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WO2021128255A1 - Charge d'affinité, son procédé de préparation et son utilisation - Google Patents

Charge d'affinité, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2021128255A1
WO2021128255A1 PCT/CN2019/129112 CN2019129112W WO2021128255A1 WO 2021128255 A1 WO2021128255 A1 WO 2021128255A1 CN 2019129112 W CN2019129112 W CN 2019129112W WO 2021128255 A1 WO2021128255 A1 WO 2021128255A1
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Prior art keywords
heparanase
sodium chloride
affinity
binding domain
domain polypeptide
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Chinese (zh)
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李锂
林森茂
冯黑妮
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Shenzhen Hepalink Pharmaceutical Group Co Ltd
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Shenzhen Hepalink Pharmaceutical Group Co Ltd
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Priority to CN201980103155.9A priority Critical patent/CN114846137B/zh
Priority to CN202410700176.1A priority patent/CN118684788A/zh
Priority to PCT/CN2019/129112 priority patent/WO2021128255A1/fr
Publication of WO2021128255A1 publication Critical patent/WO2021128255A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate

Definitions

  • This application belongs to the technical field of biological materials, and relates to an affinity filler and a preparation method and application thereof.
  • Heparan sulfate proteoglycans are a class of sugar complexes, which have two parts: a core protein and one or more heparan sulfate sugar chains covalently connected to the core protein.
  • Extracellular matrix refers to a substance secreted by cells and located in the lower layer of epithelial or endothelial cells and around connective tissue cells to provide mechanical support and physical strength for tissues and organs.
  • the cell membrane is a membrane structure located on the periphery of the protoplast and close to the cell wall, which can prevent extracellular materials from freely entering the cell and ensure the relative stability of the cell environment.
  • HSPGs are one of the main components of ECM and cell membranes, and the heparan sulfate on HSPGs is combined with a large number of growth factors, such as fibroblast growth factor, vascular endothelial growth factor, transforming growth factor and hepatocyte growth factor. HSPGs play an important role in different physiological and pathological processes such as growth, development, inflammation, invasion and infection of microorganisms and viruses, and the occurrence and development of tumors. In the body, heparan sulfate sugar chains are generally cleaved specifically by endogenous heparanase.
  • Heparanase is an endogenous ⁇ (1-4) endoglycosidase and the only endogenous glycosidase that can degrade HSPGs.
  • heparanase In normal tissue cells, heparanase is mainly distributed in the placenta, spleen, platelets, neutrophils, monocytes, activated T, B lymphocytes, and in the heart, brain, lung, skeletal muscle, kidney, and pancreas. It is not expressed in metastatic malignant tumor cells. Heparanase can promote tumor invasion and metastasis, can also inhibit tumor cell apoptosis, and participate in a series of physiological and pathological activities such as nerve axon growth, autoimmunity, and tumor angiogenesis.
  • heparanase The expression of heparanase is abnormally increased in pancreatic cancer, breast cancer, melanoma and other tumors, and its overexpression is usually positively correlated with the poor prognosis of the tumor.
  • the biological behavior of tumor cells is to promote tumor cell invasion And transfer. Heparanase can specifically cleave heparan sulfate sugar chains located on the cell surface and in the ECM, destroy the stable structure of the extracellular matrix and basement membrane, thereby making the invasion and metastasis of tumor cells easier.
  • heparanase cuts long heparan sulfate sugar chains into small fragments, which are generally composed of 20-30 sugar residues. There is evidence that these oligosaccharide chains have more biological functions than complete heparan sulfate sugar. Strong chain. Different degradation products play different roles in the development of tumors.
  • heparanase is A suitable target for cancer treatment.
  • the protein precursor is proteolyzed at two potential cleavage sites Glu109-Ser110 and GlN157-Lys158 to obtain two proteins. Subunits, 8KDa polypeptide at the amino terminus and 50KDa polypeptide at the carboxy terminus.
  • Heparin also known as unfractionated heparin, is named after it was first discovered in the liver. It is a mucopolysaccharide composed of glucuronic acid or iduronic acid and glucosamine alternately connected by ⁇ (1 ⁇ 4) glycosidic bonds. Heparin belongs to Heparin is a type of polyanionic glycosaminoglycan that is heterogeneous in structure and highly dispersed in the degree of polymerization. Heparin has a large amount of negative charge and the relative molecular mass is 1200-40000 Da. In addition to its anticoagulant effect, heparin also has a variety of biological activities and clinical uses, including anti-inflammatory, anti-angiogenesis and anti-tumor effects.
  • Heparin has a similar structure to HS, the natural substrate of heparanase, and can competitively bind heparanase to inhibit the expression of heparanase activity in tumor cells.
  • acetylase activity on the one hand, tumor cells are inhibited from degrading the extracellular matrix, thereby reducing the ability of tumor cells to invade surrounding tissues.
  • it blocks the ability of heparanase to release growth factors from the extracellular matrix, thereby inhibiting the growth of tumor cells and tumor blood vessels.
  • the heparinase inhibitory activity of heparin is low, and its anti-heparanase activity needs to be further improved.
  • the purpose of this application is to provide an affinity filler and its preparation method and its application in the preparation of glycosaminoglycan components with heparanase inhibitory activity.
  • the present application provides an affinity filler, which is a matrix coupled to a heparanase binding domain polypeptide, wherein the heparanase binding domain polypeptide is treated with an active center protecting agent.
  • the affinity filler is obtained by coupling the heparanase binding domain polypeptide protected by the active center protector on the substrate, and the affinity filler can be used to separate heparin from heparin to obtain higher heparanase inhibitory activity The components.
  • the heparanase binding domain polypeptide is KKDC, QPLK, diKKDC or KKDC-QPLK.
  • the amino acid sequence of KKDC is KMFKNSTYSRSSVDC, PI is 9.63, and M W is 1749.96.
  • the amino acid sequence of QPLK is QPRRKTAKMLK, PI is 12.02, and M W is 1356.7.
  • diKKDC is KKDC-linker-KKDC, where linker is a linker peptide containing 10 amino acids, and the amino acid sequence of linker is SLLVHKHKLI; the PI of diKKDC is 10.03, and the M W is 4651.83.
  • KKDC-QPLK is KKDC-linker-QPLK, where linker is a linker peptide containing 10 amino acids, and the amino acid sequence of linker is SLLVHKHKLI; the PI of KKDC-QPLK is 10.82, and the M W is 4258.12.
  • the active center protective agent is any one or a combination of at least two of N-heparan, heparin and heparan sulfate.
  • the method for treating the heparanase binding domain polypeptide with the active center protecting agent is: dissolving the heparanase binding domain polypeptide and the active center protecting agent in a solution containing 0.1M NaHCO 3 and 0.5M NaCl, with a pH of 8.3 In the buffer, stirring at room temperature for 0.5-2 hours (for example, 0.6 hours, 0.8 hours, 1 hour, 1.2 hours, 1.5 hours, 1.8 hours or 2 hours) to obtain the heparanase binding domain polypeptide treated with the active center protector.
  • the mass ratio of the active center protecting agent to the heparanase binding domain polypeptide is ⁇ 0.5:1, such as 0.5:1, 0.8:1, 1:1, 1.3:1, 1.5:1, 1.8:1, 2:1, 2.3:1, 2.5:1, 2.8:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc., preferably 1:1-3:1.
  • the substrate is Sepharose 4B.
  • the binding ratio of the heparanase binding domain polypeptide to the matrix is 4.50-5.60 ⁇ mol/g, for example, 4.50 ⁇ mol/g, 4.53 ⁇ mol/g, 4.63 ⁇ mol/g, 4.75 ⁇ mol/g, 4.81 ⁇ mol/g , 5.02 ⁇ mol/g, 5.48 ⁇ mol/g, 5.55 ⁇ mol/g, 5.58 ⁇ mol/g or 5.60 ⁇ mol/g.
  • the present application provides a method for preparing the affinity filler as described above, and the preparation method includes the following steps:
  • step (3) Add the pretreated substrate obtained in step (1) to the stirred in step (2) containing the heparanase binding domain polypeptide and the active center protector containing 0.1M NaHCO 3 and 0.5M NaCl, the pH is In the 8.3 buffer, react at room temperature for 0.5-2 hours (for example, 0.5 hour, 0.8 hour, 1 hour, 1.2 hours, 1.5 hours, 1.8 hours or 2 hours), and then 8-10°C (for example 8°C, 8.5°C, 9 (°C, 9.5°C or 10°C) standing overnight, suction filtered, and the filter cake was washed with a 0.1M Tris solution with pH 8.0 to obtain the affinity filler.
  • 0.5-2 hours for example, 0.5 hour, 0.8 hour, 1 hour, 1.2 hours, 1.5 hours, 1.8 hours or 2 hours
  • 8-10°C for example 8°C, 8.5°C, 9 (°C, 9.5°C or 10°C standing overnight, suction filtered, and the filter cake was washed with a 0.1M Tri
  • the mass ratio of the active center protecting agent to the heparanase binding domain polypeptide in the buffer containing the heparanase binding domain polypeptide and the active center protecting agent in step (2) is ⁇ 0.5:1, such as 0.5:1 , 0.8:1, 1:1, 1.3:1, 1.5:1, 1.8:1, 2:1, 2.3:1, 2.5:1, 2.8:1, 3:1, 3.5:1, 4:1, 4.5 :1, 5:1, etc., preferably 1:1-3:1.
  • the preparation method further includes: adding the affinity filler obtained in step (3) to a Tris solution of pH 8.0, standing for 1 to 3 hours, suction filtration, and using a solution containing 0.1M NaHCO 3 and 0.5M Wash with NaCl buffer solution with pH 8.3, then with 2mM EDTA and 10mM Tris solution, and finally with 2M NaCl with Tris-HCl solution with pH 7.0 to obtain purified affinity filler.
  • the preparation method of the affinity filler described in the present application is simple and easy to operate; the affinity filler obtained by the preparation method can separate components with higher heparanase inhibitory activity from glycosaminoglycans.
  • the present application provides a method for separating components with heparanase inhibitory activity from glycosaminoglycans by using the affinity filler as described above, and the method includes the following steps:
  • step (ii) Load the glycosaminoglycan sample onto the affinity chromatography column obtained in step (i), using chlorination with a concentration of ⁇ 1.0M (for example, 1.0M, 0.8M, 0.6M, 0.5M, 0.3M, etc.)
  • ⁇ 1.0M for example, 1.0M, 0.8M, 0.6M, 0.5M, 0.3M, etc.
  • the sodium solution is eluted, and then the sodium chloride solution with a concentration of 1.5-2.5M (for example, 1.5M, 1.8M, 2.0M, 2.2M, or 2.5M) is used for elution, and the 1.5-2.5M sodium chloride solution eluent is collected , Dialysis and concentration to obtain glycosaminoglycan component with heparanase inhibitory activity.
  • glycosaminoglycan is heparin, heparan sulfate, dermatan sulfate, hyaluronic acid or chondroitin sulfate.
  • step (ii) the elution using sodium chloride solution with a concentration ⁇ 1.0M is isocratic elution or at least two sodium chloride solutions with different concentrations within the range of concentration ⁇ 1.0M are used for gradient elution;
  • a sodium chloride solution with a concentration of 0.6M sodium chloride and a concentration of 1.0M sodium chloride is selected for elution.
  • step (ii) is to load the glycosaminoglycan sample onto the affinity chromatography column obtained in step (i), using 0.6M sodium chloride solution, 1.0M sodium chloride solution and 2.0M sodium chloride solution Gradient elution was performed, and the eluted fractions of 2.0M sodium chloride solution were collected, dialyzed, and concentrated to obtain glycosaminoglycan fractions with heparanase inhibitory activity.
  • the heparanase inhibitory activity of the components eluted with a salt solution (sodium chloride solution) with a salt concentration of 1.5-2.5M is higher than that of a salt solution with a salt concentration of ⁇ 1.0M (chlorine Sodium chloride solution) the heparanase inhibitory activity of the eluted fraction.
  • a salt solution with a salt concentration of ⁇ 1.0M chlorine Sodium chloride solution
  • the purpose of elution using sodium chloride solution with sodium chloride concentration ⁇ 1.0M is to elute low-active components.
  • the present application provides a glycosaminoglycan component with heparanase inhibitory activity, which is obtained by the method described in the third aspect.
  • the heparin component eluted with 1.5-2.5M sodium chloride eluate has higher heparanase inhibitory activity, and its inhibitory activity is about 10 times higher than that of heparin.
  • the present application provides the application of the component having heparanase inhibitory activity as described above in the preparation of anti-tumor drugs.
  • the affinity column packing material is obtained by coupling the heparanase binding domain polypeptide protected by the active center protector on the substrate.
  • the affinity packing material can be used to separate the heparanase inhibitory activity group from heparin.
  • the inhibitory activity of this component is about 10 times higher than that of heparin. It can be used for the development of anti-tumor drugs and has broad application prospects.
  • Figure 1 shows the elution curve of heparin separation using the fillers prepared in Example 1, Example 2 and Example 3.
  • 1 is the filler of Example 1
  • 2 is the filler of Example 2
  • 3 is Example 3. ⁇ filler;
  • Example 2 is the elution curve of heparin separation using the fillers prepared in Example 4 and Example 5, where A is the filler of Example 4, and B is the filler of Example 5.
  • KKDC KMFKNSTYSRSSVDC, referred to as KKDC, PI is 9.63, M W is 1749.96, prepared by Shanghai Taopu Biotechnology Co., Ltd.;
  • QPLK QPRRKTAKMLK, abbreviated as QPLK, PI is 12.02, M W is 1356.7.
  • diKKDC KKDC-linker-KKDC, in which linker is a 10 amino acid linker peptide, and its amino acid sequence is SLLVHKHKLI; the PI of diKKDC is 10.03, and the M W is 4651.83.
  • KKDC-QPLK KKDC-linker-QPLK, where linker is a 10 amino acid linker peptide with an amino acid sequence of SLLVHKHKLI; KKDC-QPLK has a PI of 10.82 and a M W of 4258.12.
  • DMB 1,9-Dimethyl-Methylene Blue, 1,9-Dimethyl-Methylene Blue.
  • DMB can react with polysaccharides in solution and can be used to determine the content of polysaccharides.
  • KKDC-I filler The preparation method of KKDC-I filler is as follows:
  • Sepharose 4B matrix 25g activated by cyanogen bromide and add water (200mL) to swell.
  • the swollen matrix was placed in a sand core funnel for suction filtration, and 1 mM HCl (5L) was continuously added to soak the matrix at the same time. The washing time was 2 hours.
  • the dilute hydrochloric acid solution in the matrix was removed by suction filtration, and the matrix (200 mL ⁇ 3) was rinsed with buffer-A solution (containing 0.1M NaHCO 3 and 0.5M NaCl, pH 8.3), and filtered with suction.
  • KKDC 250 mg
  • N-heparan 250 mg
  • step (1) is added to the reaction system of step (2) and buffer-A solution (40 mL) is added. Stir gently.
  • the volume of buffer-A solution in the reaction system and the mass ratio of the substrate are 5 mL/g. Sealed and placed on a shaker, reacted at room temperature for 1 hour, and finally allowed to stand overnight at low temperature (8-10°C). Filter with suction, and rinse the filter cake with 0.1M Tris solution (pH 8.0) (200mL ⁇ 3).
  • the binding ratio of ligand to matrix (ie, the binding ratio of KKDC to matrix) was determined: KKDC polypeptide fragment has absorption at 285 nm. As the ligand containing KKDC polypeptide fragment binds to the matrix, the supernatant The absorption value of the liquid at 285nm decreases. By measuring the absorption value at 285 nm of the solution before and after the coupling reaction between the ligand containing the KKDC polypeptide fragment and the matrix, the binding ratio of the ligand containing the KKDC polypeptide fragment to the matrix can be calculated.
  • binding ratio ⁇ [1-OD285nm(end)/OD285nm(start)]*ligand feeding amount ⁇ /matrix feeding amount.
  • the binding ratio of KKDC to the matrix in the KKDC-I filler obtained in this example is 5.58 ⁇ mol/g.
  • KKDC-II filler For the preparation of KKDC-II filler, refer to the preparation method of Example 1, wherein the amount of N-heparan in step (2) is 500 mg, that is, the mass ratio of KKDC polypeptide to N-heparan is 1:2, and finally KKDC-II filler is obtained .
  • the binding ratio was determined using the same method as in Example 1.
  • the binding ratio of KKDC to the matrix in the KKDC-II filler obtained in this example was 5.53 ⁇ mol/g.
  • KKDC-III filler For the preparation of KKDC-III filler, refer to the preparation method of Example 1, wherein the amount of N-heparan in step (2) is 0, and finally KKDC-III filler is obtained.
  • the binding ratio was determined using the same method as in Example 1.
  • the binding ratio of KKDC and the substrate prepared in this comparative example was 5.55 ⁇ mol/g.
  • diKKDC filler For the preparation of diKKDC filler, referring to the preparation method of Example 1, replace KKDC with KKDC-linker-KKDC polypeptide in step (2), and the mass ratio of diKKDC to N-heparan is 1:1, and finally diKKDC filler is obtained.
  • the binding ratio was determined using the same method as in Example 1.
  • the binding ratio of diKKDC to the matrix in the diKKDC filler obtained in this example was 4.51 ⁇ mol/g.
  • KKDC-QPLK filler For the preparation of KKDC-QPLK filler, refer to the preparation method of Example 1, replace KKDC in step (2) with KKDC-QPLK polypeptide, the mass ratio of KKDC-QPLK to N-heparan is 1:1, and finally KKDC-QPLK filler is obtained .
  • the binding ratio was determined using the same method as in Example 1.
  • the binding ratio of the KKDC-QPLK ligand to the matrix in the KKDC-QPLK filler obtained in this comparative example was 4.81 ⁇ mol/g.
  • the five fillers KKDC-I, KKDC-II, KKDC-III, diKKDC and KKDC-QPLK prepared in the examples were packed into columns ( ⁇ 2.5 ⁇ 30cm), respectively, and equilibrated with 3 column volumes of 0.2M NaCl solution.
  • the mobile phase is NaCl solution of different concentration, and the gradient concentration is set to 0.6M, 1M, 2M NaCl solution; each; The concentration is eluted by 3 column volumes, and the eluate is collected in an equal amount with an automatic fraction collector.
  • Figure 1 reflects that N-heparan as a protective agent for active centers can affect the separation ability of the affinity column; without N-heparan as a protective agent, when the elution salt concentration reaches 1M, most of it can be bound to The material on the packing is eluted. When the salt concentration is further increased to 2M, the elution curve is basically flat, indicating that the separation ability of the KKDC-III packing is insufficient. When the amount of KKDC and the protective agent is 1:1 or 1:2, the separation ability of KKDC-I and KKDC-II fillers is equivalent, and the elution curve shows three distinct elution peaks.
  • Figure 2 reflects the different separation effects produced by different types of peptide fragments.
  • the KKDC-I filler uses KKDC polypeptide
  • the diKKDC filler uses diKKDC polypeptide
  • the KKDC-QPLK filler uses KKDC-QPLK polypeptide.
  • the rest of the preparation conditions are the same. Comparing the separation effects of the three fillers in Figure 1 and Figure 2, it is found that the separation effect of diKKDC is similar to that of KKDC-I, but the separation ability of KKDC-QPLK is insufficient.
  • the elution salt concentration reaches 1M, most of the binding components have been washed
  • the salt concentration rises to 2M there is no obvious elution peak.
  • the KKDC-I filler prepared in Example 1 was used to separate heparin to obtain high heparanase inhibitory active components, and the method is as follows:
  • the assay solution (100 ⁇ L) contains 40 mM sodium acetate buffer pH 5.0 and 100 mM Fondaparinux (GlaxoSmithKline), with or without the test sample. Heparanase was added to a final concentration of 140 pM to start the assay. Seal the plate with adhesive tape and incubate at 37°C for 2-24 hours. By adding 100 ⁇ L of 1.69mM 4-[3-(4-iodophenyl)-1H-5tetrazole]-1,3-benzenedisulfonate (WST-1, Aspep, Melbourne, Australia) in 0.1M NaOH Solution to stop the measurement. The plate was resealed with adhesive tape and developed at 60°C for 60 minutes.
  • the absorbance was measured at 584 nm (Fluostar, BMG, Labtech). In each plate, in the same buffer and volume, prepare a standard curve constructed with D-galactose as a reducing sugar standard in the range of 2-100 ⁇ M, and determine the IC 50 value. As the salt concentration of the eluting mobile phase increases, the smaller the IC 50 value of the obtained component sample, the higher the activity.
  • the IC 50 test results of heparin and Hep1, Hep2, and Hep3 are shown in Table 1.
  • the high heparanase inhibitory activity component Hep3 isolated from the filler of Example 1 is taken as an example to verify its efficacy in inhibiting the lung metastasis model of melanoma in mice.
  • the method is as follows:
  • B16 cells (purchased from the cell bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) are cultured and passaged in RPMI-1640 complete medium containing 10% fetal bovine serum in a 37°C incubator containing 5% CO 2. When the proliferation is in the exponential growth period, preparation for inoculation can be carried out.
  • model control group and experimental group Take C57 black mice, females, and randomly group them according to their body weight: model control group and experimental group, the corresponding dose of samples were injected into the tail vein, and the model control group was replaced by normal saline.
  • Tumor metastasis inhibition rate (number of tumor metastases in the model control group-number of metastases in the administration group)/number of tumor metastases in the model control group ⁇ 100%.
  • Hep3 has extremely high activity of inhibiting tumor metastasis in mice. Under the mode of 7.5mg/kg one-time administration, the inhibition rate of mouse melanoma lung metastasis reached 99%.
  • the high heparanase inhibitory activity component Hep3 isolated from the filler of Example 1 is taken as an example to verify its efficacy in the mouse 4T1 breast cancer lung metastasis model.
  • the method is as follows:
  • 4T1 breast cancer cells were cultured in suspension in vitro, and the culture conditions were RPMI1640 medium with 10% heat-inactivated fetal bovine serum and 1% double antibody, cultured at 37°C, 5% CO 2 and subcultured three times a week.
  • mice inoculation On the 25th day after tumor inoculation, half of the mice in each group were randomly sacrificed. The lung tissue and spleen were removed and weighed separately. After fixation with formalin, the number of lung tumor nodules was calculated.
  • Table 3 shows the number of metastatic tumor nodules in the lung tissue of mice in each administration group; Hep3 at doses of 10 mg/kg and 20 mg/kg can significantly reduce the number of metastatic tumor nodules in the lung tissue of mice.
  • the efficacy of kg Hep3 is stronger than 10mg/kg Hep3.
  • the affinity filler of the present application can isolate components with high heparanase inhibitory activity, which can significantly inhibit tumor metastasis, and has broad prospects in the development of anti-tumor drugs.

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Abstract

Charge d'affinité, son procédé de préparation et son utilisation. La charge d'affinité est une matrice couplée à un polypeptide de domaine de liaison à l'héparanase, le polypeptide de domaine de liaison à l'héparanase étant traité avec un agent de protection de centre actif. La charge d'affinité est obtenue par couplage du polypeptide de domaine de liaison à l'héparanase sur la matrice, et un composant présentant une activité inhibitrice de l'héparanase supérieure peut être séparé et obtenu à partir de l'héparine au moyen de la charge d'affinité, l'activité inhibitrice du composant étant d'environ 10 fois celle de l'héparine, et ainsi, le composant peut être utilisé dans le développement de médicaments antitumoraux, et a une large perspective d'application.
PCT/CN2019/129112 2019-12-27 2019-12-27 Charge d'affinité, son procédé de préparation et son utilisation Ceased WO2021128255A1 (fr)

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CN113856244A (zh) * 2021-10-21 2021-12-31 国网浙江省电力有限公司检修分公司 一种具有梯度结构的多孔复合材料及其制备方法和应用
CN113856244B (zh) * 2021-10-21 2023-03-21 国网浙江省电力有限公司检修分公司 一种具有梯度结构的多孔复合材料及其制备方法和应用

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