CN102008719A

CN102008719A - Immunomicrosphere for overcoming B cell immunological tolerance and application thereof

Info

Publication number: CN102008719A
Application number: CN2010105712771A
Authority: CN
Inventors: 谈立松; 张尚权; 陈宇光
Original assignee: SHANGHAI WEIQIU BIOTECH CO Ltd
Current assignee: SHANGHAI WEIQIU BIOTECH CO Ltd
Priority date: 2010-12-02
Filing date: 2010-12-02
Publication date: 2011-04-13

Abstract

The invention relates to an immunomicrosphere for overcoming B cell immunological tolerance and application thereof. The immunomicrosphere comprises a microsphere medium, wherein the outside of the microsphere medium is coated with vaccine antigen, and the inside of the microsphere medium is coated with immune carrier protein for activating T cells. The invention also relates to application of the immunomicrosphere for overcoming the B cell immunological tolerance in preparing vaccines and immunological adjuvants. By using the technical scheme, the immunomicrosphere is combined with B cell receptor through the antigen and phagocytized by B cells, and the carrier protein is released and degraded in the cells; the immunomicrosphere is presented to Th cells through B cell MHC II to activate the TH cells to release cell factors, thereby promoting the activation and proliferation of the B cells; and high-titer antibody is generated through the affinity maturation process. The antigen protein and the immune carrier protein are respectively arranged on the surface of the microsphere and inside the microsphere, thereby preventing the antigen protein epitope and the Th cell activation epitope from competing with the B cell receptor.

Description

Overcome the immune microsphere and the application thereof of the tolerance of B cellular immunization

Technical field

The present invention relates to immune field, particularly a kind of immune microsphere and application thereof that overcomes the tolerance of B cellular immunization.

Background technology

Antigen (Ag) is meant and can combines with the TCR or the BCR of T, bone-marrow-derived lymphocyte, impels its propagation, differentiation, produces antibody or primed lymphocyte, and then the material of performance immunological effect.Antigenic sorting technique is a lot, need deny that the Th cell participates in when inducing antibody, can be divided into thymus dependent antigen (TD-Ag) and thymus independent antigen (TI-Ag).Most proteantigens all belong to TD-Ag as pathogenic microorganism, hemocyte, serum albumin etc.The inductive antibody production process of thymus dependent antigen needs the participation of T cell.The instruction that antibody produces is from two kinds of signals: the costimulating signal of antigen and B cell surface antigen receptors bind and helper T cell (Th).

The antigen protein epi-position combines with the B cell-membrane receptor and internalization takes place, and is first signal.Behind the B cytophagy immune microsphere in B intracellular (endosome) or lysosome (lysome) released antigen and by protease hydrolysis, wherein suitable segment combines with cell MHCII offers the cell to Th.Realize the Th cell activation and discharge cytokine impelling the B cell transformation to become the plasma cell of secretory antibody as secondary signal.

Vaccination is that the mankind resist and control one of effective means of infectious disease.Traditional vaccine mainly is a preventative vaccine; its application is the susceptible animal and the crowd of health population; its vaccine composition generally is the microorganism protective antigen; can in healthy human body, activate specific immune response; produce specific antibody and cytotoxic T cell (CTL); thereby obtain immunoprophylaxis ability, but can not produce efficient immune the individuality of having fallen ill to this pathogen.

Therapeutic vaccine (Therapeutic Vaccine) is the immunization therapy new ideas of setting up in recent years and growing up, and is meant to break immunologic tolerance in chronic infection person's body the vaccine that reconstruction or enhance immunity are replied.Therapeutic vaccine can be eliminated pathogen or abnormal cell in diseased individuals inducing specific immunne response, and disease is treated, and is the new treatment means of antiviral, antineoplastic.

Tumor is a treatment vaccine priority research areas.The antigen that is used to prepare vaccine has somatomedin (EGF, TGF, VEGF) and corresponding receptor (EGFR, Her2, VEGFR) tumor associated antigen CEA, MUC1 etc. to be used for treatment of solid tumor.CD20 is used for the vaccine of B cell lymphopathy.Idiotype is the another kind of B of being used for cell and T cell lymphocyte leukemia.

Merck ﹠ Co., Inc. carries out third phase clinical trial over against a kind of vaccine that is used for nonsmall-cell lung cancer (modal pulmonary carcinoma kind) treatment at present, and test plan finished in 2010.The MUC1 molecule that this vaccine is designed to bring out the cancerous cell surface produces a kind of immunoreation.1300 patients that participate in this test spread all over the world, comprising Brazil, China, India and these developing countries of Mexico.It is a global problem that this large-scale experimenter collects reflection pulmonary carcinoma.The first treatment of cancer EGF vaccine in the whole world is got permission listing in Cuba and is used for lung cancer therapy.

Switzerland medicine Sai Tuosi biotech company of group and two families of Britain Protherics company are all carrying out phase ii clinical trial to the hypertension vaccine.Be to be vaccine with the angiotensin, produce blocking antibody.

The France Neovacs Zagury of company etc. connects a kind of immunopotentiating agent with human tumour necrosis factor---keyhole-limpet hemocyanin (KLH).With this complex immunity inoculation transgenic mice, can protect mice to avoid the injury of duplicate injection tumor necrosis factor fully, be used for the rheumatoid disease treatment.Plan is in order to replace expensive monoclonal antibody and receptor therapeutic use.Vaccine at IgE is used for anaphylactic disease, may appears technological break-through suddenly.TGF is used for liver cirrhosis and arterial hypertension research.Therapeutic vaccine very likely duplicates even surmounts the splendidness in 10 years in the past of monoclonal antibody.

Above vaccine is an antigen with oneself protein in the human body all, and generations antibody is target, by the function of neutralization reaction sealing specific molecular, or ADCC effect and complement lethal effect realization therapeutic purposes by antibody

As from the foregoing, the present antigen that therapeutic vaccine adopted is selected the oneself protein of human body or animal usually for use, or claims endogenous protein.

Endogenous protein can not induced animal or the people produce high titre antibody.Be referred to as to call the tolerance of B cellular immunization.

In fact still the B cell at the oneself protein reaction still can produce on a small quantity.In secondary lymphocytic tissue, make B cell V region gene mutation because of somatic mutation mechanism.Can not produce and to be the helper T cell immunologic tolerance at the mechanism of oneself protein antibody.

Think that at present the main mechanism of keeping autoimmune tolerance status in the body is to remove in the T cell clone that central immune organ takes place, in periphery, induce self potential cell to enter incompetent (anergy) state of immunity, and the part lymphocyte is ignored (ignorance) to the immunity of oneself protein.The epi-position of endogenous protein can combine with the B cell, but can not induce the B cell to produce antibody

Existing technology is and exogenous strong antigen albumen, is referred to as to call the albumen chemical coupling of immune carrier (immunocarrier) on the immunology, introduces extrinsic protein and is used to activate the Th cell.Through carrier protein activation Th cell, just can induce the B cell to produce antibody.In immunne response, B cell recognition endogenous protein epitope, and offer the carrier protein epi-position and give the CD4+Th cell, Th cell recognition carrier protein epi-position, carrier just can couple together special T-B cell (T-B bridging) like this, and the T cell could activate the B cell.

Except with endogenous protein and immune carrier (immonocarrier) albumen Chemical Crosslinking Methods, also make up and contain the fusion rotein of immune carrier as heterologous molecule with technique for gene engineering.

Brought new problem thus: owing to B cell clonal deletion in growth course at intrinsic protein, with the abundance ratio of the bonded B cell of foreign vector albumen epi-position and the high several magnitude of abundance of the bonded B cell of endogenous protein epi-position, thus the problem that exists the immunity of crosslinking protein (or fusion rotein) intramolecularly albumen epi-position to compete.In vaccine entered human body or animal body, foreign vector albumen epi-position had competitive advantage, and B-cell receptor has higher probability in conjunction with foreign vector albumen epi-position and engulf, and produced at the proteic high titre antibody of foreign vector.And the corresponding antibodies of endogenous protein is suppressed, and can only produce low titre antibody, is difficult to realize that clinical treatment is worth.

The immune microsphere difference of reporting in the present invention and the document is: existing immune microsphere is that antigen absorption or chemical crosslinking are wrapped in microsphere inside in microsphere surface or with antigen.Function is slow releasing function or promotes macrophage phagocytic.Can not play and overcome B cellular immunization tolerance, also not be suitable for endogenous protein.

Summary of the invention

In order to address the above problem, to the invention provides a kind of immune microsphere, thereby overcome the immune race problem of epi-position in chemical crosslinking albumen in the prior art or the fusion protein molecule.

A kind of immune microsphere that overcomes the tolerance of B cellular immunization, it comprises the microsphere medium, and described microsphere medium external packets is covered with vaccine antigen, and described microsphere medium internal package is useful on the immune carrier protein of activating T cell.

The B cell epitope of described vaccine antigen is positioned at the surface of described immune microsphere, and the t cell epitope of described immune carrier protein is positioned at the inside of described immune microsphere, thereby realizes the physical isolation of B cell epitope and t cell epitope.

Described immune carrier protein is selected from: the tetanus virus albumen segment of chirp haemproteins, bovine serum albumin, ovalbumin, gene engineering expression, hepatitis B virus glutelin, meningococcal protein, HIV coat protein, cold virus albumen.

Described vaccine antigen is selected from: epidermal growth factor, angiogenesis factor, angiogenesis factor receptor, IgE, EGF-R ELISA EGFR and mutant, EGF-R ELISA tumor necrosis factor, transforming growth factor angiotensin, CD20.

Described microsphere medium is selected from: calcium alginate, polylactic acid, polylactic acid-glycollic acid, agarose, glucosan.

Described vaccine antigen is by conventional chemical method and the coupling of described microsphere medium.

Utilize difunctional cross-linking reagent to realize the coupling of amino-amino, carboxyl-amino, amino-Mercapto base.

Utilize glutaraldehyde reagent to realize amino with amino coupling.

The described application of immune microsphere on vaccine production that overcomes the tolerance of B cellular immunization.

The described application of immune microsphere on immunological adjuvant that overcomes the tolerance of B cellular immunization.

By technique scheme, immune microsphere provided by the invention is tied entirely by the B cytophagy by antigen and the thin receptor of B, and carrier protein discharges in cell, degrades; And offer cell to Th by B cell MHCII, activate the Th cell and discharge cytokine, thereby impel B cell activation, propagation; Produce high titre antibody through the affinity maturation process.By antigen protein and immune carrier protein are placed in microsphere surface and inside, avoided the competition of antigen protein epi-position and Th cell activation epi-position with B-cell receptor.

According to a preferred embodiment of the present invention, the microsphere that provides a kind of epidermal growth factor (EGF) to make up.

The EGF vaccine, this new drug with targeting character causes the concern of countries in the world the world of medicine already.This vaccine is by Cuba's molecular immunology center research and development, and clinical research confirmation is used for the treatment of nonsmall-cell lung cancer (NSCLC) treatment effectively.

This achievement in research transferred U.S. cancerVax company and carries out clinical research before 10 years.By the clinical trial of medicine, expectation will drop in following 2 to 3 years and use in the U.S..The report of EGF vaccine clinical progress has all been reported in ASCO (U.S.'s Clinical Oncology) conference for successive years.Late period, unresectable nonsmall-cell lung cancer patient was after accepting linearize treatment, was divided into two groups at random, a winding kind EGF vaccine, and another winding is subjected to best Supporting Therapy, and inoculation time is the 0th, 7,14,21,51 day, inoculation in every month afterwards 1 time.Result of study shows: test group patient improved 4～5 months than matched group patient total life cycle, and patient body quality is significantly improved, and resistance strengthens.The research of exploitation therapeutic vaccine will have the important in theory meaning, have huge economy and social benefit.

At present, Peru is carrying out the audit to medicine, estimating the end of the year can go on the market, in China, be responsible for this medicine clinical research and industrialization production by Beijing Biotech Pharmaceutical Co., Ltd., and carry out extensive clinical research in China in the end of the year 2008, further verify the curative effect of this medicine to the nonsmall-cell lung cancer patient.

It is property out of control growth that tumor cell is different from one of normal cell principal character, tumor cell itself can generate some somatomedin and promote cell unrestrictedly to grow, and EGF (epidermal growth factor) is the molecule of finding in recent years that plays an important role in cell proliferation and differentiation.The EGF vaccine uses the method for specificity active immunity just, is action target spot with EGF-EGFR (EGF-R ELISA) the system signal path of tumor tissues high expressed.After the patient inoculated this vaccine, stimulating immune system produced the antibody at EGF, and blocking-up is by the downstream signal pathway of EGF-EGFR mediation, thus inducing apoptosis of tumour cell, for the molecular targeted treatment of tumor provides the selection of upgrading.

In a preferred embodiment of the invention, be the material preparation microsphere with calcium alginate, be enclosed with the immune carrier protein (Immunocarrier) of bovine serum albumin (BSA) in the microsphere as activating T cell.Multiple the providing with chitosan in surface connects by antigenic amino.With the endogenous epidermal growth factor of mice (mEGF) is vaccine antigen, is coupled to microsphere surface by chemical method.Immune microsphere is got routine blood test ELISA method and is measured the EGF antibody titer through the tail vein injection immune mouse after 4 weeks.The result shows: the EGF antibody titer is greater than 8000; The subcutaneous immunity of conventional method, the EGF titre is less than 200.

Thereby proof can become the high titre antibody of complete antigen immunity acquisition without the mice oneself protein of chemical crosslinking.According to a preferred embodiment of the present invention, the microsphere of epidermal growth factor provided by the invention (EGF) structure provides experiment basis for EGF-microspheres vaccine treatment tumor.

According to a preferred embodiment of the present invention, the microsphere that provides a kind of angiogenesis factor (VEGF) to make up.

Tumor vessel is formed in tumor growth, invasion and attack and the transfer significant, do not bring enough oxygen and nutrition if there is new vessels to enter tumor for it, and transport metabolite, diameter of tumor will seldom surpass 2～3mm, because the blood vessel around only depending on is fully not enough to the tumor dispersion, tumor can only be in resting state or degenerate.The tumor tissues if in a single day blood vessel grows into, the mode that offers tumor tissues nutrition and oxygen is transformed into blood perfusion by disperse, and its metabolite can in time and up hill and dale be removed, and then tumor is exponential growth, shows the trend of unrestricted expansion.Growth of tumor and angiogenesis are complementary.The short angiogenesis factor that tumor cell produces can stimulating endothelial cell growth and existence, and the generation of blood vessel is not only and nutrition, oxygen is provided and transports metabolite, the factor that vascular endothelial cell also can provide growth to start to tumor.VEGF (VEGF) obtains preferably the incitant of tumor vessel formation qualitatively, and the function of blocking VEGF has become an important tool in the treatment of cancer.

In a preferred embodiment of the invention, be antigen with the thin somatomedin of blood vessel endothelium (mVEGF), with polylactic acid-glycollic acid (PLGA) preparation immune microsphere, be immune carrier protein with the chicken ovalbumin.Microsphere is multiple to provide coupling amino with chitosan.Glutaraldehyde method coupling VEGF.Immune condition is identical with the EGF microsphere.Get routine blood test ELISA method after 6 weeks and measure the mVEGF antibody titer.The result shows that the mVEGF antibody titer reaches 64000.Acquisition is than the better result of calcium alginate microsphere.Therefore, microsphere of the present invention suppresses to provide immune formulation for tumor vessel.

According to a preferred embodiment of the present invention, the microsphere that provides a kind of mice IgE C ε 3 polypeptide to make up.The general designation of the IgE mediation property disease allergic disease that to be a class caused by IgE because of its susceptible person often produces too much IgE antibody during allergen in contact environment, is gained the name with more than one allergic disease usually.After allergen enters body for the first time, the selective induction specific b cells produces the IgE antibody response, and free IgE can be under the situation of conjugated antigen not combines with mastocyte and basophilic granulocyte surface receptor by its Fc fragment and makes body be in the sensitization state in the serum.After body contacts allergen once more, multivalence allergen and two or more adjacent IgE antibodies of sensitized cell surface, it is crosslinked that the surface of cell membrane receptor is taken place, and triggers sensitized cell and take off granule, synthesize and the delivery of biologically active medium, thereby cause part or systemic anaphylaxis.IgE mediation property disease generally include atopic dermatitis (Atopic Dermatitis, AD), allergic rhinitis (Allergic Rhinitis, AR), (Asthma, AS) etc., severe symptom can cause anaphylactic shock to bronchial asthma, threat to life.The allergic disease sickness rate improves trend year by year, has seriously disturbed patient's work and life, and the burden on society that brings thus also is appreciable.Owing to the important function of IgE in the generation development link of allergic disease, become a new target spot of resisting allergic diseases treatment at present and studied focus.The molecular structure of IgE is clear, in the molecule epi-position of IgE antibody molecule and born of the same parents' maxicell receptors bind, is positioned at C ε 3 domains of antibody molecule Fc.Though FDA approval is gone on the market and demonstrated good effect in clinical practice at this regional monoclonal antibody, but, the costliness of cost and the inconvenience of using have limited its extensive use, therefore, turn to and seek the better application prospect is arranged by accepting this antibody protein passively at this proteic active immunity vaccine.Research and establishment can induce the therapeutic vaccine at IgE receptor binding site (C ε 3 domains) specificity neutralizing antibody, by on the antigen-antibody sealing IgE Fc fragment with the position of IgE Fc receptors bind, and then blocking-up is by the allergic disease effect approach of IgE mediation, for IgE mediation property treatment of diseases provides new approach.

In a preferred embodiment of the invention, the special polypeptide of C ε 3 domains that has synthesized mice IgE Fc with chemical synthesis process.Sequence is: GYGYQCIVDHPDFPKPIVRSITKTPGQR.This polypeptide corresponding monoclonal antibody is proved can block IgE and mastocyte receptors bind and effective to atopic disease therapeutics.Micro-sphere material is the PLGA/ chitosan, and the parcel chicken ovalbumin is as immune carrier.The glutaraldehyde method is with polypeptide coupling microsphere.Press the method immune mouse of example 1 and measure antibody.

The result shows: 6 week back mice IgE C ε, 3 domain polypeptide antibody titres are greater than 32000.Be better than at present commonly used promptly with BSA chemical crosslinking conventional scheme.Therefore, microsphere of the present invention provides the basis of immunization therapy for anaphylactic disease.

According to a preferred embodiment of the present invention, the microsphere that provides a kind of EGF-R ELISA III type mutant (EGFRvIII) to make up.EGF-R ELISA III mutant (EGFRvIII) is the modal a kind of depletion mutant of EGFR.For wild type E G F R albumen, thereby EGFRvIII has lacked 256 aminoacid at its extracellular fragment and has lost and the bonded function of part, but its born of the same parents' inner segment the composition phosphorylation also can take place and activates the downstream signal path under the bonded situation of discord part.Studies show that EGFRvIII only expresses on tumor cell, as cerebral glioma, breast carcinoma, ovarian cancer, carcinoma of prostate etc. are in the existence of normal cell detection less than EGFRvIII.The expression that experimental results show that EGFRvIII in the external body can promote cell transformation, tumor cell proliferation and invasion and attack, and closely related with the prognosis of tumour patient.Therefore EGFRvIII is the desirable target spot of a diagnosing tumor and treatment.

Antibody at EGFRvIII can be applied to clinical treatment, because normal structure is not expressed EGFRvIII, so the specificity of EGFRvIII antibody is stronger.Y10 is special a kind of Mus endogenous antibody at people EGFRvIII, can discern the tumour specific antigen of people and Mus simultaneously.In vitro study finds that YIO can suppress the synthetic and cell proliferation of tumor DNA, can produce complement or antibody-mediated cytotoxicity.Lumbar injection Y10 can make the B16 melanoma mouse model long term survival (n=20 of stably express EGFRvIII; P＜0.001).Y10 mechanism of action in vivo may depend on the Fc receptor.

Monoclonal antibody mAb806 prepares with the fibroblast NR6 immune mouse of expressing EGFRv m.The U87MG that mAb806 significantly suppresses to express EGFRvIII transplants the tumor growth of mice with tumor, is dosage and relies on mode; It also can suppress the tumor growth that EGFR crosses the A431 cell transplantation mice with tumor of expression simultaneously, there is not EGFRvIII in the normal structure, the tumor associated antigen epi-position that does not have EGFRvIII to contain in the relevant anti-normal structure of the tumor that is contained is so this epi-position can be used as a potential target of tumor vaccine.Reaction at the EGFRvIII epitope triggers comprises humoral immunization and cell immune response.After eimberger etc. use single peptide section coupling hemocyanin immune mouse, the subcutaneous vaccination tumor cell, mice 70% does not see tumor growth after the immunity as a result, tumor growth significantly is lower than matched group (P＜0.05).Immune mouse serum is transferred to tumor growth P＜0.05 that can stop 31% mice in the immunized mice body not), its mechanism may be relevant with antibody-mediated cytotoxicity.Ciesielski etc. are with a plurality of copies of EGFRvIII specific epitopes (LEEKK-GNYVVTDH), couple together the immune Fisher rat of multiple antigenic polypeptide (multiple antigenic peptide) that forms with lysine as bridge, and detect its cell immunoreceptor.The multiple antigenic polypeptide immune mainly produces special antitumor cell immunoreation at the F98 glioma cell of expressing the EGFRvIII target antigen.

In a preferred embodiment of the invention, at first synthesize people source EGFRVIII specific epitopes polypeptide.Sequence is: CGADSYEMEEDGVRKC.This polypeptide corresponding antibodies has confirmed oncotherapy effective.EGFRvIII polypeptide-PLGA microsphere makes up presses the enforcement of example 2 methods.Be connected in PLGA/ chitosan microball (containing BSA+OVA) as immune carrier by amino.Press the method immune mouse of example 1 and measure antibody.

The result shows: EGFRvIII specific epitopes polypeptide antibody titre after 6 weeks.Microspheres vaccine obtains good result, and serum antibody titer is greater than 32000.

According to a preferred embodiment of the present invention, the immune microsphere that provides a kind of EGF-R ELISA HER2/neu to make up.HER2/neu is second member of Epidermal Growth Factor Receptor Family.EGF-R ELISA (EGFR) family is also referred to as HER or ErbB2 family, plays a significant role in cell signalling, is the important moderator of cell growth, differentiation and survival.This family comprises EGFR (HERI or ErbBi), HER2/neu (neu or ErbB2), HER3 (ErbB3) and HER4 (tyro2 or Erb-B4), people HER2/neu gene mapping is in No. 17 the short arm of a chromosome, expression product is the strand transmembrane glycoprotein of molecular weight 185kDa, contain 1255 amino acid residues, cell inner segment (1CD) has tyrosine kinase activity.HER2/neu participates in the signal conduction by molecule in the various kinds of cell, its main downstream material comprises that mitogen activation is known has 9 kinds of parts directly to combine with EGFR, HER3 or HER4, EGFR family member single expression seldom in tissue. organize a performance but form difference, constitute homology or heterodimer, amplifying signal is transduceed and is brought into play biological action and unite more effective by the receptor complex that HER2/neu forms than other receptors.Because 1. the HER2 receptor complex has higher ligand binding capacity, and internalization rate mental retardation stops the longer time, the persistent period of prolongation receptor signal conduction on serous coat; 2. HER2 is a kind of very active tyrosine kinase, and himself sudden change and overexpression also can produce on the structure and activate.Generally, HER2/neu only expresses at fetal period, after growing up, can only find that in few tissue its low expression level is in cell surface with immunohistochemical staining.At human cancer, the molecular changes of HER2/neu is the overexpression of normal gene product normally.There is the HER2/neu overexpression in multiple human cancer,, because being positioned serous coat, HER2/neu albumen activates as breast carcinoma (25～30), ovarian cancer (25～32), adenocarcinoma of lung (30～35), constitutional renal cell carcinoma (30～40) etc. even medium level HER2 overexpression also can produce on the receptor structure.Immunohistochemical staining finds that the neighbour nearly normal breast epithelium of the HER2 protein level of breast cancer cell is high 10～100 times.Common HER2/neu amplification of human tumor and overexpression show that HER2/neu plays an important role in tumor takes place.The overexpression of HER2/neu increases transfer ability by starting multiple transfer related mechanism. comprise cell migration rate, external aggressivity, IV die cavity original enzyme activity, experimental lung metastasis etc.The HER2/rleu overexpression also influence some adhesion molecule such as epithelial cell cadherin (E-cadherin) thus etc. synthetic. promote to shift.HER2/neu overexpression breast cancer cell is insensitive to estrin treatment.HER2/neu overexpression tumor cell is also insensitive to some chemotherapeutics treatment.People such as Tsai find that in 20 kinds of non-small cell lung cancer cell systems former chemotherapy resistance and HER2/neu express significant correlation.The HER2 overexpression induces breast cancer cell to paclitaxel, Ramulus et folium taxi cuspidatae terpene drug resistance.HER2/neu albumen is the desirable target spot of cancer active immunity treatment.Because HER2/neu albumen is relevant with malignant transformation of cells, be in the cancer etiology a kind of biology associated protein; There is the HER2/neu specific antibody in some tumour patient, shows that the HER2/neu vaccine can react by induction of immunity; Proved passive immunization therapy such as HER2/neu antibody antitumor action, the prediction active immunity may also be effective.

In a preferred embodiment of the invention, at first synthetic people source HER2 specific epitopes peptide C QMWAPQWGPDC.Specific antibody at this polypeptide has been proved oncotherapy effective.It is agarose that immune microsphere makes up material.Microsphere contains BSA+OVA as immune carrier, and polypeptide antigen is connected in agarose/chitosan microball with glutaraldehyde by amino.

The result shows: the immune microsphere immune serum antibody titer that Her2 specific epitopes polypeptide makes up after 6 weeks is greater than 32000.Higher more than 5 times than conventional BSA-Her2 method.

Description of drawings

Fig. 1 is the immune microsphere simulation drawing, and wherein 1 represents immune carrier protein, 2 expression vaccine antigens.

Fig. 2 is EGF TPPA figure as a result in the microsphere immune serum that makes up of epidermal growth factor, and wherein abscissa is represented the serum dilution ratio, and vertical coordinate is represented the OD450 value, and the A in every group of block diagram represents experimental group, and B represents matched group, and C represents blank.

Fig. 3 is the West-blot testing result figure of the microsphere of epidermal growth factor (EGF) structure, and wherein 3 represent BSA, 4 expression EGF.

The specific embodiment

Further specify technical scheme of the present invention below in conjunction with the drawings and specific embodiments, but the present invention not only is confined to embodiment.

The silicon calcium alginate of embodiment 1 parcel bovine serum albumin/chitosan microball preparation

One, the silicon calcium alginate microsphere of parcel bovine serum albumin (BSA) preparation

Sodium alginate (sodium alginate) 2g is dissolved in the 90ml distilled water, adds bovine serum albumin (BSA) to whole dense 1mg/ml.0.5M NaOH transfers PH to 7.3, adds water and is settled to 100ml and is configured to 2% (m/v) solution.Calcium chloride (CaCl2) is mixed with 10% (m/v) solution.The atomizing of 2% sodium alginate soln 4ml air pump airbrush sprays in the 100ml calcium chloride solution 500 rev/mins of magnetic agitation balling-up.Magnetic agitation balance 1h, centrifugal collection microsphere, distilled water stirs balance 1h, centrifuge washing 3 times.

Microscope examination did size and microsphere homogeneity.Contrast judges that microsphere diameter is about the 5-8 micron, the microscopic examination favorable dispersibility according to the erythrocyte size.

The calcium alginate microsphere that the same procedure preparation does not contain BSA is used for controlled trial.The microsphere diameter size is to above-mentioned similar.The microscopic examination favorable dispersibility

Two, BSA assay in the microsphere

The 1ml microsphere is suspended in the phosphate buffer of 5ml, 0.1M, pH7.6.Calcium alginate microsphere molten Jie of disintegrating after two hours.BSA is released in the solution, measures the UV Absorption OD280 of BSA.The OD of concentration 1mg/ml ₂₈₀Value is 0.56.By this reckoning, BSA concentration is about 0.75mg/ml in the microsphere.

Three, the sodium alginate microcapsule preparation of cladding chitosan

The purpose of this step be the proteic outer surface of parcel introduce can coupling protein amino, overcome albumen with chitosan cladding microsphere surface simultaneously and from microsphere, spill

Chitosan (chitosan) MW:18000～20000 are got 0.5g and are dissolved in 1% acetum, are settled to 100ml, and final concentration is 0.5% (m/v).

The calcium alginate microsphere mixes with 0.5% chitosan solution, room temperature magnetic agitation 4 hours.Centrifugal collection microcapsule, agitator are broken up slow adding normal saline to suspension, centrifugal collection microcapsule, and washing repeats 5 times.

The microsphere dispersibility of cladding chitosan is slightly poor but satisfy requirement of experiment than calcium alginate microsphere.

Four, polysaccharide is quantitatively amino

1. trinitrobenzene sulfonate (TNBS) can produce orange red reaction with free amino in the chitosan, 335nm colorimetric behind the adding stop buffer.Press extinction coefficient (E335=14000) and calculate amino group concentration

2.TNBS (5%m/v) be diluted to 0.01% (m/v) working solution.Reaction buffer is the PH8.50.1M carbonic acid buffer.

3. the microsphere that does not contain BSA is used for amino mensuration, because of albumen amino develops the color simultaneously.Microsphere volume is quantitative: microsphere hangs normal saline, be with in the graduated centrifuge tube 1000 rev/mins centrifugal.Observe microsphere volume after centrifugal (below be referred to as to call hematocrit), being diluted to as required then needs concentration.The percentage concentration of following microsphere is the hematocrit percentage concentration

4,10% microsphere sample sampling 2ml adds 0.01% (m/v) TNBS 0.5ml, in 37 ℃ of water-bath 2h, adds 0.5ml 10%SDS and 0.25ml 1M hydrochloric acid again and stops.After 5 minutes in the 335nm colorimetric.Record OD335=0.75.

5, amino estimation (0.75 ÷ 14000 * 6.023 * 10 ²³) ÷ microsphere number/liter

Calcium alginate microsphere is near the erythrocyte size, and estimation is about 2,000 ten thousand/μ l, promptly by 2 * 10 ¹³/ rise calculating.Each microsphere is estimated nearly 150000 amino.The amino measuring and calculating of following microsphere is all with the method.

The polylactic acid-glycollic acid (PLGA) of example 2, parcel ovalbumin (OVA)/chitosan microball preparation

But the microsphere preparation manipulation list of references that is adopted in the present embodiment: the preparation of (1) medicine carrying microballoons, sign and the research of external slow release; Tsing-Hua University's papers written by postgraduates (2008) Wang Yiwei, Cui Fuzhai.(2) preparation of polylactic acid microsphere; Jilin University's journal (version of science) V ol.43N is 2005:842-846 o.6Nov.

One, polylactic-co-glycolic acid is purchased in SIGMA company (the composition ratio is 50: 50), and 100mg is dissolved in the 5mL organic solvent dichloromethane.Weigh 5mgOVA, dry powder uses the antibacterial dismembyator with OVA granule porphyrize.Be suspended in the organic solvent that contains PLGA.And slowly pour the aqueous phase that 50mL contains 1% polyvinyl alcohol (PVA) into, under the ice bath with 300W power ultrasonic emulsifying 30s.Subsequently emulsion is poured in the 150mL distilled water into normal pressure magnetic agitation 5h with the volatilization organic solvent, the centrifugal 10min of 15000r/min collects, deionized water wash 3 times, and lyophilization 48h removes moisture in the microsphere, in-20 ℃ of preservations.

Microscopically is observed microspherulite diameter, judges that according to vision particle diameter and micro-amplification the microsphere average grain diameter that repeats to prepare is 2.0～3.0 μ m.Favorable dispersibility

Two, microsphere parcel OVA assay

The microsphere that 1ml is used to measure is suspended from the 3ml dichloromethane, jolts gently to make the microsphere dissolving, adds the molten Jie OVA of phosphate buffer of 3ml0.1M, pH7.2.Centrifuging and taking upper strata water is measured OD280.1mg/ml is E2800.74.The result shows that OVA concentration is about 0.9mg/ml in the PLGA microsphere

Three, PLGA microsphere cladding chitosan.Operating condition such as example 1.TNBS colorimetric method for determining amino estimates that about Mei Ma microsphere has 30000 amino

Four, the PLGA microsphere operating condition of parcel BSA only replaces OVA albumen with BSA with last identical.

Five, the PLGA microsphere operating condition of parcel BSA+OVA replaces single albumen with last identical with BSA+OVA albumen.

The agarose microbeads preparation of example 3 parcel BSA

One, preparation method: low solidification temperature agarose is purchased the company in SIGMA.Agarose distilled water heat fused, concentration are 4%.Hold and place the water-bath of 45C degree.BSA is dissolved in the 0.05M PH7.2 phosphate buffer, and concentration is 0.2%.The equal-volume protein solution mixes with agarose makes ultimate density: agarose is 2%, and protein concentration is and 0.5%.

The microsphere preparation prepares with film breast method, but its concrete operations list of references: and the film emulsion process prepares the PLGA microsphere and the microcapsule of uniform particle diameter fast.Tian Rui, Wang Lianyan etc.Process engineering journal Vol.9No.4.In August, 2009.754-761。

It is that the agarose that will dissolve does not disperse to enter oil phase formation microsphere by ceramic membrane micropore (aperture 0.1-5 micron) at high pressure that the film emulsion process prepares microsphere.The oil phase composition is the edible soybean oil that contains 2%SPAN80.The ceramic membrane aperture is 0.5 micron, and the oil temperature keeps 45 degree during emulsifying.Obtain the proteic agarose microbeads that contains of 1-3 micron under the magnetic agitation.Make cool to room temperature that agarose microbeads is solidified after removing heating.Centrifugal collection microsphere.Microsphere with 25-75% progressively increase alcohol concentration at low temperatures (0-5 degree) washing remove oil phase composition and ankyrin.

Two, microsphere microscopy result: judge that according to vision particle diameter and micro-amplification the microsphere average grain diameter of preparation is 2.0-3.0 μ m.Favorable dispersibility.

Three, determining the protein quantity

The exsiccant microsphere of 100mg ethanol is suspended in the 5.0ml phosphate buffer (0.05M pH7.2).Placing in the refrigerator fully was released in the solution albumen in 48 hours.OD280 measures ovalbumin content.The result shows that the dried microsphere of every 100mg contains ovalbumin 2.5mg

Four, the result of Bao multiple chitosan

Method is identical with example 3, does the cladding amino of chitosan with not protein-contg microsphere and measures (albumen disturbs amino mensuration).Estimation Mei Ma microsphere (by 2.5 microns of mid diameters) contains amino of chitosan greater than 30000

Five, the agar microsphere operating condition of parcel OVAA only replaces BSA albumen with OVA with last identical.

Six, the agar microsphere operating condition of parcel BSA+OVA replaces single albumen with last identical with BSA+OVA albumen.

Example 4 microspheres are connected with antigen protein--and-glutaraldehyde method connects

One, with EGF is example explanation microsphere and antigenic coupling

Two, get the carbonic acid buffer (0.1M, pH8.5) that the centrifugal hematocrit 0.5ml of washed calcium alginate/chitosan microball is resuspended in 2.5ml.Add 2.5ml 0.5% glutaraldehyde, the centrifugal collection washing in mixed at room temperature 2h activation back 3 times.

Three, microsphere is suspended from the carbonic acid buffer of 3ml again.Add 500 microgram mices source mEGF (sigma company).Place on the blender refrigerator hybrid reaction after 4 hours, add ethanolamine 100 microlitres and seal remaining glutaraldehyde group.Then with normal saline washing three times.

Four, microsphere coupling EGF result:

Avidin (Avidin) the TMB chromogenic substrate working solution of anti-mice EGF antibody, horseradish peroxidase-labeled is purchased and is newly edified bio tech ltd in Wuhan.The EGF-calcium alginate microsphere is suspended in the test tube, adds Biotin-EGF antibody, HRP-avidin, TMB in succession by operating instruction.With the naked eye reach microscope and can be observed the apparent blue color of microsphere.

Example 5EGF microspheres vaccine animal test results

One. animal immune

1.20 only Kunming mouse is divided into 3 groups at random: 8 of experimental grouies, 8 of matched groups, 4 of blank groups.

2. immune microcapsule is resuspended with normal saline.The experimental group injection contains the microcapsule of BSA, the injection of 2% (v/v) microcapsule 0.1ml tail passages through which vital energy circulates; The subcutaneous multiple spot inoculation in 10% (v/v) 0.2ml back.The matched group injection does not contain the microcapsule of BSA, and method is the same.Blank group injecting normal saline, method is the same.

3. just exempt from back 14d once more immunity once, mice is put to death after 10 days, eyeball blood sampling, separation of serum.

Two .ELISA detect antibody

1.mEGF and BSA is respectively with 0.05M PH9.6 carbonic acid buffer preparation 20ug/ml coating buffer, the 0.1ml coated elisa plate that spends the night in every hole.

2.PBST wash 3 times, every hole does not have protein blocking liquid chamber temperature sealing 2h with 0.3ml.

3. wash each 0.1ml of mice serum that adds gradient dilution for 3 times respectively, hatch 2h for 37 ℃.

Add 1: 2000 every hole 0.1ml of the anti-Mus-HRP of rabbit, incubated at room 1h 4.PBST wash 5 times.Add the 0.1mlTMB colour developing after washing 5 times respectively, colour developing 15min respectively adds 50ul 1M sulphuric acid

Three, animal test results

The EGF TPPA the results are shown in Figure 2.EGF antibody average titer is 8000.

Four, West-blot detects

Do the SDS-PAGE electrophoresis 1.mEGF go up sample with 20ug respectively with BSA.

2.100mA film 1h is changeed in constant current.

3. do not have the protein blocking fluid-tight and close 2h.

4.1: 37 ℃ of 500 mice serums are in conjunction with 2h.

Add 1: 2000 anti-Mus-HRP of rabbit, incubated at room 1h 5.PBST wash 5 times.

6. wash 5 times, add the DAB colour developing

7. result such as Fig. 3, experimental mice produces anti-BSA and mEGF antibody.In BSA and the colour developing of EGF electrophoresis position.

The microsphere that embodiment 6 angiogenesis factors (VEGF) make up

One, Mus source vascular endothelial cell growth factor (mVEGF) is purchased the company in sigma.Example 2 is seen in the preparation of PLGA/ chitosan microball.MVEGF is connected with the glutaraldehyde method with the coupling of microsphere.See embodiment 4.

Two, coupling result identifies

Be used to identify the anti-mice VEGF of the fluorescein-labelled rabbit of different sulfur hydracid polyclonal antibody, purchase brilliant day biological Science and Technology Ltd. in Shanghai.

The by specification operation detects the mVEGF of microsphere surface with fluorescent-labeled antibody.Use the fluorescence microscope result.Visible microsphere is invented bright fluorescence under the fluorescence microscope visual field.

Experiment mice (the Kunming kind is from the Shanghai Experimental Animal Center) divides four groups, every group of seven mices

1,10%mVEGF-microsphere (the PLGA microsphere includes OVA) intravenous immune

2,10%mVEGF-microsphere (the blank microsphere of PLGA) intravenous immune

3, the 10 μ g mVEGF conventional subcutaneous injection immunity of Friend adjuvant

4, without any the negative serum contrast of the mice serum of handling

The low-speed centrifugal hematocrit is suspended among the 5ml PBS for the 0.5ml immune microsphere.Injection volume is a booster immunization after the 100 microlitres immune Later Zhou Dynasty, one of the Five Dynasties first time, strengthens once weekly later on.Pluck eyeball after six weeks and get blood.Conventional ELISA method is measured antibody titer in the serum.The results are shown in Table 1

Table 1VEGF microsphere immune serum TPPA (OD450 ± SD)

With mice VEGF is antigen-immunized animal, 1: 64000 titre of 6 week of immunity back acquisition.Satisfy the further technical foundation of research antitumor research.

The microsphere that embodiment 7 mice IgE C ε 3 peptides make up

One, embodiment 2 is seen in the preparation of the PLGA/ chitosan microball of parcel bovine serum albumin (BSA).

Two, mice IgE C ε 3 domain antigenic peptides are by following document chemosynthesis, its synthetic method list of references (Synthetic IgE peptide vaccine for immunotherapy of allergy Vaccine 21 (2003) 1580-1590Chang Yi Wang, Alan M.Walfield, Xinde Fang, Bruce Hammerberg, John Ye)

Mice IgE C ε 3 domain sequences are: GYGYQCIVDHPDFPKPIVRSITKTPGQR.

Three, C ε 3 peptides and BSA hand over sign authorized company to prepare with conventional glutaraldehyde method.According to company provide each BSA of data connect polypeptide 3.5 molecules,

The animal test results of the microsphere that embodiment 8 mice IgE C ε 3 make up

One, animal experiment grouping (seven every group) Kunming mouse is available from the Shanghai Experimental Animal Center

1, C ε 3 peptides-BSA, the subcutaneous immunity of Friend adjuvant

2, C ε 3 peptides-microsphere tail vein injection immunity,

3, the subcutaneous immunity of C ε 3 peptide Friend adjuvants

4, without the mice serum of any processing

Two, 0.5 milliliter of immune microsphere of immunity is suspended among the 5mlPBS.The tail vein injection amount is 100 microlitres

Immunity back two all backs booster immunizations are strengthened once weekly later on for the first time.Pluck eyeball after six weeks and get blood.

C ε 3 peptides-BSA 20 micrograms and Friend adjuvant mixing subcutaneous injection.Program is with last identical

C ε 3 peptides 20 micrograms are mixed the subcutaneous injection immunity with the Friend adjuvant.Program is with last identical

Three, antibody titer is measured: the ELISA conventional method

Conventional ELISA method is measured antibody titer body measurement result such as table 2 in the serum

Table 2IgE microsphere immune serum TPPA (OD450 ± SD)

The result shows: C ε 3 peptides-microsphere immune mouse antibody titer is greater than 30000.The immune microsphere effect is better than existing conventional method: peptide and BSA chemical crosslink technique are promptly high more than 10 times than C ε 3 peptides-BSA group titre.

Embodiment 9 EGF-R ELISA III type mutant (EGFRvIII) microspheres

One, the preparation of the PLGA/ chitosan microball of parcel bovine serum albumin (BSA+OVA) is with embodiment 2.

Two, with reference to the following document chemosynthesis EGFRvIII of authorized company epitope polypeptide

Sequence is: CGADSYEMEEDGVRKC

Its synthetic method list of references (United States Patent:Antibodies to EGF receptor epitope peptides Johns, et al.7,767,792August 3,2010)

Three, being connected of microsphere and antigen polypeptide, see example 4 with conventional glutaraldehyde method

Four, polypeptide and BSA coupling authorized company preparation.Provide each BSA of data to connect polypeptide according to company at 3.5 molecules.

The animal test results of embodiment 10 EGF-R ELISA III type mutant (EGFRvIII) microspheres

One, animal experiment grouping

28 Kunming mouses divide four groups, seven every group.Following vaccine and corresponding contrast immunity respectively.

1, PLGA microsphere-EGFR peptide 10% microsphere 100 microlitre tail vein injection immune mouses

2, EGFR peptide 20 μ g EGFR polypeptide mix subcutaneous multi-point injection with the Friend adjuvant

3, BSA-EGFR peptide 30 μ g BSA-EGFR peptides mix subcutaneous multi-point injection with the Friend adjuvant

4, the mice without any processing is a blank

Exempt from two weeks of tired back for the first time, booster immunization, totally six weeks once in a week later on.Last immunity is mice execution after back 10 days, eyeball blood sampling, separation of serum.

Two, antibody titer is measured: the ELISA conventional method

1, tested antigen is respectively with 0.05M PH9.6 carbonic acid buffer preparation 20ug/ml coating buffer, the 0.1ml coated elisa plate that spends the night in every hole.

2, the PBST washing is 3 times, and every hole does not have protein blocking liquid chamber temperature sealing 2h with 0.3ml.

3, washing adds each 0.1ml of mice serum of gradient dilution for 3 times respectively, hatches 2h for 37 ℃.

4, the PBST washing adds 1: 2000 every hole 0.1ml of the anti-Mus-HRP of rabbit, incubated at room 1h 5 times.Add the 0.1mlTMB colour developing after washing 5 times respectively, colour developing 15min respectively adds 50ul 1M sulphuric acid

Three, TPPA result such as table 3

Table 3EGFR microsphere immune serum TPPA (OD450 ± SD)

The result shows, immune microsphere-EGFRvIII peptide immune mouse, and serum antibody titer is greater than 30000.Higher more than 5 times than conventional BSA method.Provide experiment basis for further studying.

Example 11Her2 agar microspheres vaccine makes up and animal test results

One, antigen: reference literature synthesizes the Her2 epitope polypeptide.Sequence is: CQMWAPQWGPDC.But its synthetic method list of references (Generation of Peptide Mimics of the Epitope Recognized by Trastuzumab on the Oncogenic Protein Her-2/neu1.The Journal of Immunology, 2004,173:394-401.Angelika B.Riemer, Markus Klinger, Stefan Wagner, Astrid Bernhaus)

Two, microsphere: example 3 is seen in agarose/chitosan microball preparation

Three, be enclosed with BSA and OBV and pass through amino of chitosan idol sign Her2 peptide as the proteic agarose microbeads of Th

Polypeptide is connected with microsphere presses example 4 operations.

Four, polypeptide and BSA coupling conventional method glutaraldehyde method of attachment, authorized company's operation.Provide data according to company, each BSA molecule coupling polypeptide is 4.0

Five, animal experiment

1, microsphere-Her2 10% immune microsphere 0.1ml tail passages through which vital energy circulates injection

2, Her2 20 μ g Her2 peptides mix subcutaneous multi-point injection with the Friend adjuvant

3, BSA-Her2 30 μ g BSA-Her2 peptides mix subcutaneous multi-point injection with the Friend adjuvant

4, without any processing mice

Two all booster immunizations behind the initial immunity.Weekly immunity once, totally six weeks.Last immunity is mice execution after 10 days, eyeball blood sampling, separation of serum.

Six, ELISA detects antibody

Seven, experimental result: see Table 4

Table 4Her2 microsphere immune serum TPPA (OD450 ± SD)

The result shows that immune microsphere-Her2 peptide immune mouse obtains to be higher than 3200 titre antibody.Higher more than 5 times than conventional BSA-Her2 method.

Claims

1. one kind overcomes the immune microsphere that the B cellular immunization tolerates, and it comprises the microsphere medium, it is characterized in that, described microsphere medium external packets is covered with vaccine antigen, and described microsphere medium internal package is useful on the immune carrier protein of activating T cell.

2. the immune microsphere that overcomes the tolerance of B cellular immunization as claimed in claim 1, it is characterized in that, the B cell epitope of described vaccine antigen is positioned at the surface of described immune microsphere, the t cell epitope of described immune carrier protein is positioned at the inside of described immune microsphere, thereby realizes the physical isolation of B cell epitope and t cell epitope.

3. the immune microsphere that overcomes the tolerance of B cellular immunization as claimed in claim 1, it is characterized in that described immune carrier protein is selected from: the tetanus virus albumen segment of chirp haemproteins, bovine serum albumin, ovalbumin, gene engineering expression, hepatitis B virus glutelin, meningococcal protein, HIV coat protein, cold virus albumen.

4. the immune microsphere that overcomes the tolerance of B cellular immunization as claimed in claim 1, it is characterized in that described vaccine antigen is selected from: epidermal growth factor, angiogenesis factor, angiogenesis factor receptor, IgE, EGF-R ELISA EGFR and mutant, EGF-R ELISA tumor necrosis factor, transforming growth factor angiotensin, CD20.

5. the immune microsphere that overcomes the tolerance of B cellular immunization as claimed in claim 1 is characterized in that described microsphere medium is selected from: calcium alginate, polylactic acid, polylactic acid-glycollic acid, agarose, glucosan, albumen.

6. the immune microsphere that overcomes the tolerance of B cellular immunization as claimed in claim 1 is characterized in that described vaccine antigen is by conventional chemical method and the coupling of described microsphere medium.

7. the immune microsphere that overcomes the tolerance of B cellular immunization as claimed in claim 6 is characterized in that, utilizes difunctional cross-linking reagent to realize the coupling of amino-amino, carboxyl-amino, amino-Mercapto base.

8. the immune microsphere that overcomes the tolerance of B cellular immunization as claimed in claim 7 is characterized in that, utilizes glutaraldehyde reagent to realize amino with amino coupling.

9. as each described immune microsphere application on vaccine production that overcomes the tolerance of B cellular immunization among the claim 1-8.

10. as each described immune microsphere application on immunological adjuvant that overcomes the tolerance of B cellular immunization among the claim 1-8.