CN103191419A

CN103191419A - Novel anti-breast-cancer Her2 vaccine with degradable polymer as carrier

Info

Publication number: CN103191419A
Application number: CN 201210001163
Authority: CN
Inventors: 洪章勇; 尹芝南
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2012-01-04
Filing date: 2012-01-04
Publication date: 2013-07-10

Abstract

The invention discloses a preparation method of a novel anti-breast-cancer polypeptide vaccine. The method is characterized in that HER2/neu 342-350 polypeptide and E75 polypeptide are concentrated and encapsulated into nano-particles formed by a polylactic acid-glycolic acid copolymer, such that a nano-grade vaccine with a size of approximately 500nm to 2mum is formed. The vaccine can be highly efficiently taken by antigen-presenting cells (including macrophages and dendritic cells), such that Her2 antigen peptide immunogenicity can be substantially improved. According to the invention, Her2 antigen peptide is processed with the nano-grade technology, such that the Her2 antigen peptide is concentrated and encapsulated into the nano-particles formed by the degradable polymer PLGA. The nano-grade vaccine can be highly efficiently taken by antigen-presenting cells including macrophages and dendritic cells in bodies, and has high immunogenicity. Also, as an FDA-approved pharmaceutical excipient, the PLGA degradable polymer can be completely degraded in bodies, and has no toxicity. The nano-grade vaccine has the advantages of simple preparation, stable physical performance, high immunogenicity, and capability of simultaneously stimulating humoral immunity and cellular immunity. The vaccine is an ideal anti-breast-cancer Her2 vaccine.

Description

The novel degradable polymer is the anti-breast cancer Her2 vaccine of carrier

Affiliated technical field:

The invention discloses the method for production of novel anti breast carcinoma polypeptide vaccine, its feature comprises HER2/neu 342-350 polypeptide and E75 polypeptide capsule is reduced in the nano-particle that polylactic acid-glycolic guanidine-acetic acid copolymer forms, and forms the nano vaccine between about 500 nanometers to 2 of size micron.Such vaccine can be absorbed by antigen presenting cell (comprising macrophage and dendritic cell) efficiently, thereby can significantly improve the immunogenicity of her2 antigenic peptides.

Background technology:

Have every year 1200000 women that breast carcinoma takes place approximately, about 500,000 women die from breast carcinoma.North America, Northern Europe are the hotspots of breast carcinoma, and its sickness rate is about 4 times of Asia, Africa and Latin America country.Though China is the low area of sending out of breast carcinoma, its sickness rate rises year by year, and the growth rate of morbidity but exceeds national 1 to 2 percentage point occurred frequently, and especially Shanghai, capital, Tianjin and coastal area are the districts occurred frequently of China's breast carcinoma.More alarmingly be, with western countries relatively, China's patients with mastocarcinoma age of onset is lighter, the onset peak age is in 40-49 year, than the Zao 10-15 of west women.Based on the situation of sternness, the treatment of breast carcinoma has caused people's extensive concern with anti-recurrence.

The traditional therapy of breast carcinoma: 1. operative treatment: operative indication Halsted initiates radical operation of mastocarcinoma, and because performing the operation rationally, curative effect is clear and definite, becomes people in the last hundred years and treats the standard mode that breast carcinoma is followed.Since nearly half a century, breast carcinoma art formula has been carried out many explorations revised, total trend is not guarded outward and is enlarged two aspects, still debates endlessly so far.Breast local excision and full milk excision are the representativeness operations of conservative operation.2. radiotherapy: can improve the excision rate and make the inoperable patient of part obtain the surgical engine meeting again, because the vigor that radiation has suppressed tumor cell can reduce the postoperative recurrence rate and thereby the rate of transform improves survival rate and since radiation prolonged art before observing time have the case of the existing subclinical type metastasis of the part of making to avoid once unnecessary operation.3. chemotherapy: control as early as possible by chemotherapy section that cancerous cell that the micrometastasis kitchen range makes primary carcinoma and diffusion on every side thereof produces regression or part is killed to reduce postoperative recurrence and transfer, progressive stage breast carcinoma and the inflammatory type breast carcinoma limited before the enforcement art of operative treatment chemotherapy tumor is dwindled so that excision, can be according to chemotherapy effect before the tumor resection sample evaluation art select the reference of chemotherapy regimen during as postoperative or recurrence.4. the up-to-date Therapeutic Method of breast carcinoma: the tumor biotherapy technology is a kind of brand-new oncotherapy means of rising because of biotech development in recent years, be considered to the 4th kind of oncotherapy pattern except operation, chemotherapy, radiotherapy, have characteristics such as side effect is little, anti-tumor activity is high, indication is wide.It is the method for effecting a permanent cure that improves and change patient self body's immunity that biotechnology is used for oncotherapy, is considered to capture the final way of tumor, and it has a extensive future.After the raising of human tumor characterization of molecules caused tumor associated antigen to be identified by the T lymphocyte of human body, the development anti-tumor vaccine had become a kind of trend.The vaccine of tumor is by induced tumor patient's self immunologic surveillance and kills the tumor function, kill behind patient's postoperative and the chemicotherapy remaining tumor cell in the body effectively, reach the treatment tumor, the purpose of prevention of recurrence and transfer and final radical cure tumor, has high specificity, characteristics such as side effect is light just progressively become an important step in the combined therapy of tumour, also are focus and the developing direction of current oncotherapy basic research and clinical practice.

Summary of the invention:

A kind of method of production of novel anti-breast cancer Her2 polypeptide vaccine, its feature comprises antigenic peptides capsules such as HER2/neu 342-350 polypeptide and E75 polypeptide is reduced in the nano-particle that polylactic acid-glycolic guanidine-acetic acid copolymer forms, form the nano vaccine between about 500 nanometers to 2 of size micron, such vaccine can be absorbed by antigen presenting cell (comprising macrophage and dendritic cell) efficiently, thereby significantly improve the immunogenicity of her2 antigenic peptides, the method of production of this vaccine is: 1. by the synthetic HER2/neu 342-350 polypeptide of solid phase method and E75 polypeptide, concrete solid phase synthesis step is that chloromethyl polystyrene resin is as insoluble solid phase carrier, the aminoacid that at first an amino is closed group Fmoc-protection is covalently bound on solid phase carrier, under the effect of 20%pipe, the protecting group of desamidizate, first aminoacid has just been received on the solid phase carrier like this, DIEA provides the reaction environment of an alkalescence, HBTu is as condensing agent, second aminoacid is formed peptide bond with first the amino acid whose amino reaction that is connected on solid phase carrier again, on solid phase carrier, just generated a dipeptides that has protecting group like this, repeat above-mentioned peptide bond and form reaction, peptide chain is grown to the N end from the C end, until reaching needed peptide chain length, slough protecting group Fmoc-, ester bond between hydrolysis peptide chain and the solid phase carrier has just obtained synthetic good peptide; 2. the capsule contracting of polypeptide in the degradable polymer nano granule, get the aqueous solution (10mg/mlin PBS) that 0.6ml contains polypeptide, join in the dichloromethane solution of 4ml PLGA (15mg PLGA/ml dichloromethane), with the ultrasonic disruption instrument ultrasonic emulsification of sonde-type 40 seconds, cool off with ice bath in the emulsion process, then this emulsion being joined 60ml contains in the aqueous solution of polyvinyl alcohol (aqueous solution of 2.5wt%PVA), ultrasonic emulsification 2 minutes, stir then and spend the night, make the organic solvent volatilization clean, the 3000g low-speed centrifugal removed bulky grain in 25 minutes, and the 21000g ultracentrifugation was collected product in 25 minutes then, it is inferior to give a baby a bath on the third day after its birth, and lyophilizing obtains nano vaccine.

The specific embodiment:

Embodiment 1: the anti-breast cancer Her2 vaccine of synthesizing new dendrimer carrier

1. solid phase synthesis Her2 antigenic peptides

2.1. take by weighing resin in DCM (dichloromethane), argon is agitated 5min;

2.2. take by weighing Fmoc-Leu-OH 0.3mmol;

2.3.Fmoc-Leu-OH: DIEA=1: 2 (n) argon is agitated 90min;

2.4.DCM wash 5 times (inferior/min);

2.5. active DCM: DIEA: MeOH=3.4ml: the 0.2ml of sealing resin: the 0.4ml argon is agitated 10min;

2.6.DCM wash 5 times (inferior/min), DMF (dimethyl formamide) washing 5 times again (inferior/min), back 20%pipe30min;

2.7.20%pipe washing once, DMF washing 5 times is (inferior/as min), to be advisable to deviate from white Fmoc powder again;

2.8.Fmoc-Met-OH (0.3mmol): HBTu: DIEA=1: (n) argon was agitated 90min in 1: 2;

2.9.DMF wash 5 times (inferior/min);

2.10.20%pipe argon is agitated 30min;

2.11.20%pipe washing once, DMF washing 5 times (inferior/min); Add follow-up Fmoc-aminoacid successively, repeat the operation of 8-12, by the document polypeptide that is linked in sequence, use 95%TFA at last, 2.5%H2O, 2.5%TIS will synthesize good peptide section and solid-phase resin disengaging.

2. the capsule contracting of antigen polypeptide in degradable polymer PLGA nano-particle.

2.1 the PBS aqueous solution (10mg/ml in PBS) of configuration antigen polypeptide

2.2 the dichloromethane solution (15mg PLGA/ml dichloromethane) of configuration degradable polymer PLGA

2.3 get the aqueous solution that 0.6ml contains polypeptide, join in the dichloromethane solution of 4ml PLGA

2.4 with the ultrasonic disruption instrument ultrasonic emulsification of sonde-type 40 seconds, cool off with ice bath in the emulsion process.

2.5 being joined 60ml, this emulsion contains in the aqueous solution of polyvinyl alcohol (aqueous solution of 2.5wt%PVA) ultrasonic emulsification 2 minutes.

Spend the night 2.6 stir, make the organic solvent volatilization clean.

2.7 low-speed centrifugal removed bulky grain in 25 minutes under 3000g.

2.8 ultracentrifugation was collected product in 25 minutes under 21000g, it is inferior to give a baby a bath on the third day after its birth, and lyophilizing obtains nano vaccine.

Claims

1. the method for production of a novel anti-breast cancer Her2 polypeptide vaccine, its feature comprises antigenic peptides capsules such as HER2/neu 342-350 polypeptide and E75 polypeptide is reduced in the nano-particle that polylactic acid-glycolic guanidine-acetic acid copolymer forms, form the nano vaccine between about 500 nanometers to 2 of size micron, such vaccine can be absorbed by antigen presenting cell (comprising macrophage and dendritic cell) efficiently, thereby significantly improve the immunogenicity of her2 antigenic peptides, the method of production of this vaccine is: 1. by the synthetic HER2/neu 342-350 polypeptide of solid phase method and E75 polypeptide, concrete solid phase synthesis step is that chloromethyl polystyrene resin is as insoluble solid phase carrier, the aminoacid that at first an amino is closed group Fmoc-protection is covalently bound on solid phase carrier, under the effect of 20%pipe, the protecting group of desamidizate, first aminoacid has just been received on the solid phase carrier like this, DIEA provides the reaction environment of an alkalescence, HBTu is as condensing agent, second aminoacid is formed peptide bond with first the amino acid whose amino reaction that is connected on solid phase carrier again, on solid phase carrier, just generated a dipeptides that has protecting group like this, repeat above-mentioned peptide bond and form reaction, peptide chain is grown to the N end from the C end, until reaching needed peptide chain length, slough protecting group Fmoc-, ester bond between hydrolysis peptide chain and the solid phase carrier has just obtained synthetic good peptide; 2. the capsule contracting of polypeptide in the degradable polymer nano granule, get the aqueous solution (10mg/ml in PBS) that 0.6ml contains polypeptide, join in the dichloromethane solution of 4ml PLGA (15mg PLGA/ml dichloromethane), with the ultrasonic disruption instrument ultrasonic emulsification of sonde-type 40 seconds, cool off with ice bath in the emulsion process, then this emulsion being joined 60ml contains in the aqueous solution of polyvinyl alcohol (aqueous solution of 2.5wt%PVA), ultrasonic emulsification 2 minutes, stir then and spend the night, make the organic solvent volatilization clean, the 3000g low-speed centrifugal removed bulky grain in 25 minutes, and the 21000g ultracentrifugation was collected product in 25 minutes then, it is inferior to give a baby a bath on the third day after its birth, and lyophilizing obtains nano vaccine.

2. the preparation method of the anti-breast cancer nano vaccine of PLGA peptide capsule according to claim 1, it is characterized in that: Her2 polypeptide capsule is reduced among the degradable polymer PLGA, make the nano vaccine of particle diameter between 500 nanometers to 2 micron, can significantly improve the immunogenicity of antigenic peptides.

3. the preparation method of the anti-breast cancer nano vaccine of PLGA peptide capsule according to claim 1, it is characterized in that: the antigenic peptides on the employed Her2 albumen comprises HER2/neu 342-350 polypeptide and E75 polypeptide.

4. the preparation method of the anti-breast cancer nano vaccine of PLGA peptide capsule according to claim 1, it is characterized in that: in the Her2 antigenic peptides nano vaccine preparation process, the mode capsule of synthetic Her2 antigenic peptides well by two emulsifyings is reduced among the degradable polymer PLGA.