IL-2)突变体克隆及在巴斯德毕赤酵母系统中的表达与纯化”，刘堰，博士学位论文，中所述的“IL-2-C125A/L18M/L19S”)，也包括通过组织培养、蛋白质合成、细胞培养(天然、重组细胞或突变体)方法得到的产品。天然、重组IL-2或突变体的提取和分离方法是本领域技术人员所熟知的。
如本文所用，“药学上可接受”一词是指在对人施用后具有生理相容性并且不会引起肠胃失调、诸如头晕的过敏反应或类似反应。添加剂可为赋形剂、崩解剂、粘结剂、润滑剂、悬浮剂、稳定剂等等中的任一种。赋形剂的例子包括乳糖、甘露醇、益寿糖、微晶纤维素、硅化微晶纤维素、粉状纤维素等等。崩解剂的例子包括低取代羟丙基纤维素、交聚维酮、羧基乙酸淀粉钠、交联羧甲基纤维素钠、淀粉等等。粘结剂的例子包括羟丙基纤维素、羟丙甲纤维素、聚维酮、共聚维酮、预胶凝淀粉等等。润滑剂的例子包括硬脂酸、硬脂酸镁、 富马酰硬脂酸钠等等；润湿剂的例子包括聚氧乙烯山梨糖醇酐脂肪酸酯、泊洛沙姆、聚氧乙烯蓖麻油衍生物等等。悬浮剂的例子包括羟丙甲纤维素、羟丙基纤维素、聚维酮、共聚维酮、羧甲基纤维素钠、甲基纤维素等等。稳定剂的例子包括柠檬酸、富马酸、琥珀酸等等。另外，本发明的药物组合物还可包括阻凝剂、增味剂、乳化剂、防腐剂等等中的任一种。
本发明所述的药物组合物可以为片剂(包括糖衣片剂、膜包衣片剂、舌下片剂、口腔崩解片、口腔片剂等等)、丸剂、粉剂、颗粒剂、胶囊剂(包括软胶囊、微胶囊)、锭剂、糖浆剂、液体、乳剂、混悬剂、控制释放制剂(例如，瞬时释放制剂、缓释制剂、缓释微囊)、气雾剂、膜剂(例如，口服崩解膜剂、口腔粘膜-粘附膜剂)、注射剂(例如，皮下注射、静脉注射、肌内注射、腹膜内注射)、静脉滴注剂、透皮吸收制剂、软膏剂、洗剂、粘附制剂、栓剂(例如，直肠栓剂、阴道栓剂)、小药丸、鼻制剂、肺制剂(吸入剂)、眼睛滴剂等等、口服或胃肠外制剂(例如，静脉内、肌内、皮下、器官内、鼻内、皮内、滴注、脑内、直肠内等给药形式、给药至肿瘤的附近和直接给药至病变处)。优选的，所述的药物组合物为注射剂。
本发明所述的药学上可接受的辅料优选为药学上可接受的注射剂辅料，例如等渗的无菌盐溶液(磷酸二氢钠、磷酸氢二钠、氯化钠、氯化钾、氯化钙、氯化镁等，或上述盐的混合物)，或干燥的例如是冷冻干燥的组合物，其适当地通过加入无菌水或生理盐水形成可注射溶质。
本发明所述的PEG化IL-2可简写为PEG-IL-2。
附图说明
图1 IL-2在受体亲和柱上PEG化的色谱图。
图2 PEG-IL-2的电泳结果，其中1为PEG-IL-2，2为蛋白标准品。
图3 PEG-IL-2的凝胶色谱分离图谱。
图4 PEG-IL-2的SDS-PAGE结果，其中1为蛋白标准品，2为IL-2，3为峰2，4为峰1。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 IL-2α受体亲和柱的制备
S1：称取0.8g CNBr活化的琼脂糖凝胶(sehparose CNBr)加入12ml 1mM HCl，手动缓慢混合均匀，放4℃冰箱反应15min；
S2：配置偶联buffer：0.1M NaHCO 3pH 8.3与0.5M NaCl充分混合，取12ml偶联buffer洗上步溶胀的填料；
S3：取4瓶共4mg IL-2α受体，溶于2ml偶联buffer加入到步骤2溶好的填料中；
S4：室温下缓慢摇转2h；
S5：用5倍柱体积的偶联buffer洗掉多余的受体；
S6：加入0.1M pH 8.0的三(羟甲基)氨基甲烷(Tris-HCl)，室温放置2h，倒掉Tris-HCl；
S7：用5倍柱体积的含有0.5M NaCl的0.1M pH 4.0醋酸/醋酸钠缓冲液洗，然后用5倍柱体积的含有0.5M NaCl的0.1M pH 8.0的Tris-HCl缓冲液洗；
S8：重复步骤6和7，重复3次；
S9：加入0.5ml 0.1M pH 8.0的Tris-HCl；
S10：将sepharose树脂灌装至5ml的空柱子中，4℃冰箱保存。
实施例2 IL-2在受体亲和柱上PEG化
S1：将5ml受体亲和柱安装在蛋白纯化系统(AKTA purifier)上；
S2：配制流动相，A相：5mM PBS，pH7.0；B相：含有0.2M NaCl的0.2M醋酸溶液；
S3：设置流动相流速为0.45ml/min；
S4：A相平衡至UV曲线平稳；
S5：将IL-2样品经进样环进样，IL-2上的α受体结合位点与IL-2α受体结合；
S6：取适量的分子量为20KDa的甲氧基聚乙二醇-丙酸琥珀酰亚胺酯(M-SPA-20K)溶解在pH 8.0 2.5mM PB中，其终浓度为40mg/ml，抽取2ml M-SPA-20K经进样环进样，平衡至UV曲线平稳；
S7：重复步骤6，重复3次；
S8：梯度洗脱，色谱图如图1所示，收集洗脱峰，经超滤浓缩后用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行检测。
实施例3 PEG-IL-2凝胶过滤色谱分离
凝胶过滤色谱设置如下：流动相：5Mm PBS，流速：0.75ml/min，柱子：superdex 200increase 10/300GL。
分离步骤如下：S1：将色谱系统平衡至UV曲线平稳；S2：样品上样后收集洗脱峰；S3：收集组分超滤浓缩后SDS-PAGE检测，结果如图3和图4所示。
检测结果：经SDS-PAGE确认图4中的峰1为PEG-IL-2，峰2为IL-2。
实施例4 受体亲和力测试
分别收集图4中的峰1(PEG-IL-2)和峰2(IL-2)样品，浓缩后用SPR仪器进行亲和力测试。受体蛋白分别为IL-2α受体，IL-2β受体，测试结果如下：
样品 PEG-IL-2 IL-2
IL-2/IL-2Rα(uM) 0.34 0.023
IL-2/IL-2Rβ(uM) 31.96 2.80
上表结果表明，通过本发明所述的方法制备的PEG-IL-2与IL-2β受体的结合力较强。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (16)

  1. 一种制备结合位点可控的PEG化生物分子的方法，包括如下步骤：
    (1)阻隔物与生物分子中至少一个结合位点结合；
    (2)PEG化生物分子；
    (3)将阻隔物与生物分子分离；
    其中，所述步骤(1)中生物分子选自与阻隔物具有特异性结合作用的生物大分子或小分子，所述生物分子上具有至少一个阻隔物的结合位点；
    阻隔物与生物分子选自如下组合：配体与受体、DNA与其互补的DNA或RNA、酶与其底物、酶与其竞争性抑制剂、酶与其辅酶因子、维生素与其特异性结合蛋白、糖蛋白与其相应的凝集素；优选的，所述配体与受体选自：激素与受体、药物与受体。
  2. 根据权利要求1所述的制备方法，其特征在于，所述DNA与其互补的DNA或RNA选自：基因探针与碱基互补的基因序列；
    所述酶与其底物选自：蛋白酶与蛋白质、淀粉酶与淀粉、核酸酶与核酸、乳酸脱氢酶与乳酸、草酰乙酸脱羧酶与草酰乙酸；
    所述酶与其竞争性抑制剂选自：琥珀酸脱氢酶与丙二酸、二氢叶酸合成酶与磺胺类药物、胆碱酯酶与有机磷农药、巯基酶与路易士气；
    所述酶与其辅酶因子选自：丙酮酸脱氢酶与Mn2 +、过氧化氢酶和氧化还原酶与Fe2 +/Fe3 +；
    所述激素与受体选自：雌激素与雌激素受体、雄激素与雄激素受体、盐皮质激素与盐皮质激素受体、甲状腺激素与甲状腺激素受体、孕激素与孕激素受体；
    所述药物与受体选自：胰岛素、胰岛素样生长因子、上皮生长因子、血小板生长因子及淋巴因子与具有酪氨酸激酶活性的受体、肾上腺素、多巴胺、5-羟色胺、M-乙酰胆碱、阿片类、嘌呤类、前列腺素及多肽激素类药物与G-蛋白偶联受体；优选的，所述药物与受体选自：叶酸与叶酸受体、IL与IL受体；
    所述维生素与其特异性结合蛋白选自：维生素A与视黄醇结合蛋白、维生素D与维生素D结合蛋白、α-生育酚与α-生育酚转运蛋白、维生素K与脂蛋白；
    所述糖蛋白与其相应的凝集素选自：辣根过氧化物酶与刀豆凝集素A、扁豆凝集素和豌豆凝集素、木菠萝素与半乳糖、苋科凝集素与N-乙酰半乳糖胺、二聚体几丁质结合凝集素与N-乙酰氨基葡萄糖寡聚糖、双花扁豆素与血型A物质、荆豆凝集素与血型O物质2-L-岩藻糖。
  3. 根据权利要求2所述制备方法，其特征在于，所述药物与受体选自：IL-2与IL-2受体，IL-2受体包括IL-2α受体、IL-2β受体、IL-2γ受体。
  4. 根据权利要求1所述制备方法，其特征在于，所述步骤(3)中将阻隔物与生物分子分离的方法包括：离心分离、沉淀分离、过滤分离、泡沫分离、萃取分离、膜分离、层析分离、电泳分离、梯度洗脱中的一种或两种以上的组合。
  5. 根据权利要求1所述的制备方法，其特征在于，所述步骤(2)中PEG化的生物分子具有如下结构：
    Figure PCTCN2019129052-appb-100001
    m为1-12的整数，具体可为1、2、3、4、5、6、7、8、9、10、11或12；
    X为PEG与生物分子之间的连接基团，选自：-(CH 2)a-、-(CR 1R 2)a-、-(CH 2)aNH-、-NHCO(CH 2)a-、-(CH 2)aCONH-、-(CH 2)aCO-、-CO(CH 2)a-、-(CH 2)aCONH(CH 2)a-、-(CH 2)a-S-S-(CH 2)a-、-(CH 2)aCOO(CH 2)a-、-(CH 2)a-S-(CH 2)a-中的一种或多种的组合；
    a为0-10的整数；
    R 1和R 2独立地选自：-H、C 1-6的烷基、-OR'、-NHR'、-N(R') 2、-CN、-F、-Cl、-Br、-I、-COR'、-COOR'、-OCOR'、-CONHR'或-CON(R') 2中的一种或多种的组合；
    R'选自：-H、C 1-6的烷基、-F、-Cl、-Br或-I；
    所述PEG为直链、Y型、多分支的聚乙二醇残基，例如包括单甲氧基聚乙二醇(mPEG)、直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG，PEG的分子量为1-100KDa。
  6. 根据权利要求5所述的制备方法，其特征在于，所述a为0-5的整数；
    R 1和R 2独立地选自：H、C 1-3的烷基、-OH、C 1-3的烷氧基、-NH 2、-F、-Cl、-Br和-I中的一种或多种的组合；
    R'选自：-H和C 1-3的烷基；
    所述PEG为直链聚乙二醇残基，具有通式Ⅱ或Ⅲ所示的结构：
    Figure PCTCN2019129052-appb-100002
    其中p和q独立的选自1-2280的整数；
    或所述PEG为Y型聚乙二醇残基，具有通式Ⅳ或Ⅴ所示的结构：
    Figure PCTCN2019129052-appb-100003
    其中，i和h独立的选自1-1140的整数；
    或所述PEG为多分支聚乙二醇残基，具有通式Ⅵ所示的结构：
    Figure PCTCN2019129052-appb-100004
    其中，k是1-760的整数，j是3-8的整数；
    Y为封端基，选自：H、C 1-6烷基，具体可为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、正己基等、C 3-6环烷基，具体可为环丙基、环丁基、环戊基、环己基等、C 6-10芳基，具体可为苯基、萘基等、-L-T；
    L为氧(O)和端基T之间的连接基团，选自：-(CH 2) b-、-(CR 3R 4) b-、-(CH 2) bNH-、-NHCO(CH 2) b-、-(CH 2) bCONH-和-CO(CH 2) b-中的一种或多种的组合，b为0-10的整数；
    R 3和R 4独立地选自：-H、C 1-6的烷基、-OR”、-NHR”、-N(R”) 2、-CN、-F、-Cl、-Br、-I、-COR'、-COOR”、-OCOR”、-CONHR”和-CON(R”) 2中的一种或多种的组合；
    R”选自：-H、C 1-6的烷基、-F、-Cl、-Br和-I；
    T为端基，选自：H、C 1-6烷基、C 3-6环烷基、C 6-10芳基、单糖，具体可为葡萄糖、果糖、半乳糖、核糖、脱氧核糖等、低聚糖，具体可为二糖(蔗糖、乳糖等)、三糖(龙胆三糖、棉子糖等)残基；
    Q是多分支聚乙二醇的核心分子，Q选自：季戊四醇、寡聚季戊四醇、甲基葡萄糖苷、蔗糖、二甘醇、丙二醇、甘油和聚甘油的残基。
  7. 根据权利要求6所述的制备方法，其特征在于，所述T选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
    Figure PCTCN2019129052-appb-100005
    L选自：-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CONH-、-NH-、-CO-、-CONHCH 2-、-CH 2NH-、-CH 2CONH-和-COCH 2-中的一种或多种的组合；
    所述Y选自：甲基、乙基、异丙基、环丙基、环丁基、环己基、苄基、
    Figure PCTCN2019129052-appb-100006
    Figure PCTCN2019129052-appb-100007
    Q选自：季戊四醇、二聚季戊四醇和三聚季戊四醇。
  8. 根据权利要求6或7所述的制备方法，其特征在于，其特征在于，所述多分支聚乙二醇残基具有如下结构：
    Figure PCTCN2019129052-appb-100008
    所述多分支聚乙二醇残基具有如下结构：
    Figure PCTCN2019129052-appb-100009
    其中w是1-570的整数，t为1-10的整数；
    或所述多分支聚乙二醇残基具有如下结构：
    Figure PCTCN2019129052-appb-100010
    其中，s是1-280的整数，y为1-10的整数。
  9. 根据权利要求6-8任一所述的制备方法，其特征在于，所述a为0-3的整数；R 1和R 2独立地选自：H、-CH 3、-OH、-OCH 3和-OCH 2CH 3。
  10. 根据权利要求6-9任一所述的制备方法，其特征在于，所述X选自：-CH 2-、 -CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CONHCH 2-、-CH 2CONHCH 2CH 2-、-CH 2CONHCH 2CH 2NH-、-CH 2CH 2CONHCH 2-、-CH 2CO-、-CH 2CH 2CO-、-CH 2CH 2CONHCH 2CH 2-、-CH 2NH-、-CH 2CONH-、-COCH 2-、-COCH 2CH 2-、-COCH 2CH 2CH 2-、-CH 2-S-S-CH 2-、-CH 2COOCH 2-、-CH 2-S-CH 2-中的一种或多种的组合。
  11. 根据权利要求1-10所述的制备方法，其特征在于，制备一种结合位点可控的PEG化IL-2的方法，包括如下步骤：
    (1)将IL-2与IL-2α受体结合；
    (2)PEG化，PEG与IL-2偶联；
    (3)将IL-2与IL-2α受体分离。
  12. 根据权利要求11所述的制备方法，其特征在于，制备一种结合位点可控的PEG化IL-2的方法，包括如下步骤：
    (1)制备IL-2α受体亲和柱；
    (2)IL-2在亲和柱上与IL-2α受体结合；
    (3)PEG化，PEG与IL-2偶联；
    (4)通过梯度洗脱将IL-2与IL-2α受体分离。
  13. 一种权利要求11或12所述的制备方法制备的结合位点可控的PEG化IL-2，具有如下结构：
    Figure PCTCN2019129052-appb-100011
    PEG和X的限定范围如权利要求5-10所述，n为1或2，优选的，n为1。
  14. 一种权利要求11或12所述的制备方法制备的结合位点可控的PEG化IL-2与药学上可接受的载体制备的药物组合物。
  15. 一种权利要求11或12所述的制备方法制备的结合位点可控的PEG化IL-2与药学上可接受的载体制备的药物组合物在制备治疗和/或预防疾病的药物中的应用。
  16. 根据权利要求15所述的应用，其特征在于，所述的疾病为肿瘤、自身免疫疾病、 病毒性疾病或细菌性疾病。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004091517A2 (en) * 2003-04-15 2004-10-28 Smithkline Beecham Corporation Conjugates comprising human il-18 and substitution mutants thereof
CN1871254A (zh) * 2003-08-29 2006-11-29 拜耳制药公司 修饰的il－4突变蛋白受体拮抗剂
CN101584866A (zh) * 2008-05-21 2009-11-25 北京双鹭药业股份有限公司 聚乙二醇修饰的人白介素-2、其制备方法及应用
WO2010023670A2 (en) * 2008-08-28 2010-03-04 Insight Biopharmaceuticals Ltd. Methods for covalently attaching a polymer to a methionine residue in proteins and peptides
US20170369547A1 (en) * 1997-07-14 2017-12-28 Bolder Biotechnology, Inc. Cysteine variants of interleukin-11 and methods of use thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6548644B1 (en) * 1997-03-10 2003-04-15 Immunex Corporation Site protected protein modification
WO2010045321A2 (en) * 2008-10-15 2010-04-22 Baxter International Inc. Pegylation of recombinant blood coagulation factors in the presence of bound antibodies
CN101591649B (zh) * 2009-07-10 2013-02-13 江苏泰康生物医药有限公司 甲氧基聚乙二醇修饰的精氨酸脱亚氨酶及其制备与应用
US9724396B2 (en) * 2013-03-15 2017-08-08 Massachusetts Institute Of Technology Use of antagonists of growth hormone or growth hormone receptor to prevent or treat stress-sensitive psychiatric illness
SI3180020T1 (sl) * 2014-08-11 2019-04-30 Delinia, Inc. Modificirane variante IL-2, ki selektivno aktivirajo regulatorne celice T, za zdravljenje avtoimunih bolezni

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170369547A1 (en) * 1997-07-14 2017-12-28 Bolder Biotechnology, Inc. Cysteine variants of interleukin-11 and methods of use thereof
WO2004091517A2 (en) * 2003-04-15 2004-10-28 Smithkline Beecham Corporation Conjugates comprising human il-18 and substitution mutants thereof
CN1871254A (zh) * 2003-08-29 2006-11-29 拜耳制药公司 修饰的il－4突变蛋白受体拮抗剂
CN101584866A (zh) * 2008-05-21 2009-11-25 北京双鹭药业股份有限公司 聚乙二醇修饰的人白介素-2、其制备方法及应用
WO2010023670A2 (en) * 2008-08-28 2010-03-04 Insight Biopharmaceuticals Ltd. Methods for covalently attaching a polymer to a methionine residue in proteins and peptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3896079A4 *

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