CN103191423A - Novel anti-breast-cancer Her2 vaccine with dendrimer as carrier - Google Patents

Abstract

The invention discloses a preparation method of a novel anti-breast-cancer polypeptide vaccine. The method is characterized in that antigen peptides such as HER2/neu 342-350 polypeptide and E75 polypeptide are connected to the surface of a dendrimer, and immunogenicity is improved by improving vaccine molecular weight; also, a Toll-like receptor (TLR) ligand highly efficiently inducing in-vivo immune response and an antigen stimulating CD4+Th cell proliferation (Th antigen) are both connected to the surface of the dendrimer, such that vaccine immunogenicity is further enhanced.

Description

The novel dendritic chemical compound 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 the surface that antigenic peptides such as HER2/neu 342-350 polypeptide and E75 polypeptide is connected to dendrimer (dendrimer), improves its immunogenicity by the molecular weight that improves vaccine; Simultaneously with the aglucon of immunoreactive Toll sample receptor (TLR) in can efficient inductor with stimulate CD4 ⁺The antigen of Th cell proliferation (Th antigen) is connected to the surface of dendrimer together, further strengthens the immunogenicity of vaccine.

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:

The invention discloses novel anti breast carcinoma polypeptide vaccine; it is characterized in that comprising an E75 polypeptide fragment that derives from HER2/neu (EGF-R ELISA); an effector T cell (Th; t helper cell) epitope; a TLR (toll-like receptor; toll like cell receptor) aglucon; with the cysteine that is connected this three component; the method of production of this vaccine is: 1. be substrate with the cysteine, the Oxidation by perchloric acid forms the cysteine of dimerization, adds tert-butyl acetate and amino acid whose carboxyl esterification; add Hexadecanoyl chloride; form acylation reaction at the amino place, the reduction disulfide bond also adds epoxy prapanol, forms the oxidation reaction of open loop oxygen at the sulfydryl place; add Hexadecanoyl chloride again; at two hydroxyl places acylation reaction taking place, remove the protecting group on the initial cysteine, just makes Pam ₃The Cys chemical compound; 2. 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; 3. last, the carboxyl on the Pam3Cys is connected by condensation reaction with amino on the E75 polypeptide.

The present invention compares with existing similar vaccine, novel structure, and synthetic method is original, is with a wide range of applications.

The specific embodiment:

Embodiment 1: the novel dendritic chemical compound is the anti-breast cancer Her2 vaccine of carrier

1. solid phase synthesis Th epitope, the Her2 antigenic peptides, and contain Pam ₃Polypeptide

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.Increase a cysteine but the N-that is noted that at Th polypeptide and Her2 antigen polypeptide is terminal, in order to utilize its sulphur atom to realize and being connected of dendrimer.And the synthetic polypeptide that contains Pam3Cys, the chemical compound (it is terminal to be free carboxylic acid) that will contain PamsCys is the same as common amino acid, connects in solid phase.

2. polypeptide and dendrimer compound is connected

The polypeptide that contains Pam3Cys, the Th polypeptide, the Her2 antigenic peptides is dissolved in the mixed solvent of PBS buffer/DMSO by a certain percentage, the dendrimer that adds G4 or G5 generation, add the SMCC coupling agent then, room temperature reaction spends the night, dialysis purification (semipermeable membrane by molecular weight 8K).

3. the sign that connects product.

By the sign that high performance liquid chromatography and the nuclear magnetic resonance analyser of analytical type are carried out gross product, be sure of being connected of each polypeptide and dendrimer.

Claims (5)

1. the method for production of a novel anti-breast cancer Her2 polypeptide vaccine, its feature comprises the surface that antigenic peptides such as HER2/neu 342-350 polypeptide and E75 polypeptide is connected to dendrimer (dendrimer), improves its immunogenicity by the molecular weight that improves vaccine; Simultaneously with the aglucon of immunoreactive Toll sample receptor (TLR) in the efficient inductor of energy and the antigen (Th antigen) of stimulation CD4+Th cell proliferation, be connected to the surface of dendrimer together, further strengthen the immunogenicity of vaccine, the method for production of this vaccine is: 1. synthesize and contain Pam ₃The aglucon of Toll sample receptor; be substrate with the cysteine, form the cysteine of dimerization by the Oxidation of perchloric acid, add tert-butyl acetate and amino acid whose carboxyl esterification; add Hexadecanoyl chloride; form acylation reaction at the amino place, the reduction disulfide bond also adds epoxy prapanol, forms the oxidation reaction of open loop oxygen at the sulfydryl place; add Hexadecanoyl chloride again; at two hydroxyl places acylation reaction taking place, remove the protecting group on the initial cysteine, just makes Pam ₃The Cys chemical compound; 2. by the synthetic polypeptide of solid phase method, comprise the antigenic peptides of Her2, antigenic peptides and the Pam of Th ₃Connect the preparation of polypeptide such as SKKKK at carbon teminal, and the end at each polypeptide increases a cysteine, be used for the usefulness that is connected with dendrimer, 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, repeated 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-, the ester bond between hydrolysis peptide chain and the solid phase carrier has just obtained synthetic good peptide; 3. the coupling of polypeptide and dendrimer is selected terminal dendrimer for the G4 of free amino group or G5 generation for use, by the SMCC coupling agent, at H ₂O/DMSO does under the solvent, with Pam ₃The antigenic peptides of polypeptide, Th polypeptide, Her2 etc. will be gone into by a certain percentage in reactant liquor, be connected on the surface of dendrimer, and product is by the dialysis purification.

2. dendrimer according to claim 1 is the preparation method of the vaccine of carrier, it is characterized in that: the Her2 polypeptide vaccine is the immunogenicity that improves it by dendrimer as carrier.

3. dendrimer according to claim 1 is the preparation method of the vaccine of carrier, it is characterized in that: this vaccine is also by connecting the aglucon of Toll sample receptor on dendrimer surface, and means such as Th antigen further improve the immunogenicity of vaccine.

4. dendrimer according to claim 1 is the preparation method of the vaccine of carrier, it is characterized in that: antigenic peptides, Th polypeptide or Pam3 polypeptide etc. by SMCC as bridging agent, water/DMSO as mixed solvent in, be connected on the dendrimer.

5. dendrimer according to claim 1 is the preparation method of the vaccine of carrier, it is characterized in that: antigenic peptides, Th polypeptide and Pam3 polypeptide shift to an earlier date mixing by a certain percentage, are connected to the dendrimer carrier then simultaneously.