CN110483647B

CN110483647B - Anti-tumor polypeptide and application thereof

Info

Publication number: CN110483647B
Application number: CN201910762095.3A
Authority: CN
Inventors: 叶果; 张成瑶; 杨鑫; 孙冬梅; 孙有俊; 范彩云; 鲍芳丽
Original assignee: Chongqing University Cancer Hospital
Current assignee: Chongqing University Cancer Hospital
Priority date: 2019-08-19
Filing date: 2019-08-19
Publication date: 2021-02-09
Anticipated expiration: 2039-08-19
Also published as: CN110483647A

Abstract

The invention discloses an anti-tumor polypeptide and application thereof, which have higher targeting property and affinity to tumors and can also increase the sensitivity of the tumors to other treatment methods; compared with blocking antibodies, the blocking antibody has low immunogenicity and low adverse reaction rate, can be synthesized by a chemical method, and has simpler preparation and purification processes and high production efficiency; the polypeptide of the invention has various anti-tumor effects, can have higher inhibition rate on the proliferation of tumor cells of various origins, and has the inhibition rate of more than 60 percent on melanoma, gastric cancer and liver cancer under the effective administration concentration; under the effective administration concentration, the tumor inhibition rate on esophageal squamous cell carcinoma, non-small cell lung cancer, colon cancer, oral squamous cell carcinoma and breast cancer can reach more than 50%.

Description

Anti-tumor polypeptide and application thereof

Technical Field

The invention relates to the technical field of polypeptide medicines, and particularly relates to an anti-tumor polypeptide and application thereof.

Background

The polypeptide is a compound formed by connecting one or more amino acids together by peptide bonds, and is generally formed by dehydration condensation of 10-100 amino acid molecules, and the molecular weight of the compound is less than 10000 Da. The polypeptide is an active group for the protein to play a role, is a bioactive substance related to various cell functions in an organism and participates in regulating various physiological functions, so the polypeptide has very important development value in clinical application.

The source of active polypeptide is divided into natural active polypeptide, artificially modified polypeptide based on natural product and artificially synthesized active polypeptide. The natural polypeptide has wide source, and can be divided into animal bioactive polypeptide and plant polypeptide. With the development of science and technology, natural polypeptides have not been the only source of polypeptides, but instead, genetically recombinant polypeptides and chemically synthesized polypeptides.

Methods for the synthesis of bioactive peptides include liquid phase synthesis, solid phase synthesis, enzymatic synthesis, and combinatorial biosynthesis. The liquid phase synthesis has 2 strategies of gradual synthesis and fragment synthesis, is convenient and quick for synthesizing the polypeptide with less amino acids, has high purity and can be synthesized in large quantity. Solid phase synthesis is a method in which the C-terminus of an amino acid is immobilized on an insoluble resin, and then the amino acids are condensed in sequence. This method simplifies the work-up procedure for each reaction step, and has a high yield, with the disadvantages that the intermediate products in each reaction step cannot be purified, and the final product must be purified by a reliable separation means. Both liquid phase synthesis and solid phase synthesis belong to chemical synthesis, and 90% of polypeptide drugs developed and marketed at present are chemically synthesized, so that the application is wide. The enzymatic synthesis is a biosynthesis method which utilizes certain specific enzymes to catalyze reactions, has mild reaction conditions and strong stereospecificity, and has the defects of many adverse reactions and easy denaturation and inactivation of the enzymes. Combinatorial biosynthesis is the manipulation of genes encoding enzymes in the metabolic pathways of microorganisms to obtain new products. The method has the problems of high cost, easy formation of improper folding and posttranslational modification of eukaryotic gene expression and the like, thereby preventing the wide application of the method.

Currently, local cancers, while successfully treated with surgery and radiation therapy, are still the first choice for conventional treatment of advanced or metastatic tumors. The chemotherapy drugs have the defects of low selectivity, large adverse reaction, multi-drug resistance and the like, so the use of the chemotherapy drugs is limited to a great extent. Although the monoclonal antibody tumor targeted therapy solves the problem of targeting, the monoclonal antibody tumor targeted therapy has large protein molecular weight, high immunogenicity, easy generation of allergy and immunological cross reaction, high selling price due to high research and development cost, high research and development difficulty, limited productivity and the like, common cancer patients are difficult to popularize and use, and meanwhile, the monoclonal antibody drugs have the problems of single dosage form, most of administration modes are intravenous injection or instillation, oral administration cannot be realized, and the like, so that the drug application is very inconvenient.

The half-life of most antitumor peptide drugs is shorter under the restriction of technical conditions, but the antitumor polypeptide has the unique advantages of higher affinity and stronger specificity to target tumors, low adverse reaction and capability of increasing the sensitivity of the tumors to other treatment methods compared with small molecular chemical drugs; compared to antibodies, they are more easily penetrated into tissues due to their small volume; can be chemically synthesized, and can be chemically modified by various means, which is helpful for designing and researching novel active polypeptide.

The novel anti-tumor active polypeptide has the characteristics of high affinity, strong specificity and low adverse reaction, and most of the novel anti-tumor active polypeptide also has the property of selectively targeting tumor cells, so the novel anti-tumor active polypeptide has very important development value in clinical application and has better prospect as an anti-tumor compound.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: aiming at the defects of the existing monoclonal antibody medicines and chemotherapy medicines in treating tumors, the invention provides an anti-tumor polypeptide and application thereof in preparing anti-tumor medicines.

In order to solve the technical problems, the invention provides the following technical scheme:

an antitumor polypeptide comprises CCNDEGLEMRIK and GDASLKMDKSDAV amino acid sequences, wherein the amino acid sequence of the polypeptide is CCNDEGLEMRIK-Linker-GDASLKMDKSDAV; or GDASLKMDKSDAV-Linker-CCNDEGLEMRIK; or GDASLKMDKSDAVCCNDEGLEMRIK.

Preferably, the amino acid sequence of the Linker is DPTGG or G₃And S is repeated for 1-2 times.

Preferably, the polypeptide amino acid sequence is (1) CCNDEGLEMRIK-DPTGG-GDASLKMDKSDAV; or (2) GDASLKMDKSDAV-DPTGG-CCNDEGLEMRIK; or (3) CCNDEGLEMRIK-GGGSGGGS-GDASLKMDKSDAV; or (4) GDASLKMDKSDAV-GGGSGGGS-CCNDEGLEMRIK; or (5) CCNDEGLEMRIK-GGGS-GDASLKMDKSDAV; or (6) GDASLKMDKSDAV-GGGS-CCNDEGLEMRIK or (7) GDASLKMDKSDAVCCNDEGLEMRIK.

Preferably, the polypeptide is prepared by adopting Fmoc solid phase synthesis method, which comprises the following specific steps:

(1) weighing a proper amount of Fmoc-Ala (otBu) -Wang resin, adding the Fmoc-Ala-Wang resin into a polypeptide solid phase synthesis tube, adding DCM, and swelling the resin for 30 min;

(2) swelling, draining, adding DMF, washing for three times, draining again, and adding a decapping solution;

(3) sealing, placing the mixture into a shaking table, oscillating for reaction for 20min, draining, adding 50mL of DMF every time, washing for 5 times, and draining;

(4) adding 30g of the treated resin into an optometry agent, heating in a water bath kettle at 100 ℃ for 1.5min, and observing the color;

(5) after color testing is passed, carrying out condensation of a second amino acid according to the amino acid sequence of the polypeptide, reacting at 35 ℃ for 60min at the molar ratio of the material amount to the resin of 3:1, draining, washing with 50mL of DMF for 5 times, and draining;

(6) adding 30g of condensed resin into an optometry agent, heating at 100 ℃ for 90s, wherein the resin is colorless and transparent, and the solution is pale yellow, namely the optometry passes;

(7) repeating the steps (4) to (6) until the last amino acid is condensed, removing the protecting group Fmoc, washing with 50mL of DMF for 9 times, then washing with 50mL of DCM and 50mL of anhydrous methanol for three times respectively, pumping, and drying in vacuum to obtain the dry peptide resin;

(8) adding 10mL of cutting fluid into 1g of dry peptide resin, carrying out closed reaction for 2h at room temperature, carrying out suction filtration by using a sand core funnel, and collecting filtrate;

(9) adding the filtrate into anhydrous ether, wherein the volume ratio of the filtrate to the anhydrous ether is 1:10, performing suction filtration after polypeptide is separated out, collecting a filter cake, and performing vacuum drying at 35 ℃ for 4 hours to obtain a crude polypeptide product;

(10) purifying the crude polypeptide product by reversed phase liquid chromatography with column C₁₈Reversed phase silica gel column.

The application of the antitumor polypeptide in preparing antitumor drugs.

Preferably, the polypeptide can be used for treating solid tumors, including gastric cancer, esophageal squamous carcinoma, non-small cell lung cancer, colon cancer, liver cancer, melanoma, oral squamous carcinoma, and breast cancer.

Preferably, the anti-rheumatoid arthritis drug comprises an effective dose of anti-rheumatoid arthritis polypeptide and other pharmaceutically acceptable excipients or carriers, the drug carriers comprise organic and inorganic carriers, the oral and intestinal administration routes are suitable for adding the organic carriers, the organic carriers comprise one or more of starch, lactose, calcium oxide stearate, vegetable oil and organic solubilizing agents, the organic solubilizing agents comprise one or more of polyethylene glycol, glycerol and water-soluble organic and inorganic bases, the anti-rheumatoid arthritis drug is prepared into a solid form or a liquid form, and the anti-rheumatoid arthritis drugs in various forms are sterilized after being prepared.

Preferably, the dosage form of the anti-tumor drug is tablets, injections, sprays, capsules or coated pills, and the administration mode of the injections is intravenous injection, subcutaneous injection or intramuscular injection.

Preferably, the administration site of the antitumor drug comprises systemic administration and local administration, wherein the systemic administration is oral administration, intravenous injection or drip infusion, subcutaneous injection or intramuscular injection, and the local administration is tumor tissue internal injection and tumor peripheral tissue injection.

The invention has the following beneficial effects:

(1) the polypeptide of the invention can target to tumor neovascularization, inhibit the formation of the neovascularization and achieve the aim of preventing or treating tumors; the polypeptide can also block an immune check point by CD47-SIRP alpha, relieve the inhibition of tumor cells on immune cells, inhibit immune escape, inhibit angiogenesis and promote the attack of an autoimmune system on tumors, thereby achieving the purpose of preventing or treating the tumors;

(2) the polypeptide of the invention has higher targeting property and affinity to tumors, and can also increase the sensitivity of tumors to other treatment methods; compared with blocking antibodies, the blocking antibody has low immunogenicity and low adverse reaction rate, can be synthesized by a chemical method, and has simpler preparation and purification processes and high production efficiency;

(3) the polypeptide of the invention has small molecular weight, can penetrate various physiological barriers, and is beneficial to the diffusion and targeting of the medicine at a target position during systemic administration.

(4) The amino acid sequence of the invention adopts flexible peptide segments to connect two functional segments, which can effectively improve the drug effect, enhance the drug stability and prolong the half-life period.

(5) The polypeptide of the invention has various anti-tumor effects, can have higher inhibition rate on the proliferation of tumor cells of various origins, and has the inhibition rate of more than 60 percent on melanoma, gastric cancer and liver cancer under the effective administration concentration; under the effective administration concentration, the tumor inhibition rate on esophageal squamous cell carcinoma, non-small cell lung cancer, colon cancer, oral squamous cell carcinoma and breast cancer can reach more than 50%.

Drawings

FIG. 1: blank set flow cytometry fluorescence intensity plots;

FIG. 2: PE labeling sequence 2 polypeptide group flow cytometry fluorescence intensity map;

FIG. 3: PE-tagged sequence 4 polypeptide group flow cytometry fluorescence intensity map;

FIG. 4: PE marker sequence 6 polypeptide group flow cytometry fluorescence intensity map;

FIG. 5: PE-tagged sequence 7 polypeptide group flow cytometry fluorescence intensity map;

FIG. 6: mouse anti-human SIRP alpha monoclonal antibody + PE mark sequence 2 polypeptide group flow cytometry fluorescence intensity map;

FIG. 7: mouse anti-human SIRP alpha monoclonal antibody + PE mark sequence 4 polypeptide group flow cytometry fluorescence intensity map;

FIG. 8: mouse anti-human SIRP alpha monoclonal antibody + PE mark sequence 6 polypeptide group flow cytometry fluorescence intensity map;

FIG. 9: mouse anti-human SIRP alpha monoclonal antibody + PE mark sequence 7 polypeptide group flow cytometry fluorescence intensity map;

Detailed Description

The CCNDEGLEMRIK sequence is derived from 86-93 amino acid residues of human VEGF165 protein CCNDEGLE, MRIK is 107-110 amino acid residues, both are surface binding sites of VEGF165 and receptors thereof, and the amino acid sequences of the two peptide segments are integrated and artificially synthesized into a new amino acid sequence which can effectively bind VEGF receptors, but has no receptor activation structural domain, thus having no receptor activation capability and competitively inhibiting VEGF signal pathways, thereby inhibiting strong angiogenesis in tumor tissues.

The GDASLKMDKSDAV sequence is derived from 94-106 amino acid residues of human CD47 protein, the sequence is located in an immunoglobulin-like domain of an extracellular segment of CD47, the immunoglobulin-like domain is a region where CD47 and a ligand SIRP-alpha thereof recognize and combine mutually, an artificially synthesized GDASLKMDKSDAV sequence can be specifically combined on the SIRP-alpha protein on the surface of macrophage to competitively inhibit the combination of the SIRP-alpha and CD47, and after the immune check point of CD47 and the SIRP-alpha is blocked, the inhibition effect of tumor cells on the macrophage can be relieved, so that the macrophage recognizes and attacks the tumor cells, thereby achieving the purposes of killing and inhibiting the proliferation of the tumor cells.

After the anti-tumor polypeptide is combined with the SIRP-alpha molecule on the surface of the macrophage in a targeted manner, the macrophage is promoted to phagocytose tumor cells and is simultaneously combined with VEGFR2 on vascular endothelial cells in tumor tissues, angiogenesis in the tumor tissues is further inhibited, blood supply in the tumor tissues is blocked, the tumor tissues lack oxygen and nutrition supply, the microenvironment of the tumor tissues is changed, and finally, the proliferation of the tumor cells is inhibited and the tumor cells are killed.

The following examples are included to provide further detailed description of the present invention and to provide those skilled in the art with a more complete, concise, and exact understanding of the principles and spirit of the invention.

Example 1: the anti-tumor polypeptide is prepared by adopting an FMOC solid phase synthesis method, and the specific method is as follows:

(1) weighing 5g of Fmoc-Ala (otBu) -Wang resin, adding into a polypeptide solid phase synthesis tube, adding 50mL of DCM, and swelling the resin for 30 min;

(2) swelling, draining, adding 50mL of DMF, washing for three times, draining again, and adding 50mL of decapping solution;

(4) adding 30g of the treated resin into an optometry agent, heating in a water bath kettle at 100 ℃ for 1.5min, observing the color, wherein if the resin is brownish yellow and the solution is bluish purple, the Fmoc is removed, and if the resin is colorless and the solution is light in color, the uncapping step is repeated until the optometry passes;

(6) adding 30g of condensed resin into color testing agents (100 mu L of ninhydrin, phenol and pyridine respectively), heating at 100 ℃ for 90s, wherein the resin is colorless and transparent, and the solution is pale yellow, namely the color testing is passed;

(7) and (5) repeating the steps (4) to (6), and condensing the amino acid raw materials one by one according to the amino acid sequences in the table 1 until the last amino acid condensation is completed. Removing a protecting group Fmoc, washing with 50mL of DMF for 9 times, then washing with 50mL of DCM and 50mL of anhydrous methanol for three times respectively, pumping, and drying in vacuum to obtain dry peptide resin;

(10) purifying the crude polypeptide product by reversed phase liquid chromatography with column C₁₈Reverse phase silica gel columns (see Zhu X, Robertson J T, Sacks H S, Dohan F C, Tseng JL, Desiderio D M. peptides, 1995,16(6): 1097-1107).

TABLE 1 polypeptide amino acid sequence Listing

Numbering	Amino acid sequence
		1	CCNDEGLEMRIK-DPTGG-GDASLKMDKSDAV
2	GDASLKMDKSDAV-DPTGG-CCNDEGLEMRIK
		3	CCNDEGLEMRIK-GGGSGGGS-GDASLKMDKSDAV
4	GDASLKMDKSDAV-GGGSGGGS-CCNDEGLEMRIK
		5	CCNDEGLEMRIK-GGGS-GDASLKMDKSDAV
6	GDASLKMDKSDAV-GGGS-CCNDEGLEMRIK
		7	GDASLKMDKSDAVCCNDEGLEMRIK

Wherein DPTGG, GGGSGGGS and GGGS are all flexible connecting peptides.

The purity of the purified polypeptide is 92.3 percent and is used for the subsequent antitumor drug effect experiment in animals.

Example 2: preparation of antitumor drug

The composition tablets were prepared according to the following formulation:

TABLE 2 antitumor drug component Table

Components	Content (wt.)
		Amino acid sequence 1 polypeptide	50mg
Corn starch	278mg
		Lactose	70mg
Calcium oxide stearate	2mg
		In total	400mg

The polypeptide prepared in example 1 (amino acid sequence 1) and lactose were used to form granules by binding with corn starch paste, after drying, corn starch and calcium oxide stearate were added, and the mixture was compressed by a tableting machine into tablets 2 mm thick, 400mg by weight, 9SCE hardness.

Example 3: preparation of antitumor drug

The composition suppository is prepared according to the following formula:

TABLE 3 antitumor drug component Table

Components	Content (wt.)
		Amino acid sequence 2 polypeptide	50mg
Talcum powder	250mg
		Vegetable oil	25mg
Sodium bicarbonate	25mg
		Polyethylene glycol having an average molecular weight of 4000	50mg
In total	400mg

Suppositories containing the polypeptide (amino acid sequence 2) prepared in example 1 as the main active ingredient were prepared according to the conventional pharmaceutical preparation method (refer to "modern pharmacy").

Example 4: preparation of antitumor drug

The composition capsules were prepared as follows:

TABLE 4 antitumor drug component Table

Capsules containing the polypeptide (amino acid sequence 3) prepared in example 1 as the main active ingredient were prepared according to a conventional pharmaceutical preparation method (refer to "modern pharmacy") using edible gelatin as the capsule shell.

Example 5: preparation of antitumor drug injection

The injection mother solution has the following formula:

TABLE 5 antitumor drug component Table

Components	Content (wt.)
		Amino acid sequence 4 polypeptide	10mg
Glycerol	0.01mL
		Diethanolamine (DEA)	0.005mL
Physiological saline	0.985mL

Mixing all the components thoroughly, filtering with 0.22 μm filter membrane for sterilization, aseptically packaging into ampoule bottle filled with nitrogen, and diluting the mother liquor 1000 times with normal saline as working solution.

Example 6: preparation of antitumor drug injection

The injection mother solution has the following formula:

TABLE 6 antitumor drug component Table

Components	Content (wt.)
		Amino acid sequence 5 polypeptide	25mg
Glycerol	0.1mL
		Diethanolamine (DEA)	0.15mL
Physiological saline	0.75mL

The components are fully mixed, prepared, filtered and sterilized by a 0.22-micron filter membrane, aseptically subpackaged into ampoules filled with nitrogen, and when in use, the mother liquor is diluted 1000 times by adopting normal saline to serve as working solution.

Example 7: preparation of antineoplastic medicine emulsion

The composition formula is as follows:

TABLE 7 antitumor drug component Table

Components	Content (wt.)
		Amino acid sequence 6 polypeptide	1mg
Glycerol	0.2mL
		Physiological saline	0.8mL
Libaolining medicine	9mL

Accurately weighing polypeptide of amino acid sequence 6, dissolving in physiological saline, adding glycerol and LIBAOLINING, stirring, packaging, and sterilizing by irradiation.

Example 8: preparation of antineoplastic injection

The composition formula is as follows:

TABLE 8 antitumor drug component Table

Components	Content (wt.)
		Amino acid sequence 7 polypeptide	40mg
Mannitol	0.2mL
		Physiological saline	1.8mL

The preparation and use methods are the same as example 6.

Example 9: an antitumor in vivo experiment was performed using the antitumor agent prepared in example 2.

Experimental materials: 50 male nude mice of 18-22 g;the tumor cell strain is gastric cancer MGC-803The antitumor drug prepared in example 2 was dissolved in sterile physiological saline to a final concentration of 0.2mg/mL, filtered through a 0.22 μm filter membrane, aseptically dispensed, and stored at 4 ℃ for further use. Avastin and docetaxel injection were purchased from Jiangsu Xiansui Zaokang pharmaceutical Co.

The experimental method comprises the following steps:

(1) MGC-803 cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator to a density of above 80%, using 10% FBS-containing DMEM high-sugar medium, digesting with 0.25% trypsin digestion solution to collect cells, centrifuging at 1000rpm, discarding supernatant, washing with normal saline for three times, and counting to 5 × 10⁷mL, stored at 4 ℃ until use.

(2) Inoculating the tumor cell suspension to the axilla of the forelimb of a nude mouse, wherein the suspension is 0.1 mL/mouse, a blank control group is not treated, and the tumor condition is observed every three days after inoculation, and the tumor volume reaches 0.1mm³The administration is divided into groups.

(3) A blank control group, a model group, an administration group in example 2 and a positive control group are arranged, each group comprises 8 mice, the positive drug adopts Avastin and docetaxel injection, the administration dose of Avastin is 15mg/kg, the administration is carried out once every three days, the administration dose of docetaxel injection is 5mg/kg, the administration is carried out once every 7 days, and the administration modes of the positive drug are intravenous injection. The blank control group is not treated, the model group is subjected to intragastric administration by using physiological saline after the model is constructed, the administration volume is 0.1mL/10g, the administration group is subjected to intragastric administration by using an antitumor drug solution, the administration volume is 0.1mL/10g, and the administration is carried out once a day.

(4) Tumor volume was measured 21 days after administration, and the tumor inhibition rate was calculated as follows:

tumor inhibition rate (%) (tumor volume in model group-tumor volume in administration group)/tumor volume in model group

The experimental results are as follows:

TABLE 9 antitumor effect of each group of drugs

Group of	Active ingredient	Tumor inhibition Rate (%)
			Example 2	Sequence 1 polypeptide	68.6
Avastin	Monoclonal antibody	61.2
			Docetaxel injection	Paclitaxel	51.9

Example 10: an antitumor in vivo experiment was performed using the antitumor agent prepared in example 3.

Experimental materials: 50 male nude mice of 18-22 g; tumor cell strainKYSE-30 for esophageal squamous carcinomaThe antitumor agent prepared in example 3 was dissolved in sterile physiological saline to a final concentration of 0.2mg/mL, filtered through a 0.22 μm filter membrane, aseptically dispensed, and stored at 4 ℃ for further use. Avastin and docetaxel injection were purchased from Jiangsu Xiansui Zaokang pharmaceutical Co.

The experimental method comprises the following steps: same as example 1

The experimental results are as follows:

TABLE 10 antitumor effect of each group of drugs

Example 11: an antitumor in vivo experiment was performed using the antitumor agent prepared in example 4.

Experimental materials: 50 male nude mice of 18-22 g; tumor cell strainMelanoma B16F10The antitumor agent prepared in example 4 was dissolved in sterile physiological saline to a final concentration of 0.2mg/mL, filtered through a 0.22 μm filter membrane, aseptically dispensed, and stored at 4 ℃ for further use. Avastin and docetaxel injection were purchased from Jiangsu Xiansui Zaokang pharmaceutical Co.

The experimental method comprises the following steps: same as example 1

The experimental results are as follows:

TABLE 11 antitumor effect of each group of drugs

Group of	Active ingredient	Tumor inhibition Rate (%)
			Example 4	Sequence 3 polypeptide	66.4
Avastin	Monoclonal antibody	65.9
			Docetaxel injection	Paclitaxel	55.8

Example 12: an antitumor in vivo experiment was performed using the antitumor agent prepared in example 5.

Experimental materials: 60 male nude mice of 18-22 g; tumor cell strainColon cancer HCT-116The antitumor drug prepared in example 5 is diluted 1000 times by sterile physiological saline and then administered, the final concentration of the drug is 10 mug/mL, and the drug is sterilized and subpackaged after being filtered by a 0.22 micron filter membrane and stored at 4 ℃ for later use. Avastin and docetaxel injection were purchased from Jiangsu Xiansui Zaokang pharmaceutical Co.

The experimental method comprises the following steps:

(1) HCT-116 cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator to a density of above 80%, using 10% FBS-containing DMEM high-sugar medium, digesting with 0.25% trypsin digestion solution to collect cells, centrifuging at 1000rpm, discarding supernatant, washing with normal saline for three times, and counting to 5 × 10⁷mL, stored at 4 ℃ until use.

(3) A blank control group, a model group, an administration group in example 5, a solvent group and a positive control group are arranged, 8 mice in each group are used as positive drugs, Avastin and docetaxel injection are adopted as the positive drugs, the administration dose of Avastin is 20mg/kg, the administration is carried out once every three days, the administration dose of docetaxel injection is 5mg/kg, the administration is carried out once every 7 days, and the administration modes of the positive drugs are tail vein injection. The blank control group is not treated, the tail vein of the model group is injected with normal saline after the model is made, the administration volume is 0.1mL/10g, the solvent group and the administration group are respectively injected with solvent and anti-tumor drug injection in the tail vein, the administration volume is 0.1mL/10g, and the administration is carried out once a day.

tumor inhibition rate (%) (model group tumor volume-experimental group tumor volume)/model group tumor volume

The experimental results are as follows:

TABLE 12 antitumor effect of each group of drugs

Group of	Active ingredient	Tumor inhibition Rate (%)
			Example 5	Sequence 4 polypeptide	58.4
Solvent group	Is free of	-0.5
			Avastin	Monoclonal antibody	65.9
Docetaxel injection	Paclitaxel	55.8

Example 13: an antitumor in vivo experiment was performed using the antitumor agent prepared in example 6.

Experimental materials: 60 male nude mice of 18-22 g; tumor cell strainLiver cancer SMMC-7721The antitumor drug prepared in example 6 is diluted 1000 times by sterile physiological saline and then administered, the final concentration of the drug is 25 mug/mL, and the drug is sterilized and subpackaged after being filtered by a 0.22 micron filter membrane and stored at 4 ℃ for later use. Avastin and docetaxel injection, both purchased fromJiangsu Xiansui Zaokang medicine Co.

The experimental method is different from that of example 12 in that the mode of administration of the model group, the vehicle group and the administration group is subcutaneous injection.

The experimental results are as follows:

TABLE 13 antitumor effect of each group of drugs

Group of	Active ingredient	Tumor inhibition Rate (%)
			Example 6	Sequence 5 Polypeptides	65.9
Solvent group	Is free of	1.1
			Avastin	Monoclonal antibody	60.2
Docetaxel injection	Paclitaxel	53.1

Example 14: an antitumor in vivo experiment was conducted using the antitumor agent prepared in example 7.

Experimental materials: 60 male nude mice of 18-22 g; the tumor cell strain is lung cancer A549 with a final concentration of 100 μ g/mL, and is sterilized and packaged after being filtered by a 0.22-micron filter membrane, and stored at 4 ℃ for later use. Avastin and docetaxel injection were purchased from Jiangsu Xiansui Zaokang pharmaceutical Co.

The experimental method is different from that of example 12 in that the administration modes of the model group, the vehicle group and the administration group are hind limb muscle injection administration. The administration volume was 0.02mL/10 g.

The experimental results are as follows:

TABLE 14 antitumor effect of each group of drugs

Group of	Active ingredient	Tumor inhibition Rate (%)
			Example 7	Sequence 6 polypeptide	59.1
Solvent group	Is free of	2.5
			Avastin	Monoclonal antibody	59.9
Docetaxel injection	Paclitaxel	61.2

Example 15: an antitumor in vivo experiment was conducted using the antitumor agent prepared in example 8.

Experimental materials: 60 male nude mice of 18-22 g; tumor cell strainOral squamous carcinoma TCA8113The antitumor drug prepared in example 8 is diluted 1000 times by sterile physiological saline and then administered, the final concentration of the drug is 20 mug/mL, and the drug is sterilized and subpackaged after being filtered by a 0.22 micron filter membrane and stored at 4 ℃ for later use. Avastin and docetaxel injection were purchased from Jiangsu Xiansui Zaokang pharmaceutical Co.

The experimental method differs from that of example 12 in that the administration modes of the model group, the vehicle group and the administration group are tumor tissue injection administration. The administration volume was 0.01mL/10 g.

The experimental results are as follows:

TABLE 15 antitumor effect of each group of drugs

Group of	Active ingredient	Tumor inhibition Rate (%)
			Example 8	Sequence 7 polypeptide	51.2
Solvent group	Is free of	0.3
			Avastin	Monoclonal antibody	65.4
Docetaxel injection	Paclitaxel	60.7

Example 16: an antitumor in vivo experiment was conducted using the antitumor agent prepared in example 8.

Experimental materials: 60 female nude mice of 18-22 g; the tumor cell strain is breast cancer MDA-MB-231, the antitumor drug prepared in the embodiment 8 is diluted by 1000 times by adopting sterile physiological saline and then is administrated, the final concentration of the drug is 20 mug/mL, and the drug is sterilized and subpackaged after being filtered by a 0.22 micron filter membrane and is preserved for standby at 4 ℃. Avastin and docetaxel injection were purchased from Jiangsu Xiansui Zaokang pharmaceutical Co.

The experimental method is different from that of example 12 in that the administration modes of the model group, the vehicle group and the administration group are subcutaneous injection administration of peritumoral tissues. The administration volume was 0.1mL/10 g.

The experimental results are as follows:

TABLE 16 antitumor effect of each group of drugs

Group of	Active ingredient	Tumor inhibition Rate (%)
			Example 8	Sequence 7 polypeptide	50.5
Solvent group	Is free of	3.3
			Avastin	Monoclonal antibody	66.8
Docetaxel injection	Paclitaxel	62.3

According to the experimental results, the polypeptide has various anti-tumor effects, can have higher inhibition rate on the proliferation of tumor cells of various origins, and has the inhibition rate of more than 60 percent on melanoma, gastric cancer and liver cancer under the effective administration concentration; under the effective administration concentration, the tumor inhibition rate on esophageal squamous cell carcinoma, non-small cell lung cancer, colon cancer, oral squamous cell carcinoma and breast cancer can reach more than 50%.

Flow cytometry detection of binding of polypeptides to macrophages:

a human THP-1 cell strain with high SIRP alpha expression is taken as an experimental macrophage.

Taking THP-1 cells cultured in suspension, adding PMA with final concentration of 100ng/mL to induce differentiation for 12h for adherence, collecting cells after trypsinization, washing twice with 1 × PBS, resuspending and adjusting cell concentration to 10⁷and/mL, dividing the cells into 9 groups, namely a blank group, a PE labeling polypeptide 2 group, a PE labeling polypeptide 4 group, a PE labeling polypeptide 6 group, a PE labeling polypeptide 7 group, a mouse anti-human SIRP alpha monoclonal antibody + PE labeling polypeptide 2 group, a mouse anti-human SIRP alpha monoclonal antibody + PE labeling polypeptide 4 group, a mouse anti-human SIRP alpha monoclonal antibody + PE labeling polypeptide 6 group, a mouse anti-human SIRP alpha monoclonal antibody + PE labeling polypeptide 7 group, taking 100 mu L of cell suspension from each group, and adding 20 mu LPBS into the blank group to incubate the cells for 2.5 h.

Adding 10 mu LPBS into each group of PE labeled polypeptide, incubating the cells for 2h at 37 ℃, adding 10 mu L of PE labeled polypeptide for light-shielding reaction for 30min, wherein the final concentration of the PE labeled polypeptide is 1 mu g/mu L;

10 mu L of mouse anti-human SIRP alpha monoclonal antibody (10 mu g/mL) is added into each group of SIRP alpha monoclonal antibody and PE marking polypeptide, 10 mu L of PE marking polypeptide is added for light-shielding reaction for 30min after the cells are incubated for 2h at 37 ℃, and the final concentration of the PE marking polypeptide is 1 mu g/mu L;

the 9 groups of cells were washed with pre-chilled 1 XPBS and centrifuged (1000rmp/10min)3 times, then resuspended in 200. mu.L of pre-chilled 1 XPBS, and each group of cells was examined separately with a flow cytometer.

The related statistical analysis is completed by SPSS10.0 software, and the main method is one-factor analysis of variance. If P is less than 0.05, the difference is statistically significant, and the result is shown in FIGS. 1-9.

The results of fig. 1-5 show that the fluorescence intensity of the cell surface is significantly increased after the PE-labeled polypeptide is incubated alone, indicating that a large amount of the PE-labeled polypeptide is bound to the cell surface, while fig. 6-9 show that when the PE-labeled polypeptide is incubated after the cells are treated with sirpa monoclonal antibody in advance, the fluorescence intensity approaches that of the blank control group, indicating that little or no PE-labeled polypeptide is bound to the cell surface, and the binding sites of the sirpa monoclonal antibody and the polypeptide are the same, so that the polypeptide of the present invention can specifically bind to sirpa on the macrophage surface.

Macrophage and tumor cell co-culture experiment:

taking a human THP-1 cell strain with high SIRP alpha expression as an experimental macrophage and taking MGC-803 cells with high CD47 expression as tumor cells;

collecting suspension cultured THP-1 cells, washing with 1 × PBS twice, re-suspending, counting, adding cell suspension into 24-well plate, and controlling total THP-1 number to 10⁴Adding PMA with the final concentration of 100ng/mL into each well, inducing differentiation for 12h for adherence, adding harvested and counted MGC-803 cells, wherein the ratio of total number of THP-1 to MGC-803 cells is 1:15, simultaneously adding SIRPa monoclonal antibody (with the final concentration of 10 mug/mL) and polypeptide with the sequence of 1-7 (with the final concentration of 10 mug/mL) respectively, adding only 1mLPBS into a blank control group, adding only the same amount of MGC-803 cells into a tumor cell group, and carrying out no treatment, wherein each group is provided with three multiple wells.

37℃，5％CO₂After the cells were co-cultured for 48h under the conditions, the cell viability was measured by the MTT method, and the tumor inhibition rate was calculated:

tumor inhibition rate (tumor cell group absorbance average-experimental group absorbance average)/tumor cell group absorbance average 100%

The results are shown in Table 17:

TABLE 17 Effect of polypeptides on macrophage and tumor cell Co-culture

The results show that the polypeptide blocks a CD47 and SIRP-alpha signal channel by combining with SIRP-alpha on the surface of macrophage, relieves the immunosuppressive action of tumor cells on the macrophage, effectively promotes the inhibitory action of the macrophage on the tumor cells, and ensures that the tumor inhibition rate of each polypeptide is obviously higher than that of a co-culture blank control group.

Chick embryo chorioallantoic membrane angiogenesis inhibition experiment

Taking 11-day-old chick embryos, marking an air chamber and a fetal position under an egg examination lamp, drawing a mark near the fetal position without great vessels with iodine, disinfecting the top of the air chamber and the mark with ethanol, drilling a small hole at the top of the air chamber, and grinding egg shells at the mark with an egg grinder to form a crack parallel to a longitudinal axis without damaging shell membranes. Laying the egg flat, removing the shell of the crack without damaging the shell membrane, pricking a small slit on the shell membrane without damaging the underlying chorioallantoic membrane, and dripping a drop of sterile physiological saline on the shell membrane. The air in the air chamber is slowly sucked from the small hole at the end of the air chamber by using a rubber nipple to generate negative pressure of the air chamber, and at the moment, the physiological saline sinks, the chorioallantoic membrane sinks, and an artificial air chamber is formed between the shell membrane and the allantoic membrane.

Uncovering the shell membrane on the artificial air chamber, exposing the chorioallantoic membrane (CAM), dripping 0.2-0.5mL of drug suspension on the chorioallantoic membrane, adding the sample, sealing the window with sterile transparent adhesive tape, incubating for 3 days, observing once every 12 hours, and sealing with sterile adhesive tape. Laying the chick embryo horizontally, and incubating at 37 ℃ to avoid turning over so as to avoid the displacement of the artificial air chamber. And taking out after 5 days for harvesting. Disinfecting the inoculated part and the periphery with iodine and ethanol, tearing off the egg shell at the closed part, enlarging the opening with a sterile forceps, slightly clamping the chorioallantoic membrane, shearing off the inoculated surface and the membranes at the periphery with sterile scissors, and fixing with formaldehyde.

And taking the fixed CAM membrane, dehydrating with ethanol, embedding in paraffin, and continuously slicing along the direction parallel to the CAM, wherein the thickness is 8 mu m. 0.5% toluidine blue stain. The cross section of the microvessels was counted under 250-fold visual field of the specimen, and the mean value of the counted microvessels was taken as the MVD (microvessel density) per unit area of the specimen as follows:

angiogenesis inhibition rate (blank group MVD value-experimental group MVD value)/blank group MVD value 100%

TABLE 18 determination of angiogenesis inhibiting drug efficacy

The results in Table 11 show that the invention can not only block CD47 and SIRP-alpha signal channel, but also obviously inhibit angiogenesis, and the effect is close to or better than that of VEGF monoclonal antibody (bevacizumab).

The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby, and any modification made on the basis of the technical scheme according to the technical idea proposed by the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.

SEQUENCE LISTING

<110> Nanjing Germination bacteria industry Co., Ltd

<120> an anti-tumor polypeptide and application thereof

<130> 11204

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 30

<212> PRT

<213> Artificial sequence

<400> 1

Cys Cys Asn Asp Glu Gly Leu Glu Met Arg Ile Lys Asp Pro Thr Gly

1 5 10 15

Gly Gly Asp Ala Ser Leu Lys Met Asp Lys Ser Asp Ala Val

20 25 30

<210> 2

<211> 30

<212> PRT

<213> Artificial sequence

<400> 2

Gly Asp Ala Ser Leu Lys Met Asp Lys Ser Asp Ala Val Asp Pro Thr

1 5 10 15

Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Met Arg Ile Lys

20 25 30

<210> 3

<211> 33

<212> PRT

<213> Artificial sequence

<400> 3

Cys Cys Asn Asp Glu Gly Leu Glu Met Arg Ile Lys Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Asp Ala Ser Leu Lys Met Asp Lys Ser Asp Ala

20 25 30

Val

<210> 4

<211> 33

<212> PRT

<213> Artificial sequence

<400> 4

Gly Asp Ala Ser Leu Lys Met Asp Lys Ser Asp Ala Val Gly Gly Gly

1 5 10 15

Ser Gly Gly Gly Ser Cys Cys Asn Asp Glu Gly Leu Glu Met Arg Ile

20 25 30

Lys

<210> 5

<211> 29

<212> PRT

<213> Artificial sequence

<400> 5

Cys Cys Asn Asp Glu Gly Leu Glu Met Arg Ile Lys Gly Gly Gly Ser

1 5 10 15

Gly Asp Ala Ser Leu Lys Met Asp Lys Ser Asp Ala Val

20 25

<210> 6

<211> 29

<212> PRT

<213> Artificial sequence

<400> 6

Gly Asp Ala Ser Leu Lys Met Asp Lys Ser Asp Ala Val Gly Gly Gly

1 5 10 15

Ser Cys Cys Asn Asp Glu Gly Leu Glu Met Arg Ile Lys

20 25

<210> 7

<211> 25

<212> PRT

<213> Artificial sequence

<400> 7

Gly Asp Ala Ser Leu Lys Met Asp Lys Ser Asp Ala Val Cys Cys Asn

1 5 10 15

Asp Glu Gly Leu Glu Met Arg Ile Lys

20 25

Claims

1. An anti-tumor polypeptide, comprising: comprises CCNDEGLEMRIK and GDASLKMDKSDAV amino acid sequences, and the amino acid sequence of the polypeptide is CCNDEGLEMRIK-Linker-GDASLKMDKSDAV; or GDASLKMDKSDAV-Linker-CCNDEGLEMRIK; or GDASLKMDKSDAVCCNDEGLEMRIK.

2. An anti-tumor polypeptide according to claim 1, wherein: the amino acid sequence of the Linker is DPTGG or G₃And S is repeated for 1-2 times.

3. An anti-tumor polypeptide according to claim 2, wherein: the amino acid sequence is (1) CCNDEGLEMRIK-DPTGG-GDASLKMDKSDAV; or (2) GDASLKMDKSDAV-DPTGG-CCNDEGLEMRIK; or (3) CCNDEGLEMRIK-GGGSGGGS-GDASLKMDKSDAV; or (4) GDASLKMDKSDAV-GGGSGGGS-CCNDEGLEMRIK; or (5) CCNDEGLEMRIK-GGGS-GDASLKMDKSDAV; or (6) GDASLKMDKSDAV-GGGS-CCNDEGLEMRIK or (7) GDASLKMDKSDAVCCNDEGLEMRIK.

4. A method of synthesizing the anti-tumor polypeptide of claim 1, wherein: the polypeptide is synthesized by adopting an Fmoc solid phase synthesis method, and comprises the following specific steps:

(9) adding the filtrate into anhydrous ether, wherein the volume ratio of the filtrate to the anhydrous ether is 1:10, performing suction filtration after polypeptide is separated out, collecting a filter cake, and performing vacuum drying at 35 ℃ for 4h to obtain a crude polypeptide product.

5. The use of the antitumor polypeptide of claim 1 in the preparation of an antitumor medicament, wherein: the polypeptide is used for preparing antitumor drugs for inhibiting gastric cancer, esophageal squamous carcinoma, non-small cell lung cancer, colon cancer, liver cancer, melanoma, oral squamous carcinoma and breast cancer.

6. The use of an anti-tumor polypeptide according to claim 5, wherein: the anti-tumor medicine comprises an effective dose of anti-tumor polypeptide and other pharmaceutically acceptable auxiliary materials or carriers, the medicine carriers comprise organic and inorganic carriers, the organic carriers are suitable to be added in oral and intestinal administration routes, the organic carriers comprise one or more of starch, lactose, calcium oxide stearate, vegetable oil and organic solubilizing agents, the organic solubilizing agents comprise one or more of polyethylene glycol, glycerol and water-soluble organic and inorganic bases, the anti-tumor medicine is prepared into a solid form or a liquid form, and the anti-tumor medicines in various forms are sterilized after being prepared.

7. The use of the antitumor polypeptide of claim 5 in the preparation of an antitumor medicament, wherein: the dosage form of the anti-tumor medicine is tablets, injections, sprays, capsules or coated pills, and the administration mode of the injections is intravenous injection, subcutaneous injection or intramuscular injection.

8. The use of the antitumor polypeptide of claim 5 in the preparation of an antitumor medicament, wherein: the administration part of the anti-tumor drug comprises systemic administration and local administration, wherein the systemic administration is oral administration, intravenous injection or drip infusion, subcutaneous injection or intramuscular injection, and the local administration is tumor tissue internal injection and tumor peripheral tissue injection.