WO2018086603A1 - 聚乙二醇化血管内皮抑制素类似物及其应用 - Google Patents

聚乙二醇化血管内皮抑制素类似物及其应用 Download PDF

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WO2018086603A1
WO2018086603A1 PCT/CN2017/110532 CN2017110532W WO2018086603A1 WO 2018086603 A1 WO2018086603 A1 WO 2018086603A1 CN 2017110532 W CN2017110532 W CN 2017110532W WO 2018086603 A1 WO2018086603 A1 WO 2018086603A1
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endostatin
polyethylene glycol
analog
lysine
complex
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French (fr)
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周代福
王文超
李辉
常国栋
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Protgen Ltd
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Protgen Ltd
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Priority to JP2019524188A priority Critical patent/JP7140454B2/ja
Priority to CA3043380A priority patent/CA3043380A1/en
Priority to EP17870533.1A priority patent/EP3539570B1/en
Priority to AU2017357327A priority patent/AU2017357327B2/en
Priority to US16/349,191 priority patent/US11098104B2/en
Priority to CN202210948599.6A priority patent/CN115721730B/zh
Priority to CN201780069252.1A priority patent/CN109963597B/zh
Publication of WO2018086603A1 publication Critical patent/WO2018086603A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel recombinant protein drugs.
  • the invention relates to PEGylated endostatin analogs and uses thereof.
  • Neovascularization refers to the formation of new capillaries on the original blood vessels. Tumor growth and migration depend on the formation of new blood vessels, and microvascular endothelial cells in tumors as a target for cancer treatment provide a treatment for tumors (Folkman J.N Engl J Med 1971; 285: 1182-1186).
  • Endostatin is a 20kDa fragment of the carboxy terminus of collagen XVIII.
  • Professor Judah Folkman of Harvard University and others found this protein in the culture medium of hemangioendothelioma cells, which has the activity of inhibiting vascular endothelial cell proliferation, migration and angiogenesis in vivo. Further experiments showed that recombinant endostatin can inhibit the growth and migration of various tumors in mice, and even completely cure the tumor, and does not produce drug resistance.
  • the mechanism of its activity is that it inhibits the growth of vascular endothelial cells, inhibits the formation of new blood vessels near the tumor tissue, makes the tumor tissue unable to obtain the large amount of nutrients and oxygen necessary for growth, and finally stops growth or necrosis (Folkman J. Et al. Cell 1997; 88: 277-285; Folkman J. et al. Nature 1997; 390: 404-407).
  • endostatin can significantly inhibit tumor-associated lymphangiogenesis (Yongzhang Luo. et al. Journal of Pathology 2010; 222: 249–260; Yongzhang Luo. Front. Med. 5:336–340.).
  • Recombinant human endostatin prepared by genetic engineering can be used as a tumor therapeutic drug, and its clinical experiments show that it can effectively inhibit tumor growth.
  • clinical trials with non-small cell lung cancer as the main indication have shown that its therapeutic effect on tumors is more prominent.
  • chemotherapy chemotherapy
  • radiation therapy radiation therapy
  • protein drugs have less toxic side effects and do not produce drug resistance, but administration through the gastrointestinal tract produces a severe first pass effect.
  • protein drugs are usually administered by intravenous or subcutaneous injection.
  • intravenous or subcutaneous injection For proteins with small molecular weight, the half-life of the drug is usually short after intravenous administration.
  • small protein can be quickly eliminated by renal filtration.
  • Proteins with a hydraulic radius in excess of albumin or a molecular weight greater than 66,000 daltons (66 kDa) in the blood are usually stably retained in the circulatory system, while small protein drugs are rapidly eliminated by glomerular filtration, so For effective therapeutic concentrations in the blood, patients need to receive injections or drip frequently. Although this treatment can achieve the purpose of treatment, it brings serious inconvenience and pain to the patient, and also seriously affects the patient's compliance with drug treatment, and also increases the cost of medication. Moreover, the long-term frequent use of certain drugs may also have some side effects, such as an immune response.
  • Endothelin which is a protein drug, also has the disadvantage of short half-life and high clearance in vivo.
  • the main clinical means of administration are frequent administration to maintain an effective blood concentration, usually daily, and long-term medication, which makes the patient's mental and economic burden large.
  • the object of the present invention is to improve the in vivo metabolic characteristics of the protein, to have higher stability and longer in vivo half-life, and even higher therapeutic effect, thereby reducing the frequency of administration, reducing the cost of medication, and reducing the economy of patients. The purpose of the burden.
  • Modification of proteins with high molecular polymers is a common method for altering and controlling the kinetic properties of drugs such as half-life, immunological characteristics, and toxicological properties.
  • polyethylene glycol is the most widely used amphiphilic polymer, which has the characteristics of good solubility, good biocompatibility, non-toxicity and no immunogenicity. It is included in the US FDA and China SFDA. Polymers approved for drug preparation approved by drug regulatory agencies in many countries and regions. Coupling proteins with hydrophilic polymers such as polyethylene glycol can reduce proteolysis, increase protein stability, reduce non-specific adsorption and antigenicity, and can greatly increase when the hydraulic radius of the conjugate reaches the lower limit of glomerular filtration.
  • a monomethoxy polyethylene glycol succinimide active ester modifier such as mPEG-SC, mPEG-SCM, mPEG-SPA, mPEG-NHS, etc.
  • a monomethoxy polyethylene glycol succinimide active ester modifier such as mPEG-SC, mPEG-SCM, mPEG-SPA, mPEG-NHS, etc.
  • mPEG-Aldehyde can react specifically with the N-terminal ⁇ -amino group of a protein or polypeptide under weakly acidic conditions to form an unstable Schiff base, further by sodium cyanoborohydride.
  • the mono-methoxy polyethylene glycol maleimide modifier (such as mPEG-MAL, mPEG2-MAL, etc.) is used to selectively modify the free cysteine in the protein or polypeptide.
  • polyethylene glycol modification technology has been successfully applied to a variety of proteins.
  • the FDA has approved the marketed polyethylene glycol modified protein drugs such as PEG-adenosine deiminase (1990) and PEG-asparaginase (1994).
  • PEG- ⁇ 2b interferon (2001), PEG-granulocyte colony-stimulating factor (2002), PEG- ⁇ 2a interferon (2002), PEG- ⁇ 2a interferon and ribavirin (2004) ), PEG-erythropoietin (2007), PEG-TNF ⁇ antibody (2008), PEG- ⁇ 2b interferon and ribavirin mixed preparation (2008), PEG-uric acid oxidase (2010).
  • Representative polyethylene glycol modified protein drugs that are undergoing clinical research are PEG-arginine deiminase (Polaris Group, Phase III), PEG-growth hormone (Changchun Jinsai Pharmaceutical, Phase III), PEG- 11a interferon (Biogen Idec, stage III) and the like.
  • polyethylene glycol modified protein In addition to the polyethylene glycol modified protein to achieve extended drug half-life, there are other polymer modifiers that can be used, such as dextran, sucrose, starch, polyalanine, copolymers of oxalic acid and malonic acid, carboxymethyl Cellulose, polyvinylpyrrolidone, poly1,3-ethylene glycol methine ether, copolymer of ethylene and maleic hydrazide, polysialic acid, cyclodextrin, and the like.
  • polymer modifiers such as dextran, sucrose, starch, polyalanine, copolymers of oxalic acid and malonic acid, carboxymethyl Cellulose, polyvinylpyrrolidone, poly1,3-ethylene glycol methine ether, copolymer of ethylene and maleic hydrazide, polysialic acid, cyclodextrin, and the like.
  • the present inventors have found that conjugated polyethylene glycol on lysine which is far from the nucleolin-binding region on endostatin can significantly increase protein activity, and therefore, the present invention provides a polyethylene glycol-modified endostatin analogue,
  • the endostatin analog is conjugated to polyethylene glycol in a region distant from the nucleolin binding region, This results in an increase in the biological activity of inhibiting tumor cell migration.
  • the polyethylene glycol-modified endostatin analog of the present invention has higher stability and longer in vivo half-life than unmodified endostatin, and its inhibition of neovascularization activity is remarkably improved.
  • a first aspect of the invention provides a Vas Endostatin Analog and a Polyethylene Glycol Coupling Complex; wherein the Endostatin Analog is at 96th of SEQ ID NO. 1 corresponding to the natural endostatin amino acid sequence a position having a lysine residue at a position other than a lysine residue at any other position; and the endostatin analog is only on the lysine residue and polyethylene glycol Coupling.
  • a second aspect of the invention provides a Vas Endostatin Analog and a Polyethylene Glycol Coupling Complex; wherein the Endostatin Analog is at 96th of SEQ ID NO. 1 corresponding to the natural endostatin amino acid sequence a position having a lysine residue at a position other than a lysine residue at any other position; and the endostatin analog at the N-terminus and the lysine residue Glycol coupling.
  • the endostatin analogue is natural endothelin amino acid sequence SEQ ID NO. 1 at 76, 107, 118 and 184 The lysine residue at position is mutated.
  • the sequence of the endostatin analog is set forth in SEQ ID NO:3.
  • a third aspect of the invention provides a vascular endostatin analog and a polyethylene glycol conjugate complex; wherein the endostatin analogue is at 96th nucleotide corresponding to the natural endostatin amino acid sequence of SEQ ID NO. a position having a lysine residue at a position other than a lysine residue at any other position; and the endostatin analog has an inserted amino acid sequence at its N-terminus; and, the vascular endothelium Inhibin analogs are only coupled to polyethylene glycol on this lysine residue.
  • a fourth aspect of the invention provides a vascular endostatin analog and a polyethylene glycol conjugate complex; wherein the endostatin analogue is at 96th nucleotide corresponding to the natural endostatin amino acid sequence of SEQ ID NO. a position having a lysine residue at a position other than a lysine residue at any other position, and the endostatin analog has an inserted amino acid sequence at its N-terminus; and, the vascular endothelium The inhibin analog is coupled to the polyethylene glycol at the N-terminus and the lysine residue.
  • the endostatin analogue is the natural endostatin amino acid sequence of SEQ ID NO. 1, 76, 107, 118 and A lysine residue at position 184 is mutated and is formed by inserting an amino acid sequence at its N-terminus.
  • the inserted amino acid sequence is the insertion of GGSHHHHH between the N-terminal methionine M and histidine H.
  • the sequence of the endostatin analog is set forth in SEQ ID NO: 8.
  • the endostatin analogue of the conjugated complex of the invention has a lysine at positions 76, 107, 118 and 184 of the natural endostatin amino acid sequence of SEQ ID NO.
  • the residues are mutated to X1, X3, X4, X5, respectively, wherein X1, X3, X4 or X5 are independently any natural amino acid other than lysine.
  • X1, X3, X4 or X5 is independently preferably a water-soluble amino acid, further preferably any of the charged amino acids arginine, histidine, glutamic acid and aspartic acid, further preferably a positively charged amino acid Any of arginine and histidine is most preferably arginine.
  • the end of the endostatin analog is inserted into the N-terminus of the complex of the invention.
  • the base acid sequence is MGGSHHHHH.
  • the endostatin analog is covalently coupled to polyethylene glycol in the complex of the invention.
  • the polyethylene glycol has an average molecular weight of from 5,000 to 40,000 Daltons.
  • the polyethylene glycol has an average molecular weight of from 20,000 to 40,000 Daltons.
  • the polyethylene glycol is monomethoxy polyethylene glycol, monoglucose polyethylene glycol or monogalactose polyethylene glycol.
  • the polyethylene glycol is linear or branched.
  • the polyethylene glycol is monomethoxy polyethylene glycol.
  • the polyethylene glycol is coupled to an amino group of a endostatin analog.
  • the polyethylene glycol is coupled to a lysine analog lysine side chain ⁇ -amino group.
  • the polyethylene glycol is coupled to the N-terminal a-amino group of the endostatin analog and the lysine residue ⁇ -amino group.
  • the amino coupling of the polyethylene glycol to the endostatin analog can be carried out using monomethoxy polyethylene glycol propionaldehyde (mPEG-ALD), monomethoxy polyethylene glycol butyl Aldehyde (mPEG-ButyrALD), monomethoxy polyethylene glycol succinimide carbonate (mPEG-SC), monomethoxy polyethylene glycol succinimide acetate (mPEG-SCM), monomethyl Oxypolyethylene glycol succinimidyl propionate (mPEG-SPA), monomethoxy polyethylene glycol succinimide butyrate (mPEG-SBA), monomethoxy polyethylene glycol succinyl
  • the imine ⁇ -methylbutyrate (mPEG-SMA) and the monomethoxypolyethylene glycol NHS ester (mPEG-NHS) were subjected to a coupling reaction.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the endostatin analog of the present invention and a polyethylene glycol coupling complex as described above and a pharmaceutically acceptable carrier.
  • the present invention also provides a method of treating a disease caused by neovascularization or neonatal lymphatic administration comprising administering to a patient an endostatin analogue of the present invention and a polyethylene glycol coupling complex or a pharmaceutical composition as described above.
  • the present invention also provides the use of the endostatin analog of the present invention and a polyethylene glycol conjugate complex or a pharmaceutical composition for treating diseases caused by neovascularization or neoplastic lymphatic vessels.
  • the disease caused by the neovascular or neoplastic lymphatic is a tumor.
  • the invention also provides the use of a conjugated complex of the invention in the manufacture of a medicament for inhibiting the production of neovascular or neoplastic lymphatic vessels in vivo or in vitro.
  • the complex of the present invention When the complex of the present invention is used for inhibiting the formation of neovascular or neoplastic lymphatic vessels in vivo, its half-life is significantly prolonged compared with endostatin, and its inhibition rate is significantly increased compared with endostatin under the same administration conditions.
  • Figure 1 Results of SDS-PAGE electrophoresis analysis of modified samples of 20kDamPEG-AL N-terminally modified endostatin and subsequent chromatographically purified samples.
  • the first lane is a low molecular weight standard, which is 116, 66, 45, 30, 25, 18.4, and 14.4 kDa from top to bottom;
  • the second lane is a sample modified with 20 kDa mPEG-ALD for endostatin;
  • Lanes 7 were fractions 1, 2, 3, 4, and 5, respectively, eluted at 100, 150, 200, 350, and 500 mM NaCl for chromatographic purification, wherein the 200 mM NaCl elution fraction (component 3) in lane 5 was N-terminal.
  • Single modified product component is a low molecular weight standard, which is 116, 66, 45, 30, 25, 18.4, and 14.4 kDa from top to bottom
  • the second lane is a sample modified with 20 kDa mPEG
  • Figure 2 Results of SDS-PAGE electrophoresis analysis of modified samples of 20 kDa mPEG-ALDN terminal single modified endostatin K1 analog and subsequent chromatographic purification samples.
  • the first lane is a low molecular weight standard, which is 116, 66, 45, 30, 25, 18.4, and 14.4 kDa from top to bottom;
  • the second lane is a sample modified with 20 kDam PEG-ALD for the endostatin K1 analog;
  • Lanes 3-7 were fractions 1, 2, 3, 4, and 5, respectively, eluted at 100, 150, 150, 350, and 500 mM NaCl during chromatographic purification.
  • the lanes of lanes 4 and 5 were eluted with 150 mM NaCl (components). 2, 3) are 20 kDa polyethylene glycol N-terminal single modified endostatin K1 analog.
  • FIG. 3 Electrophoresis analysis of 20kDam PEG-SPA double-modified vasopressin K1 analogue lysine side chain amino group and N-terminal amino double modification and purified sample.
  • the first lane is a low molecular weight standard, which is 116, 66, 45, 30, 25, 18.4, and 14.4 kDa from top to bottom;
  • the second lane is endothelin with 20 kDam PEG-SPA modified sample; lanes 3-8 Components 1, 2, 3, 4, 5, 6 were eluted at 25, 50, 75, 100, 200, and 500 mM NaCl for chromatographic purification, respectively.
  • the 6 MW 100 mM NaCl elution fraction (component 4) was K1.
  • the analog double modifies the product component.
  • Figure 4 Results of SDS-PAGE electrophoresis of modified samples and subsequent purified samples of 20 kDa without PEG-MAL modified endostatin ESC analog thiol.
  • Lane 1 is a sample modified with 20 kDam PEG-MAL for endostatin ESC analog; lanes 2 to 5 are eluted with 100, 150, 200 and 500 mM NaCl for chromatographic purification, respectively, and lane 6 is a low molecular weight standard.
  • the product is 116, 66, 45, 30, 25, 18.4, 14.4 kDa from top to bottom; wherein the 200 mM NaCl elution component (component 3) in the fourth lane is the ESC analog thiol single modified product component.
  • Figure 5 Results of SDS-PAGE electrophoresis analysis of modified samples of 20 kDam PEG-ALD modified vascular endostatin K1 analog lysine flank amino group and subsequent purified samples.
  • the first lane is a low molecular weight standard, which is 116, 66, 45, 30, 25, 18.4, and 14.4 kDa from top to bottom;
  • the second lane is endothelin with 20 kDam PEG-ALD modified sample;
  • lanes 3-7 The fractions were eluted with 50, 100, 125, 200, and 500 mM NaCl, respectively.
  • the lanes 4 and 5 were 20 kDa polyethylene glycol single modified endostatin analogs, and two samples were modified.
  • Figure 6 Tertiary structure diagram of human endostatin, indicating the N-terminus and C-terminus of human endostatin protein and the lysine residue K1 at position 76, and the lysine residue K2 at position 96 The position of the 107th lysine residue K3, the 118th lysine residue K4, and the binding region of the present protein to the receptor Nucleolin.
  • Figure 7 Tertiary structure diagram of human endostatin, indicating the N-terminus, C-terminus of the human endostatin protein and the lysine residue K1 at position 76, the lysine residue K2 at position 96 The position of the 107th lysine residue K3, the 118th lysine residue K4, and the position of the 127th asparagine residue.
  • Figure 8 Sequence of an endostatin analog of the present invention.
  • Endostatin may refer to natural endostatin, preferably human endostatin, for example, the sequence having SEQ ID NO: 1, but is not limited thereto, for example, It is a natural endostatin from other mammals such as mice, rats, pigs, dogs, rabbits, sheep, goats, cats and the like.
  • Endostatin may also refer to a functional variant of endostatin, such as an engineered functional variant that has one or a few amino acid substitutions, deletions or additions compared to native endostatin. And have substantially the same biological functions, such as inhibition of vascular endothelial cell proliferation, migration and angiogenesis in vivo.
  • Endostatin may also refer to a derivative or modified product of native endostatin or a functional variant thereof, such as a polyethylene glycol modified product.
  • the term "functional variant” as used herein, includes one or more (eg, 1-5, 1-10, or 1-15, in the amino acid sequence, specifically, for example, may be 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12 or more) mutants of amino acid substitutions, deletions or additions of endostatin, and the mutants are similar to endostatin
  • the biological activity of a "functional variant" of endostatin may be, for example, natural endostatin, such as 30% or higher, 50% or higher, 60% or higher of natural human endostatin, 70% or higher, 80% or higher or 90% or higher.
  • the "functional variant” may be a naturally occurring mutant or an artificial mutant such as a mutant obtained by site-directed mutagenesis or a mutant produced by a genetic recombination method.
  • the biological activity of the "functional variant” can be detected by methods well known in the art for detecting endostatin activity.
  • HMEC cells can be selected, and the inhibition rate of migration of HMEC cells by functional variants is analyzed by Migration (Tranwell Assay) method.
  • Migration Tranwell Assay
  • the number of cells reflects the activity of the protein (refer to Luo yongzhang et al., Endostatin inhibits tumourly mphangiogenesis and lymphatic metastasis via cell surface nucleolin on Lymphangiogenic endothelial cells (J Pathol 2010; 222: 249-260).
  • a “endostatin analog” of the invention has a lysine residue at a position corresponding to position 96 of native human endostatin (eg, SEQ ID NO: 1), while in other There is no lysine residue at any position such that polyethylene glycol is only coupled to the lysine residue of the endostatin analog or simultaneously coupled to the amino terminus and the Lai Coupling on the residue of the amino acid without any other amino acid residues.
  • the thus obtained coupling complex has higher stability and longer in vivo half-life than the natural human endostatin or the natural end-end monoethylene glycol-conjugated complex of human endostatin. Moreover, it inhibits the angiogenic activity significantly.
  • the endostatin analog of the present invention can be obtained by engineering a natural endostatin (for example, natural human endostatin or natural mammalian endostatin), specifically, natural human endostatin.
  • a lysine residue at positions 76, 107, 118, and 184 in e.g., SEQ ID NO: 1 is mutated, only the endostatin analog obtained at the 96th lysine residue is retained, or
  • the lysine residues corresponding to positions 76, 107, 118, and 184 in the natural human endostatin for example, SEQ ID NO: 1 are mutated, and only the corresponding residues are retained.
  • Natural human endostatin eg An endostatin analog obtained from the lysine residue at position 96 of SEQ ID NO: 1.
  • the endostatin analogs of the invention may also be obtained by engineering a known naturally occurring or synthetic endostatin functional variant, in particular, if the endostatin functional variant is corresponding A lysine residue at position 96 of the natural endostatin retains the lysine residue and mutates all other lysine residues of the endostatin functional variant.
  • the amino acid at that position is mutated to a lysine residue and the blood vessel is made All other lysine residues of the endostatin functional variant are mutated.
  • An endostatin functional variant that can be engineered to obtain an endostatin analog of the invention includes an ES variant in which the first amino acid M is randomly deleted when human ES is recombinantly expressed in E. coli.
  • the endostatin functional variant that can be engineered to obtain the endostatin analog of the present invention also includes an N-terminal deletion of 4 amino acids due to random cleavage of the N-terminus when ES is recombinantly expressed in yeast.
  • Functional variants of the endostatin that can be engineered to obtain the endostatin analogs of the invention include YH-16, which is an ES change obtained by adding 9 additional amino acids (MGGSHHHHH) to the N-terminus of the ES.
  • endostatin functional variants that can be engineered to obtain the endostatin analogs of the invention also include those endothelin mutants such as ES006, ES008, ES011 disclosed in PCT International Application No. PCT/CN2012/081210. , S02, S09, Z006, Z008, ZN1, etc.
  • the endostatin functional variants that can be engineered to obtain the endostatin analogs of the invention also include those endostatin mutants disclosed in PCT International Publication No. WO 2016/070798, such as 003, 007, Z101, 009, S03, 36, 249, 381, 57, 114, 124, 125, 160, 163, 119 (the entire contents of which are incorporated herein by reference).
  • sequence corresponding to the natural endostatin amino acid sequence of SEQ ID NO: 1 means that the endostatin functional variant and the natural endostatin amino acid are expressed by software or algorithms well known in the art. Sequence SEQ ID NO: 1 is the position corresponding to the corresponding amino acid residue on SEQ ID NO: 1 on the functional variant of endostatin after alignment.
  • the software or algorithms include, but are not limited to, BLAST, FASTA.
  • the "polyethylene glycol (PEG)" described in the present invention may be monomethoxypolyethylene glycol, monoglucose polyethylene glycol or monogalactose polyethylene glycol.
  • the polyethylene glycol used may be linear or branched and may have a molecular weight of, for example, from about 5 kD to about 50 kD, from about 20 kD to about 40 kD, or such as about 20 kD.
  • polyethylene glycol modification or "polyethylene glycol coupling” as used herein may refer to a polyethylene glycol modifier that chemically couples a polyethylene glycol modifier molecule to a protein molecule and participates in a coupling reaction.
  • the group is that it is alive
  • the group of the protein is mainly a free amino group, a mercapto group or the like, preferably an amino group.
  • Coupling with polyethylene glycol (PEG) can extend the half-life of the coupled complex in vivo, avoid protease degradation or increase solubility. Methods for modifying proteins with polyethylene glycol are well known to those skilled in the art.
  • polyethylene glycol modifier includes, but is not limited to, a monomethoxypolyethylene glycol modifier which cleaves one hydroxyl group at one end of the polyethylene glycol molecule with a methoxy group and activates the other end with a suitable activation method. Activated polyethylene glycol obtained after one hydroxyl group. Since the reactivity of the hydroxyl group itself is very low, the reactivity of the activated polyethylene glycol molecule is greatly improved, and it is called a "polyethylene glycol modifier".
  • the selection of activation groups, the mechanism of activation, and the modification reaction mechanism of the resulting polyethylene glycol modifiers are well known in the art and have been reported in many literatures. Polyethylene glycol modifiers are commercially available.
  • Polyethylene glycol modifiers that may be used include, but are not limited to, polyethylene glycol monomethoxy polyethylene glycol propionaldehyde (mPEG-ALD), monomethoxy polyethylene glycol butyraldehyde (mPEG-Butyr ALD), single Methoxy polyethylene glycol succinimide carbonate (mPEG-SC), monomethoxy polyethylene glycol succinimide acetate (mPEG-SCM), monomethoxy polyethylene glycol succinyl Imine propionate (mPEG-SPA), monomethoxy polyethylene glycol succinimide butyrate (mPEG-SBA), monomethoxy polyethylene glycol succinimide ⁇ -methylbutyric acid Ester (mPEG-SMA), monomethoxy polyethylene glycol NHS ester (mPEG-NHS).
  • mPEG-ALD polyethylene glycol monomethoxy polyethylene glycol propionaldehyde
  • mPEG-Butyr ALD single Methoxy poly
  • the polyethylene glycol is coupled to the amino group of the endostatin analog, for example, to the lysine analog lysine side chain ⁇ -amino group, or to the endostatin analog N-terminal ⁇ . - Amino and lysine residues ⁇ -amino coupling.
  • the endostatin K1 analog described in the present invention refers to an endostatin analog having the amino acid sequence of SEQ ID NO. 2, which is derived from the amino acid sequence SEQ ID NO.
  • the lysine residues at positions 96, 107, 118, and 184 at the N-terminus were mutated to X2, X3, X4, and X5, respectively.
  • X2, X3, X4 and X5 are any natural amino acid other than lysine, preferably a water-soluble amino acid, further preferably a charged amino acid arginine, histidine, glutamic acid and aspartic acid.
  • the endostatin K2 analog described in the present invention refers to an endostatin analogue having the amino acid sequence of SEQ ID NO. 3, which is derived from the amino acid sequence SEQ ID NO.
  • the lysine residues at positions 76, 107, 118, and 184 at the N-terminus were mutated to X1, X3, X4, and X5, respectively.
  • X1, X3, X4 and X5 are any natural amino acid other than lysine, preferably a water-soluble amino acid, further preferably a charged amino acid arginine, histidine, glutamic acid and aspartic acid.
  • the endostatin K3 analog described in the present invention refers to an endostatin analogue having the amino acid sequence of SEQ ID NO. 4, which is derived from the amino acid sequence SEQ ID NO.
  • the lysine residues at positions 76, 96, 118, and 184 at the N-terminus were mutated to X1, X2, X4, and X5, respectively.
  • X1, X2, X4, and X5 are any natural amino acid other than lysine, preferably a water-soluble amino acid, further Preferred is one of the charged amino acids arginine, histidine, glutamic acid and aspartic acid, further preferably one of the positively charged amino acids arginine and histidine, most preferably arginine .
  • the endostatin K4 analog described in the present invention refers to an endostatin analog having the amino acid sequence of SEQ ID NO. 5, which is the endothelin amino acid sequence SEQ ID NO.
  • the lysine residues at positions 76, 96, 107, and 184 at the N-terminus were mutated to X1, X2, X3, and X5, respectively.
  • X1, X2, X3 and X5 are any natural amino acid other than lysine, preferably a water-soluble amino acid, further preferably a charged amino acid arginine, histidine, glutamic acid and aspartic acid.
  • the endostatin ESC analog described in the present invention refers to an endostatin analog having the amino acid sequence of SEQ ID NO. 6, which is the endothelin amino acid sequence of SEQ ID NO. A cysteine residue C is attached to the C-terminus.
  • the endostatin NK1, NK2, NK3, NK4, and NESC analogs described in the present invention respectively refer to the amino acid sequences of SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO.
  • the endostatin NK1, NK2, NK3, NK4, and NESC analogs are the methionine M and histidine H at the N-terminus of the above-described endostatin K1, K2, K3, K4, and ESC analogs, respectively.
  • the amino acid sequence GGSHHHHH was inserted.
  • the endostatin ESK analog described in the present invention refers to an endostatin analogue having the amino acid sequence of SEQ ID NO. 12, which is derived from the amino acid sequence of SEQ ID NO.
  • the lysine residues at positions 76, 96, 107, 118, and 184 at the N-terminus were mutated to X1, X2, X3, X4, and X5, respectively, and the residue No. 127 of asparagine was mutated to lysine.
  • X1, X2, X3, X4, X5 are any natural amino acid other than lysine, preferably a water-soluble amino acid, further preferably a charged amino acid arginine, histidine, glutamic acid and aspartame
  • One of the acids is further preferably one of the positively charged amino acids arginine and histidine, and most preferably arginine.
  • the endostatin NESK analog described in the present invention refers to an endostatin analog having the amino acid sequence of SEQ ID NO. 13, which is at the N-terminus of the above-described endostatin ESK analog.
  • the amino acid sequence GGSHHHHH was inserted between methionine M and histidine H.
  • single modification refers to a product obtained by modifying a vascular endostatin or an endostatin analog with a PEG molecule.
  • the present invention also provides a pharmaceutical composition comprising the endostatin analog of the present invention and a polyethylene glycol coupling complex as described above for use in the treatment of diseases caused by neovascularization or neoplastic lymphatic vessels.
  • the disease caused by the neovascular or neoplastic lymphatic is a tumor, including but not limited to lung cancer, breast cancer, and the like.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a solid or liquid diluent, filler, antioxidant, stabilizer, and the like that can be safely applied.
  • various carriers well known in the art can be administered, including, but not limited to, sugars, starches, celluloses and derivatives thereof, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols. Alginic acid, phosphate buffer, emulsifier, isotonic saline, and/or pyrogen-free water.
  • the pharmaceutical composition may also be a sustained release formulation in a form selected from the group consisting of microcapsules, hydrogels, microspheres, micro-osmotic pumps or liposomes.
  • the present invention also provides a kit comprising the endostatin analog of the present invention and a polyethylene glycol-conjugated complex as described above and instructions for use.
  • the invention also provides a method of treating a disease caused by neovascularization or neoplastic lymphatic administration comprising administering to the subject a therapeutically effective amount of an endostatin analogue of the invention as described above and a polyethylene glycol conjugate complex.
  • the disease caused by the neovascular or neoplastic lymphatic is a tumor, including but not limited to lung cancer, breast cancer, colon cancer, and the like.
  • terapéuticaally effective amount refers to an amount of active compound that is sufficient to cause a biological or medical response desired by a clinician in a subject.
  • the "therapeutically effective amount” of the endostatin analog to the polyethylene glycol conjugate complex can be determined by one skilled in the art based on the route of administration, the subject's weight, age, condition, and the like. For example, a typical daily dose may range from 0.01 mg to 100 mg of active ingredient per kg of body weight.
  • the medicament provided by the present invention can be formulated into a clinically acceptable dosage form such as a powder or an injection.
  • the pharmaceutical composition of the present invention can be administered to a subject using any suitable route, for example, by oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal, and the like. Route administration.
  • N-terminal amino polyethylenes of endostatin and endostatin K1, K2, K3, K4, ESC, NK1, NK2, NK3, NK4, NESC, ESK, and NESK analogs were prepared and purified, respectively.
  • the mutated lysine was mutated from lysine to refined ammonia. acid.
  • Example 1 Coupling of 20 kDa polyethylene glycol with the N-terminal amino group of native human endostatin
  • the natural human endostatin was dialyzed into a 30 mM sodium acetate solution having a pH of 5.0 ⁇ 1.0, and the protein concentration was measured, and the protein concentration was adjusted to be between 5 and 15 mg/ml.
  • the dosage is 20 mM.
  • Example 2 Purification of 20 kDa polyethylene glycol with N-terminal amino coupling product of natural human endostatin
  • the solution of the monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) and the natural human endostatin coupling reaction product in Example 1 was purified by a cation chromatography column. Specifically, GE Healthcare SPFF medium was selected, and the reaction solution was adjusted to a pH of 5.0 to 7.0, and the cation column was equilibrated with 20 mM NaH 2 PO 4 (pH to 5.0 to 7.0) and loaded. Gradient elution was further carried out using 20 mM NaH 2 PO 4 , 500 mM NaCl pH 5.0 to 7.0), and different components were collected according to 280 nm ultraviolet absorption.
  • mPEG-ALD monomethoxypolyethylene glycol propionaldehyde
  • Example 3 Coupling of 20 kDa polyethylene glycol with N-terminal amino group of endostatin K1 analog
  • the endostatin K1 analog was dialyzed into a 30 mM sodium acetate solution having a pH of 5.0 ⁇ 1.0, the protein concentration was determined, and the protein concentration was adjusted to be between 5 and 15 mg/ml. Calculate the amount of 20kDa monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) to be added according to the molar ratio of the target protein to polyethylene glycol 1:3, and calculate the reducing agent cyanoborohydride according to the final solution volume. The amount of sodium used was 20 mM.
  • mPEG-ALD monomethoxypolyethylene glycol propionaldehyde
  • Example 4 Purification of the N-terminal amino coupling product of 20 kDa polyethylene glycol and endostatin K1 analog
  • the solution of the coupling reaction product of monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) and endostatin K1 analog in Example 3 was purified by a cation chromatography column.
  • GE Healthcare SPFF medium was selected, and the reaction solution was adjusted to a pH of 5.0 to 7.0, and the cation column was equilibrated with 20 mM NaH 2 PO 4 (pH to 5.0 to 7.0) and loaded. Gradient elution was further carried out using 20 mM NaH 2 PO 4 , 500 mM NaCl pH 5.0 to 7.0), and different components were collected according to 280 nm ultraviolet absorption. And SDS-PAGE analysis was carried out, and the analysis results are shown in Fig. 2.
  • both components 2 and 3 are products of a 20 kDa polyethylene glycol mono-modified endostatin K1 analog, and based on the same modification principle of Example 2, it can be considered that the single modified product is an N-terminal single modification. product.
  • Example 5 20 kDa polyethylene glycol and vascular endostatin K1 analog lysine side chain amino group and N-terminal amino double coupling
  • the endostatin K1 analog was dialyzed into a 20 mM NaH 2 PO 4 solution at pH 8.5, the protein concentration was determined, and the protein concentration was adjusted to between 5 and 15 mg/ml. Calculate the amount of 20kDa monomethoxypolyethylene glycol succinimidyl propionate to be added according to the molar ratio of the target protein to polyethylene glycol 1:10, add to the target protein, stir evenly and let stand at room temperature. 60 to 120 minutes, the electrophoresis pattern of the modification results is shown in Fig. 3. Each band of the electrophoresis gel was analyzed by Bio-Rad's 1D gel quantification software Quantity One and compared with the molecular weight standard.
  • the ratio of the endostatin K1 analog was double modified (molecular weight 60 kDa) at 50. More than %, one endostatin K1 analog was modified with two monomethoxy polyethylene glycols having a molecular weight of 20 kDa.
  • Example 6 Purification of 20 kDa polyethylene glycol with vascular endostatin K1 analog lysine side chain amino group and N-terminal amino double coupling product
  • Example 5 The solution of the coupling reaction product of monomethoxypolyethylene glycol succinimidyl propionate and endostatin K1 analog in Example 5 was purified by a cation chromatography column. GE Healthcare SPFF medium was selected, and the reaction solution was adjusted to a pH of 5.0 to 7.0, and the cation column was equilibrated with 20 mM NaH 2 PO 4 (pH to 5.0 to 7.0) and loaded. Gradient elution was further carried out using 20 mM NaH 2 PO 4 , 500 mM NaCl pH 5.0 to 7.0), and different components were collected according to 280 nm ultraviolet absorption. And SDS-PAGE analysis was carried out, and the analysis results are shown in Fig. 3. As can be seen from Figure 3, component 4 is the product of a 20 kDa polyethylene glycol double modified endostatin K1 analog.
  • Example 7 Coupling and purification of 20 kDa polyethylene glycol with N-terminal amino group of endostatin K2 analogue
  • the N-terminal coupling was carried out by the method of Example 3, and the modified product was purified by the method of Example 4 to obtain similar experimental results.
  • Example 8 Coupling and purification of 20 kDa polyethylene glycol with vasopressin K2 analog lysine side chain amino group and N-terminal amino group
  • Example 9 Coupling and purification of 20 kDa polyethylene glycol with N-terminal amino group of endostatin K3 analog
  • the N-terminal coupling was carried out by the method of Example 3, and the modified product was purified by the method of Example 4 to obtain similar experimental results.
  • Example 10 Coupling and purification of 20 kDa polyethylene glycol with vasopressin K3 analog lysine side chain amino group and N-terminal amino group
  • Example 11 Coupling and purification of 20 kDa polyethylene glycol with N-terminal amino group of endostatin K4 analog
  • the N-terminal coupling was carried out by the method of Example 3, and the modified product was purified by the method of Example 4 to obtain similar experimental results.
  • Example 12 Coupling and purification of 20 kDa polyethylene glycol with vasopressin K4 analog lysine side chain amino group and N-terminal amino group
  • N-terminal coupling was carried out by the method of Example 5, and the modified product was purified by the method of Example 6, and similar experimental results were obtained.
  • Example 13 Coupling of 20 kDa polyethylene glycol with endostatin ESC analog
  • the endostatin ESC analog was dialyzed into a 20 mM NaH 2 PO 4 solution at pH 7.5, the protein concentration was determined, and the protein concentration was adjusted to between 5 and 15 mg/ml. Calculate the amount of 20kDa monomethoxypolyethylene glycol maleimide (mPEG-MAL) to be added according to the molar ratio of the target protein to polyethylene glycol 1:5, add to the target protein, and mix well. After standing at room temperature for 6-8 hours, the electrophoresis pattern of the modification results is shown in Fig. 4. Each band of the electrophoresis gel was analyzed by Bio-Rad's 1D gel quantification software Quantity One and compared with the molecular weight standard.
  • mPEG-MAL monomethoxypolyethylene glycol maleimide
  • the ratio of the endostatin ESC analog was single modified (molecular weight 40 kDa) at 80. Above 100%, one endostatin ESC analog molecule was modified with a monomethoxy polyethylene glycol maleimide molecule having a molecular weight of 20 kDa. Since monomethoxy polyethylene glycol maleimide can only react with free sulfhydryl groups, it can be considered that the polyethylene glycol modification site of the present product is a free sulfhydryl group of the endostatin ESC analog.
  • Example 14 Purification of a coupling product of 20 kDa polyethylene glycol and an endostatin ESC analog
  • the solution of the coupling reaction product of monomethoxypolyethylene glycol maleimide (mPEG-MAL) and endostatin ESC analog in Example 13 was purified by a cation chromatography column.
  • GE Healthcare SPFF medium was selected, and the reaction solution was adjusted to a pH of 5.0 to 7.0, and the cation column was equilibrated with 20 mM NaH 2 PO 4 (pH to 5.0 to 7.0) and loaded. Further, gradient elution was carried out using 20 mM NaH 2 PO 4 , 500 mM NaCl (pH 5.0 to 7.0), and different components were collected according to 280 nm ultraviolet absorption.
  • the SDS-PAGE analysis was carried out, and the analysis results are shown in Fig. 4.
  • component 3 is the product of a 20 kDa polyethylene glycol mono-modified endostatin ESC analog.
  • Example 15 Coupling and purification of 20 kDa polyethylene glycol with N-terminal amino groups of endostatin NK1, NK2, NK3, NK4, ESK, and NESK analogs
  • the N-terminal coupling was carried out by the method of Example 3, and the modified product was purified by the method of Example 4 to obtain similar experimental results.
  • Example 16 Coupling and purification of 20 kDa polyethylene glycol with endostatin NK1, NK2, NK3, NK4, ESK, NESK analog lysine side chain amino group and N-terminal amino group
  • Example 17 Purification of 20 kDa polyethylene glycol with vascular endostatin K1 analog lysine side chain amino coupling product
  • the endostatin K1 analog was dialyzed into a 20 mM sodium dihydrogen phosphate solution having a pH of 8.5 ⁇ 0.5, the protein concentration was determined, and the protein concentration was adjusted to be between 10 and 20 mg/ml. Calculate the amount of 20kDa monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) to be added according to the molar ratio of the target protein to polyethylene glycol 1:1, and calculate the reducing agent cyanoborohydride according to the final solution volume.
  • mPEG-ALD monomethoxypolyethylene glycol propionaldehyde
  • the amount of sodium is 10 mM, and the required monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) and sodium cyanoborohydride are weighed, added to the target protein, stirred uniformly, and allowed to stand at room temperature for 6-8. hour.
  • the reaction product solution was purified by a cation chromatography column, specifically selected from GE Healthcare Macrocap SP medium, and the reaction solution was adjusted to a pH of 4.5 ⁇ 0.5.
  • the cation column was equilibrated with 30 mM NaAc (pH to 4.0 to 5.0) and loaded.
  • Example 18 Coupling and purification of 20 kDa polyethylene glycol with lysine side chain amino groups of endostatin K2, K3, NK1, NK2, NK3 analogs
  • six of the endostatin molecules can be modified with polyethylene glycol, which are N-terminal amino groups and 5 lysine side chain amino groups.
  • the fifth lysine residue is located at the C-terminus, adjacent to the N-terminus, and it is difficult to achieve a single modification to the fifth lysine side chain amino group due to steric hindrance.
  • the thiol modification by adding a cysteine residue behind it reduces the preparation difficulty.
  • the second lysine residue is the farthest from the nucleolin binding region of the protein, followed by the N-terminus, and the polyethylene glycol modified ethylene glycol long chain is used for the binding region.
  • the effect is minimal, so the single- or double-modification of the N-terminal amino group and the second lysine side-chain amino group is superior to the other site modification scheme for the migration inhibition rate of the HMEC cell, indicating that the selection is far from the nucleolin binding region. Modification is the best choice for endostatin.
  • the effect of polyethylene glycol modification on the activity of protein on different surface of ES molecule is different.
  • the inhibition of N-terminal single modification and K2 single modification product on inhibiting migration of HMEC cells is obvious.
  • the point double-modification product had the most obvious inhibition on the inhibition of migration of HMEC cells.
  • K2 was the farthest from the nucleolin binding region.
  • the single modification inhibited the migration of HMEC cells by protein, while the K1, K3, K4 and C-terminal modification products inhibited the protein.
  • the HMEC cell migration activity increased little or decreased, and the double modified product also obtained similar results, indicating that K2 far from the nucleolin binding region is the best modification choice.
  • the N-terminal and K2 double modification is used. best choice.
  • a point mutation was performed on the ES natural structure N127 to form a new modification site, and relevant experiments were carried out, and the results confirmed that the region was confirmed.
  • the modified product (either single or double-modified with the N-terminus) increased the migration activity of HMEC cells less than the corresponding modified product of K2. It is fully demonstrated that the K2 and N-terminal double modifications are the best choice for increased protein activity and extended half-life.

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Abstract

本发明提了供聚乙二醇修饰的血管内皮抑制素类似物及其应用，所述血管内皮抑制素类似物在远离nucleolin结合区域的赖氨酸与聚乙二醇偶联或者在远离nucleolin结合区域的赖氨酸以及N端的氨基与聚乙二醇偶联。

Description

聚乙二醇化血管内皮抑制素类似物及其应用 技术领域
本发明涉及新的重组蛋白质药物。具体而言，本发明涉及聚乙二醇化血管内皮抑制素类似物及其应用。
背景技术
新生血管形成是指新的毛细血管在原有的血管上的生成。肿瘤的生长和迁移依赖于新生血管的生成，将肿瘤中的微血管内皮细胞作为癌症治疗的靶点为治疗肿瘤提供了一种治疗方式(Folkman J.N Engl J Med 1971；285:1182-1186)。
血管内皮抑制素(Endostatin)是胶原XVIII羧基端的一段分子量为20kDa的酶切产物。1997年美国哈佛大学Judah Folkman教授等人在血管内皮瘤细胞的培养基中发现此蛋白，它具有抑制血管内皮细胞增殖、迁移和体内血管生成的活性。进一步实验发现重组血管内皮抑制素可以抑制老鼠体内多种肿瘤的生长、迁移，甚至能完全治愈肿瘤，而且不产生耐药性。其发挥活性的机理在于它通过抑制血管内皮细胞的生长，抑制了肿瘤组织附近的新生血管的生成，使得肿瘤组织得不到生长所必需的大量营养和氧气，最后停止生长或坏死(Folkman J.et al.Cell 1997；88:277-285；Folkman J.et al.Nature 1997；390：404-407)。2010年清华大学罗永章教授等人发现血管内皮抑制素还可以显著抑制肿瘤相关的淋巴管生成(Yongzhang Luo.et al.Journal of Pathology 2010；222：249–260；Yongzhang Luo.Front.Med.2011,5:336–340.)。通过基因工程制备的重组人血管内皮抑制素可以作为肿瘤治疗药物，其临床试验表明它可以有效的抑制肿瘤生长。在中国，以非小细胞肺癌为主要适应症的临床试验表明它对于肿瘤的治疗效果比较突出。
多年来肿瘤治疗主要依靠小分子化合物治疗(化疗：chemotherapy)和放射治疗(放疗：radiotherapy)。这两种方法虽然都非常有效，但是也存在一些毒副反应。相比之下，蛋白类药物毒副作用更小，而且不产生耐药性，但是采用经胃肠道给药会产生严重的首过效应(first pass effect)。为了达到最高的体内活性，提高生物利用度，减少药物体内降解，通常蛋白类药物采用静脉或皮下注射给药方式。对于分子量小的蛋白来说，通常静脉给药后药物体内半衰期很短，其中一个重要的原因是小分子蛋白能够通过肾过滤的途径快速消除。在血液中水力半径超过白蛋白或者分子量大于66,000道尔顿(66kDa)的蛋白通常可以稳定的保留在循环系统中，而小分子蛋白药物却会通过肾小球过滤被快速消除，因此为了维持在血液中的有效治疗浓度，患者需要频繁接受注射或者点滴。这种治疗方式虽然能够达到治疗的目的，但是却给患者带来了严重的不便和痛苦，也严重影响了患者对于药物治疗的依从性，也提高了用药成本。而且某些药物长期频繁使用还有可能会产生一些副作用，如免疫反应。
作为蛋白药物的血管内皮抑制素同样存在半衰期短、体内清除率高的缺点。目前临床用药手段主要为频繁给药以维持有效血药浓度，通常为每天给药，而且需要长期用药，这使得患者的精神和经济负担都很大。本发明的目的就在于改善该蛋白的体内代谢特征，使其具有更高的稳定性和更长的体内半衰期，甚至具有更高的疗效，从而达到减少用药频率，降低用药成本，减轻患者的经济负担的目的。
用高分子聚合物对蛋白进行修饰是改变和控制药物的动力学特性如半衰期、免疫学特征以及毒理特性的常用方法。其中聚乙二醇是应用最为广泛的一种双亲性高分子聚合物，具有溶解性好、生物相容性好、无毒无免疫原性的特点，是被包括美国FDA、中国SFDA在内的很多国家和地区的药品管理机构批准的可用于药物制备的高分子。将蛋白质与亲水高分子如聚乙二醇偶联，可以降低蛋白酶解、增加蛋白稳定性、减少非特异性吸附和抗原性，当偶联物水力学半径达到肾小球滤过下限时可以大大降低肾脏清除效率，是延长蛋白质药物体内半衰期的有效方法(Gianfranco Pasut et.al.Advanced Drug Delivery Reviews 60(2008)69-78；F.M.Veronese et al.Milestones in Drug Therapy 2009,11-31)。随着聚乙二醇修饰技术的发展，蛋白质分子上的多种基团都可以被PEG修饰以满足不同的需求。例如，利用单甲氧基聚乙二醇琥珀酰亚胺活性酯类修饰剂(如mPEG-SC、mPEG-SCM、mPEG-SPA、mPEG-NHS等)随机修饰蛋白质或者多肽N末端α氨基或者赖氨酸侧链的ε氨基，实现对蛋白质的多位点修饰。利用单甲氧基聚乙二醇醛(mPEG-Aldehyde)在弱酸性条件下可以特异性的与蛋白质或者多肽的N端α氨基反应，形成不稳定的希夫碱，通过氰基硼氢化钠进一步还原，生成稳定的仲胺(secondary amine)实现对N末端的定点修饰。利用单甲氧基聚乙二醇马来酰亚胺类修饰剂(如mPEG-MAL、mPEG2-MAL等)选择性修饰蛋白或者多肽中游离半胱氨酸。截至目前聚乙二醇修饰技术已经成功应用于多种蛋白，FDA已经批准上市的聚乙二醇修饰蛋白药物有PEG-腺苷脱亚胺酶(1990年)、PEG-天冬酰胺酶(1994年)、PEG-α2b干扰素(2001年)、PEG-粒细胞集落刺激因子(2002年)、PEG-α2a干扰素(2002年)、PEG-α2a干扰素与利巴韦林混合制剂(2004年)、PEG-红细胞生成素(2007年)、PEG-TNFα抗体(2008年)、PEG-α2b干扰素与利巴韦林混合制剂(2008年)、PEG-尿酸氧化酶(2010年)。正在进行临床研究的代表性的聚乙二醇修饰蛋白药物有PEG-精氨酸脱亚胺酶(Polaris Group，III期)、PEG-生长激素(长春金赛药业，III期)、PEG-β1a干扰素(Biogen Idec，III期)等。
除了聚乙二醇修饰蛋白实现延长药物半衰期以外，还有其它高分子修饰剂可以使用，如葡聚糖、聚蔗糖、淀粉、聚丙氨酸、乙二酸与丙二酸的共聚物、羧甲基纤维素、聚乙烯吡咯烷酮、聚1,3-乙二醇次甲基醚、乙烯和顺丁烯二酰肼的共聚物、多聚唾液酸以及环糊精等。
发明内容
本发明人发现，在血管内皮抑制素上远离nucleolin结合区域的赖氨酸上偶联聚乙二醇能够明显提高蛋白活性，因此，本发明提供聚乙二醇修饰的血管内皮抑制素类似物，所述血管内皮抑制素类似物在远离nucleolin结合区域的赖氨酸与聚乙二醇偶联， 由此使得其抑制肿瘤细胞迁移的生物学活性提高。本发明的聚乙二醇修饰的血管内皮抑制素类似物与未修饰的血管内皮抑制素相比具有更高的稳定性和更长的体内半衰期，而且其抑制新生血管生成活性显著提高。
本发明的第一方面提供血管内皮抑制素类似物与聚乙二醇偶联复合物；其中所述血管内皮抑制素类似物在相应于天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第96位的位置上具有赖氨酸残基，而在任何其它位置上均不具有赖氨酸残基；并且，所述血管内皮抑制素类似物仅在该赖氨酸残基上与聚乙二醇偶联。
本发明的第二方面提供血管内皮抑制素类似物与聚乙二醇偶联复合物；其中所述血管内皮抑制素类似物在相应于天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第96位的位置上具有赖氨酸残基，而在任何其它位置上均不具有赖氨酸残基；并且，所述血管内皮抑制素类似物在N末端和该赖氨酸残基上与聚乙二醇偶联。
在优选的实施方案中，在上述第一方面和第二方面的复合物中，所述血管内皮抑制素类似物是天然血管内皮抑制素氨基酸序列SEQ ID NO.1第76、107、118和184位的赖氨酸残基发生突变而形成的。在一个具体方案中，所述血管内皮抑制素类似物的序列如SEQ ID NO:3所示。
本发明的第三方面提供血管内皮抑制素类似物与聚乙二醇偶联复合物；其中所述血管内皮抑制素类似物在相应于天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第96位的位置上具有赖氨酸残基，而在任何其它位置上均不具有赖氨酸残基；并且所述血管内皮抑制素类似物在其N端具有插入氨基酸序列；并且，所述血管内皮抑制素类似物仅在该赖氨酸残基上与聚乙二醇偶联。
本发明的第四方面提供血管内皮抑制素类似物与聚乙二醇偶联复合物；其中所述血管内皮抑制素类似物在相应于天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第96位的位置上具有赖氨酸残基，而在任何其它位置上均不具有赖氨酸残基，并且所述血管内皮抑制素类似物在其N端具有插入氨基酸序列；并且，所述血管内皮抑制素类似物在N末端和该赖氨酸残基上与聚乙二醇偶联。
在优选的实施方案中，在上述第三方面和第四方面的复合物中，所述血管内皮抑制素类似物是天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第76、107、118和184位的赖氨酸残基发生突变，并且在其N端插入氨基酸序列而形成的。在更优选的实施方案中，所插入的氨基酸序列是在N端的甲硫氨酸M和组氨酸H之间插入GGSHHHHH。在更优选的实施方案中，所述血管内皮抑制素类似物的序列如SEQ ID NO:8所示。
在优选的实施方案中，本发明的偶联复合物中的血管内皮抑制素类似物中，天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第76、107、118和184位的赖氨酸残基分别被突变为X1、X3、X4、X5，其中X1、X3、X4或X5独立地为除赖氨酸以外的任何一种天然氨基酸。X1、X3、X4或X5独立地优选是水溶性氨基酸，进一步优选是带电荷氨基酸精氨酸、组氨酸、谷氨酸和天冬氨酸的任一种，进一步优选是带正电荷的氨基酸精氨酸和组氨酸的任一种，最优选为精氨酸。
在优选的实施方案中，在本发明的复合物中，血管内皮抑制素类似物N端插入氨 基酸序列是MGGSHHHHH。
在优选的实施方案中，本发明的复合物中，血管内皮抑制素类似物与聚乙二醇为共价键偶联。
在优选的实施方案中，所述聚乙二醇的平均分子量为为5,000到40,000道尔顿。
在更优选的实施方案中，所述聚乙二醇的平均分子量为20,000到40,000道尔顿。
在优选的实施方案中，所述聚乙二醇为单甲氧基聚乙二醇、单葡萄糖聚乙二醇或者单半乳糖聚乙二醇。
在优选的实施方案中，所述聚乙二醇为线性的或者分支的。
在优选的实施方案中，所述聚乙二醇为单甲氧基聚乙二醇。
在一些实施方案中，聚乙二醇与血管内皮抑制素类似物的氨基偶联。
在一些实施方案中，聚乙二醇与血管内皮抑制素类似物赖氨酸侧链ε-氨基偶联。
在一些实施方案中，聚乙二醇与血管内皮抑制素类似物N末端α-氨基和赖氨酸残基ε-氨基偶联。
在更优选的实施方案中，聚乙二醇与血管内皮抑制素类似物的氨基偶联可以采用单甲氧基聚乙二醇丙醛(mPEG-ALD)、单甲氧基聚乙二醇丁醛(mPEG-ButyrALD)、单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC)、单甲氧基聚乙二醇琥珀酰亚胺乙酸酯(mPEG-SCM)、单甲氧基聚乙二醇琥珀酰亚胺丙酸酯(mPEG-SPA)、单甲氧基聚乙二醇琥珀酰亚胺丁酸酯(mPEG-SBA)、单甲氧基聚乙二醇琥珀酰亚胺α-甲基丁酸酯(mPEG-SMA)、单甲氧基聚乙二醇NHS酯(mPEG-NHS)进行偶联反应。
本发明还提供药物组合物，其包含如上所述的本发明的血管内皮抑制素类似物与聚乙二醇偶联复合物和药学可接受的载体。
本发明还提供治疗新生血管或者新生淋巴管导致的疾病的方法，包括给患者施用如上所述的本发明的血管内皮抑制素类似物与聚乙二醇偶联复合物或药物组合物。
本发明还提供本发明的血管内皮抑制素类似物与聚乙二醇偶联复合物或药物组合物在治疗新生血管或者新生淋巴管导致的疾病中的应用。
在优选的实施方案中，所述新生血管或者新生淋巴管导致的疾病是肿瘤。
本发明还提供本发明的偶联复合物在制备体内或体外用于抑制新生血管或者新生淋巴管的生成的药物中的应用。
本发明的复合物用于体内抑制新生血管或者新生淋巴管的生成时，其半衰期较血管内皮抑制素显著延长，在相同给药条件下其抑制率较血管内皮抑制素显著增加。
附图说明
图1：20kDamPEG-AL N端单修饰血管内皮抑制素的修饰样品和后续层析纯化样品的SDS-PAGE电泳分析结果。第1泳道为低分子量标准品，由上到下依次为116、66、45、30、25、18.4、14.4kDa；第2泳道为血管内皮抑制素用20kDa mPEG-ALD修饰的样品；第3～7泳道分别为层析纯化时100、150、200、350以及500mMNaCl洗脱获得的组分1、2、3、4、5，其中第5泳道200mMNaCl洗脱组分(组分3)为N端单修饰产物组分。
图2：20kDa mPEG-ALDN端单修饰血管内皮抑制素K1类似物的修饰样品和后续层析纯化样品的SDS-PAGE电泳分析结果。第1泳道为低分子量标准品，由上到下依次为116、66、45、30、25、18.4、14.4kDa；第2泳道为血管内皮抑制素K1类似物用20kDamPEG-ALD修饰的样品；第3～7泳道分别为层析纯化时100、150、150、350以及500mMNaCl洗脱获得的组分1、2、3、4、5，其中第4、5泳道150mM NaCl洗脱组分(组分2、3)均为20kDa聚乙二醇N端单修饰血管内皮抑制素K1类似物。
图3：20kDamPEG-SPA双修饰血管内皮抑制素K1类似物赖氨酸侧链氨基以及N端氨基双修饰和纯化样品电泳分析结果。第1泳道为低分子量标准品，由上到下依次为116、66、45、30、25、18.4、14.4kDa；第2泳道为血管内皮抑制素用20kDamPEG-SPA修饰样品；第3～8泳道分别为层析纯化时25、50、75、100、200以及500mMNaCl洗脱组分1、2、3、4、5、6，其中第6泳道100mM NaCl洗脱组分(组分4)为K1类似物双修饰产物组分。
图4：20kDa没PEG-MAL修饰血管内皮抑制素ESC类似物巯基的修饰样品和后续纯化样品的SDS-PAGE电泳结果。第1泳道为血管内皮抑制素ESC类似物用20kDamPEG-MAL修饰的样品；第2～5泳道分别为层析纯化时100、150、200以及500mM NaCl洗脱组分，第6泳道为低分子量标准品，由上到下依次为116、66、45、30、25、18.4、14.4kDa；其中第4泳道200mMNaCl洗脱组分(组分3)为ESC类似物巯基单修饰产物组分。
图5：20kDamPEG-ALD修饰血管内皮抑制素K1类似物赖氨酸侧连氨基的修饰样品和后续纯化样品的SDS-PAGE电泳分析结果。第1泳道为低分子量标准品，由上到下依次为116、66、45、30、25、18.4、14.4kDa；第2泳道为血管内皮抑制素用20kDamPEG-ALD修饰样品；第3～7泳道分别层析纯化时为50、100、125、200以及500mM NaCl洗脱组分，其中第4、5泳道均为20kDa聚乙二醇单修饰血管内皮抑制素类似物，两个样品进行修饰位点分析确认第4泳道样品为聚乙二醇N端单修饰产物以及少量赖氨酸侧连氨基单修饰产物，第5泳道样品为血管内皮抑制素K1类似物赖氨酸侧连氨基单修饰产物。
图6：人血管内皮抑制素的三级结构图，标示了人血管内皮抑制素蛋白的N端、C端以及第76位赖氨酸残基K1、第96位赖氨酸残基K2、第107位赖氨酸残基K3、第118位赖氨酸残基K4的位置以及本蛋白与受体Nucleolin结合区域。
图7：人血管内皮抑制素的三级结构图，标示了人血管内皮抑制素蛋白的N端、C端以及第76位赖氨酸残基K1、第96位赖氨酸残基K2、第107位赖氨酸残基K3、第118位赖氨酸残基K4的位置以及第127位天冬酰胺残基的位置。
图8：本发明涉及的血管内皮抑制素类似物的序列。
具体实施方式
本文使用的术语“血管内皮抑制素(Endostatin,ES)”可以指天然血管内皮抑制素，优选是人血管内皮抑制素，例如具有SEQ ID NO:1的序列，但不限于此，例如，还可 以是来自于其它哺乳动物，例如小鼠、大鼠、猪、狗、兔、绵羊、山羊、猫等的天然血管内皮抑制素。“血管内皮抑制素”还可以指血管内皮抑制素的功能性变体，例如工程化的功能性变体，它与天然血管内皮抑制素相比具有一个或几个氨基酸的取代、缺失或添加，并具有基本相同的生物学功能，例如抑制血管内皮细胞增殖、迁移和体内血管生成的活性。“血管内皮抑制素”还可以指天然血管内皮抑制素或其功能性变体的衍生物或修饰产物，例如聚乙二醇修饰产物。
本文使用的术语“功能性变体”包括在氨基酸序列中含有一个或多个(例如1-5个、1-10个或者1-15个，具体地，例如可以是1、2、3、4、5、6、7、8、9、10、11、12个甚至更多个)氨基酸的取代、缺失或添加的血管内皮抑制素的突变体，并且所述突变体具有与血管内皮抑制素类似的抑制血管内皮细胞增殖、迁移和体内血管生成的生物学活性。血管内皮抑制素的“功能性变体”的生物学活性例如可以是天然血管内皮抑制素，例如天然人血管内皮抑制素的30％或更高、50％或更高、60％或更高、70％或更高、80％或更高或90％或更高。所述“功能性变体”可以是自然产生的突变体，也可以是人工突变体，例如通过定点诱变获得的突变体，或通过基因重组方法产生的突变体。
所述“功能性变体”的生物学活性可以通过本领域众所周知的用于检测血管内皮抑制素活性的方法来检测。例如可以选择HMEC细胞，采用Migration(Tranwell Assay)方法分析功能性变体对HMEC细胞迁移的抑制率，计细胞数反映蛋白活性(可参考Luo yongzhang等，Endostatin inhibits tumourlymphangiogenesis and lymphatic metastasis via cell surface nucleolin on lymphangiogenic endothelial cells(J Pathol 2010；222:249–260)。
本发明中，“血管内皮抑制素类似物”和“血管内皮抑制素功能性变体”可以互换使用。在一些实施方案中，本发明的“血管内皮抑制素类似物”在相应于天然人血管内皮抑制素(例如SEQ ID NO:1)第96位的位置上具有赖氨酸残基，而在其它任何位置上均不具有赖氨酸残基，以使得聚乙二醇仅仅被偶联在所述血管内皮抑制素类似物的该赖氨酸残基上或者被同时偶联在氨基末端和该赖氨酸残基上，而不与其它任何氨基酸残基偶联。由此获得的偶联复合物与天然人血管内皮抑制素或天然人血管内皮抑制素的氨基末端单聚乙二醇偶联复合物相比，具有更高的稳定性和更长的体内半衰期，而且其抑制新生血管生成活性显著提高。
本发明的血管内皮抑制素类似物可以通过对天然血管内皮抑制素(例如天然人血管内皮抑制素或天然哺乳动物血管内皮抑制素)的改造获得，具体而言，是使天然人血管内皮抑制素(例如SEQ ID NO:1)中的第76、107、118、184位的赖氨酸残基发生突变，仅保留第96位的赖氨酸残基获得的血管内皮抑制素类似物，或者使天然的其它哺乳动物的血管内皮抑制素中相应于天然人血管内皮抑制素(例如SEQ ID NO:1)中的第76、107、118、184位的赖氨酸残基发生突变，仅保留相应于天然人血管内皮抑制素(例如 SEQ ID NO:1)中的第96位的赖氨酸残基获得的血管内皮抑制素类似物。本发明的血管内皮抑制素类似物也可以通过对已知的天然产生的或人工合成的血管内皮抑制素功能性变体的改造获得，具体而言，如果血管内皮抑制素功能性变体在相应于天然血管内皮抑制素第96位的位置上是赖氨酸残基，则保持该赖氨酸残基不变，使该血管内皮抑制素功能性变体的所有其它赖氨酸残基发生突变；如果血管内皮抑制素功能性变体在相应于天然血管内皮抑制素第96位的位置上不是赖氨酸残基，则使该位置上的氨基酸突变为赖氨酸残基，并使该血管内皮抑制素功能性变体的所有其它赖氨酸残基发生突变。
可被改造从而获得本发明的血管内皮抑制素类似物的血管内皮抑制素功能性变体包括当人ES在大肠杆菌中重组表达时，其第一个氨基酸M被随机删除产生的ES变体。可被改造从而获得本发明的血管内皮抑制素类似物的血管内皮抑制素功能性变体还包括当ES在酵母中重组表达时由于N末端发生随机剪切产生的N末端缺失4个氨基酸的ES变体。可被改造从而获得本发明的血管内皮抑制素类似物的血管内皮抑制素功能性变体包括YH-16，它是在ES的N-末端添加了9个额外氨基酸(MGGSHHHHH)而得到的ES变体，以便于增加可溶性表达和方便纯化(Fu Y et al.IUBMB Life 2009；61:613-626；Wang J et al.Zhongguo fei ai za zhi 2005；8:283-290；Han B et al.J Thorac Oncol2011；6(6):1104-1109，通过合并的方式将其全部内容合并入本文)。可被改造从而获得本发明的血管内皮抑制素类似物的血管内皮抑制素功能性变体还包括PCT国际申请PCT/CN2012/081210中公开的那些血管内皮抑制素突变体，如ES006、ES008、ES011、S02、S09、Z006、Z008、ZN1等(通过引用的方式将其全部内容合并入本文)。可被改造从而获得本发明的血管内皮抑制素类似物的血管内皮抑制素功能性变体还包括PCT国际公开号WO2016/070798中公开的那些血管内皮抑制素突变体，如003、007、Z101、009、S03、36、249、381、57、114、124、125、160、163、119(通过引用的方式将其全部内容合并入本文)。
本发明所述“相应于天然血管内皮抑制素氨基酸序列SEQ ID NO:1的……位置”是指利用本领域众所周知的软件或算法将血管内皮抑制素功能性变体与天然血管内皮抑制素氨基酸序列SEQ ID NO:1进行比对后，血管内皮抑制素功能性变体上与SEQ ID NO:1上相应氨基酸残基对应的位置。所述软件或算法包括但不限于BLAST、FASTA。
本发明所述的“聚乙二醇(PEG)”可以是单甲氧基聚乙二醇、单葡萄糖聚乙二醇或者单半乳糖聚乙二醇。所使用的聚乙二醇可以是线性的或分支的，可以具有例如约5kD至约50kD、约20kD至约40kD或例如约20kD的分子量。
本文使用的术语“聚乙二醇修饰”或“聚乙二醇偶联”可以指将聚乙二醇修饰剂分子与蛋白质分子化学偶联到一起，参与偶联反应的聚乙二醇修饰剂的基团是其在活 化过程中引入的活化基团，蛋白质的基团主要是其中游离的氨基基团、巯基基团等，优选氨基基团。与聚乙二醇(PEG)偶联可以延长偶联复合物在体内的半衰期、避免蛋白酶降解或增加可溶性。用聚乙二醇修饰蛋白质的方法是本领域技术人员所熟知的。
术语“聚乙二醇修饰剂”包括但不限于单甲氧基聚乙二醇修饰剂，它是用甲氧基封闭聚乙二醇分子一端的一个羟基，而用适宜的活化方法活化另一端的一个羟基后所得到的活化聚乙二醇。由于羟基自身的反应活性很低，所以经过活化后的聚乙二醇分子的反应活性有了很大的提高，被称为“聚乙二醇修饰剂”。关于活化基团的选择、活化的机理以及活化所得的聚乙二醇修饰剂的修饰反应机理是本领域众所周知的，在许多文献中已有报道。聚乙二醇修饰剂可以通过商购获得。可以使用的聚乙二醇修饰剂包括但不限于聚乙二醇单甲氧基聚乙二醇丙醛(mPEG-ALD)、单甲氧基聚乙二醇丁醛(mPEG-ButyrALD)、单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC)、单甲氧基聚乙二醇琥珀酰亚胺乙酸酯(mPEG-SCM)、单甲氧基聚乙二醇琥珀酰亚胺丙酸酯(mPEG-SPA)、单甲氧基聚乙二醇琥珀酰亚胺丁酸酯(mPEG-SBA)、单甲氧基聚乙二醇琥珀酰亚胺α-甲基丁酸酯(mPEG-SMA)、单甲氧基聚乙二醇NHS酯(mPEG-NHS)。
本发明中，聚乙二醇与血管内皮抑制素类似物的氨基偶联，例如与血管内皮抑制素类似物赖氨酸侧链ε-氨基偶联，或者与血管内皮抑制素类似物N末端α-氨基和赖氨酸残基ε-氨基偶联。
本发明(例如实施例)中所述的血管内皮抑制素K1类似物是指氨基酸序列为SEQ ID NO.2的血管内皮抑制素类似物，是将血管内皮抑制素氨基酸序列SEQ ID NO.1从N端起第96、107、118、184位的赖氨酸残基分别突变为X2、X3、X4、X5。其中X2、X3、X4、X5为除赖氨酸以外的任何一种天然氨基酸，优选为水溶性氨基酸，进一步优选为带电荷氨基酸精氨酸、组氨酸、谷氨酸和天冬氨酸的一种，进一步优选为带正电荷的氨基酸精氨酸和组氨酸的一种，最优选为精氨酸。
本发明(例如实施例)中所述的血管内皮抑制素K2类似物是指氨基酸序列为SEQ ID NO.3的血管内皮抑制素类似物，是将血管内皮抑制素氨基酸序列SEQ ID NO.1从N端起第76、107、118、184位的赖氨酸残基分别突变为X1、X3、X4、X5。其中X1、X3、X4、X5为除赖氨酸以外的任何一种天然氨基酸，优选为水溶性氨基酸，进一步优选为带电荷氨基酸精氨酸、组氨酸、谷氨酸和天冬氨酸的一种，进一步优选为带正电荷的氨基酸精氨酸和组氨酸的一种，最优选为精氨酸。
本发明(例如实施例)中所述的血管内皮抑制素K3类似物是指氨基酸序列为SEQ ID NO.4的血管内皮抑制素类似物，是将血管内皮抑制素氨基酸序列SEQ ID NO.1从N端起第76、96、118、184位的赖氨酸残基分别突变为X1、X2、X4、X5。其中X1、X2、X4、X5为除赖氨酸以外的任何一种天然氨基酸，优选为水溶性氨基酸，进一步 优选为带电荷氨基酸精氨酸、组氨酸、谷氨酸和天冬氨酸的一种，进一步优选为带正电荷的氨基酸精氨酸和组氨酸的一种，最优选为精氨酸。
本发明(例如实施例)中所述的血管内皮抑制素K4类似物是指氨基酸序列为SEQ ID NO.5的血管内皮抑制素类似物，是将血管内皮抑制素氨基酸序列SEQ ID NO.1从N端起第76、96、107、184位的赖氨酸残基分别突变为X1、X2、X3、X5。其中X1、X2、X3、X5为除赖氨酸以外的任何一种天然氨基酸，优选为水溶性氨基酸，进一步优选为带电荷氨基酸精氨酸、组氨酸、谷氨酸和天冬氨酸的一种，进一步优选为带正电荷的氨基酸精氨酸和组氨酸的一种，最优选为精氨酸。
本发明(例如实施例)中所述的血管内皮抑制素ESC类似物是指氨基酸序列为SEQ ID NO.6的血管内皮抑制素类似物，是在血管内皮抑制素氨基酸序列SEQ ID NO.1的C末端连接一个半胱氨酸残基C。
本发明(例如实施例)中所述的血管内皮抑制素NK1、NK2、NK3、NK4、NESC类似物分别是指氨基酸序列为SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ IDNO.10、SEQ ID NO.11的血管内皮抑制素类似物。血管内皮抑制素NK1、NK2、NK3、NK4、NESC类似物分别是在上述的血管内皮抑制素K1、K2、K3、K4、ESC类似物的N末端的甲硫氨酸M和组氨酸H之间插入氨基酸序列GGSHHHHH。
本发明(例如实施例)中所述的血管内皮抑制素ESK类似物是指氨基酸序列为SEQ ID NO.12的血管内皮抑制素类似物，为将血管内皮抑制素氨基酸序列SEQ ID NO.1从N端起第76、96、107、118、184位的赖氨酸残基分别突变为X1、X2、X3、X4和X5，并且将第127号残基天冬酰胺突变为赖氨酸。其中X1、X2、X3、X4、X5为除赖氨酸以外的任何一种天然氨基酸，优选为水溶性氨基酸，进一步优选为带电荷氨基酸精氨酸、组氨酸、谷氨酸和天冬氨酸的一种，进一步优选为带正电荷的氨基酸精氨酸和组氨酸的一种，最优选为精氨酸。
本发明(例如实施例)中所述的血管内皮抑制素NESK类似物是指氨基酸序列为SEQ ID NO.13的血管内皮抑制素类似物，是在上述血管内皮抑制素ESK类似物的N末端的甲硫氨酸M和组氨酸H之间插入氨基酸序列GGSHHHHH。
本文使用的术语“单修饰”是指一个PEG分子修饰一个血管内皮抑制素或血管内皮抑制素类似物得到的产物。
本文使用的术语“双修饰”是指两个PEG分子修饰一个血管内皮抑制素或血管内皮抑制素类似物得到的产物。
本发明还提供药物组合物，其包含如上所述的本发明的血管内皮抑制素类似物与聚乙二醇偶联复合物，用于治疗新生血管或者新生淋巴管导致的疾病。在一些实施方案中，所述新生血管或者新生淋巴管导致的疾病是肿瘤，包括但不限于肺癌、乳腺癌等。适当地，所述药物组合物进一步包含药学可接受的载体。
本文所使用的术语“药学可接收的载体”是指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质。依照给药途径，可以施用本领域众所周知的各种不同的载体，包括，但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水、和/或无热原水等。
所述药物组合物还可以是缓释制剂，其形式选自微胶囊、水凝胶、微球、微型渗透泵或脂质体。
本发明还提供包含如上所述的本发明的血管内皮抑制素类似物与聚乙二醇偶联复合物和使用说明的试剂盒。
本发明还提供治疗新生血管或者新生淋巴管导致的疾病的方法，包括给受试者施用治疗有效量的如上所述的本发明的血管内皮抑制素类似物与聚乙二醇偶联复合物。在一些实施方案中，所述新生血管或者新生淋巴管导致的疾病是肿瘤，包括但不限于肺癌、乳腺癌、结肠癌等。
本文所使用的术语“治疗有效量”指的是足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。血管内皮抑制素类似物与聚乙二醇偶联复合物的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg活性成分。
本发明所提供的药物可以制成粉末、注射剂等临床可接受的剂型。可以使用任何适当的途径给受试者施用本发明的药物组合物，例如可通过口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径给药。
除非另有说明，否则本说明书中所使用的科学及技术术语应具有本领域普通技术人员通常所了解的含义。通常，本说明书中所使用的与细胞及组织培养、分子生物学、免疫学、微生物学、遗传学及蛋白质与核酸化学有关的命名及其技术是本领域公知和常用的。
除非另有说明，否则本说明书中所使用的方法及技术一般根据本领域公知的和常规的方法和本说明书中所阐述的或引用的各种参考文献中所述的方式来进行。
下面的实施例中，分别制备、纯化血管内皮抑制素以及血管内皮抑制素K1、K2、K3、K4、ESC、NK1、NK2、NK3、NK4、NESC、ESK、NESK类似物的N端氨基聚乙二醇单修饰产物、N端氨基和赖氨酸氨基聚乙二醇双修饰产物、赖氨酸氨基聚乙二醇单修饰产物和C端巯基聚乙二醇单修饰产物，并检测它们对HMEC细胞迁移的抑制率。下述实施例中所使用的血管内皮抑制素K1、K2、K3、K4、NK1、NK2、NK3、NK4、ESK、NESK类似物中，突变的赖氨酸均是由赖氨酸突变为精氨酸。
实施例1：20kDa聚乙二醇与天然人血管内皮抑制素的N端氨基偶联
将天然人血管内皮抑制素透析到pH5.0±1.0的30mM醋酸钠溶液中，测定蛋白浓度，并将蛋白浓度调整至5～15mg/ml之间。按照目的蛋白与聚乙二醇的摩尔比1:3计算需要加入的20kDa的单甲氧基聚乙二醇丙醛(mPEG-ALD)量，同时按照最终溶液体积计算还原剂氰基硼氢化钠的用量，其浓度为20mM，称取需要的单甲氧基聚乙二醇丙醛(mPEG-ALD)和氰基硼氢化钠，加入到目的蛋白中，搅拌均匀后室温静置6～8小时，修饰结果电泳图见附图1。利用Bio-Rad的1D凝胶定量软件Quantity One对电泳凝胶的每一条带进行含量分析并与分子量标准比对，结果显示此时血管内皮抑制素被单一修饰(产物分子量40kDa)的比例在60％以上，即一个血管内皮抑制素被一个分子量为20kDa的单甲氧基聚乙二醇修饰。
实施例2：20kDa聚乙二醇与天然人血管内皮抑制素的N端氨基偶联产物的纯化
将实施例1中单甲氧基聚乙二醇丙醛(mPEG-ALD)与天然人血管内皮抑制素偶联反应产物溶液用阳离子层析柱纯化。具体选择GE Healthcare公司SPFF介质，并将反应液调整pH值至5.0～7.0，阳离子柱用20mM NaH2PO4(pH值至5.0～7.0)平衡并上样。再采用20mM NaH2PO4、500mM NaCl pH5.0～7.0)进行梯度洗脱，根据280nm紫外吸收收集不同的组分。并进行SDS-PAGE分析，分析结果见图1。由图1可知，组分3即为20kDa聚乙二醇单修饰血管内皮抑制素的产物(分子量40kDa)。由于N末端氨基与赖氨酸侧链氨基pKa值分别为8.0和10.5，在pH5.0±1.0条件下修饰时N末端氨基的反应活性是赖氨酸侧链氨基活性的10^(10.5-8.0)＝316倍，因此可以认为该单修饰产物为N端单修饰产物。
实施例3：20kDa聚乙二醇与血管内皮抑制素K1类似物N端氨基偶联
将血管内皮抑制素K1类似物透析到pH5.0±1.0的30mM醋酸钠溶液中，测定蛋白浓度，并将蛋白浓度调整至5～15mg/ml之间。按照目的蛋白与聚乙二醇的摩尔比1:3计算需要加入的20kDa的单甲氧基聚乙二醇丙醛(mPEG-ALD)的量，同时按照最终溶液体积计算还原剂氰基硼氢化钠的用量，其浓度为20mM。称取需要的单甲氧基聚乙二醇丙醛(mPEG-ALD)和氰基硼氢化钠，加入到目的蛋白中，搅拌均匀后室温静置6～8小时，修饰结果电泳图见图2。利用Bio-Rad的1D凝胶定量软件Quantity One对电泳凝胶的每一条带进行含量分析并与分子量标准比对，结果显示此时血管内皮抑制素K1类似物被单一修饰的比例在60％以上，即一个血管内皮抑制素K1类似物被一个分子量为20kDa的单甲氧基聚乙二醇修饰。
实施例4：20kDa聚乙二醇与血管内皮抑制素K1类似物N端氨基偶联产物的纯化
将实施例3中单甲氧基聚乙二醇丙醛(mPEG-ALD)与血管内皮抑制素K1类似物偶联反应产物溶液用阳离子层析柱纯化。选择GE Healthcare公司SPFF介质，并将反应液调整pH值至5.0～7.0，阳离子柱用20mM NaH2PO4(pH值至5.0～7.0)平衡并上样。再采用20mM NaH2PO4、500mM NaCl pH5.0～7.0)进行梯度洗脱，根据280nm紫外吸收收集不同的组分。并进行SDS-PAGE分析，分析结果见图2。由图2可知，组分2、3均为20kDa聚乙二醇单修饰血管内皮抑制素K1类似物的产物，基于实施例2相同的修饰原理分析，可以认为该单修饰产物为N端单修饰产物。
实施例5：20kDa聚乙二醇与血管内皮抑制素K1类似物赖氨酸侧链氨基以及N端氨基双偶联
将血管内皮抑制素K1类似物透析到pH8.5的20mM NaH2PO4溶液中，测定蛋白浓度，并将蛋白浓度调整至5～15mg/ml之间。按照目的蛋白与聚乙二醇的摩尔比1:10计算需要加入的20kDa的单甲氧基聚乙二醇琥珀酰亚胺丙酸酯的量，加入到目的蛋白中，搅拌均匀后室温静置60～120分钟，修饰结果电泳图见图3。利用Bio-Rad的1D凝胶定量软件Quantity One对电泳凝胶的每一条带进行含量分析并与分子量标准比对，此时血管内皮抑制素K1类似物被双修饰(分子量60kDa)的比例在50％以上，即一个血管内皮抑制素K1类似物被两个分子量为20kDa的单甲氧基聚乙二醇修饰。
实施例6：20kDa聚乙二醇与血管内皮抑制素K1类似物赖氨酸侧链氨基以及N端氨基双偶联产物的纯化
将实施例5中单甲氧基聚乙二醇琥珀酰亚胺丙酸酯与血管内皮抑制素K1类似物偶联反应产物溶液用阳离子层析柱纯化。选择GE Healthcare公司SPFF介质，并将反应液调整pH值至5.0～7.0，阳离子柱用20mM NaH2PO4(pH值至5.0～7.0)平衡并上样。再采用20mM NaH2PO4、500mM NaCl pH5.0～7.0)进行梯度洗脱，根据280nm紫外吸收收集不同的组分。并进行SDS-PAGE分析，分析结果见图3。由图3可知，组分4为20kDa聚乙二醇双修饰血管内皮抑制素K1类似物的产物。
实施例7：20kDa聚乙二醇与血管内皮抑制素K2类似物N端氨基偶联以及纯化
采用实施例3的方法进行N端偶联，采用实施例4的方法进行修饰产物纯化，得到类似的实验结果。
实施例8：20kDa聚乙二醇与血管内皮抑制素K2类似物赖氨酸侧链氨基以及N端氨基偶联以及纯化
采用实施例5的方法进行偶联，采用实施例6的方法进行修饰产物纯化，得到类似的实验结果。
实施例9：20kDa聚乙二醇与血管内皮抑制素K3类似物N端氨基偶联以及纯化
采用实施例3的方法进行N端偶联，采用实施例4的方法进行修饰产物纯化，得到类似的实验结果。
实施例10：20kDa聚乙二醇与血管内皮抑制素K3类似物赖氨酸侧链氨基以及N端氨基偶联以及纯化
采用实施例5的方法进行偶联，采用实施例6的方法进行修饰产物纯化，得到类似的实验结果。
实施例11：20kDa聚乙二醇与血管内皮抑制素K4类似物N端氨基偶联以及纯化
采用实施例3的方法进行N端偶联，采用实施例4的方法进行修饰产物纯化，得到类似的实验结果。
实施例12：20kDa聚乙二醇与血管内皮抑制素K4类似物赖氨酸侧链氨基以及N端氨基偶联以及纯化
采用实施例5的方法进行N端偶联，采用实施例6的方法进行修饰产物纯化，得到类似的实验结果。
实施例13：20kDa聚乙二醇与血管内皮抑制素ESC类似物偶联
将血管内皮抑制素ESC类似物透析到pH7.5的20mM NaH2PO4溶液中，测定蛋白浓度，并将蛋白浓度调整至5～15mg/ml之间。按照目的蛋白与聚乙二醇的摩尔比1:5计算需要加入的20kDa的单甲氧基聚乙二醇马来酰亚胺(mPEG-MAL)的量，加入到目的蛋白中，搅拌均匀后室温静置6～8小时，修饰结果电泳图见图4。利用Bio-Rad的1D凝胶定量软件Quantity One对电泳凝胶的每一条带进行含量分析并与分子量标准比对，此时血管内皮抑制素ESC类似物被单一修饰(分子量40kDa)的比例在80％以上，即一个血管内皮抑制素ESC类似物分子被一个分子量为20kDa的单甲氧基聚乙二醇马来酰亚胺分子修饰。由于单甲氧基聚乙二醇马来酰亚胺只能与自由巯基反应，因此可以认为本产物的聚乙二醇修饰位点为血管内皮抑制素ESC类似物的自由巯基。
实施例14：20kDa聚乙二醇与血管内皮抑制素ESC类似物偶联产物的纯化
将实施例13中单甲氧基聚乙二醇马来酰亚胺(mPEG-MAL)与血管内皮抑制素ESC类似物偶联反应产物溶液用阳离子层析柱纯化。选择GE Healthcare公司SPFF介质，并将反应液调整pH值至5.0～7.0，阳离子柱用20mM NaH2PO4(pH值至5.0～7.0)平衡并上样。再采用20mM NaH2PO4、500mM NaCl(pH5.0～7.0)进行梯度洗脱，根据280nm紫外吸收收集不同的组分。并进行SDS-PAGE分析，分析结果见图4。由图4可知，组分3即为20kDa聚乙二醇单修饰血管内皮抑制素ESC类似物的产物。
实施例15：20kDa聚乙二醇与血管内皮抑制素NK1、NK2、NK3、NK4、ESK、NESK类似物N端氨基偶联以及纯化
采用实施例3的方法进行N端偶联，采用实施例4的方法进行修饰产物纯化，得到类似的实验结果。
实施例16：20kDa聚乙二醇与血管内皮抑制素NK1、NK2、NK3、NK4、ESK、NESK类似物赖氨酸侧链氨基以及N端氨基偶联以及纯化
采用实施例5的方法进行偶联，采用实施例6的方法进行修饰产物纯化，得到类似的实验结果。
实施例17：20kDa聚乙二醇与血管内皮抑制素K1类似物赖氨酸侧链氨基偶联产物纯化
将血管内皮抑制素K1类似物透析到pH8.5±0.5的20mM磷酸二氢钠溶液中，测定蛋白浓度，并将蛋白浓度调整至10～20mg/ml之间。按照目的蛋白与聚乙二醇的摩尔比1:1计算需要加入的20kDa的单甲氧基聚乙二醇丙醛(mPEG-ALD)的量，同时按照最终溶液体积计算还原剂氰基硼氢化钠的用量，其浓度为10mM，称取需要的单甲氧基聚乙二醇丙醛(mPEG-ALD)和氰基硼氢化钠，加入到目的蛋白中，搅拌均匀后室温静置6～8小时。反应产物溶液用阳离子层析柱纯化，具体选择GE Healthcare公司Macrocap SP介质，并将反应液调整pH值至4.5±0.5。阳离子柱用30mMNaAc(pH值至4.0～5.0)平衡并上样。再采用30mMNaAc、500mM NaCl(pH4.0～5.0)进行梯度洗脱，根据280nm紫外吸收收集不同的组分。将修饰样品以及后续纯化收集的各个组分进行SDS-PAGE电泳分析，得到的分析结果见图5。对收集到的两个单一修饰组分第4和第5泳道样品采用trypsin酶切后质谱分析的方法进行修饰位点分析后确认第5泳道样品组分为赖氨酸侧链氨基单一修饰产物。
实施例18：20kDa聚乙二醇与血管内皮抑制素K2、K3、NK1、NK2、NK3类似物赖氨酸侧链氨基偶联以及纯化
采用实施例17的方法进行偶联和修饰产物纯化，得到类似的实验结果。
实施例19：修饰产物活性测定
对实施例1至实施例18获得的全部目标纯化产物进行细胞活性测定，从中筛选最优修饰方案。选择HMEC细胞，采用Migration(Tranwell Assay)方法，计细胞数反映蛋白活性(可参考Luo yongzhang等，Endostatin inhibits tumourlymphangiogenesis and lymphatic metastasis via cell surface nucleolin on lymphangiogenic endothelial cells(J Pathol 2010；222:249–260)，分析各修饰产物对HMEC细胞迁移的抑制率，结果见表1：
表1：各修饰产物对HMEC细胞迁移的抑制率的测定结果
测活样品名称 抑制率(％)
天然人血管内皮抑制素 21.5
天然人血管内皮抑制素N端氨基单修饰修饰产物 61.3
血管内皮抑制素K1类似物N端氨基单修饰产物 32.3
血管内皮抑制素K1类似物赖氨酸侧链氨基单修饰产物 39.8
血管内皮抑制素K1类似物赖氨酸侧链氨基和N端氨基双修饰产物 41.9
血管内皮抑制素K2类似物N端氨基单修饰产物 48.9
血管内皮抑制素K2类似物赖氨酸侧链氨基单修饰产物 74.2
血管内皮抑制素K2类似物赖氨酸侧链氨基和N端氨基双修饰产物 79.0
血管内皮抑制素K3类似物N端氨基单修饰产物 31.7
血管内皮抑制素K3类似物赖氨酸侧链氨基单修饰产物 23.2
血管内皮抑制素K3类似物赖氨酸侧链氨基和N端氨基双修饰产物 7.0
血管内皮抑制素K4类似物N端氨基单修饰产物 15.0
血管内皮抑制素K4类似物赖氨酸侧链氨基和N端氨基双修饰产物 5.0
血管内皮抑制素ESC类似物C端巯基偶联产物 42.0
血管内皮抑制素NK1类似物N端氨基单修饰产物 30.0
血管内皮抑制素NK1类似物赖氨酸侧链氨基单修饰产物 37.2
血管内皮抑制素NK1类似物赖氨酸侧链氨基和N端氨基双修饰产物 38.2
血管内皮抑制素NK2类似物N端氨基单修饰产物 44.5
血管内皮抑制素NK2类似物赖氨酸侧链氨基单修饰产物 72.1
血管内皮抑制素NK2类似物赖氨酸侧链氨基和N端氨基双修饰产物 75.0
血管内皮抑制素NK3类似物N端氨基单修饰产物 27.9
血管内皮抑制素NK3类似物赖氨酸侧链氨基单修饰产物 20.2
血管内皮抑制素NK3类似物赖氨酸侧链氨基和N端氨基双修饰产物 5.4
血管内皮抑制素NK4类似物N端氨基单修饰产物 12.0
血管内皮抑制素NK4类似物赖氨酸侧链氨基和N端氨基双修饰产物 3.6
血管内皮抑制素ESK类似物N端氨基单修饰产物 32.0
血管内皮抑制素ESK类似物赖氨酸侧链氨基和N端氨基双修饰产物 56.0
血管内皮抑制素NESK类似物N端氨基单修饰产物 29.3
血管内皮抑制素NESK类似物赖氨酸侧链氨基和N端氨基双修饰产物 50.4
上述活性结果显示，所有类似物N端单修饰产物对HMEC细胞迁移抑制率均低于血管内皮抑制素N端单修饰产物，说明突变对于蛋白活性有一定的影响；赖氨酸侧链氨基单一修饰结果显示对第二个赖氨酸侧链氨基单修饰(即血管内皮抑制素K2类似物)产物对HMEC细胞迁移抑制率最高，设置高于血管内皮抑制素N端单修饰产物；对N末端氨基和赖氨酸侧链氨基进行双修饰产物中，对第二个赖氨酸侧链氨基和N末端氨基进行双修饰(即血管内皮抑制素K2类似物)产物对HMEC细胞迁移抑制率在所有产物中最高。
结合血管内皮抑制素分子的三级结构(图6)进行综合分析，血管内皮抑制素分子中有6个可以进行聚乙二醇修饰氨基，分别为N末端氨基和5个赖氨酸侧链氨基，其中第5个赖氨酸残基位于C末端，与N末端靠近，由于空间位阻的原因难以实现针对第5个赖氨酸侧链氨基的单一修饰。通过在其后面添加一个半胱氨酸残基进行巯基修饰降低制备难度。在上述6个进行修饰的位点中，第二个赖氨酸残基与该蛋白的nucleolin结合区域距离最远，其次为N末端，进行聚乙二醇修饰乙二醇长链对于该结合区域的影响最小，因此对N末端氨基和第二个赖氨酸侧链氨基进行单一修饰或者双修饰，产物对HMEC细胞迁移抑制率都优于对其它位点修饰方案，充分说明选择远离nucleolin结合区域进行修饰是对于血管内皮抑制素的最佳选择。
为了验证上述结论，我们根据血管内皮抑制素分子的三级结构特点，在同样远离nucleolin结合区域同时远离第二个赖氨酸残基的三级结构表面选择第127位引入一个赖氨酸残基(即血管内皮抑制素ESK类似物)进行修饰，结果显示，该类似物的双修饰 产物HMEC细胞迁移抑制率低于血管内皮抑制素K2类似物，说明对血管内皮抑制素K2类似物进行双修饰对于血管内皮抑制素或者其类似物来说是最佳的选择。
参见图6和图7，在ES分子表面不同区域进行聚乙二醇修饰对蛋白活性的影响不同，N端单修饰和K2单修饰产物对抑制HMEC细胞迁移活性抑制率增加明显，这两个位点双修饰产物对抑制HMEC细胞迁移活性抑制率增加最明显，K2距离nucleolin结合区域最远，单一修饰对蛋白抑制HMEC细胞迁移活性增加最大，而K1、K3、K4以及C端修饰产物对蛋白抑制HMEC细胞迁移活性增加较小，或者降低，而双修饰产物也获得了相似的结果，说明远离nucleolin结合区域的K2是最佳修饰选择，为了进一步增加产物的半衰期，采用N端和K2双修饰是最佳的选择。为了确认在K2区域远离K3、K4一侧区域是否存在与K2相似的可插入修饰位点，针对ES天然结构N127进行点突变，形成一个新的修饰位点，进行相关实验，结果证实，该区域修饰产物(无论是单修饰还是和N端一起双修饰产物)HMEC细胞迁移活性增加均小于K2相对应的修饰产物的影响。充分说明了K2和N端双修饰对于蛋白活性增加以及半衰期延长是最佳的选择。

Claims (23)

  1. 血管内皮抑制素类似物与聚乙二醇偶联复合物；其中所述血管内皮抑制素类似物在相应于天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第96位的位置上具有赖氨酸残基，而在任何其它位置上均不具有赖氨酸残基；并且，所述血管内皮抑制素类似物仅在该赖氨酸残基上与聚乙二醇偶联。
  2. 血管内皮抑制素类似物与聚乙二醇偶联复合物；其中所述血管内皮抑制素类似物在相应于天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第96位的位置上具有赖氨酸残基，而在任何其它位置上均不具有赖氨酸残基；并且，所述血管内皮抑制素类似物在N末端和该赖氨酸残基上与聚乙二醇偶联。
  3. 权利要求1或2的偶联复合物，其中所述血管内皮抑制素类似物是天然血管内皮抑制素氨基酸序列SEQ ID NO.1第76、107、118和184位的赖氨酸残基发生突变而形成的。
  4. 血管内皮抑制素类似物与聚乙二醇偶联复合物；其中所述血管内皮抑制素类似物在相应于天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第96位的位置上具有赖氨酸残基，而在任何其它位置上均不具有赖氨酸残基，并且所述血管内皮抑制素类似物在其N端具有插入氨基酸序列的血管内皮抑制素功能性变体；并且，所述血管内皮抑制素类似物仅在该赖氨酸残基上与聚乙二醇偶联。
  5. 血管内皮抑制素类似物与聚乙二醇偶联复合物；其中所述血管内皮抑制素类似物在相应于天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第96位的位置上具有赖氨酸残基，而在任何其它位置上均不具有赖氨酸残基，并且所述血管内皮抑制素类似物在其N端具有插入氨基酸序列；并且，所述血管内皮抑制素类似物在N末端和该赖氨酸残基上与聚乙二醇偶联。
  6. 权利要求4或5的偶联复合物，其中所述血管内皮抑制素类似物是天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第76、107、118和184位的赖氨酸残基发生突变，并且在其N端的甲硫氨酸M和组氨酸H之间插入GGSHHHHH而形成的。
  7. 权利要求1、2、4或5的偶联复合物，其中N端插入氨基酸序列是MGGSHHHHH。
  8. 权利要求3或6任一项的偶联复合物，其中天然血管内皮抑制素氨基酸序列SEQ ID NO.1的第76、107、118和184位的赖氨酸残基分别被突变为X1、X3、X4、X5，其中X1、X3、X4或X5独立地为除赖氨酸以外的任何一种天然氨基酸。
  9. 权利要求8的偶联复合物，其中X1、X3、X4或X5独立地为水溶性氨基酸。
  10. 权利要求9的偶联复合物，其中X1、X3、X4或X5独立地为精氨酸、组氨酸、谷氨酸和天冬氨酸的任一种。
  11. 权利要求10的偶联复合物，其中X1、X3、X4或X5独立地为精氨酸和组氨酸的任一种。
  12. 权利要求11的偶联复合物，其中X1、X3、X4或X5均为精氨酸。
  13. 权利要求1-12任一项的偶联复合物，其中血管内皮抑制素类似物与聚乙二醇为共价键偶联。
  14. 权利要求1-13任一项的偶联复合物，其中所述聚乙二醇的平均分子量为为5,000到40,000道尔顿。
  15. 权利要求14的偶联复合物，其中所述聚乙二醇的平均分子量为20,000到40,000道尔顿。
  16. 权利要求1-15任一项的偶联复合物，其中所述聚乙二醇为单甲氧基聚乙二醇、单葡萄糖聚乙二醇或者单半乳糖聚乙二醇。
  17. 权利要求16的偶联复合物，其中所述聚乙二醇为单甲氧基聚乙二醇。
  18. 权利要求1-17任一项的偶联复合物，其中所述聚乙二醇为线性的或者分支的。
  19. 权利要求1-18任一项的偶联复合物，其中聚乙二醇与血管内皮抑制素类似物的氨基用单甲氧基聚乙二醇丙醛(mPEG-ALD)、单甲氧基聚乙二醇丁醛(mPEG-ButyrALD)、单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC)、单甲氧基聚乙二醇琥珀酰亚胺乙酸酯(mPEG-SCM)、单甲氧基聚乙二醇琥珀酰亚胺丙酸酯(mPEG-SPA)、单甲氧基聚乙二醇琥珀酰亚胺丁酸酯(mPEG-SBA)、单甲氧基聚乙二醇琥珀酰亚胺α-甲基丁酸酯(mPEG-SMA)、单甲氧基聚乙二醇NHS酯(mPEG-NHS)进行偶联反应。
  20. 药物组合物，其包含权利要求1-19任一项的偶联复合物和任选的药学可接受的载体。
  21. 治疗新生血管或者新生淋巴管导致的疾病的方法，包括给患者施用权利要求1-19任一项的偶联复合物或权利要求20的药物组合物。
  22. 权利要求1-19任一项的偶联复合物或权利要求20的药物组合物在治疗新生血管或者新生淋巴管导致的疾病中的应用。
  23. 权利要求1-19任一项的偶联复合物在制备体内或体外用于抑制新生血管或者新生淋巴管的生成的药物中的应用。
PCT/CN2017/110532 2016-11-10 2017-11-10 聚乙二醇化血管内皮抑制素类似物及其应用 Ceased WO2018086603A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023016410A1 (zh) * 2021-08-09 2023-02-16 刘鹏 一种抗血管生成药物

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3539570B1 (en) * 2016-11-10 2022-04-13 Beijing Protgen Ltd. Pegylated endostatin analogue and application thereof
CN111166869A (zh) * 2019-12-30 2020-05-19 江苏大学 一种量子点与重组人内皮抑制素偶联物及其制备工艺
US20250032629A1 (en) * 2020-05-19 2025-01-30 Anygen Co., Ltd. Novel nucleolin-binding peptide and use thereof
CN114438047B (zh) * 2020-11-05 2024-11-22 重庆派金生物科技有限公司 制备聚乙二醇修饰的尿酸氧化酶的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007082483B1 (en) * 2006-01-20 2007-09-07 Univ Tsinghua Medicament for treatment of tumor and the use thereof
CN101219206A (zh) * 2008-02-01 2008-07-16 山东先声麦得津生物制药有限公司 重组人血管内皮抑制素在制药中的应用
CN101596320A (zh) * 2006-01-20 2009-12-09 清华大学 一种治疗肿瘤的药物及其应用
WO2016070798A1 (zh) 2014-11-03 2016-05-12 清华大学 一种抑制脂肪细胞分化和胰岛素耐受的药物

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
US6080728A (en) * 1996-07-16 2000-06-27 Mixson; A. James Carrier: DNA complexes containing DNA encoding anti-angiogenic peptides and their use in gene therapy
CN103083681A (zh) * 2006-01-20 2013-05-08 清华大学 一种治疗肿瘤的药物及其应用
CN101411879B (zh) * 2008-11-28 2011-02-09 暨南大学 SiO2/Au核壳结构纳米材料-生物蛋白药物复合物及其制备方法
JP2013532126A (ja) * 2010-05-11 2013-08-15 フレッド ハッチンソン キャンサー リサーチ センター クロロトキシン変異体、コンジュゲート、およびそれらを使用する方法
US20130023479A1 (en) * 2011-07-22 2013-01-24 Protgen Ltd. Use of nucleolin as a biomarker for lymphangiogenesis in cancer prognosis and therapy
JP6336389B2 (ja) * 2011-09-09 2018-06-06 チンファ ユニヴァーシティＴｓｉｎｇｈｕａ Ｕｎｉｖｅｒｓｉｔｙ Ａｔｐ結合部位に変異を有するエンドスタチン変異体
CN104926933B (zh) * 2014-03-19 2018-09-07 北京仁和天通生物科技有限公司 Endostatin突变体、Endostatin突变体与聚乙二醇的交联物以及它们的应用
CN105709209A (zh) * 2016-01-17 2016-06-29 新乡医学院第一附属医院 一种治疗肿瘤的药物及其制备方法
EP3539570B1 (en) * 2016-11-10 2022-04-13 Beijing Protgen Ltd. Pegylated endostatin analogue and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007082483B1 (en) * 2006-01-20 2007-09-07 Univ Tsinghua Medicament for treatment of tumor and the use thereof
CN101596320A (zh) * 2006-01-20 2009-12-09 清华大学 一种治疗肿瘤的药物及其应用
CN101219206A (zh) * 2008-02-01 2008-07-16 山东先声麦得津生物制药有限公司 重组人血管内皮抑制素在制药中的应用
WO2016070798A1 (zh) 2014-11-03 2016-05-12 清华大学 一种抑制脂肪细胞分化和胰岛素耐受的药物

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
F.M. VERONESE ET AL., MILESTONES IN DRUG THERAPY, 2009, pages 11 - 31
FOLKMAN J. ET AL., CELL, vol. 88, 1997, pages 277 - 285
FOLKMAN J. ET AL., NATURE, vol. 390, 1997, pages 404 - 407
FOLKMAN J., N ENGL J MED, vol. 285, 1971, pages 1182 - 1186
FU Y ET AL., IUBMB LIFE, vol. 61, 2009, pages 613 - 626
GIANFRANCO PASUT ET AL., ADVANCED DRUG DELIVERY REVIEWS, vol. 60, 2008, pages 69 - 78
HAN B ET AL., J THORAC ONCOL, vol. 6, no. 6, 2011, pages 1104 - 1109
LUO YONGZHANG ET AL.: "Endostatin inhibits tumourlymphangiogenesis and lymphatic metastasis via cell surface nucleolin on lymphangiogenic endothelial cells", J PATHOL, vol. 222, 2010, pages 249 - 260
See also references of EP3539570A4
WANG J ET AL., ZHONGGUO FEI AI ZA ZHI, vol. 8, 2005, pages 283 - 290
YONGZHANG LUO ET AL., JOURNAL OF PATHOLOGY, vol. 222, 2010, pages 249 - 260
YONGZHANG LUO, FRONT. MED., vol. 5, 2011, pages 336 - 340

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023016410A1 (zh) * 2021-08-09 2023-02-16 刘鹏 一种抗血管生成药物

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