WO2021128255A1 - Affinity filler, preparation method therefor and use thereof - Google Patents

Abstract

Disclosed are an affinity filler, a preparation method therefor and the use thereof. The affinity filler is a matrix coupled with 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 means of coupling the heparanase binding domain polypeptide on the matrix, and a component with higher heparanase inhibitory activity can be separated and obtained from heparin by means of using the affinity filler, wherein the inhibitory activity of the component is approximately 10 times that of heparin, and thus, the component can be used in the development of anti-tumor drugs, and has a broad application prospect.

Description

Affinity filler and its preparation method and application Technical field

This application belongs to the technical field of biological materials, and relates to an affinity filler and a preparation method and application thereof.

Background technique

Heparan sulfate proteoglycans (HSPGs) 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 (ECM) 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 β(1-4) endoglycosidase and the only endogenous glycosidase that can degrade HSPGs. 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.

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.

At the same time, 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.

In addition, after heparan sulfate on the extracellular matrix and cell membrane is cut into small fragments by heparanase, various production factors bound to the sugar chain are released to promote tumor cell growth and tumor angiogenesis.

As early as the 1980s, people began to study the role of heparanase-induced sugar chain degradation in the occurrence and development of tumors. At present, there has been a clearer understanding, and more and more evidences have confirmed that heparanase is A suitable target for cancer treatment. Studies have shown that the heparanase cDNA encodes a polypeptide composed of 543 amino acids with a relative molecular weight of about 65KDa. 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. Two protein subunits are combined through a non-covalent bond to form a heterodimer, which together purifies and binds into an active heparanase. At present, studies have determined that the three potential heparin binding domains on the 50KDa protein subunit of heparanase are Lys158-Asp171 (KKFKNSTYSRSSVDC, referred to as KKDC), Gln270-Lys280 (ie QPRRKTAKMLK, referred to as QPLK) and Lys411- Arg432.

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 α(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. By inhibiting 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. On the other hand, 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. However, the heparinase inhibitory activity of heparin is low, and its anti-heparanase activity needs to be further improved.

Summary of the invention

In view of the shortcomings of the prior art, 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.

In order to achieve the purpose of this application, this application adopts the following technical solutions:

In the first aspect, 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.

In this application, 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.

Preferably, the heparanase binding domain polypeptide is KKDC, QPLK, diKKDC or KKDC-QPLK.

In this application, 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.

Preferably, the active center protective agent is any one or a combination of at least two of N-heparan, heparin and heparan sulfate.

Preferably, 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.

Preferably, 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.

In a specific embodiment, the substrate is Sepharose 4B.

Preferably, the binding ratio of the heparanase binding domain polypeptide to the matrix is 4.50-5.60 μmol/g, for example, 4.50 μmol/g, 4.53 μmol/g, 4.63 μmol/g, 4.75 μmol/g, 4.81 μmol/g , 5.02μmol/g, 5.48μmol/g, 5.55μmol/g, 5.58μmol/g or 5.60μmol/g.

In the second aspect, the present application provides a method for preparing the affinity filler as described above, and the preparation method includes the following steps:

(1) Swell the matrix activated by cyanogen bromide in water, and then immerse the swollen matrix with hydrochloric acid with a concentration of 0.5-2mM for 1-3 hours (1 hour, 1.2 hours, 1.5 hours, 1.8 hours, 2 Hours, 2.2 hours, 2.5 hours, 2.8 hours or 3 hours), and then _{wash the substrate with a buffer containing 0.1M NaHCO 3} and 0.5M NaCl with a pH of 8.3 to obtain a pretreated substrate;

(2) Dissolve the heparanase binding domain polypeptide and active center protecting agent in a _{buffer containing 0.1M NaHCO 3} and 0.5M NaCl, pH 8.3, and stir at room temperature for 0.5-2 hours (e.g., 0.5 hours, 0.8 hours, 1 hour, 1.2 hours, 1.5 hours, 1.8 hours or 2 hours); and

(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.

Preferably, 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.

In a specific embodiment, 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.

In the third aspect, 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:

(i) Pack the column with the affinity filler and equilibrate with a sodium chloride solution with a sodium chloride concentration of 0.2M to obtain an affinity chromatography column; and

(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.) 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.

In this application, the glycosaminoglycan is heparin, heparan sulfate, dermatan sulfate, hyaluronic acid or chondroitin sulfate.

In 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;

Preferably, during the 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.

Preferably, 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.

In this application, 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. The purpose of elution using sodium chloride solution with sodium chloride concentration ≤1.0M is to elute low-active components.

In the fourth aspect, the present application provides a glycosaminoglycan component with heparanase inhibitory activity, which is obtained by the method described in the third aspect.

In this application, 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.

In the fifth aspect, the present application provides the application of the component having heparanase inhibitory activity as described above in the preparation of anti-tumor drugs.

Compared with the prior art, this application has the following beneficial effects:

In this application, 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. In addition, 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.

Description of the drawings

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, and ③ is Example 3.的filler;

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.

Detailed ways

The technical solutions of the present application will be further explained through specific implementations below. It should be understood by those skilled in the art that the described embodiments are merely to help understand the application and should not be regarded as specific limitations to the application.

The raw materials used in the examples are as follows:

(1) KKDC: KMFKNSTYSRSSVDC, referred to as KKDC, PI is 9.63, M _W is 1749.96, prepared by Shanghai Taopu Biotechnology Co., Ltd.;

(2) QPLK: QPRRKTAKMLK, abbreviated as QPLK, PI is 12.02, M _W is 1356.7.

(3) 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.

(4) 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.

(5) 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.

Example 1

The preparation method of KKDC-I filler is as follows:

(1) Matrix pretreatment

Take the 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.

(2) Preparation of ligand

KKDC (250 mg) and N-heparan (250 mg) were dissolved in buffer-A solution (85 mL) and stirred at room temperature for 0.5 hours.

(3) Coupling reaction between ligand and substrate

The substrate of 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).

(4) Elute unreacted impurities

Add 400mL Tris solution (pH 8.0) to the product in step (3), let stand at room temperature for 2 hours, filter with sand core funnel, rinse with 500mL buffer-A solution, and then use 10mM Tris solution (500mL) containing 2mM EDTA rinse. Finally, wash with 1L Tris-HCl solution (pH 7.0) containing 2M NaCl to obtain KKDC-I filler.

For the filler prepared in this example, 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.

Calculation formula: 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 μmol/g.

Example 2

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 μmol/g.

Example 3

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 μmol/g.

Example 4

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 μmol/g.

Example 5

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 μmol/g.

Example 6

In this example, the separation effect of different fillers is evaluated, and the method is as follows:

The five fillers KKDC-I, KKDC-II, KKDC-III, diKKDC and KKDC-QPLK prepared in the examples were packed into columns (Φ2.5×30cm), respectively, and equilibrated with 3 column volumes of 0.2M NaCl solution.

(2) Separate unfractionated heparin with the five affinity chromatography columns obtained in step (1), 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.

(3) Take 10μL sample from each tube of eluent, drop it into 96-well microtiter plate, add 150μL DMB solution, test the absorbance value at 525nm with a microplate reader, and make a dot-line graph. The results are shown in Figure 1 and Figure 2. (It can be seen from the mobile phase curve that the mobile phase uses 0.6M, 1M, 2M NaCl solution, and each platform represents the elution mobile phase used by the sample with the indicated tube number).

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, and 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. When the elution salt concentration reaches 1M, most of the binding components have been washed When the salt concentration rises to 2M, there is no obvious elution peak.

Example 7

In this example, 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:

(1) Take the KKDC-I packing obtained in Example 1 and pack it into a column (Φ2.5×30cm), and equilibrate with 2 column volumes of 0.2M NaCl solution.

(2) Dissolve 50 mg of heparin in 1 mL of pure water and load the sample, eluting with 0.6M NaCl, 1M NaCl, 2M NaCl solution for about 3 column volumes, and collect the eluate with an automatic partial collector. The collector is set to 1 min/tube, and the volume of each tube is 5 mL.

(3) Take 10μL of sample from each tube of eluent, drop it into 96-well microtiter plate, add 150μL of DMB solution, test the absorbance at 525nm with a microplate reader, and make a dot-line graph.

(4) Collect the eluted fractions of 0.6M NaCl, 1.0M NaCl and 2.0M NaCl solutions, respectively, and perform desalting post-treatment. For high-salt components eluted with a mobile phase with a salt concentration greater than 2.0M NaCl, dialyze first, and then rotate. For the eluted fractions of 0.6M NaCl and 1.0M NaCl, the eluted fractions were first concentrated by rotary evaporation, then desalted by dialysis, and then concentrated by rotary evaporation. Pass each concentrated fraction through a Bio-Gel P-2 column (Φ2.5×100cm), eluting with water as the mobile phase, and use DMB (dimethyl methylene blue) dye to identify whether the eluate contains glycosaminoglycans , And then use AgNO _{3 to} confirm that it does not contain NaCl, collect the corresponding components, rotary steam, concentrate, and freeze-dry to obtain salt-free samples of components with different binding abilities to KKDC peptide fragments, according to the peak sequence They were named Hep1 (corresponding to the elution component of 0.6M NaCl solution), Hep2 (corresponding to the elution component of 1.0M NaCl solution) and Hep3 (corresponding to the elution component of 2.0M NaCl solution).

Refer to "Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening" (Anal Biochem. 396(1), 2010., 112-116) for the method for determining heparanase inhibitory activity in vitro. Test the heparanase inhibitory activity of Hep1, Hep2 and Hep3, the method is as follows:

The assay solution (100 μL) 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 μL 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 μM, 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.

Table 1

sample name	IC 50 (ng/mL)
heparin	37
Hep1	47
Hep2	30
Hep3	3.8

From the results in Table 1, it can be seen that the heparanase inhibitory activity of Hep3 is about 10 times higher than that of heparin, indicating that the affinity column using KKDC-I packing can separate the heparanase inhibitory activity from heparin. Components.

Example 8

In this example, 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:

1. Experimental steps

(1) 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.

(2) 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.

(3) Tail vein inoculation of melanoma cells was performed 30 minutes after administration. Observe the mice's activity status, weight changes, and whether there are any abnormalities every day.

(4) End the experiment on the 14th day after inoculation. All the experimental mice were sacrificed. Fresh lungs were taken and weighed. The lung tissues of the mice were fixed with Bouin fixative to observe the distribution of lung tumor metastases in the mice and calculate the lung tumors in the mice Metastasis inhibition rate.

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%.

2. Experimental results

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%.

Example 9

In this example, 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:

1. Experimental steps

(1) 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.

(2) When the cells are in the exponential growth phase, the cells are collected, counted, and inoculated in the fourth fat pad of the abdomen of BALB/c mice.

(3) Randomly divide into 3 groups according to body weight and tumor inoculation order, including 1 blank control group and 2 administration groups. The administration group started daily administration (intraperitoneal injection) on the second day after inoculation. On the 13th day of tumor cell inoculation, the tumor in situ was surgically removed. No administration was given on the same day. The administration group continued administration on the second day after the operation. The dosage regimen of each group is shown in Table 2 below.

Table 2 Specific dosing schedule

Serial number	Number of nude mice	drug	Dose (mg/kg)	Route of administration		Dosing frequency
1	12	Normal saline	Equal dose	Intraperitoneal injection	Once a day
2	12	Hep3	10	Intraperitoneal injection	Once a day
3	12	Hep3	20	Intraperitoneal injection	Once a day

(4) 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.

2. Experimental results

The experimental results are shown in Table 3, which 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.

table 3

Therefore, it can be seen that 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.

This application uses the above-mentioned examples to illustrate the affinity filler of this application, its preparation method and application, but this application is not limited to the above-mentioned process steps, which does not mean that this application must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement to this application, the equivalent replacement of raw materials selected in this application, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

Claims (15)

1. An affinity filler, which is a matrix coupled with a heparanase binding domain polypeptide; wherein the heparanase binding domain polypeptide is treated with an active center protecting agent.
2. The affinity filler according to claim 1, wherein the heparanase binding domain polypeptide is KKDC, QPLK, diKKDC or KKDC-QPLK.
3. The affinity filler according to claim 1 or 2, wherein the active center protecting agent is any one or a combination of at least two of N-heparan, heparin and heparan sulfate.
4. The affinity filler according to any one of claims 1 to 3, wherein the method for treating the heparanase binding domain polypeptide with the active center protecting agent is: combining the heparanase binding domain polypeptide with the active center protecting agent Dissolved in a _{buffer solution containing 0.1M NaHCO 3} and 0.5M NaCl, pH 8.3, and stirred at room temperature for 0.5-2 hours to obtain a heparanase binding domain polypeptide treated with an active center protectant.
5. The affinity filler according to any one of claims 1 to 4, wherein the mass ratio of the active center protecting agent to the heparanase binding domain polypeptide is ≥0.5:1, preferably 1:1-3:1.
6. The affinity filler according to any one of claims 1 to 5, wherein the matrix is Sepharose 4B.
7. The affinity filler according to any one of claims 1 to 6, wherein the binding ratio of the heparanase binding domain polypeptide to the matrix is 4.50-5.60 μmol/g.
8. A method for preparing the affinity filler according to any one of claims 1-7, which comprises the following steps:

   (1) Swell the matrix activated by cyanogen bromide in water, then immerse the swollen matrix with 0.5-2mM hydrochloric acid for 1-3 hours, and then use 0.1M NaHCO ₃ and 0.5M NaCl, pH Wash the substrate with 8.3 buffer to obtain the pretreated substrate;

   (2) Dissolve the heparanase binding domain polypeptide and the active center protecting agent in a _{buffer containing 0.1M NaHCO 3} and 0.5M NaCl, pH 8.3, and stir at room temperature for 0.5-2 hours; and

   (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, and then stand at 8-10°C overnight, and then filter with suction. The filter cake is washed with 0.1M Tris solution with pH 8.0 to obtain the affinity filler.
9. The preparation method according to claim 8, wherein the mass ratio of the active center protecting agent to the heparanase binding domain polypeptide in step (2) is ≥0.5:1, preferably 1:1-3:1.
10. The preparation method according to claim 8 or 9, wherein the preparation method further comprises: 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 Washed with a buffer solution of pH 8.3 containing 0.1M NaHCO ₃ and 0.5M NaCl, then washed with a solution containing 2mM EDTA and 10mM Tris, and finally washed with a Tris-HCl solution containing 2M NaCl with a pH of 7.0 to obtain the purified affinity And filler.
11. A method for separating components with heparanase inhibitory activity from glycosaminoglycans by using the affinity filler according to any one of claims 1-7, which comprises the following steps:

    (i) Pack the column with the affinity filler and equilibrate with 0.2M sodium chloride solution to obtain an affinity chromatography column; and

    (ii) Load the glycosaminoglycan sample onto the affinity chromatography column obtained in step (i), eluting with a sodium chloride solution with a concentration of ≤1.0M, and then use a sodium chloride solution with a concentration of 1.5-2.5M For elution, the 1.5-2.5M sodium chloride solution eluate is collected, dialyzed, and concentrated to obtain glycosaminoglycan components with heparanase inhibitory activity.
12. The method according to claim 11, wherein the glycosaminoglycan is heparin, heparan sulfate, dermatan sulfate, hyaluronic acid, or chondroitin sulfate.
13. The method according to claim 11 or 12, wherein in step (ii), the elution with a sodium chloride solution with a concentration ≤1.0M is isocratic elution or at least two different elutions in the range of a concentration ≤1.0M are used. Concentration of sodium chloride solution for gradient elution;

    Preferably, in the 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;

    Preferably, 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.
14. A glycosaminoglycan component with heparanase inhibitory activity, which is obtained by the method of any one of claims 11-13.
15. The use of the glycosaminoglycan component of claim 14 in the preparation of anti-tumor drugs.

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