CN110483647A - A kind of antineoplastic polypeptide and its application - Google Patents
A kind of antineoplastic polypeptide and its application Download PDFInfo
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Abstract
The invention discloses a kind of antineoplastic polypeptide and its applications, higher to targeting, the compatibility of tumour, can also increase tumour to the susceptibility of other treatment method；Compared with blocking antibody, immunogenicity is low, and adverse reaction rate is low, can be by being chemically synthesized, and preparation and purification process is more simple, high production efficiency；Polypeptide of the invention have a variety of antitumor efficacies, can the tumor cell proliferation inhibiting rate with higher to a variety of Different Origins 60% or more is reached to the inhibiting rate of melanoma, gastric cancer, liver cancer under effective administration concentration；Under effective administration concentration, to esophageal squamous cell carcinoma, non-small cell lung cancer, colon cancer, oral squamous cell carcinomas, breast cancer tumour inhibiting rate up to 50% or more.
Description
Technical field
The present invention relates to polypeptide drugs technical field, in particular to a kind of antineoplastic polypeptide and its application.
Background technique
Polypeptide is by one or more amino acid with the compound that peptide bond links together and is formed, usually by 10~100
A amino acid molecular dehydrating condensation forms, their molecular weight < 10000Da.Polypeptide is the active group that protein plays a role,
It is to be related to the bioactive substance of various cell functions in organism, participates in various physiological functions, thus in clinical application
It is upper that there is very important Development volue.
The source of active peptides is divided into natural biological polypeptide, the manually modified polypeptide based on natural products and artificial synthesized
Active peptides.The source of natural polypeptides is very extensive, can be divided into animal organism active peptides and plant polypeptide.With science and technology
Development, natural polypeptides has not been unique polypeptide source instead genetically modified polypeptide and chemically synthesized polypeptide.
The synthetic method of biologically active peptide includes liquid phase synthesis, synthesis in solid state, enzyme' s catalysis and Combinatorial biosynthesis.Liquid phase
Synthesis has gradually synthesis and segment to synthesize 2 kinds of strategies, for synthesizing the polypeptide fast and easy of less amino acid, purity is high, and energy
A large amount of synthesis.Synthesis in solid state is that the C-terminal of amino acid is fixed on insoluble resin, is then successively condensed the side of amino acid
Method.This process simplify the post-processing operations of every single step reaction, have higher yields, the disadvantage is that every step intermediate product cannot be pure
Change, and final product must be purified by reliable separation means.Liquid phase synthesis and synthesis in solid state belong to chemical synthesis, mesh
In the polypeptide drugs of preceding exploitation listing, 90% is chemical synthesis, is widely used.Enzyme' s catalysis is urged using certain specific enzymes
Change the biological synthesis method of reaction, reaction condition is mild, stereocpecificity is strong, the disadvantage is that there are many adverse reactions, enzymes to be easy
Deactivation.Combinatorial biosynthesis is by operating to some enzyme coding genes in microbial metabolism approach, to obtain
New product.Since this method is expensive, and there is eukaryotic gene expression easily to form inappropriate folding and posttranslational modification etc.
Problem, to hinder its extensive use.
Although localized cancer can successfully use surgical operation and radiation therapy treatment at present, chemotherapy is still normal
The first choice of rule treatment advanced stage or metastatic tumo(u)r.It is lacked since chemotherapeutics has low selectivity, erious adverse reaction, multidrug resistance etc.
It falls into, largely limits its use.Although monoclonal antibody class cancer target therapy solves the problems, such as targeting, but its albumen point
Son amount is big, and immunogenicity is high, is also easy to produce allergy and immunological cross-reaction, and at high cost due to researching and developing, research and development difficulty is big, and production capacity has
The reasons such as limit cause its price high, and common cancers patient is difficult universal use, while that there are dosage forms is more single for monoclonal antibody class drug
One, administration mode is mostly to be injected intravenously or instil, the problems such as can not taking orally, so that medicinal application is inconvenient.
It is restricted by technical conditions, the half-life period of most antitumor peptide medicaments is shorter, but antineoplastic polypeptide has it solely
Special advantage: compared with small-molecule chemical drug, there is higher affinity and stronger specificity to target tumor, and not
Good reaction is low, can also increase tumour to the susceptibility of other treatment method；Compared with antibody, since their volumes are small, because
This is easier to penetrate into tissue；Can be with chemical synthesis, and can be chemically modified with multiple means, help to design
With research novel active polypeptide.
New antitumoral active peptides have the characteristics that high-affinity, strong specificity, low adverse reaction, furthermore most of to go back
Have the property of selectively targeting tumour cell, thus there is very important Development volue in clinical application, as antitumor
Compound has preferable prospect.
Summary of the invention
The technical problems to be solved by the invention: it is deposited in treatment tumour for current monoclonal antibody class drug and chemotherapy class drug
Shortcomings, the present invention provides a kind of antineoplastic polypeptide and application in preparation of anti-tumor drugs.
In order to solve the above technical problems, the present invention provides technical solution below:
A kind of antineoplastic polypeptide includes two sections of amino acid sequences of CCNDEGLEMRIK and GDASLKMDKSDAV, the ammonia of polypeptide
Base acid sequence is CCNDEGLEMRIK-Linker-GDASLKMDKSDAV；Or GDASLKMDKSDAV-Linker-
CCNDEGLEMRIK；Or GDASLKMDKSDAVCCNDEGLEMRIK.
Preferably, the amino acid sequence of the Linker is DPTGG or G3S is repeated 1-2 times.
Preferably, polypeptid 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 using Fmoc solid-phase synthesis, is comprised the following specific steps that:
(1) it weighs appropriate Fmoc-Ala (otBu)-Wang resin Solid-phase synthesis peptides pipe is added and DCM is added, be swollen
Resin 30min；
(2) it is drained after being swollen, DMF washing is added and drains three times and again, liquid of raising one's hat is added；
(3) it is put into concussion reaction 20min in shaking table after sealing, 50mL DMF is added after draining every time and washs 5 times and drains；
(4) toner is added in the resin for taking 30g to handle well, and 1.5min is heated in 100 DEG C of water-baths, observes color；
(5) it tests after color passes through and carries out the condensation of second amino acid, inventory and resin according to the amino acid sequence of polypeptide
Molar ratio be 3:1, drain after 35 DEG C of reaction 60min, 50mL DMF washing 5 times is simultaneously drained；
(6) toner is added in the resin 30g after taking condensation, and 100 DEG C of heating 90s, resin is colorless and transparent, and solution is faint yellow i.e.
Color is tested to pass through；
(7) step (4)~(6) are repeated, until the last one amino acid condensation is completed, slough blocking group Fmoc,
50mL DMF is respectively washed three times with 50mL DCM and 50mL anhydrous methanol again after washing 9 times, is drained, and is dried in vacuo up to dry peptide
Resin；
(8) take the dry peptide resin of 1g that 10mL cutting liquid is added, in room temperature confined reaction 2h, sand core funnel is filtered, and collects filter
Liquid；
(9) filtrate being added in anhydrous ether, the volume ratio of filtrate and anhydrous ether is 1:10, and polypeptide filters after being precipitated,
Filter cake is collected, 35 DEG C of vacuum drying 4h obtain polypeptide crude product；
(10) polypeptide crude product is purified using reversed-phase liquid chromatography, chromatographic column uses C18Reverse phase silica gel column.
Above-mentioned antineoplastic polypeptide application in preparation of anti-tumor drugs.
Preferably, the polypeptide can be used for treating solid tumor, and the solid tumor includes gastric cancer, esophageal squamous cell carcinoma, non-small cell
Lung cancer, colon cancer, liver cancer, melanoma, oral squamous cell carcinomas, breast cancer.
Preferably, the resisting rheumatoid arthritis drug include effective dose resisting rheumatoid arthritis polypeptide and its
His pharmaceutically acceptable auxiliary material or carrier, the pharmaceutical carrier include organic and inorganic carrier, are taken orally and intestinal canal administration approach
It is suitble to that organic carrier is added, organic carrier includes starch, lactose, calcium oxide stearate, vegetable oil, one in organic solubilized agent
Kind or it is a variety of, the organic solubilized agent includes one of polyethylene glycol, glycerol, water-soluble organic and inorganic base or a variety of,
Resisting rheumatoid arthritis drug is made into solid form or liquid form, these various forms of resisting rheumatoid arthritis medicines
It is sterilized after the completion of object preparation.
Preferably, the dosage form of the anti-tumor drug is tablet, injection, spray, capsule or coated pills, the note
The administration mode for penetrating agent is vein, subcutaneously or intramuscularly injects.
Preferably, the medicine-feeding part of the anti-tumor drug includes Formulations for systemic administration and local administration, Formulations for systemic administration be it is oral,
Intravenous injection or instillation, subcutaneous injection or intramuscular injection, local administration are injected for injection, tumour perienchyma in tumor tissues.
It is that the present invention obtains the utility model has the advantages that
(1) polypeptide of the invention can inhibit new vessels to be formed, reach prevention or treatment targeted to tumor neogenetic blood vessels
The purpose of tumour；Polypeptide of the invention can release tumour cell to immunocyte with CD47-SIRP α-block immunologic test point
Inhibition, inhibit immunologic escape, promote attack of the self immune system to tumour while angiogenesis inhibiting, reach prevention
Or the purpose for the treatment of tumour；
(2) polypeptide of the invention is higher to targeting, the compatibility of tumour, can also increase tumour to other treatment method
Susceptibility；Compared with blocking antibody, immunogenicity is low, and adverse reaction rate is low, can be prepared and purified by being chemically synthesized
Process is more simple, high production efficiency；
(3) polypeptide molecular weight of the invention is smaller, can wear a variety of physiologic barriers, is conducive to when Formulations for systemic administration drug in target
The diffusion and targeting of position.
(4) two function fragments are connected using flexible peptide fragment in amino acid sequence of the invention, drug effect can be effectively improved, increased
Strong medicine stability extends half-life period.
(5) polypeptide of the invention has a variety of antitumor efficacies, can have to the tumor cell proliferation of a variety of Different Origins
Higher inhibiting rate reaches 60% or more to the inhibiting rate of melanoma, gastric cancer, liver cancer under effective administration concentration；Effective
Under administration concentration, to esophageal squamous cell carcinoma, non-small cell lung cancer, colon cancer, oral squamous cell carcinomas, breast cancer tumour inhibiting rate up to 50% with
On.
Detailed description of the invention
Fig. 1: blank group flow cytometry fluorescence intensity figure；
2 polypeptide group flow cytometry fluorescence intensity figure of Fig. 2: PE flag sequence；
4 polypeptide group flow cytometry fluorescence intensity figure of Fig. 3: PE flag sequence；
6 polypeptide group flow cytometry fluorescence intensity figure of Fig. 4: PE flag sequence；
7 polypeptide group flow cytometry fluorescence intensity figure of Fig. 5: PE flag sequence；
Fig. 6: the anti-human 2 polypeptide group flow cytometry fluorescence intensity figure of SIRP α monoclonal antibody+PE flag sequence of mouse；
Fig. 7: the anti-human 4 polypeptide group flow cytometry fluorescence intensity figure of SIRP α monoclonal antibody+PE flag sequence of mouse；
Fig. 8: the anti-human 6 polypeptide group flow cytometry fluorescence intensity figure of SIRP α monoclonal antibody+PE flag sequence of mouse；
Fig. 9: the anti-human 7 polypeptide group flow cytometry fluorescence intensity figure of SIRP α monoclonal antibody+PE flag sequence of mouse；
Specific embodiment
It is 86~93 amino acids that CCNDEGLEMRIK sequence in the present invention, which derives from human vascular endothelial growth factor 165 CCNDEGLE,
Residue, MRIK are 107~110 amino acids residues, and the two is all the surface binding site of VEGF165 Yu its receptor, by two
Artificial synthesized new amino acid sequence can effectively combine vegf receptor after the amino acid sequence integration of peptide fragment, but without receptor activation knot
Structure domain, thus the ability without receptor activation, emulative inhibition VEGF signal path, to inhibit blood strong in tumor tissues
Pipe is newborn.
GDASLKMDKSDAV sequence derives from people CD47 protein 94~106 amino acids residues, this sequence is located at CD47 born of the same parents
The immunoglobulin like domain of outer segment, immunoglobulin like domain are that CD47 and its ligand SIRP- α is mutually distinguishable and combines
Region, artificial synthesized GDASLKMDKSDAV sequence can specificity the SIRP- α albumen for being incorporated into Macrophage Surface on,
The emulative combination for inhibiting SIRP- α and CD47, CD47 and SIRP- alpha immunization checkpoint can release tumour cell to huge after blocking
The inhibiting effect of phagocyte, so that macrophage identifies and attack tumour cell, to reach killing and tumour cell is inhibited to increase
The purpose grown.
After antineoplastic polypeptide targeting combines the SIRP- alpha molecule of Macrophage Surface, promoting macrophage phagocytosis tumour thin
While born of the same parents in conjunction with the VEGFR2 on the vascular endothelial cell in tumor tissues, the blood vessel further suppressed in tumor tissues is new
It is raw, the blood supply in tumor tissues is blocked, tumor tissues is made to lack oxygen and nutrition supply, changes tumor tissues microenvironment, finally
Inhibit tumor cell proliferation and killing tumor cell.
Below by the description to embodiment, specific embodiments of the present invention will be described in further detail, with side
Those skilled in the art is helped to have more complete, accurate and deep understanding to inventive concept of the invention, technical solution.
Embodiment 1: antineoplastic polypeptide is prepared using FMOC solid-phase synthesis in the present invention, and the specific method is as follows:
(1) 5g Fmoc-Ala (otBu)-Wang resin is weighed Solid-phase synthesis peptides pipe is added and 50mL DCM is added,
Swellable resins 30min；
(2) it is drained after being swollen, 50mL DMF washing is added and drains three times and again, the liquid 50mL that raises one's hat is added；
(3) it is put into concussion reaction 20min in shaking table after sealing, 50mL DMF is added after draining every time and washs 5 times and drains；
(4) toner is added in the resin for taking 30g to handle well, and 1.5min is heated in 100 DEG C of water-baths, observes color, if
Resin brown color, solution bluish violet illustrate that Fmoc has been removed, if resin is colourless, solution colour is very light to need repetition above-mentioned
Step of raising one's hat passes through to color is tested；
(5) it tests after color passes through and carries out the condensation of second amino acid, inventory and resin according to the amino acid sequence of polypeptide
Molar ratio be 3:1, drain after 35 DEG C of reaction 60min, 50mL DMF washing 5 times is simultaneously drained；
(6) toner (each 100 μ L of ninhydrin, phenol, pyridine) is added in the resin 30g after taking condensation, 100 DEG C of heating 90s,
Resin is colorless and transparent, and faint yellow solution is to test color to pass through；
(7) step (4)~(6) are repeated, are condensed amino acid starting material one by one according to the amino acid sequence in table 1, until will most
The latter amino acid condensation is completed.Blocking group Fmoc is sloughed, 50mL DMF is anhydrous with 50mL DCM and 50mL again after washing 9 times
Methanol respectively washs three times, drains, and is dried in vacuo up to dry peptide resin；
(8) take the dry peptide resin of 1g that 10mL cutting liquid is added, in room temperature confined reaction 2h, sand core funnel is filtered, and collects filter
Liquid；
(9) filtrate being added in anhydrous ether, the volume ratio of filtrate and anhydrous ether is 1:10, and polypeptide filters after being precipitated,
Filter cake is collected, 35 DEG C of vacuum drying 4h obtain polypeptide crude product；
(10) polypeptide crude product is purified using reversed-phase liquid chromatography, chromatographic column uses C18Reverse phase silica gel column is (specific pure
Change method and condition referring to Zhu X, Robertson J T, Sacks H S, Dohan F C, Tseng JL, Desiderio D
M.Peptides., 1995,16 (6): 1,097 1 1107).
1 polypeptid acid sequence table of table
| Number | 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, GGGS are flexible peptide linker.
The purity of polypeptide is 92.3% after purification, for antitumor effect experiment in subsequent animal body.
Embodiment 2: the preparation of anti-tumor drug
Composition tablet is prepared according to the following formulation:
2 antineoplastic component table of table
| Component | Content |
| 1 polypeptide of amino acid sequence | 50mg |
| Cornstarch | 278mg |
| Lactose | 70mg |
| Calcium oxide stearate | 2mg |
| It is total | 400mg |
It bonds to form particle by corn starch paste using the polypeptide (amino acid sequence 1) and lactose that prepare in embodiment 1,
After drying, cornstarch and calcium oxide stearate is added, mixture is pressed into 2 millimeters thicks, 400mg weight, 9SCE by pelleter
The tablet of hardness.
Embodiment 3: the preparation of anti-tumor drug
Composition suppository is prepared according to the following formulation:
3 antineoplastic component table of table
| Component | Content |
| 2 polypeptide of amino acid sequence | 50mg |
| Talcum powder | 250mg |
| Vegetable oil | 25mg |
| Sodium bicarbonate | 25mg |
| The polyethylene glycol that average molecular weight is 4000 | 50mg |
| It is total | 400mg |
Polypeptide (amino of the preparation to prepare in embodiment 1 (is referred to " Modern Pharmaceutics ") according to traditional drug formulations method
Acid sequence 2) be main active suppository.
Embodiment 4: the preparation of anti-tumor drug
Composition capsule is prepared according to the following formulation:
4 antineoplastic component table of table
Using edible gelatin as capsule shells, preparation (is referred to " Modern Pharmaceutics ") to implement according to traditional drug formulations method
The polypeptide (amino acid sequence 3) prepared in example 1 is the capsule of main active.
Embodiment 5: the preparation of antineoplastic composition injection
Injection mother liquor formula is as follows:
5 antineoplastic component table of table
| Component | Content |
| 4 polypeptide of amino acid sequence | 10mg |
| Glycerol | 0.01mL |
| Diethanol amine | 0.005mL |
| Physiological saline | 0.985mL |
Each component be sufficiently mixed dissolution after 0.22 μm of membrane filtration degerming, it is aseptic subpackaged enter the ampoule bottle filled with nitrogen
In, mother liquor is diluted 1000 times as working solution using physiological saline when using.
Embodiment 6: the preparation of antineoplastic composition injection
Injection mother liquor formula is as follows:
6 antineoplastic component table of table
| Component | Content |
| 5 polypeptide of amino acid sequence | 25mg |
| Glycerol | 0.1mL |
| Diethanol amine | 0.15mL |
| Physiological saline | 0.75mL |
Each component be sufficiently mixed preparation after the completion of 0.22 μm of membrane filtration degerming, it is aseptic subpackaged enter the ampoule filled with nitrogen
In bottle, mother liquor is diluted 1000 times as working solution using physiological saline when using.
Embodiment 7: the preparation of anti-tumor drug emulsion
Composite formula is as follows:
7 antineoplastic component table of table
| Component | Content |
| 6 polypeptide of amino acid sequence | 1mg |
| Glycerol | 0.2mL |
| Physiological saline | 0.8mL |
| Lipofundin | 9mL |
The polypeptide of accurate weighing amino acid sequence 6, is dissolved in physiological saline, adds glycerol and Lipofundin is sufficiently stirred
After dispense, irradiation sterilization.
Embodiment 8: the preparation of antineoplastic composition injection
Composite formula is as follows:
8 antineoplastic component table of table
| Component | Content |
| 7 polypeptide of amino acid sequence | 40mg |
| Mannitol | 0.2mL |
| Physiological saline | 1.8mL |
Method of preparation and use is the same as embodiment 6.
Embodiment 9: antitumor experiment in vivo is carried out using the anti-tumor drug prepared in embodiment 2.
Experimental material: 18~22g Male nude mice 50；Tumor cell line is Gastric Cancer MGC -803, will be made in embodiment 2
Standby anti-tumor drug is dissolved in sterile saline, makes final concentration of 0.2mg/mL, sterile point after the filtering of 0.22 micron membrane filter
Dress, 4 DEG C save backup.Avastin and docetaxel injection are purchased from Jiangsu first sign Zai Kang Pharmaceuticals Ltd.
Experimental method:
(1) by MGC-803 cell at 37 DEG C, 5%CO2Culture is to 80% or more density in incubator, culture medium be containing
The DMEM high glucose medium of 10%FBS collects cell with 0.25% tryptic digestive juice digestion, and 1000rpm centrifugation is abandoned
Clearly, it is resuspended after brine three times and counts up to 5 × 107/ mL, 4 DEG C save backup.
(2) tumor cell suspension being seeded to nude mouse forelimb oxter, only, blank control group is not handled 0.1mL/,
Tumor situation is observed after inoculation every three days, reaches 0.1mm in gross tumor volume3When grouping administration.
(3) blank control group, model group, 2 administration group of embodiment and positive controls, every group of 8 mouse, positive drug are set
Using Avastin and docetaxel injection, the dosage of Avastin is 15mg/kg, is administered once every three days, more west he
The dosage of match injection is to be administered once for 5mg/kg every 7 days, and the administration mode of positive drug is intravenous injection.Blank control
Group is without any processing, and model group gives physiological saline stomach-filling after modeling, and administered volume 0.1mL/10g, administration group is given anti-
Tumour medicine solution stomach-filling, administered volume 0.1mL/10g, by daily single.
(4) gross tumor volume is measured after being administered 21 days, calculates tumour inhibiting rate, and calculation formula is as follows:
Tumour inhibiting rate (%)=(model group gross tumor volume-administration group gross tumor volume)/model group gross tumor volume
Experimental result is as follows:
9 each group drugs against tumor drug effect of table
| Group | Effective component | Tumour inhibiting rate (%) |
| Embodiment 2 | 1 polypeptide of sequence | 68.6 |
| Avastin | Monoclonal antibody | 61.2 |
| Docetaxel injection | Taxol | 51.9 |
Embodiment 10: antitumor experiment in vivo is carried out using the anti-tumor drug prepared in embodiment 3.
Experimental material: 18~22g Male nude mice 50；Tumor cell line isEsophageal squamous cell carcinoma KYSE-30, by embodiment 3
The anti-tumor drug of middle preparation is dissolved in sterile saline, makes final concentration of 0.2mg/mL, nothing after the filtering of 0.22 micron membrane filter
Bacterium packing, 4 DEG C save backup.Avastin and docetaxel injection are purchased from Jiangsu first sign Zai Kang Pharmaceuticals Ltd.
Experimental method: with embodiment 1
Experimental result is as follows:
10 each group drugs against tumor drug effect of table
Embodiment 11: antitumor experiment in vivo is carried out using the anti-tumor drug prepared in embodiment 4.
Experimental material: 18~22g Male nude mice 50；Tumor cell line isMelanin tumour b16 F10, will be in embodiment 4
The anti-tumor drug of preparation is dissolved in sterile saline, makes final concentration of 0.2mg/mL, sterile after the filtering of 0.22 micron membrane filter
Packing, 4 DEG C save backup.Avastin and docetaxel injection are purchased from Jiangsu first sign Zai Kang Pharmaceuticals Ltd.
Experimental method: with embodiment 1
Experimental result is as follows:
11 each group drugs against tumor drug effect of table
| Group | Effective component | Tumour inhibiting rate (%) |
| Embodiment 4 | 3 polypeptide of sequence | 66.4 |
| Avastin | Monoclonal antibody | 65.9 |
| Docetaxel injection | Taxol | 55.8 |
Embodiment 12: antitumor experiment in vivo is carried out using the anti-tumor drug prepared in embodiment 5.
Experimental material: 18~22g Male nude mice 60；Tumor cell line isColon cancer HCT-116, will be in embodiment 5
The anti-tumor drug of preparation is administered after diluting 1000 times using sterile saline, drug final concentration of 10 μ g/mL, and 0.22 micron
Aseptic subpackaged after membrane filtration, 4 DEG C save backup.Avastin and docetaxel injection are purchased from Jiangsu first sign health medicine again
Co., Ltd.
Experimental method:
(1) by HCT-116 cell at 37 DEG C, 5%CO2Culture is to 80% or more density in incubator, culture medium be containing
The DMEM high glucose medium of 10%FBS collects cell with 0.25% tryptic digestive juice digestion, and 1000rpm centrifugation is abandoned
Clearly, it is resuspended after brine three times and counts up to 5 × 107/ mL, 4 DEG C save backup.
(2) tumor cell suspension being seeded to nude mouse forelimb oxter, only, blank control group is not handled 0.1mL/,
Tumor situation is observed after inoculation every three days, reaches 0.1mm in gross tumor volume3When grouping administration.
(3) blank control group, model group, 5 administration group of embodiment, solvent group and positive controls are set, it is every group 8 small
Mouse, positive drug use Avastin and docetaxel injection, and the dosage of Avastin is 20mg/kg, and one is administered every three days
Secondary, the dosage of docetaxel injection is to be administered once for 5mg/kg every 7 days, and the administration mode of positive drug is tail vein note
It penetrates.Blank control group is without any processing, model group tail vein injection saline after modeling, administered volume 0.1mL/10g,
Solvent group and administration group difference tail vein injection solvent and antineoplastic composition injection, administered volume 0.1mL/10g are administered daily
Once.
(4) gross tumor volume is measured after being administered 21 days, calculates tumour inhibiting rate, and calculation formula is as follows:
Tumour inhibiting rate (%)=(model group gross tumor volume-experimental group gross tumor volume)/model group gross tumor volume
Experimental result is as follows:
12 each group drugs against tumor drug effect of table
| Group | Effective component | Tumour inhibiting rate (%) |
| Embodiment 5 | 4 polypeptide of sequence | 58.4 |
| Solvent group | Nothing | -0.5 |
| Avastin | Monoclonal antibody | 65.9 |
| Docetaxel injection | Taxol | 55.8 |
Embodiment 13: antitumor experiment in vivo is carried out using the anti-tumor drug prepared in embodiment 6.
Experimental material: 18~22g Male nude mice 60；Tumor cell line isLiver cancer SMMC-7721, will be in embodiment 6
The anti-tumor drug of preparation is administered after diluting 1000 times using sterile saline, drug final concentration of 25 μ g/mL, and 0.22 micron
Aseptic subpackaged after membrane filtration, 4 DEG C save backup.Avastin and docetaxel injection are purchased from Jiangsu first sign health medicine again
Co., Ltd.
Experimental method with embodiment 12 is the difference is that model group, solvent group and administration group administration mode are subcutaneous note
Penetrate administration.
Experimental result is as follows:
13 each group drugs against tumor drug effect of table
| Group | Effective component | Tumour inhibiting rate (%) |
| Embodiment 6 | 5 polypeptide of sequence | 65.9 |
| Solvent group | Nothing | 1.1 |
| Avastin | Monoclonal antibody | 60.2 |
| Docetaxel injection | Taxol | 53.1 |
Embodiment 14: antitumor experiment in vivo is carried out using the anti-tumor drug prepared in embodiment 7.
Experimental material: 18~22g Male nude mice 60；Tumor cell line is lung cancer A549, final concentration of 100 μ of drug
G/mL, aseptic subpackaged after the filtering of 0.22 micron membrane filter, 4 DEG C save backup.Avastin and docetaxel injection, are purchased from river
Su Xiansheng Zai Kang Pharmaceuticals Ltd.
Experimental method with embodiment 12 is the difference is that model group, solvent group and administration group administration mode are muscle of posterior limb
Meat drug administration by injection.Administered volume is 0.02mL/10g.
Experimental result is as follows:
14 each group drugs against tumor drug effect of table
| Group | Effective component | Tumour inhibiting rate (%) |
| Embodiment 7 | 6 polypeptide of sequence | 59.1 |
| Solvent group | Nothing | 2.5 |
| Avastin | Monoclonal antibody | 59.9 |
| Docetaxel injection | Taxol | 61.2 |
Embodiment 15: antitumor experiment in vivo is carried out using the anti-tumor drug prepared in embodiment 8.
Experimental material: 18~22g Male nude mice 60；Tumor cell line isOral squamous cell carcinomas TCA8113, by embodiment 8
The anti-tumor drug of middle preparation is administered after diluting 1000 times using sterile saline, and the final concentration of 20 μ g/mL of drug, 0.22 is micro-
Aseptic subpackaged after rice membrane filtration, 4 DEG C save backup.Avastin and docetaxel injection, being purchased from Jiangsu first sign, health is cured again
Medicine Co., Ltd.
Experimental method with embodiment 12 is the difference is that model group, solvent group and administration group administration mode are tumor group
Knit inner injecting and administering.Administered volume is 0.01mL/10g.
Experimental result is as follows:
15 each group drugs against tumor drug effect of table
| Group | Effective component | Tumour inhibiting rate (%) |
| Embodiment 8 | 7 polypeptide of sequence | 51.2 |
| Solvent group | Nothing | 0.3 |
| Avastin | Monoclonal antibody | 65.4 |
| Docetaxel injection | Taxol | 60.7 |
Embodiment 16: antitumor experiment in vivo is carried out using the anti-tumor drug prepared in embodiment 8.
Experimental material: 18~22g female nude mice 60；Tumor cell line is breast cancer MDA-MB-231, by embodiment 8
The anti-tumor drug of middle preparation is administered after diluting 1000 times using sterile saline, and the final concentration of 20 μ g/mL of drug, 0.22 is micro-
Aseptic subpackaged after rice membrane filtration, 4 DEG C save backup.Avastin and docetaxel injection, being purchased from Jiangsu first sign, health is cured again
Medicine Co., Ltd.
Experimental method with embodiment 12 is the difference is that model group, solvent group and administration group administration mode are tumour week
Enclose hermetical drug administration by injection.Administered volume is 0.1mL/10g.
Experimental result is as follows:
16 each group drugs against tumor drug effect of table
| Group | Effective component | Tumour inhibiting rate (%) |
| Embodiment 8 | 7 polypeptide of sequence | 50.5 |
| Solvent group | Nothing | 3.3 |
| Avastin | Monoclonal antibody | 66.8 |
| Docetaxel injection | Taxol | 62.3 |
According to above-mentioned experimental result, polypeptide of the invention has a variety of antitumor efficacies, can swell to a variety of Different Origins
Tumor cell proliferation inhibiting rate with higher reaches the inhibiting rate of melanoma, gastric cancer, liver cancer under effective administration concentration
60% or more；Suppression under effective administration concentration, to esophageal squamous cell carcinoma, non-small cell lung cancer, colon cancer, oral squamous cell carcinomas, breast cancer
Ratio of outflow is up to 50% or more.
The combination of Flow cytometry polypeptide and macrophage:
Take the highly expressed Human THP-1 cells strain of SIRP α as experiment macrophage.
The THP-1 cell for suspending and cultivating is taken, the PMA induction differentiation 12h that final concentration 100ng/mL is added is adherent, trypsase
Cell is collected after digestion, 1 × PBS is resuspended after washing twice and adjusts cell concentration to 107Cell is divided into 9 groups, respectively by/mL
For blank group, 2 groups of PE labeling polypeptide, 4 groups of PE labeling polypeptide, 6 groups of PE labeling polypeptide, 7 groups of PE labeling polypeptide, the anti-human SIRP α of mouse
2 groups of monoclonal antibody+PE labeling polypeptide, 4 groups of the anti-human SIRP α monoclonal antibody+PE labeling polypeptide of mouse, the anti-human SIRP α monoclonal antibody+PE labeling polypeptide 6 of mouse
Group, 7 groups of the anti-human SIRP α monoclonal antibody+PE labeling polypeptide of mouse, every group takes 100 μ L of cell suspension, then blank group that 20 μ LPBS are added and are incubated for
Cell 2.5h.
Addition 10 μ L of PE labeling polypeptide is protected from light after 10 μ LPBS, 37 DEG C of incubated cell 2h are added in PE labeling polypeptide each group
The final concentration of 1 μ g/ μ L of 30min, PE labeling polypeptide；
The 10 anti-human SIRP α monoclonal antibodies of μ L mouse (10 μ g/mL) are first added in SIRP α monoclonal antibody+PE labeling polypeptide each group, and 37 DEG C are incubated for carefully
10 μ L of PE labeling polypeptide is added after born of the same parents 2h and is protected from light 30min, the final concentration of 1 μ g/ μ L of PE labeling polypeptide；
Centrifugation (1000rmp/10min) 3 times after above-mentioned 9 groups of cells washs with 1 × PBS of pre-cooling, 1 is pre-chilled with 200 μ L ×
PBS is resuspended, and group of cells is detected respectively with flow cytometer.
Correlation statistical analysis is completed by SPSS10.0 software, and main method is one-way analysis of variance.If P < 0.05 is prompted
Difference is statistically significant, the result is shown in Figure 1~9.
After Fig. 1~5 are the results show that be individually incubated for PE labeling polypeptide, cell surface fluorescence intensity is dramatically increased, and shows that PE is marked
Note polypeptide is largely incorporated into cell surface, and Fig. 6~9 is shown, is incubated for PE label again after handling cell with SIRP α monoclonal antibody in advance
When polypeptide, fluorescence intensity shows that cell surface is less or combines without PE labeling polypeptide, SIRP α monoclonal antibody close to blank control group
It is identical as the binding site of polypeptide, therefore polypeptide of the present invention may specifically bind on the SIRP α of Macrophage Surface.
Macrophage and tumour cell co-culture experiments:
It takes the highly expressed Human THP-1 cells strain of SIRP α as experiment macrophage, takes the highly expressed MGC-803 of CD47
Cell is as tumour cell；
The THP-1 cell for the culture that suspends is collected, 1 × PBS is resuspended after washing twice and counts, and cell suspension is added in 24 orifice plates
And controlling THP-1 sum is 104/ hole, the PMA induction differentiation 12h that final concentration 100ng/mL is added is adherent, after addition harvest counts
MGC-803 cell, the ratio between total number of cells of THP-1 and MGC-803 are 1:15, while being separately added into SIRP α monoclonal antibody (final concentration
10 μ g/mL), 1~7 polypeptide of sequence (10 μ g/mL of final concentration), 1mLPBS is only added in blank control group, and tumour cell group is only added
The MGC-803 cell of equivalent, without any processing, above-mentioned every group sets three multiple holes.
37 DEG C, 5%CO2Under the conditions of after co-cultured cell 48h, mtt assay measures cell viability, and calculates tumor control rate:
Tumor control rate=(tumour cell group absorbance values-experimental group absorbance values)/tumour cell group is inhaled
Luminosity average value * 100%
It the results are shown in Table 17:
The influence that 17 polypeptide of table co-cultures macrophage and tumour cell
The above results show polypeptide of the present invention by blocking CD47 and SIRP- α in conjunction with the SIRP- α of Macrophage Surface
Signal path relieves tumour cell to the immunosuppressive action of macrophage, it is thin to tumour to have effectively promoted macrophage
The inhibiting effect of born of the same parents, so that the tumor control rate of each polypeptide is significantly higher than co-cultivation blank control group.
The experiment of chick chorioallantoic membrane Agiogenesis inhibition
Instar chicken embryo on the 11st is taken, gas chamber and the position of foetus are marked under ovoscopy lamp, and without one mark of picture at big blood vessel near the position of foetus
With the tincture of iodine, at ethanol disinfection plenum roof and mark, then bore an aperture in plenum roof, at the same with mill egg apparatus at mark general
Chorion grinds a slight crack parallel with the longitudinal axis, does not injure shell membrane.Ovum is laid flat, gently chorion at slight crack is removed, does not injure shell
The drop of sterile saline one is added dropwise on shell membrane in puncturing a crack on shell membrane but not injuring following chorioallantoic membrane in film.With
Rubber pacifier slowly sucks gas room air from the aperture of gas chamber end, causes gas chamber negative pressure, and visible physiological saline sinks at this time,
Chorioallantoic membrane is sunk, and artifical-air cell is formed between shell membrane and chorioallantoic membrane.
The shell membrane on artifical-air cell is opened, villus allantois (CAM) film at this is exposed, 0.2-0.5mL drug suspension is added dropwise
In in chorioallantoic membrane, after sample is added, with sterile transparent adhesive tape closed window, it is incubated for 3 days, observation in every 12 hours is primary, uses
Strile gauze dressing sealing.By chicken embryo accumbency, it is incubated in 37 degree, avoids stirring, in case artifical-air cell shifts.Harvest is taken out after 5 days.With
The tincture of iodine, ethanol disinfection inoculation position and surrounding tear chorion at closing off and gently pick up villus at aseptic nipper enlarged openings
Chorioallantoic membrane cuts the film of inoculating surfaces and surrounding with sterile scissors, and formaldehyde is fixed.
Take it is fixed after CAM film, ethanol dehydration, paraffin embedding, along in parallel with the direction CAM, serial section, 8 μm of thickness.
0.5% Toluidine blue staining.Sample is taken to count the cross section of capilary under 250 times of visuals field, respectively random 6 unduplicated height
Times visual field, takes its average value as follows as MVD (microvessel density) result under the unit area of the sample:
Agiogenesis inhibition rate=(blank group MVD value-experimental group MVD value)/blank group MVD value * 100%
18 angiogenesis of table inhibits the evaluation of pesticide effectiveness
It is new can also to significantly inhibit blood vessel the result shows that the present invention can not only block CD47 and SIRP- alpha signal access for table 11
Raw, effect is close or is better than VEGF monoclonal antibody (bevacizumab) group.
The above examples only illustrate the technical idea of the present invention, and this does not limit the scope of protection of the present invention, all
According to the technical idea provided by the invention, any changes made on the basis of the technical scheme each falls within the scope of the present invention
Within；The technology that the present invention is not directed to can be realized by the prior art.
SEQUENCE LISTING
<110>Nanjing Meng Meng Bacteria Co., Ltd.
<120>a kind of antineoplastic polypeptide and its application
<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 (9)
1. a kind of antineoplastic polypeptide, it is characterised in that: include two sections of amino acid sequences of CCNDEGLEMRIK and GDASLKMDK SDAV
Column, the amino acid sequence of polypeptide are CCNDEGLEMRIK-Linker-GDASL KMDKSDAV；Or GDASLKMDKSDAV-
Linker-CCNDEGLEMRIK；Or GDASLK MDKSDAVCCNDEGLEMRIK.
2. a kind of antineoplastic polypeptide according to claim 1, it is characterised in that: the amino acid sequence of the Linker is
DPTGG or G3S is repeated 1-2 times.
3. a kind of antineoplastic polypeptide according to claim 2, it is characterised in that: its amino acid sequence is (1)
CCNDEGLEMRIK-DPTGG-GDASLKMDKSDAV；Or (2) GDASLKMDKS DAV-DPTGG-CCNDEGLEMRIK；Or (3)
CCNDEGLEMRIK-GGGSGGGS-GDA SLKMDKSDAV；Or (4) GDASLKMDKSDAV-GGGSGGGS-
CCNDEGLEMRIK；Or (5) CCNDEGLEMRIK-GGGS-GDASLKMDKSDAV；Or (6) GDASLKMDK SDAV-GGGS-
CCNDEGLEMRIK or (7) GDASLKMDKSDAVCCNDEGLEMRI K.
4. a kind of synthetic method of antineoplastic polypeptide described in claim 1, it is characterised in that: the polypeptide is solid using Fmoc
Phase synthesi synthesis, comprises the following specific steps that:
(1) appropriate Fmoc-Ala (otBu)-Wang resin is weighed Solid-phase synthesis peptides pipe is added and DCM, swellable resins are added
30min；
(2) it is drained after being swollen, DMF washing is added and drains three times and again, liquid of raising one's hat is added；
(3) it is put into concussion reaction 20min in shaking table after sealing, 50mL DMF is added after draining every time and washs 5 times and drains；
(4) toner is added in the resin for taking 30g to handle well, and 1.5min is heated in 100 DEG C of water-baths, observes color；
(5) it tests after color passes through and carries out the condensation of second amino acid according to the amino acid sequence of polypeptide, inventory and resin rub
You drain, 50mL DMF is washed 5 times and drained than being 3:1 after 35 DEG C of reaction 60min；
(6) toner is added in the resin 30g after taking condensation, and 100 DEG C of heating 90s, resin is colorless and transparent, and solution is faint yellow to test color
Pass through；
(7) step (4)~(6) are repeated, until the last one amino acid condensation is completed, slough blocking group Fmoc, 50mL
DMF is respectively washed three times with 50mL DCM and 50mL anhydrous methanol again after washing 9 times, is drained, and is dried in vacuo up to dry peptide resin；
(8) take the dry peptide resin of 1g that 10mL cutting liquid is added, in room temperature confined reaction 2h, sand core funnel is filtered, and collects filtrate；
(9) filtrate is added in anhydrous ether, the volume ratio of filtrate and anhydrous ether is 1:10, and polypeptide filters after being precipitated, and is collected
Filter cake, 35 DEG C of vacuum drying 4h, obtains polypeptide crude product.
5. antineoplastic polypeptide application in preparation of anti-tumor drugs according to claim 1.
6. antineoplastic polypeptide application in preparation of anti-tumor drugs according to claim 5, it is characterised in that: described
For polypeptide for treating solid tumor, the solid tumor includes gastric cancer, esophageal squamous cell carcinoma, non-small cell lung cancer, colon cancer, liver cancer, black
Plain tumor, oral squamous cell carcinomas, breast cancer.
7. the application of resisting rheumatoid arthritis polypeptide according to claim 5, it is characterised in that: the resisting rheumatoid disease
Property arthritis drug include effective dose resisting rheumatoid arthritis polypeptide and other pharmaceutically acceptable auxiliary materials or carrier,
The pharmaceutical carrier includes organic and inorganic carrier, and oral and intestinal canal administration approach is suitble to that organic carrier, organic carrier packet is added
One of starch, lactose, calcium oxide stearate, vegetable oil, organic solubilized agent or a variety of are included, the organic solubilized agent includes
One of polyethylene glycol, glycerol, water-soluble organic and inorganic base are a variety of, and resisting rheumatoid arthritis drug is made into solid
Body form or liquid form, these various forms of resisting rheumatoid arthritis medicine preparations are sterilized after the completion.
8. antineoplastic polypeptide application in preparation of anti-tumor drugs according to claim 5, it is characterised in that: described
The dosage form of anti-tumor drug is tablet, injection, spray, capsule or coated pills, and the administration mode of the injection is quiet
Arteries and veins is subcutaneously or intramuscularly injected.
9. antineoplastic polypeptide application in preparation of anti-tumor drugs according to claim 5, it is characterised in that: described
The medicine-feeding part of anti-tumor drug includes Formulations for systemic administration and local administration, and Formulations for systemic administration is oral, intravenous injection or instils, is subcutaneous
Injection or intramuscular injection, local administration are injection in tumor tissues, the injection of tumour perienchyma.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111729074A (en) * | 2020-06-02 | 2020-10-02 | 遵义医科大学珠海校区 | Application of polypeptide RL25 in preparation of antitumor drugs |
| CN114344481A (en) * | 2022-01-11 | 2022-04-15 | 郑州大学 | Polypeptide self-assembly nano-drug and preparation method and application thereof |
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| CN102924578A (en) * | 2011-08-09 | 2013-02-13 | 哈药集团技术中心 | Anti-tumor polypeptide, preparation method and anti-tumor applications thereof |
| CN105713095A (en) * | 2016-03-14 | 2016-06-29 | 南京安吉生物科技有限公司 | Multifunctional fusion polypeptide and preparation method and application thereof |
| CN106117321A (en) * | 2016-06-29 | 2016-11-16 | 大连医科大学 | A kind of antineoplastic polypeptide and its preparation method and application |
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2019
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| WO2000059935A1 (en) * | 1999-04-05 | 2000-10-12 | Mayo Foundation For Medical Education And Research | Anti-apoptotic fusion polypeptide |
| US20060019887A1 (en) * | 1999-09-03 | 2006-01-26 | Amgen Inc. | Compositions and methods for the prevention or treatment of cancer and bone loss associated with cancer |
| CN102924578A (en) * | 2011-08-09 | 2013-02-13 | 哈药集团技术中心 | Anti-tumor polypeptide, preparation method and anti-tumor applications thereof |
| CN105713095A (en) * | 2016-03-14 | 2016-06-29 | 南京安吉生物科技有限公司 | Multifunctional fusion polypeptide and preparation method and application thereof |
| CN106117321A (en) * | 2016-06-29 | 2016-11-16 | 大连医科大学 | A kind of antineoplastic polypeptide and its preparation method and application |
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| CN111729074A (en) * | 2020-06-02 | 2020-10-02 | 遵义医科大学珠海校区 | Application of polypeptide RL25 in preparation of antitumor drugs |
| CN111729074B (en) * | 2020-06-02 | 2021-03-26 | 遵义医科大学珠海校区 | Application of polypeptide RL25 in preparation of antitumor drugs |
| CN114344481A (en) * | 2022-01-11 | 2022-04-15 | 郑州大学 | Polypeptide self-assembly nano-drug and preparation method and application thereof |
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