CN1498658A - Preparation of high-performance noncellular vaccine and usage - Google Patents
Preparation of high-performance noncellular vaccine and usage Download PDFInfo
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- CN1498658A CN1498658A CNA021450226A CN02145022A CN1498658A CN 1498658 A CN1498658 A CN 1498658A CN A021450226 A CNA021450226 A CN A021450226A CN 02145022 A CN02145022 A CN 02145022A CN 1498658 A CN1498658 A CN 1498658A
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Abstract
A novel vaccine for preventing and treating tumor and injectious diseases is a thermally induced exolapaxis of tumor cell or pathogen and its infectious cell, which is rich in antigen moleculae, MHC moleculae, various chemotactic factors, adhesion moleculae, coirritation moleculae and heat shock protein. Its advantages are high specificity and curative effect.
Description
Technical field
The present invention relates to biology and medical domain, relate more specifically to a kind of preparation method and the application of such vaccine in all kinds of tumors and infectious disease prevention and treatment of new and effective vaccine.
Background technology
The initiative immunization therapy of tumor and infectious disease is the effective means of treatment and prophylaxis of tumours or infectious disease, since the mankind adopt inoculation cowpox prevention variola, human at infectious disease prevention and control on obtained very big progress, infectious disease such as variola, poliomyelitis have been eliminated substantially in China at present, but aspect the treatment of other major diseases,, still lack effective means as the prevention and the treatment aspect of diseases such as tumor and acquired immune deficiency syndrome (AIDS), viral hepatitis.
The immunization therapy of tumor is the new treatment pattern of after operation, radiotherapy, chemotherapy, and the key link of immunotherapy of tumors induces body to produce specificity and nonspecific antineoplastic immune exactly.Adopt tumor vaccine to remove the antineoplastic immune of inducing specific, for suppressing growth of tumor and shift even more importantly, although more known tumor antigens at present, for most of tumors, tumor antigen is still unknown.Tumor vaccine at present commonly used comprises the dendritic cell-based tumor vaccines, recombinant vaccine of the lysate that adopts full cell tumor vaccine, tumor cell, tumor antigen sensitization etc., has obtained certain curative effect, but has also obviously existed not enough.
1. tumor associated antigen peptide vaccine.The mankind have obtained four class tumor associated antigens at present, and (1) tumor specific antigen comprises MAGE-1, MAGE-3, GAGE, RAGE etc., and this class antigen is not expressed except testis and Placenta Hominis in normal structure, only express in tumor cell; (2) tumor differentiation antigen comprises MART-1, gp-100, tyrosine kinase related protein-1, is distributed in melanocytoma; (3) the mutant gene encoding proteins is extensive as these gene distribution such as MUM-1, but sudden change and become tumor antigen in tumor tissues; (4) at a class antigen of tumor tissues high expressed, as HER2-neu etc.Adopt the mode of synthetic antigenic peptide or gene engineering expression and dna vaccination to prepare such vaccine at present.
2. dendritic cell-based tumor vaccines.Dendritic cell is a sole duty antigen presenting cell the strongest in the body, be the perpetrator of anti-tumor immune response, adopt tumor cell lysate or tumor associated antigen peptide sensitization dendritic cell to feed back in the body, immunne response that can inducing antigen-specific, such therapy has entered the clinical I-III phase and has tested at present
3. anti-idiotype antibody vaccine.Anti-idiotype antibody can imitate albumen and antigenic peptides, people can use anti-idiotype antibody and induce specific immunoreation at tumor associated antigen in part test, and the advantage of such vaccine is to produce the immunne response stronger than antigenic peptides.
4. the tumor vaccine of genetic modification.Adopting virus or genetic modification tumor cell is the more class tumor vaccine of research as tumor vaccine, mainly is the immunogenicity that adopts cytokine gene, costimulatory molecules etc., wishes to improve tumor cell, and inducing antitumor immunity is replied.
Yet, at present all there is obvious deficiency in above 4 kinds of tumor vaccines, and many tumor antigens are indeterminate, CTL that can't inducing tumor-specific, and also there are factors such as manipulation in vitro complexity, difficult quality are controlled, curative effect is limited in the tumor cell tumor vaccine of employing dendritic cell-based tumor vaccines and genetic modification.
Therefore, this area presses for the new good effect of exploitation, new tumor vaccine easy and simple to handle.
Summary of the invention
Purpose of the present invention just provides a kind of vaccine composition that is used for tumor and infectious disease treatment and prevention efficiently, and this vaccine composition is to adopt the efflux body of cell after thermal induction;
Another object of the present invention provides the method for making and the purposes of described efflux body.
In a first aspect of the present invention, a kind of efflux body is provided, described efflux body be by the secretion of the generation of cell after thermal induction to extracellular vesicle, wherein said cell is selected from down group: tumor cell, pathogen and by the cell of pathogenic infection;
Described vesicle has following feature:
(a) has the vesicle structure that diameter is 50-100nm;
(b) contain the immune molecule that is selected from down group: tumor antigen or pathogen antigen, MHC molecule, adhesion molecule and costimulatory molecules;
(c) contain the chemotactic factor that is selected from down group: MIP-1 α, MIP-1 β, MIP-3 β, MCP-1 and IP-10;
(d) contain the heat shock protein molecule.
In a preference, described efflux body external can chemotactic and immune cell activated, and inducing antigen-specific and nonspecific immune response in vivo.
In another preference, efflux body of the present invention prepares with the following method:
(1) with cell at 43 ± 3 ℃ of following thermal induction 0.5-10 hours, under 37 ± 2 ℃, hatched 2-24 hour then, the collecting cell culture supernatant, wherein said cell is selected from down group: tumor cell, pathogen and by the cell of pathogenic infection;
(2) 300-500 * g low-speed centrifugal is removed cell, collects supernatant;
(3) centrifugal removal cell debris of 1000-3000 * g middling speed and organelle are collected supernatant;
(4) 7000-12000 * g high speed centrifugation is removed minicell device and impurity, collects supernatant;
(5) 80000-120000 * centrifugal 0.5-5 of g ultrahigh speed hour, collecting precipitation was efflux body.
In another preference, described cell is a tumor cell, especially people's tumor cell.
In another preference, described efflux body contains tumor antigen or pathogen antigen, MHC molecule, adhesion molecule, costimulatory molecules, MIP-1 α, MIP-1 β, MIP-3 β, MCP-1IP-10 and heat shock protein molecule simultaneously.
In a second aspect of the present invention, a kind of pharmaceutical composition is provided, contain the efflux body of the present invention and the pharmaceutically acceptable carrier of effective dose.Pharmaceutical composition of the present invention can be curative or preventative.In a preference, it is a vaccine.
In a third aspect of the present invention, a kind of method for preparing efflux body is provided, may further comprise the steps:
(1) with cell at 43 ± 3 ℃ of following thermal induction 0.5-10 hours, under 37 ± 2 ℃, hatched 2-24 hour then, the collecting cell culture supernatant, wherein said cell is selected from down group: tumor cell, pathogen and by the cell of pathogenic infection;
(2) 300-500 * g low-speed centrifugal is removed cell, collects supernatant;
(3) centrifugal removal cell debris of 1000-3000 * g middling speed and organelle are collected supernatant;
(4) 7000-12000 * g high speed centrifugation is removed minicell device and impurity, collects supernatant;
(5) 80000-120000 * centrifugal 0.5-5 of g ultrahigh speed hour, collecting precipitation was efflux body.
In a preference, the condition of each step is as follows:
(1) places 41-45 ℃ of thermal induction about 1 ± 0.5 hour, hatched the collecting cell culture supernatant 4-8 hour at 37 ℃ then;
(2) the centrifugal 5-10 of 300-500 * g minute, collect supernatant;
(3) 1000-3000 * g is centrifugal 20 ± 10 minutes, collects supernatant;
(4) 7,000-12, centrifugal 30 ± 15 minutes of 0000 * g collects supernatant;
(5) 80,000-120, centrifugal 1 ± 0.5 hour of 000 * g, collecting precipitation, PBS washing, efflux body after the acquisition thermal induction.
In a fourth aspect of the present invention, the purposes of efflux body of the present invention is provided, it is used to preparation prevention or treatment tumor and infectious disease medicament.
In a fifth aspect of the present invention, the method for a kind of prevention or treatment tumor and infectious disease is provided, this method comprises, uses the thermal induction efflux body of the present invention of safe and effective amount to individuality.
Description of drawings
Fig. 1 has shown the electron microscopic observation of efficient vaccine structure of the present invention.
Fig. 2 is the evaluation of efficient vaccine of the present invention.
Fig. 3 has shown efficient vaccine expression chemotactic factor, MHC molecule and heat shock protein of the present invention.
Fig. 4 has shown the chemotaxis of efficient vaccine of the present invention to dendritic cell and T cell.
Fig. 5 has shown that efficient vaccine of the present invention stimulates the T cell or strengthens dendritic cell stimulation T cell proliferation test.
Fig. 6 has shown the inhibitory action of efficient vaccine of the present invention to tumor.
The specific embodiment
The inventor is through extensive and deep research, find that the efflux body (exosome) of cell after thermal induction is a kind of new tumor and infectious disease efficient vaccine of can be used for, can solve the deficiency that disease antigen is indeterminate, be difficult to the inducing specific immunne response well, simultaneously can obviously improve curative effect, have easy, the characteristics such as specificity is high, induce immune response is strong, good effect of preparation.Finished the present invention on this basis.
Efflux body is that the diameter that cell discharges is the small film vesicle of 50-100nm, and sophisticated erythrocyte is discharged extracellular, for example TfR (TfR) or acetylcholine esterase etc. with the form of efflux body with the unwanted serous coat albumen of function institute.By the further investigation of Electronic Speculum, confirm that efflux body is not formed by the direct blastogenesis of cell membrane, be called many vesicles body (multivesicular bodies, the structure that forms by endocytosis process MVBs) but derive from.This vesicles appears in the MVBs inner chamber, is likely the film blastogenesis by the MVBs medial surface.By the direct fusion of MVBs and endochylema film, these vesicles are released in the extracellular substrate, promptly are called as efflux body at last.The discovered in recent years efflux body is not merely by the unique secretion of reticulocyte of final period differentiation, and the someone has confirmed that the bone-marrow-derived lymphocyte of Epstein-Barr virus transfection can secrete same this vesicles.In bone-marrow-derived lymphocyte and antigen presenting cell, comprise MVBs this participation endocytosis process structure just polypeptide in conjunction with the position of mhc class ii molecule.Epstein-Barr virus source contain polypeptide mhc class ii molecular complex, and it directly can be and pass CD4
+The T lymphocyte.Zitvogel etc. have reported that DC equally also can secrete efflux body, and the efflux body in this DC source contains MHCI class and mhc class ii molecule.When the polypeptide with the tumor cell source impacts the efflux body that obtains behind the DC, can induce the anti tumor immune response of CTL mediation, cause the tumor growth of tumor-bearing mice obviously to suppress.Recently, the tumor cell that the Zitvogel laboratory has been reported people and mice again all can the continuous release efflux body, passs DC and produces significant CD8 thereby the efflux body in these tumor cells sources contains MHCI quasi-molecule and LAMP1 and tumor antigen can be
+The anti tumor immune response that the T cell relies on, and this immunological effect has the cross-fire treatment effect to the tumor-bearing mice of homologous and different system, and the efflux body that the prompting tumor is originated may be closely related with tumour immunity.Efflux body can be used as the preparation and the tumor vaccine of oncotherapy, and mediation is at the tumour immunity protective effect of homology mice and non-homology mice.
In the present invention, compare with the efflux body (EXO) that discharges without the tumor cell of heat shock, the change of matter has taken place in tumor cell " efflux body after heat shock (HS EXO) ", contains the chemotactic factor of multiple high concentration.Chemotactic factor is the cellular products that leukocyte is had the small molecule of chemotaxis as a superfamily in the cytokine.Chemotactic factor and receptor thereof be in inflammatory reaction, immunne response, and infection all plays very critical effect in the antitumor.But multiple chemotactic factor chemotactic immunologically competent cell and inhibition angiogenesis are resisted growth of tumor and transfer.
Thermal induction efflux body of the present invention has fundamental characteristics and self unique biological characteristic of general efflux body:
(1) be the vesicle spline structure of 50-100nm, size is homogeneous relatively, structural integrity;
(2) contain heat shock protein molecules such as antigen (comprise tumor antigen or derived from the antigen of microorganism and other infection cells), MHCI/II quasi-molecule, costimulatory molecules, TfR, LAMP-1 and HSC70;
(3) contain multiple chemotactic factor, as MIP-1 α (CCL3), MIP-1 β (CCL4), MIP-3 β (CCL19), MCP-1 (CCL2) and IP-10 (CXCL10);
(4) can chemotactic, activation dendritic cell and T cell.
Experiment confirm of the present invention, significantly chemotactic DC and T cell of efflux body after the heat shock, chemotactic factor can be brought into play chemotactic activity in the prompting efflux body.Contain chaperone HSP70 that high-caliber MHCI class and II quasi-molecule and multiple costimulatory molecules, adhesion molecule, tumor antigen offer etc. among the HS EXO, and the most of immune molecule that is contained among the HS EXO compares remarkable rise with EXO, and prompting HS EXO has stronger immunologic competence than EXO.With HS EXO and EXO treatment tumor, find that HS EXO has better oncotherapy effect, induce the reaction of stronger CTL and specificity antineoplastic immunity., can protect homology and non-homology lotus tumor host to avoid the attack of tumor cell better, and the established tumor of lotus tumor host is also had the obvious treatment effect as vaccine with HS EXO.The 4h tumor area promptly has the infiltration of DC behind injection HS EXO, to 24h, promptly reach many levels, the chemotactic factor that can utilize wherein after the injection in the prompting HS EXO tumor body to be contained attracts DC to arrive local the gathering rapidly, adhere to by its surperficial adhesion molecule and DC, then in the presence of costimulatory molecules, rely on the entrained endogenous oncopeptide of self HSP albumen to give DC with tumor specific antigen and the total angtigen presentation of tumor, then impel the ripe of DC and return lymph node, start the specific immune response reaction.Injection can be raised CD4 in the tumor body of HS EXO
+And CD8
+Thereby the T lymphocyte form effective anti tumor immune response in the part.
The present invention also provides the preparation method of this new and effective vaccine, and it mainly comprises step:
(a) thermal induction a period of time under the thermal induction temperature, recovery then.For example, cell at 43 ± 3 ℃ of following thermal induction 0.5-10 hours, was hatched under 37 ± 2 ℃ 2-24 hour then, the collecting cell culture supernatant, wherein said cell is selected from down group: tumor cell, pathogen and by the cell of pathogenic infection;
(b) separate acquisition thermal induction efflux body.Preferably, this separation comprises step: (i) low-speed centrifugal is removed cell,, (ii) the centrifugal removal cell debris of middling speed and organelle, (iii) high speed centrifugation is removed minicell device and impurity and (iv) ultrahigh speed is centrifugal, collecting precipitation (being efflux body).The actual conditions that should be understood that these steps can be protected to some extent, as long as isolate efflux body.A kind of preferred separation method condition is
(i) low-speed centrifugal, 300-500 * g low-speed centrifugal is removed cell, collects supernatant;
(ii) centrifugal removal cell debris of 1000-3000 * g middling speed and organelle are collected supernatant;
(iii) 7000-12000 * g high speed centrifugation is removed minicell device and impurity, collects supernatant;
(iv) 80000-120000 * centrifugal 0.5-5 of g ultrahigh speed hour, collecting precipitation was efflux body.
In a preference, the condition of each step is as follows:
(1) cell that will prepare efflux body is placed in 41-45 ℃ thermal induction and hatched 4-8 hour the collecting cell culture supernatant then about 1 hour in 37 ℃;
The centrifugal 5-10 of (2) 300 * g minute, collect supernatant;
Centrifugal 20 minutes of (3) 1200 * g collect supernatant;
(4) 10, centrifugal 30 minutes of 000 * g collects supernatant;
(5) 10, centrifugal 1 hour of 0000 * g, collecting precipitation, PBS are washed 2 times, efflux body after the acquisition thermal induction.
The present invention also provides a kind of pharmaceutical composition or immune composition.In described compositions, contain the thermal induction efflux body of the present invention of pharmaceutically acceptable carrier (comprising diluent, excipient etc.) and effective dose.The quantity of thermal induction efflux body is generally 10 micrograms-100 milligram/agent, preferably is 100-1000 microgram/agent.
Term used herein " effective dose " refers to the therapeutic agent treatment, alleviates or prevent the amount of target disease or situation, or shows the amount of detectable treatment or preventive effect.Depend on the nature and extent of the build of this object and health status, disease and the therapeutic agent selecting to give and/or the combination of therapeutic agent for the accurate effective dose of a certain object.Therefore, specifying accurately in advance, effective dose is useless.Yet, for certain given situation, can determine this effective dose with normal experiment, the clinicist can judge.
For the purposes of the present invention, effective dosage is for giving individual about 0.01 mg/kg to 50 mg/kg, preferably the thermal induction efflux body of 0.05 mg/kg to 10 mg/kg body weight.
Pharmaceutical composition also can contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent (for example tumor antigen) administration.This term refers to some medicament carriers like this; They itself do not induce generation to accepting the individual deleterious antibody of said composition, and do not have undue toxicity after the administration.These carriers are well known to those of ordinary skill in the art.(Mack Pub.Co. can find discussing fully about pharmaceutically acceptable excipient in N.J.1991) at Remington ' s Pharmaceutical Sciences.
Acceptable carrier can contain liquid on the therapeutic composition Chinese materia medica, as water, saline, glycerol and ethanol.In addition, also may there be complementary material in these carriers, as wetting agent or emulsifying agent, pH buffer substance etc.In addition, can also contain immunological adjuvant in the immune composition.
Usually, therapeutic composition can be made injectable agent, for example liquid solution or suspension; Also can be made into before injection, be fit to allocate in solution or the suspension, the solid form of liquid-carrier.
In case be made into compositions of the present invention, can directly give object with it.The object of waiting to prevent or treating can be an animal; Especially people.
Treatment or the prophylactic medicament (comprising vaccine) that contains the thermal induction efflux body of the present invention, can oral administration, in subcutaneous, the Intradermal, intracavity, tumor or modes such as diseased region, lymph node, intravenous injection or heeling-in use.The therapeutic dose scheme can be single agent scheme or multi-agent scheme.
Thermal induction efflux body of the present invention can be used for treatment and prophylaxis of tumours and infectious disease to be taken place, and tumor and the infectious disease that has taken place had therapeutical effect.Representational example comprises (but being not limited to): various tumors such as pulmonary carcinoma, breast carcinoma, hepatocarcinoma, gastric cancer, the esophageal carcinoma, cancer of pancreas, colorectal cancer, melanoma, renal carcinoma, malignant lymphoma, leukemia, cervical cancer, ovarian cancer, nasopharyngeal carcinoma, oral cancer, osteosarcoma, cerebral glioma, bladder cancer, multiple myeloma etc. and all kinds of infectious disease such as antibacterial, virus, fungus, parasitic infection comprise the prevention and the treatment of acquired immune deficiency syndrome (AIDS), various viral hepatitis etc.
Major advantage of the present invention is:
(1) the HS EXO HSP molecule that contains multiple chemotactic factor, costimulatory molecules, MHC molecule and carry tumor antigen information, the feature of all vaccines before therefore it includes substantially, be a kind of artificial antigen presentation vesicle (artificial antigen presenting vesicles, AAPV).Because this efflux body comes from tumor cell, carries tumor antigen, and the characteristic of the necessary costimulatory signal of efficient tumor vaccine of nearly all present development is arranged again, therefore can be used as a kind of new generation vaccine of acellular.The new generation vaccine that this acellular is structural can effectively induce body to produce anti-tumor immune response efficiently, has effectively excited specificity and non-specific antineoplastic immune, and its recurrence and transfer are treated and prevented to tumor.
(2) this new and effective tumor vaccine imports without genetic modification and exogenous gene, and is therefore little to the effect of body potential hazard, do not have the difficult problem of ethics, no individual limit.Prepare simple, be novel biological agent and the vaccine that a kind of utmost point has potential applicability in clinical practice, having important use is worth and good market prospect, to effective control tumor to develop, improve the treating malignant tumor present situation significant, great social benefit and economic benefit will be arranged.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The preparation of thermal induction efflux body
Get good (80% is paved with) the human lung adenocarcinoma cell A549 of growth conditions, carry out 43 ℃ of heat shock 1h earlier after, 37 ℃ are recovered 4h, collect culture supernatant, collect the tumor cell culture supernatant without heat shock simultaneously.4 step centrifuging are separated.I.e. 4 ℃ of centrifugal 300g, 10 minutes; 1,200g, 30 minutes; 10,000g, 30 minutes, last, ultracentrifugation 100,000g 60 minutes, the precipitation that is obtained is thermoinducible cell efflux body.This thermal induction efflux body can be directly as tumor vaccine or its main active efficiently.
Repeat above-mentioned steps, make the thermal induction efflux body with colon cancer cell CT26, mouse lung adenocarcinoma cell 3LL cell, mouse melanin tumor cell B16, mouse hypertrophy cell tumor T815 respectively.
Embodiment 2
The evaluation of thermal induction efflux body
The efflux body suspension is washed 2 times with PBS, through the fixing 1h of 4 ℃ of fixatives (2% paraformaldehyde, 0.25% glutaraldehyde), washes with PBS and to make suspension one time and drip sheet, and 1% osmic acid is fixed, gradient alcohol dehydration, and transmission electron microscope observing is taken the photograph sheet after the plumbous uranium dyeing of ultrathin section.The result as shown in Figure 1, this efflux body is the vesicle of the 50-100nm of homogeneous.
Adopting Western-blot to analyze its albumen forms, the result as shown in Figure 2, this efflux body is expressed MHC I quasi-molecule, Mac-1, TfR (Transferin receptor, TfR), the lysosome related membrane protein (lysosome-associated membrane protein, LAMP) and HSC70.
Embodiment 3
The thermal induction efflux body contains chemotactic factor, MHC molecule and heat shock protein molecule
Efflux body is surveyed protein concentration through the BCA method, adds sample-loading buffer.Separate in the 15%SDS-polyacrylamide gel electrophoresis, every hole adds efflux body 30ug, electrotransfer is to nitrocellulose filter (0.45u) (Bio-Rad company), different one resist in 4 ℃ in conjunction with 12h after, add corresponding two resistive connections and close 2h, develop by enhanced chemiluminescence test kit (Amersham Life Sciences) description.
The result contains a large amount of chemotactic factors, MHC molecule and heat shock protein molecule as shown in Figure 3 in the efflux body.
Embodiment 4
The thermal induction efflux body is to the chemotaxis of dendritic cell and T cell
Get 1 * 10
6Dendritic cell or T cell are resuspended among the RPMI 1640 of the 0.1 usefulness l that contains 0.5%BSA, add Transwell chamber (the 5 μ pore size that are paved with the 107B cell monolayer in advance; Purchase company in Costar) to go up in the hole, in the hole, total amount was 0.6ml under different dilution culture supernatant added after above-mentioned efflux body stimulated.In 37 ℃ of 5%CO
2After incubator was hatched 4h, the cell under collecting in the hole behind the FACS labelling, counted on flow cytometer.
The result as shown in Figure 4, efflux body chemotactic DC (CD11c
+) and the quantity of T cell obviously increase.
Embodiment 5
The thermal induction efflux body stimulates the facilitation of T cell proliferation to dendritic cell
Present embodiment adopts the conventional 3H-TdR method of mixing to analyze, and method is as follows.Separate the normal circumference blood lymphocyte, suspend, inject T cell nylon hair post, put 37 ℃, 5%CO2 incubator and cultivated 1 hour, go out NA cell, regulate cell concentration to 2 * 10 as reacting cells (being the T cell of purification) with complete medium
6/ ml adds culture plate at the bottom of 96 hole circles, 100 μ l/ holes; Get the 7th day DC of the thermal induction efflux body 20 μ g/ml sensitization 24h of preparation among the embodiment 1, after the deactivation of 4000rad lonizing radiation, with 2 * 10
3, 6 * 10
3, 2 * 10
4/ hole/100 μ l add each hole of having contained reacting cells respectively, cumulative volume 200 μ l, 3 every group multiple holes.Put and cultivate 5 days (120 hours) in 37 ℃, 5%CO2 incubator, finish to add in preceding 18 hours 3H-TdR (0.5 μ Ci/ hole) in cultivating.Collecting cell, liquid scintillation counter detects the cpm value, and the result represents with 3 hole meansigma methodss.
The result as shown in Figure 5.This thermal induction efflux body can directly stimulate or strengthen dendritic cell and stimulate the Allogeneic T proliferation of lymphocytes.
Embodiment 6
The thermal induction efflux body is to the inhibitory action of tumor growth
The right shank subcutaneous vaccination 2 * 10 of C57BL/6 mice
5The CT-26 mouse colonic cell, random packet, 10 every group, respectively at the efflux body in offside shank subcutaneous injection CT-26 cell source, 15 μ g/100 μ l/ are only after 3 days.Carried out 1 time in 2 days at every interval, totally 4 times.4 week of treatment, mice was put to death in the back, and the title tumor is heavy
The result as shown in Figure 6.Adopt novel tumor efficient vaccine provided by the invention (thermal induction efflux body), can obviously suppress growth of tumor.
In addition, for making the thermal induction efflux body with colon cancer cell CT26, mouse lung adenocarcinoma cell 3LL cell, mouse melanin tumor cell B16, mouse hypertrophy cell tumor T815 among the embodiment 1, also obtained similar result.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. an efflux body is characterized in that, it be by the secretion of the generation of cell after thermal induction to extracellular vesicle, wherein said cell is selected from down group: tumor cell, pathogen and by the cell of pathogenic infection;
Described vesicle has following feature:
(a) has the vesicle structure that diameter is 50-100nm;
(b) contain the immune molecule that is selected from down group: tumor antigen or pathogen antigen, MHC molecule, adhesion molecule and costimulatory molecules;
(c) contain the chemotactic factor that is selected from down group: MIP-1 α, MIP-1 β, MIP-3 β, MCP-1 and IP-10;
(d) contain the heat shock protein molecule.
2. efflux body as claimed in claim 1 is characterized in that, described efflux body external can chemotactic and immune cell activated, and inducing antigen-specific and nonspecific immune response in vivo.
3. efflux body as claimed in claim 1 is characterized in that, prepares with the following method:
(1) with cell at 43 ± 3 ℃ of following thermal induction 0.5-10 hours, under 37 ± 2 ℃, hatched 2-24 hour then, the collecting cell culture supernatant, wherein said cell is selected from down group: tumor cell, pathogen and by the cell of pathogenic infection;
(2) 300-500 * g low-speed centrifugal is removed cell, collects supernatant;
(3) centrifugal removal cell debris of 1000-3000 * g middling speed and organelle are collected supernatant;
(4) 7000-12000 * g high speed centrifugation is removed minicell device and impurity, collects supernatant;
(5) 80000-120000 * centrifugal 0.5-5 of g ultrahigh speed hour, collecting precipitation.
4. efflux body as claimed in claim 1 is characterized in that described cell is a tumor cell.
5. efflux body as claimed in claim 1, it is characterized in that described efflux body contains tumor antigen or pathogen antigen, MHC molecule, adhesion molecule, costimulatory molecules, MIP-1 α, MIP-1 β, MIP-3 β, MCP-1IP-10 and heat shock protein molecule simultaneously.
6. a pharmaceutical composition is characterized in that, contains the described efflux body of claim 1 and the pharmaceutically acceptable carrier of effective dose.
7. pharmaceutical composition as claimed in claim 7 is characterized in that it is a vaccine.
8. a method for preparing efflux body is characterized in that, may further comprise the steps:
(1) with cell at 43 ± 3 ℃ of following thermal induction 0.5-10 hours, under 37 ± 2 ℃, hatched 2-24 hour then, the collecting cell culture supernatant, wherein said cell is selected from down group: tumor cell, pathogen and by the cell of pathogenic infection;
(2) 300-500 * g low-speed centrifugal is removed cell, collects supernatant;
(3) centrifugal removal cell debris of 1000-3000 * g middling speed and organelle are collected supernatant;
(4) 7000-12000 * g high speed centrifugation is removed minicell device and impurity, collects supernatant;
(5) 80000-120000 * centrifugal 0.5-5 of g ultrahigh speed hour, collecting precipitation was efflux body.
9. method as claimed in claim 8, the condition of each step is as follows:
(1) places 41-45 ℃ of thermal induction about 1 ± 0.5 hour, hatched the collecting cell culture supernatant 4-8 hour at 37 ℃ then;
(2) the centrifugal 5-10 of 300-500 * g minute, collect supernatant;
(3) 1000-3000 * g is centrifugal 20 ± 10 minutes, collects supernatant;
(4) 7,000-12, centrifugal 30 ± 15 minutes of 0000 * g collects supernatant;
(5) 80,000-120, centrifugal 1 ± 0.5 hour of 000 * g, collecting precipitation, PBS washing, efflux body after the acquisition thermal induction.
10. the purposes of efflux body as claimed in claim 1 is characterized in that, is used for preparation prevention or treatment tumor and infectious disease medicament.
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| CN101461942B (en) * | 2008-08-27 | 2010-07-21 | 时宏珍 | Dendritic cell vaccine carrying recombinant human HSP70 polypeptide complexes, preparation method and application |
| WO2013178079A1 (en) * | 2012-05-31 | 2013-12-05 | 湖北盛齐安生物科技有限公司 | Tumor vaccine and preparation method thereof |
| CN104726410A (en) * | 2013-12-23 | 2015-06-24 | 浙江大学 | Exosome with immunosuppression function and application thereof |
| WO2018130108A1 (en) * | 2017-01-12 | 2018-07-19 | 湖北盛齐安生物科技股份有限公司 | Oral tumor vaccine and use thereof |
| CN109310712A (en) * | 2016-06-08 | 2019-02-05 | 莱赛特制药有限公司 (莱赛特制药) | For vesica outside the human blood platelets lysate derived cell of medicine |
| CN110846281A (en) * | 2018-08-20 | 2020-02-28 | 中国科学院过程工程研究所 | Exosome-based anti-tumor vaccine |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2785543B1 (en) * | 1998-11-05 | 2003-02-28 | Inst Nat Sante Rech Med | MODIFIED EXOSOMES AND USES |
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| WO2013178079A1 (en) * | 2012-05-31 | 2013-12-05 | 湖北盛齐安生物科技有限公司 | Tumor vaccine and preparation method thereof |
| CN103446580A (en) * | 2012-05-31 | 2013-12-18 | 湖北盛齐安生物科技有限公司 | Tumor vaccine and preparation method thereof |
| CN103446580B (en) * | 2012-05-31 | 2016-08-03 | 湖北盛齐安生物科技有限公司 | A kind of tumor vaccine and preparation method thereof |
| US9844508B2 (en) | 2012-05-31 | 2017-12-19 | Hubei Soundny Biotechnology Co., Ltd. | Tumor vaccine and method for producing the same |
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| CN109310712A (en) * | 2016-06-08 | 2019-02-05 | 莱赛特制药有限公司 (莱赛特制药) | For vesica outside the human blood platelets lysate derived cell of medicine |
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| CN108295250A (en) * | 2017-01-12 | 2018-07-20 | 湖北盛齐安生物科技股份有限公司 | A kind of oral tumor vaccine and application thereof |
| CN110846281A (en) * | 2018-08-20 | 2020-02-28 | 中国科学院过程工程研究所 | Exosome-based anti-tumor vaccine |
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